grapevine carbohydrate and nitrogen allocation during ...€¦ · grapevine carbohydrate and...

Grapevine carbohydrate and nitrogen allocation during berry maturation:

Implications of source-sink relations and water supply

Gerhard C. Rossouw

BSc (Agric), University of Stellenbosch

MSc (Agric), University of Stellenbosch

Submitted in total fulfilment of the requirements of the degree of

Doctor of Philosophy

March 2017

National Wine and Grape Industry Centre

School of Agricultural and Wine Sciences - Charles Sturt University

Faculty of Science

Wagga Wagga, NSW, Australia

Table of contents

Table of contents

Certificate of authorship ........................................................................................................ i

Editorial note ........................................................................................................................ii

Acknowledgements.............................................................................................................. iii

Statement of contribution to publications ...............................................................................iv

Abstract ..............................................................................................................................vi

Chapter 1: General introduction and research aims ................................................................. 1

1.1. Introduction and aims ............................................................................................ 1

1.2. References ............................................................................................................ 7

Chapter 2: Literature review ................................................................................................. 9

2.1. Introduction .......................................................................................................... 9

2.2. Grapevine non-structural carbohydrates................................................................... 10

2.2.1. Roles of carbohydrate reserves in grapevines ................................................... 12

2.2.2. Mobilisation of carbohydrate reserves ........................................................... 13

2.2.3. Soluble sugar specific roles .......................................................................... 15

2.2.4. Minor sugars and sugar alcohols ................................................................... 19

2.2.5. Factors influencing carbohydrate reserve distribution in grapevines .................. 23

2.3. Grapevine nitrogen............................................................................................... 30

2.3.1. Roles of N in grapevines ............................................................................... 32

2.3.2. Fruit N accumulation .................................................................................... 33

2.3.3. Factors influencing N distribution .................................................................. 34

2.3.4. Amino acids ................................................................................................. 37

Table of contents

2.4. Concluding remarks .............................................................................................40

2.5. Literature cited ....................................................................................................41

Chapter 3: Paper 1 ............................................................................................................... 52

3.1. Main objective for paper 1 .....................................................................................52

Carbohydrate distribution during berry ripening of potted grapevines: Impact of water

availability and leaf-to-fruit ratio ......................................................................................53

Chapter 4: Paper 2 ............................................................................................................... 64


Implications of the presence of maturing fruit on carbohydrate and nitrogen distribution in

grapevines under postveraison water constraints ..................................................................65

Chapter 5: Paper 3 ............................................................................................................... 79


5.2. Supporting information ........................................................................................79

Vitis vinifera root and leaf metabolic composition during fruit maturation: Implications of

defoliation ......................................................................................................................80

Chapter 6: Paper 4 ............................................................................................................. 122

6.1. Main objective for paper 4 ................................................................................... 122

6.2. Supplementary material..................................................................................... 122

Impact of post-véraison leaf source limitation on the metabolic profile of Vitis vinifera cv.

Shiraz berries................................................................................................................ .123

Chapter 7: General conclusions and future work .................................................................... 158

Appendix ..........................................................................................................................166

Certificate of authorship

i

Certificate of authorship

I, Gerhard Rossouw, hereby declare that this submission is my own work and that, to

the best of my knowledge and belief, it contains no material previously published or

written by another person nor material which to a substantial extent has been accepted

for the award of any other degree or diploma at Charles Sturt University or any other

educational institution, except where due acknowledgment is made in the thesis.Any

contribution made to the research by colleagues with whom I have worked at Charles

Sturt University or elsewhere during my candidature is fully acknowledged.

I agree that this thesis be accessible for the purpose of study and research in accordance

with the normal conditions established by the Executive Director, Library Services,

Charles Sturt University or nominee, for the care, loan and reproduction of thesis,

subject to confidentiality provisions as approved by the University.

Gerhard Rossouw

10 March 2017

Editorial note

ii

Editorial note

Thesis structure

This thesis contains two accepted and two submitted peer review publications. The

English style in the thesis is mainly Australian English in accordance with Charles Sturt

University`s academic manual available at http://www.csu.edu.au/acad_sec/academic-

manual/hcontm.htm (section 4: Regulations for presentations of print theses, other

examinable print works and the written component of examinable multi-media work).

However, American English is used in three of the publications (chapters 3, 4 and 6), in

accordance with the preference of the respective journals.

Chapters 1 and 2 have been referenced based on the American Psychological

Association (APA 6th edition), in line with Charles Sturt University`s referencing

style. The four publications (chapters 3, 4, 5 and 6) are referenced in accordance to

the respective journal’s preferred referencing style.

The APA 6th edition is available at:

http://www.csu.edu.au/division/library/ereserve/pdf/apa-6ed.pdf.

http://www.csu.edu.au/acad_sec/academic-manual/hcontm.htm

http://www.csu.edu.au/acad_sec/academic-manual/hcontm.htm

http://www.csu.edu.au/division/library/ereserve/pdf/apa-6ed.pdf

Acknowledgements

iii

Acknowledgements

I would like to acknowledge my principle supervisor, Dr Bruno Holzapfel, for

mentoring me and offering me guidance during my PhD studies. I also thank my

co-supervisors, Dr Jason Smith, Dr Celia Barril and Prof Alain Deloire for their

valuable support and contributions during this research.

I thank Robert Lamont, David Foster and Peter Carey for providing me with technical

assistance during the three pot experiments. I also thank Beverley Orchard for her

contributions toward the statistical planning of my experiments and for conducting

statistical analyses. I thank Katja Suklje for her contributions to the GC/MS analysis.

I would also like to acknowledge Wine Australia for financially supporting my PhD

project through stipend and operating fund contributions. Likewise, I would like to

thank the National Wine and Grape Industry Centre, Charles Sturt University, for

providing me with a postgraduate scholarship.

Finally, I would like to thank my family and friends for continuously supporting me

throughout my studies.

Statement of contribution to publications

iv


The following publications are included in this thesis:

Rossouw, G. C.,Smith, J. P., Barril, C., Deloire, A., & Holzapfel, B. P. (2017).

Carbohydrate distribution during berry ripening of potted grapevines: Impact of water

availability and leaf-to-fruit ratio. Scientia Horticulturae, 216, 215-225.

Rossouw, G. C.,Smith, J. P., Barril, C., Deloire, A., & Holzapfel, B. P. (2017).

Implications of the presence of maturing fruit on carbohydrate and nitrogen distribution

in grapevines under postveraison water constraints. Journal of the American Society for

Horticultural Science, 142(2), 71-84.

Rossouw, G. C.,Orchard, B. A.,Suklje, K., Smith, J. P., Barril, C., Deloire, A., &

Holzapfel, B. P. (2107). Vitis vinifera root and leaf metabolic composition during fruit

ripening: Implications of defoliation. Physiologia Plantarum (Under review).

Rossouw, G. C.,Suklje, K., Orchard, B. A., Smith, J. P., Barril, C., Deloire, A., &

Holzapfel, B. P. (2107). Impact of post-véraison leaf source limitation on the metabolic

profile of Vitis vinifera cv. Shiraz berries. Plant Physiology and

Biochemistry (Under review).


v

All papers were written by Gerhard Rossouw as first author and contributions by co-

authors during the writing process were made on the understanding that these papers

would contribute to this thesis and should therefore represent the work of Gerhard

Rossouw. As such the papers do represent the first author's development of concepts,

hypothesis formation, experimental design and implementation, data analysis and

interpretation. All co-authors support the use of the papers as experimental chapters of

this thesis.

Date

Prof Alain Deloire

Dr Jason Smith Date

Dr CeliEi Barril Date

Dr Katja Suklje

Ms Beverley Orchard Date

V

( Dr Bruno Holzapfel

Date

Date

Abstract

vi

Abstract

The post-véraison period is characterised by rapid berry sugar accumulation, and

therefore, a substantial carbon (C) sink demand. In contrast to sugar accumulation, berry

nitrogen (N) incorporation is often variable at distinct stages of the season, and does not

necessarily predominate after véraison. Nevertheless, important alterations in fruit N

content and composition likely occur during the post-véraison period. The fruit sugar

requirement is sourced from leaf photoassimilation, however, total non-structural

carbohydrate (TNC) remobilisation from perennial tissues may provide an alternative C

source when photoassimilation is insufficient. N is translocated from the roots, leaves

and shoots to the berries after véraison, when soil N uptake is expected to be restricted.

The grapevine leaf-to-fruit ratio and water availability are major factors influencing

canopy photoassimilation, and subsequently, the allocation of TNC among perennial

organs and the fruit. Likewise, the leaf area and water supply also affect N distribution

between the perennial and reproductive organs. Restricting the post-véraison leaf area

and/or vine water availability may induce reserve TNC and N utilisation, and could

subsequently be detrimental toward TNC and N storage. Therefore, the overall aim of

this study was to evaluate the post-véraison distribution and partitioning of TNC and N

among the different grapevine organs, as influenced by source-sink relationships and

water supply.

Three distinct pot experiments, using three-year-old own rooted Vitis vinifera

grapevines, were conducted during the post-véraison period. For the first experiment

(2013-14), within each leaf-to-fruit ratio treatment (full and 50% leaves), grapevines

were grown under full or 50% reduced irrigation. Changes in dry biomass, and starch

Abstract

and total sugar concentrations were monitored in the roots, trunks, shoots and leaves.

Berry sugar and anthocyanin accumulation were also assessed. During the second

experiment (2014-15), grapevines were grown with or without fruit from véraison, with

water constraints sustained throughout the experiment. The root, trunk, shoot and leaf

structural biomass, starch, total soluble sugar, total N, and amino N concentrations were

determined, while the fruit sugar and N accumulation were also assessed. The root

glucose, fructose and sucrose contents were additionally measured through high

performance liquid chromatography. The final experiment (2015-16) consisted of a full

leaf area control (100 primary shoot leaves and all laterals) and two defoliation

treatments (25 primary leaves only and no leaves), and the vines were well watered

throughout. Changes in fruit sugar, anthocyanin and N content, and juice yeast

assimilable N (YAN) concentration were monitored. The root and leaf starch and N

concentrations were also determined, and primary metabolite abundance in the fruit,

roots and leaves was measured via untargeted gas chromatography/mass spectrometry

analysis.

A sustained post-véraison water constraint caused root starch depletion, concurrent with

the phase of rapid fruit sugar accumulation. As soon as fruit sugar accumulation slowed,

roots accumulated starch reserves. When water constraints were sustained, root sucrose

accumulation coincided with starch hydrolysis during peak fruit sugar accumulation.

Leaf N depletion corresponded with fruit N accumulation, while the roots of defruited

vines stored N reserves. Fruit sugar and anthocyanin accumulation continued during the

post-véraison period in completely defoliated vines, albeit at reduced rates. However,

defoliation had little impact on the fruit total N content, although the juice YAN

increased after complete and partial defoliation. Most sugars and organic acids depleted

vii

Abstract

in the roots after defoliation, while many amino acids accumulated in roots and

remaining leaves. Defoliation suppressed the root and leaf myo-inositol concentration,

and similarly affected its sugar and organic acids derivatives. Shikimate pathway-

derived amino acids accumulated in roots after full defoliation, while organic acids

derived from this pathway depleted in remaining leaves after partial defoliation.

Although restricted, glucose, fructose and sucrose, and most minor sugars and sugar

alcohols still accumulated in the fruit under leaf source restriction or absence. Fruit

arginine accumulated after partial or full defoliation, while the content of various

shikimate pathway products (such as anthocyanins and phenolic acids) increased or

decreased to different extents in response to leaf area availability.

Among the different grapevine organs, TNC reserves were most abundant in the roots.

Root TNC reserves subsequently supported the post-véraison fruit sugar content when

canopy photoassimilation restriction was induced by water constraints and/or a reduced

leaf area. The root starch reserves were replenished as soon as fruit sugar accumulation

slowed, even occurring from a few weeks before harvest. During sustained post-

véraison water constraints, root starch hydrolysis during peak fruit sugar accumulation

yielded sucrose, which is subsequently transportable to the berries to support the fruit

sugar content. The concentration of myo-inositol and its derivatives (e.g. galactinol and

raffinose), in addition to that of amino acids derived from the shikimate pathway (e.g.

phenylalanine), were significantly affected in roots during a post-véraison leaf C source

limitation or absence. Myo-inositol metabolism seemingly played a distinct role during

root starch remobilisation while berry sugar accumulation occurred. In contrast to the

roots being an important TNC source when photoassimilation was restricted by limited

water supply, leaf N seemed to be a significant contributor to fruit N content during the

viii

Abstract

ix

sustained water constraints. The presence of ripening fruit in conjunction with water

constraints, subsequently prevented root N storage between véraison and fruit maturity.

While root total N concentration was little affected by defoliation, root amino N

composition was altered, for example prompting arginine accumulation. Arginine also

subsequently accumulated in the fruit, effectively increasing the juice YAN of

defoliated vines. This study provided a novel illustration of post-véraison TNC and N

partitioning and distribution under differing water availability and/or source-sink

relationships. The results contribute to the understanding of grapevine reserve TNC

utilisation during a period of substantial fruit C demand. Furthermore, although the

extent of fruit N sink requirement after véraison is not as clear as the corresponding fruit

C demand, the study contributes to understanding grapevine N reserve utilisation during

fruit maturation.

Chapter 1: General introduction and research aims

1


1.1. Introduction and aims

The period between the onset of berry softening (véraison) and fruit maturity of

grapevines is characterised by berry sugar accumulation and cell expansion. Berry sugar

accumulation occurs rapidly from the start véraison, while slower accumulation could

take place towards the latter stages of berry maturation (McCarthy & Coombe, 1999).

Nitrogenous and phenolic compounds, such as amino acids and anthocyanins, are some

of the other major metabolites incorporated into the berries during berry maturation.

Some organic acids (predominantly malic acid) which accumulates earlier in berry

development, are in contrast degraded after véraison (Iland, Dry, Proffitt, & Tyerman,

2011). During this period of berry sugar accumulation, the grapevine water supply and

the relationship between the vegetative and reproductive organ biomasses, are major

determinants of non-structural carbohydrate (TNC) (Holzapfel, Smith, Field, & Hardie,

2010) and nitrogen (N) (Roubelakis-Angelakis & Kliewer, 1992) allocation between the

perennial (mainly the roots) and reproductive (fruit) grapevine structures.

Carbon (C) assimilated during leaf photosynthesis is primarily translocated to the

ripening berries (Williams, 1996). However, when the canopy leaf area is low or

deliberately reduced in relation to the total fruit biomass, TNC reserves are thought to

be remobilised to support the fruit sugar content (Candolfi-Vasconcelos, Candolfi, &

Koblet, 1994). Grapevines store a large proportion of their TNC in the roots (Holzapfel

et al., 2010), and the root system could provide an alternative source of C to supplement

the supply from canopy photosynthesis. However, as the major perennial structure,

TNC reserve storage in the roots is also essential prior to dormancy, and the roots


2

therefore start to replenish TNC reserves from flowering (Holzapfel et al., 2010). These

reserves are subsequently utilised towards vegetative and reproductive development

from budburst the next season (Bennett, Javis, Creasy, & Throught, 2005; J. P. Smith &

Holzapfel, 2009). Water constraints (Escalona, Flexas, & Medrano, 1999) and/or a low

leaf-to-fruit ratio (Petrie, Trought, & Howell, 2000) are detrimental to overall canopy

photoassimilation and can, therefore, increase the allocation of the restricted available C

to the fruit in the expense of root reserve replenishment. Root TNC are mainly stored as

immobile starch molecules, and in order to facilitate the remobilisation of carbohydrate

reserves, the starch is hydrolysed, resulting in the accumulation of phloem mobile

soluble sugars (A. M. Smith, Zeeman, & Smith, 2005). Conditions leading to TNC

reserve remobilisation, therefore, reduce the ratio of starch to soluble sugars in the

storage tissues, and subsequently alter the composition of carbohydrates within the

related organs, such as the roots.

Amino acid incorporation enables the accumulation of organic N in the berries. During

the post-véraison period, the soil N uptake is likely restricted or absent, and

translocation of N occurs from the roots, leaves and shoots towards the berries

(Conradie, 1991). The mature leaves and roots are, however, the major sources of amino

acids in higher plants, from where the amino acids are translocated to sink organs to

support metabolism or development (Rentsch, Schmidt, & Tegeder, 2007). Therefore,

similar to TNC, the source requirements placed upon the roots and leaves to supply

towards the fruit N sink, may be affected by the biomass ratio of the perennial and

annual structure. Furthermore, water supply during berry maturation can influence the

allocation of N between the perennial and annual structure (Holzapfel, Watt, Smith,

Suklje, & Rogiers, 2015). Also similar to TNC, the perennial structure of grapevines


3

stores N reserves, and trunk and root N storage start before berry maturation

(Roubelakis-Angelakis & Kliewer, 1992). These N reserves are also utilised during

early shoot growth after budburst the following season. Storage proteins are degraded in

the N source organs of plants, resulting in the accumulation of free amino acids

(Masclaux-Daubresse et al., 2010), which are subsequently transportable to sink organs

such as the ripening fruit. The grapevine source-sink biomass ratio and water status,

could therefore alter the composition of N containing metabolites in the source organs

(especially mature leaves and the roots) and in the berries.

Ultimately, the grapevine leaf-to-fruit ratio and water supply are major determinants of

important fruit quality parameters during berry maturation. In addition to the fruit sugar

and N content, intermediates of C metabolism (e.g. organic acids ) are also likely

impacted (López-Bucio, Nieto-Jacobo, Ramırez-Rodrıguez, & Herrera-Estrella, 2000).

These compounds are important precursors for the biosynthesis of secondary

metabolites, such as phenolic compounds (e.g. anthocyanins). These secondary

metabolites are important fruit quality parameters, and contribute to wine colouration

and aromatic potential. For the winemaking process and desired wine style, berry sugar

content is crucial for alcoholic fermentation, while berry N is assimilated by the yeasts

and contribute to the development of aromatic compounds. The partitioning, allocation

and distribution of TNC and N during the period between véraison and fruit maturity

are essential for both, their accumulation in the fruit and in the storage tissues.

The main aim of this research project was to determine the impact of both grapevine

water status, and the ratio between the sizes of vegetative and reproductive organs, on

the allocation of TNC and N between the reserve organs and the fruit during berry


4

maturation. In the context of this study, the allocation includes compound partitioning

and distribution. The current literature regarding this research topic is discussed in

chapter 3. However, the outcomes of the present study contribute to the following gaps

in the literature:

Gap 1:

Carbohydrate reserve remobilisation from perennial tissues (roots and trunks) towards

post-véraison berries, when the canopy leaf area is restricted, has been illustrated

through 14

C studies. However, the extent or importance of the contribution from TNC

reserves towards the fruit sugar content when leaf photoassimilation is restricted, still

needs to be quantified. To the best of our knowledge, the contribution of TNC reserves

to berry sugar accumulation has only been shown when canopy photoassimilation is

limited by a restricted leaf area. Inhibition of grapevine canopy photoassimilation by

water constraints, however, also commonly occurs during berry maturation, especially

in warmer climate regions. Furthermore, to investigate the effects of the grapevine crop

load or post-véraison water constraints on TNC reserve dynamics, prior studies only

evaluated the TNC reserve abundance on a concentration basis, and exclusively at a

particular stage of the season (mostly at budburst). The kinetics of TNC allocation

between the different grapevine organs during berry maturation still requires

investigation. In order to contribute to the above mentioned gaps in the literature, the

following research objective was addressed:

1. To investigate the interactive effects of the leaf-to-fruit ratio and grapevine water

status during two phases of berry sugar accumulation (rapid and slow) on the

carbohydrate distribution between the different grapevine organs (Chapter 3).


5

Gap 2:

Grapevine berries are a post-véraison sink for both, TNC and N. The extent of the

utilisation of TNC and N reserves during berry maturation, especially sourced from the

roots, largely determines starch and N reserve replenishment by fruit maturity. The

kinetics of TNC and N content development in the different grapevine organs during

berry maturation, have not yet been studied. Sustained water constraints during berry

maturation are likely, a major determinant of the extent of root TNC and N reserve

contribution towards berry sugar and N content. Water constraints may influence the

partitioning of TNC (starch and soluble sugars) and N (total and amino N) in source and

sink organs, which contributes to their mobilisation between the different organs. More

work is required to better understand the implications of a sustained post-véraison water

constraint on the composition of TNC and N within the perennial (especially the roots)

grapevine organs. The subsequent consequences on reserve starch and N replenishment

by fruit maturity, additionally, needs clarification. The following research objective was

aimed at contributing to the above mentioned gaps:

2. To determine how the presence or absence of fruit during sustained post-véraison

water constraints influences the allocation of carbohydrates and N between the

different grapevine organs (Chapter 4).

Gap 3:

Carbon and N metabolism in grapevine source organs are crucial during the process of

TNC and N distribution to sink organs. The root system and leaves of grapevines

represent two major sources of TNC and N. When the leaf area is restricted during berry


6

maturation, the source demand placed upon the roots is likely amplified. The

composition of primary metabolites within the roots and leaves during the post-véraison

period could regulate C and N translocation towards sink organs, such as the berries. To

the best of our knowledge, no previous studies have evaluated the profiles of primary

grapevine metabolites in C and N source organs during the berry maturation period.

Yet, an untargeted evaluation of the change in source organ primary metabolite

abundance may be beneficial towards understanding the underlying functioning of

primary metabolism during reserve TNC and N utilisation. Restricting the C and N

source availability through defoliation, may enable a novel assessment of root C and N

metabolism and the subsequent utilisation of these compounds during berry maturation.

The following objective was established in order to enhance the understanding of

grapevine source organ C and N metabolism during berry maturation:

3. To assess the implications of defoliation on fruit sugar and N accumulation in

conjunction with the carbohydrate, N and primary metabolite composition of the

major grapevine source organs (roots and leaves) (Chapter 5).

Gap 4:

The post-véraison canopy leaf area availability affects the development of berry

composition. As an important C and N source, limiting the grapevine leaf area can be

detrimental towards berry sugar, N and anthocyanin accumulation. Although a

few previous studies observed primary berry metabolism during the post-véraison

period, these studies were mostly aimed at comparing different genotypes. More work is

needed to observe the implications of limiting or eliminating the leaf C and N

source, on primary metabolism in the berries. Studying alterations in the contents of


7

untargeted primary metabolites within maturing berries, may contribute to

understanding the changing berry composition as influenced by a restricted leaf

area availability. The following objective is intended to contribute to the above

mentioned gaps:

4. To study the implications of defoliation on the post-véraison metabolic composition

of grapevine berries (Chapter 6).

Overall, this study is intended to improve the understanding of TNC and N reserve

utilisation during berry maturation, and how the extent of this utilisation ultimately

affects berry composition. The implications of water supply, and the relationship

between reproductive and vegetative grapevine organ sizes during berry maturation are

of particular interest.

1.2. References

Bennett, J., Javis, P., Creasy, G. L., & Throught, M. C. T. (2005). Influence of

defoliation on overwintering carbohydrate reserves, return bloom, and yield of

mature Chardonnay grapevines. American Journal of Enology and Viticulture,

56(4), 386-393.

Candolfi-Vasconcelos, M. C., Candolfi, M. P., & Koblet, W. (1994). Retranslocation of

carbon reserves from the woody storage tissues into the fruit as a response to

defoliation stress during the ripening period in Vitis vinifera L. Planta, 192(4),

567-573.

Conradie, W. (1991). Distribution and translocation of nitrogen absorbed during early

summer by two-year-old grapevines grown in sand culture. American Journal of

Enology and Viticulture, 42(3), 180-190.

Escalona, J. M., Flexas, J., & Medrano, H. (1999). Stomatal and non-stomatal

limitations of photosynthesis under water stress in field-grown grapevines.

Australian Journal of Plant Physiology, 26, 421-433.

Holzapfel, B. P., Smith, J. P., Field, S. K., & Hardie, W. J. (2010). Dynamics of

carbohydrate reserves in cultivated grapevines. Horticutural Reviews, 37, 143-

211.

Holzapfel, B. P., Watt, J., Smith, J. P., Suklje, K., & Rogiers, S. Y. (2015). Timing of N

application and water constraints on N accumulation and juice amino N

concentration in Chardonnay grapevines. Vitis, 54(4), 203-211.


8

Iland, P., Dry, P., Proffitt, T., & Tyerman, S. D. (2011). The grapevine: from the

science to the practice of growing vines for wine. Adelaide, Australia: Patrick

Iland Wine Promotions Pty Ltd.

López-Bucio, J., Nieto-Jacobo, M. A. F., Ramırez-Rodrıguez, V., & Herrera-Estrella, L.

(2000). Organic acid metabolism in plants: from adaptive physiology to

transgenic varieties for cultivation in extreme soils. Plant Science, 160(1), 1-13.

McCarthy, M. G., & Coombe, B. G. (1999). Is weight loss in ripening grape berries cv.

Shiraz caused by impeded phloem transport? Australian Journal of Grape and

Wine Research, 5(1), 17-21.

Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., Chardon, F., Gaufichon, L.,

& Suzuki, A. (2010). Nitrogen uptake, assimilation and remobilization in plants:

Challenges for sustainable and productive agriculture. Annals of Botany, 105,

1141-1157.

Petrie, P., Trought, M., & Howell, S. (2000). Influence of leaf ageing, leaf area and crop

load on photosynthesis, stomatal conductance and senescence of grapevines

(Vitis vinifera L. cv. Pinot Noir) leaves. Vitis, 39(1), 31-36.

Rentsch, D., Schmidt, S., & Tegeder, M. (2007). Transporters for uptake and allocation

of organic nitrogen compounds in plants. FEBS letters, 581(12), 2281-2289.

Roubelakis-Angelakis, K. A., & Kliewer, W. M. (1992). Nitrogen metabolism in

grapevine. Horticultural Reviews, 14, 407-452.

Smith, A. M., Zeeman, S. C., & Smith, S. M. (2005). Starch degradation. Annual

Review of Plant Biology, 56, 73-98.

Smith, J. P., & Holzapfel, B. P. (2009). Cumulative responses of Semillon grapevines to

late season perturbation of carbohydrate reserve status. American Journal of


Williams, L. E. (1996). Grape. In E. Zamski & A. Schaffer (Eds.), Photoassimilate

distribution in plants and crops. Source-sink relationships (pp. 851-881). New

York, NY: Marcel Dekker.

Chapter 2: Literature review

9


2.1. Introduction

The period of grapevine berry maturation (from véraison to fruit maturity) coincides

with strong competition between the different grapevine organs for the utilisation of

photoassimilates and nutrients. During this period, temporary sinks (e.g. the fruit) and

permanent sinks (e.g. the roots) are competing for the accumulation of both non-

structural carbohydrates (TNC) (Holzapfel, Smith, Field, & Hardie, 2010) and nitrogen

(N) (Cheng, Xia, & Bates, 2004). Abiotic conditions, such as water constraints, and

vegetative and reproductive organ biomass balances, are major determinants of the

utilisation and distribution of TNC and N within grapevines (Cheng et al., 2004;

Escalona, Flexas, & Medrano, 1999; Petrie, Trought, & Howell, 2000; Roubelakis-

Angelakis & Kliewer, 1992).

The overall aim of this review is to summarise the existing literature regarding

grapevine TNC and N allocation between the different organs during berry maturation.

In the context of this review, the allocation of these compounds includes their respective

distribution, partitioning and utilisation within the different organs. This review is

focussed on grapevine related literature, however, information from other plant species

is mentioned when available evidence from grapevine related literature is insufficient.

In this review, a separate emphasis is placed upon the distributions of TNC and N in

grapevines. It is, nevertheless, important to note that inorganic N is required to allow

carbon (C) to be utilised for structural tissue growth, while TNC breakdown provides

adenosine triphosphate (ATP) and C skeletons to support the accumulation of inorganic


10

N (Stitt & Krapp, 1999). Although both TNC and N have distinct and essential roles

during berry ripening in plants, it is clear that the metabolisms of these compounds are

strongly linked.

As the grapevine water supply (Escalona et al., 1999), crop load (Poni, Lakso, Turner,

& Melious, 1994) and leaf area (Petrie, Trought, Howell, & Buchan, 2003) are major

determinants of leaf C assimilation, and because of the impact of vine water supply

(Araujo, Williams, & Matthews, 1995) and cropping (Rodriguez-Lovelle & Gaudillere,

2002) on N distribution, specific emphases are placed upon the effects of grapevine

water status and source-sink relations within this review. The goals are to summarise,

during fruit ripening: a) the physiological roles and utilisation of TNC, b) the

distribution of TNC, c) the functions of N, and d) the distribution of N within

grapevines. Information on TNC and N distribution during other stages of the annual

grapevine development cycle is included to enable a thorough description of the roles of

TNC and N in grapevines. The overall intention is to clarify the existing information,

and to identify literature gaps, thereby improving the knowledge regarding the effects of

TNC and N distribution on short- and long-term grapevine development and

productivity.

2.2. Grapevine non-structural carbohydrates

Carbohydrates in plants consist of structural carbohydrates (cellulose and

hemicellulose) and total non-structural carbohydrates (TNC, predominantly starch and

soluble sugars). Carbohydrates are synthesised during the day through the process of

photosynthesis in the leaves (photoassimilation), when atmospheric carbon dioxide

(CO2) is converted into carbohydrates. In grapevines, most assimilated C is allocated


11

towards the biosynthesis of structural cellulose (Winkler & Williams, 1938), which

cannot be further utilised, as Ccannot be recovered from cellulose (Kozlowski &

Pallardy, 1997). TNC are therefore accumulated, and utilised towards C distribution and

as a resource for reproductive and vegetative development. In the leaves, sucrose is

vastly biosynthesised during photoassimilation, and then becomes available for

distribution from the leaves to the rest of the plant, via the osmotic gradient of the

phloem vascular system. Some photoassimilates are also stored in the leaves during the

day as starch and, at night, this stored C is hydrolysed and further distributed within the

plant to continue the mobilisation of leaf assimilates in the absence of photosynthesis

(A. M. Smith & Stitt, 2007). Most of the TNC in plants are present as starch and stored

in different parts of the plant, especially in the roots, while soluble sugars generally only

represent a small part of total TNC (Scholefield, Neales, & May, 1978; Zufferey et al.,

2012). The various types of TNC found in plant tissues include sugar alcohols (e.g.

inositol and mannitol), monosaccharides (e.g. fructose, glucose and galactose),

disaccharides (e.g. sucrose and maltose), oligosaccharides (e.g. raffinose) and

polysaccharides (e.g. starch).

Fruit sugar accumulation initiates rapidly at the start of berry maturation, when the

berries start to soften (véraison) (Davies & Robinson, 1996). However, the

replenishment of TNC reserves in the permanent grapevine structure (especially the

roots) could start around flowering, and may continue throughout berry maturation,

dependent on the grapevine crop load (Holzapfel et al., 2010).


12

2.2.1. Roles of carbohydrate reserves in grapevines

The distribution of TNC in plants provides resources that are used towards generating

energy and structure, or are stored as TNC reserves (mainly as starch) in perennial

tissues (roots and trunks). In deciduous plants like the grapevine, TNC reserves play a

role in cold hardiness and the maintenance of metabolism during dormancy (Loescher,

McCamant, & Keller, 1990). TNC reserves also aid in the replenishment of damaged

tissues, contribute to fruit ripening, and improve the defence of plants against pests and

diseases (Holzapfel et al., 2010). TNC reserves in grapevines are also important for the

annual reestablishment of the vegetative growth of leaves and shoots, and can contribute

in determining the reproductive capacity (fruit yield) of grapevines for the following

season (Bennett, Javis, Creasy, & Throught, 2005; J. P. Smith & Holzapfel, 2009;

Zufferey et al., 2012).

During early season grapevine reproductive development, TNC reserves are utilised

during inflorescence development (J. P. Smith & Holzapfel, 2009) and aid in flower

induction, while TNC are also utilised during the processes of pollination, fertilisation,

and fruit set (Zapata, Deléens, Chaillou, & Magné, 2004). The TNC reserve availability

will therefore contribute to the fruitfulness and quality of fruiting canes in grapevines,

with the reserve status therefore playing an important role in determining the

fruitfulness of grapevines (Bennett et al., 2005). Later in the season, TNC reserves

could also contribute to fruit sugar accumulation. It was, for example, shown through

14C labelling that TNC reserves are directed from perennial plant organs towards the

fruit, when vines are excessively defoliated at the onset of fruit maturation. This

mobilisation of TNC reserves peaks during mid fruit ripening, a period when the berry


13

TNC sink requirement is especially strong (Candolfi-Vasconcelos, Candolfi, & Koblet,

1994).

The contribution of TNC reserves towards early season vegetative development (shoot

and leaf expansion), and towards root growth, is crucial until leaf photosynthesis

becomes the primary source of C later in the season (as they only contribute little to C

accumulation early on). In fact, it was previously suggested that new shoots are

generally dependent on TNC reserves until newly developing leaves are about half their

full size, when they start to act as sources for photoassimilates (Hale & Weaver, 1962).

Insufficient abundance of TNC reserves leads to reduced shoot and root growth

(Candolfi-Vasconcelos et al., 1994), and to reduced leaf size and lateral shoot

development (J. P. Smith & Holzapfel, 2009). The roles of soluble sugars in the early

vegetative and reproductive development of grapevines are further discussed later in

this review.

2.2.2. Mobilisation of carbohydrate reserves

Starch is the predominant TNC in woody grapevine tissues, and is especially abundant

in the roots (J. P. Smith & Holzapfel, 2009). Starch is a large molecule, and is not

osmotically active, and can therefore not be transported between different parts of the

plant, while sugars can, by generating a reduction in osmotic potential when it

accumulates (Gibson, 2005; Zufferey et al., 2012). When a TNC deficiency develops in

plants, starch reserve deposits are degraded (solubilised), yielding C containing

intermediates, such as glucans, maltose and glucose, which are ultimately restructured

as sucrose (A. M. Smith, Zeeman, & Smith, 2005). Sucrose can then be transported in

the phloem from source to sink organs (Ruan, Jin, Yang, Li, & Boyer, 2010).


Insufficient leaf photoassimilation leads to an undersupply of assimilated C towards the

requirements of TNC sinks, and thereby creates a TNC deficiency in the plant. Such

a deficiency can easily be generated during the berry maturation period, as the berries

are a strong sink for TNC during this stage. The degradation of starch reserves during

berry maturation therefore leads to increased concentrations of soluble sugars in the

source organ, allowing for TNC remobilisation towards the maturing berries

(Candolfi-Vasconcelos et al., 1994).

Plant sugar abundance is suggested to be involved in the signalling towards the

regulation of reserve starch degradation or storage. The expression of numerous plant

genes responds to TNC abundance or shortage (Eveland & Jackson, 2012; Rolland,

Moore, & Sheen, 2002). Many of these genes encode enzymes involved in

photosynthesis, and in sugar and starch metabolism (S. M. Smith et al., 2004). When a

plant sugar deficiency occurs, genes involved in photosynthesis, TNC remobilisation

and export, and in N metabolism, tend to be up-regulated (Eveland & Jackson, 2012).

Low sugar status therefore leads to enhanced photosynthesis, as well as reserve TNC

mobilisation and exportation from source tissues (Gupta & Kaur, 2005). However,

when sugars are abundant, altered gene expression enhances typical sink organ

activities, like TNC importation, and further utilisation (for e.g., vegetative growth or

storage) (Eveland & Jackson, 2012).

High grapevine crop load, insufficient canopy leaf area, and/or sustained water

constraints, are major conditions that increase the sink (fruit) TNC requirement, relative

to the supply from canopy photoassimilation. Such an imbalance of C availability then

induces the remobilisation of reserves from perennial vine parts during berry maturation

14


15

(Candolfi-Vasconcelos et al., 1994). The TNC reserve mobilisation is one adaptive

mechanism for grapevines to adjust to limitations in photoassimilation during fruit

maturation, while compensatory photosynthesis by the intact leaves is also another

regulatory feature to adapt to the insufficient C assimilation, especially after defoliation

treatments (Petrie et al., 2000; Scholefield et al., 1978). The increased photosynthetic

rate of the remaining leaves does not always compensate for the reduced leaf area, as

excessive leaf removal could still easily reduce the overall canopy photosynthesis rate

(Poni, Bernizzoni, & Civardi, 2008).

2.2.3. Soluble sugar specific roles

Apart from the mentioned role in TNC transportation between the source and sink

organs of grapevines, soluble sugars are also involved in various other functions within

the plant. These roles include the utilisation of sugars as structural components, as cell

nutrients, and as potential signals in plant growth and development (Çakir et al., 2003).

Sugars are also used in the biosynthesis of polysaccharides, such as starch and cellulose,

in plants (Gupta & Kaur, 2005). Sucrose is the major TNC transported from

photosynthetic tissue to sinks, where it can be degraded to hexoses or their derivatives,

which are useful for various metabolic or biosynthetic processes (Ruan et al., 2010).

However, raffinose family oligosaccharides (e.g. raffinose) and sugar alcohols (e.g.

mannitol) also play important transport roles in many plants (Noiraud, Maurousset, &

Lemoine, 2001).

2.2.3.1. Signalling and gene expression

As briefly mentioned earlier, sugars can act as signalling molecules that control distinct

aspects of plant development. These signals provide information on the prevailing


16

internal and external conditions, and contribute to signalling networks in response to the

abundance of metabolites (e.g. sugars or amino acids) (Smeekens, Ma, Hanson, &

Rolland, 2010). Such signalling networks create a link between C assimilation, C

storage, and plant growth (A. M. Smith & Stitt, 2007), and can significantly contribute

to yield and crop quality in plants (Smeekens et al., 2010).

Sugar signals can either be generated by TNC abundance and ratios to other metabolites

(e.g., C-to-N ratios), or by the flux through sugar specific sensors and/or transporters in

the plant (Eveland & Jackson, 2012). Sensor proteins are responsible for the sensing of

plant sugar status. The interaction between a sugar molecule and a sensor protein causes

a signal to be generated (Gupta & Kaur, 2005), and this allows plants to modulate site-

specific and whole-plant growth, potentially to coordinate developmental programmes

with available TNC (Eveland & Jackson, 2012). Plants therefore monitor their own

sugar status in order to optimally utilise available sugar for growth and development

(Smeekens et al., 2010).

Sucrose can be sensed as a signal directly or through an indirect signal that arises via its

hexose cleavage products, i.e., glucose and fructose (Eveland & Jackson, 2012; Koch,

2004). Invertase and sucrose synthase are enzymes responsible for sucrose cleavage

(Koch, 2004), and the activities of these enzymes contribute to sugar signalling. Hexose

sugars generally have greater signalling potential in promoting organ growth and cell

proliferation, while sucrose is associated with cell differentiation and maturation

(Eveland & Jackson, 2012). Various minor sugars and sugar alcohols, e.g., myo-inositol

(Valluru & Van den Ende, 2011), trehalose (Iturriaga, Suárez, & Nova-Franco, 2009;

Smeekens et al., 2010), raffinose (Valluru & Van den Ende, 2011) and galactinol


17

(Valluru & Van den Ende, 2011), were also previously indicated as being involved in

plant sugar signalling networks, and are discussed later.

2.2.3.2. Osmotic regulation and protection

As osmotically active solutes, plant sugars can play distinct roles during abiotic stress

conditions, such as a drought (Rodrigues et al., 1993). In fact, the biosynthesis and

accumulation of osmotic regulators (e.g. sugars) is a well-known adaptive mechanism

for plants in response to osmotic constraints (Shulaev, Cortes, Miller, & Mittler, 2008).

Sugars are also important osmoprotectants, as they stabilise proteins and cell

membranes (Rontein, Basset, & Hanson, 2002). Indeed, many sugars interact with

proteins and cell membranes through hydrogen bonding, and thereby importantly

prevent protein denaturation (Devi & Sujatha, 2014). The accumulation of soluble

solids corresponds to the increase of drought tolerance in plants (Hoekstra, Golovina, &

Buitink, 2001), and certain plant species, or even within specie varieties with a higher

abiotic stress tolerance, are known to accumulate higher volumes of sugar metabolites

related to osmotic regulation and cell membrane stabilisation (Morsy, Jouve, Hausman,

Hoffmann, & Stewart, 2007).

During abiotic stress conditions (e.g., dry or saline conditions), accumulated sugars can

raise the osmotic pressure in plant cells at the site of their accumulation (e.g., the roots

or leaves) (Rontein et al., 2002). Sugar accumulation in plant organs thereby contributes

to the maintenance of a cell turgor potential, and thus generates a gradient for water

uptake into the plant cells (Rhodes & Samaras, 1994). Grapevine leaves (Patakas &

Noitsakis, 1999) and roots (Düring & Dry, 1995) are known to accumulate sugars in

order to undergo osmotic adjustment during water constraints. However, as it provides


18

the point of contact between the plant and the soil, the osmotic regulation of grapevine

roots cells is especially important, and root sugar accumulation could be associated with

increased root osmolarity when the soil moisture content decreases (Rogiers, Holzapfel,

& Smith, 2011). Root soluble sugar accumulation can also play a role in embolism

repair, stimulating the osmotic movement of water into embolised vessels, to refill and

restore water flow (Canny, 1998; Salleo, Trifilò, Esposito, Nardini, & Lo Gullo, 2009).

Sucrose accumulation is often suggested to be related to osmotic regulation in many

plants that are exposed to water stress (Quick, Siegl, Neuhaus, Feil, & Stitt, 1989;

Rogiers, Holzapfel, et al., 2011). Sucrose is also thought to play a role in the protection

of cellular macromolecules or cell membranes (Bray, 1997; Leopold, 1990).

Nevertheless, sucrose may be cleaved into fructose and glucose during abiotic stress

conditions, and invertase activity is related to genetic variation, where more stress

tolerant varieties may have greater invertase activity (Morsy et al., 2007). In fact, high

hexose (glucose and fructose) levels are suggested to drive lower cell water potential

and maintain turgor pressure during water stress (Sturm, 1999). Many other sugars (e.g.

raffinose) and sugar alcohols (e.g. myo-inositol), that are usually present in relatively

lower abundance in plants, also play crucial roles in plant osmotic regulation and

osmoprotection (Krasensky & Jonak, 2012; Morsy et al., 2007; Rontein et al., 2002).

These aspects are discussed later.

2.2.3.3. Vegetative and reproductive development

As briefly mentioned earlier, reserve TNC plays important roles in the reestablishment

of vegetative spring growth. When the abundance of sugars that were synthesised in the

leaves exceeds the TNC requirement of temporary sink organs (e.g., the fruit), sugars

are translocated to woody plant parts (e.g., the trunks and roots), where they can be


19

converted into starch (Scholefield et al., 1978). This happens especially late in the

growing season, when fruit sugar accumulation slows down. This reserve starch is then

the first form of TNC used for new shoot growth the following season (Scholefield et

al., 1978).

Sugars utilised for reproductive development are either derived from TNC reserves or

synthesised through photoassimilation in the leaves or inflorescence, depending on the

stage of annual grapevine development (Lebon et al., 2008). However, inadequate sugar

supply due to insufficient reserve TNC availability at budburst could inhibit flower

initiation and, subsequently, also shoot fruitfulness (J. P. Smith & Holzapfel, 2009).

Sugars therefore play an important role in flowering and in the formation of plant

reproductive organs and structures, by providing essential energy resources (Lebon et

al., 2008). Low sugar abundance in the grapevine may cause flower abortion, especially

when this limited sugar availability is found during female meiosis (Lebon et al., 2008).

Furthermore, fruit set, and ultimately the fruit yield, is suppressed by low sugar

availability (Bennett et al., 2005; Intrieri, Filippetti, Allegro, Centinari, & Poni, 2008).

A further role of sugars towards the reproductive development is the possible

contribution towards berry sugar accumulation, as discussed earlier.

2.2.4. Minor sugars and sugar alcohols

Apart from sucrose and the hexose sugars, a range of other sugars and sugar alcohols

were previously reported as being present within grapevine tissues. However, most of

the early studies related to the presence of minor sugars in grapevines were only

conducted during the cold winter periods. In these studies, the minor sugars that were

reported to be present in grapevines included raffinose (Hamman, Dami, Walsh, &


20

Stushnoff, 1996; Jones, Paroschy, McKersie, & Bowley, 1999; Kliewer, 1966; Koussa,

Cherrad, Bertrand, & Broquedis, 1998), stachyose (Hamman et al., 1996; Kliewer,

1966; Panczel, 1962), maltose (Kliewer, 1966; Panczel, 1962), galactose (Kliewer,

1966), inositol (McArtney & Ferree, 1999; Ndung'u, Shimizu, Okamoto, & Hirano,

1997) and melibiose (Kliewer, 1966; Panczel, 1962). With more recent developments in

the methods to profile the metabolic composition of plant tissues, various late studies

have successfully identified the presence of various other minor sugars in grapevines

(Cuadros-Inostroza et al., 2016; Degu et al., 2014; Hochberg, Batushansky, Degu,

Rachmilevitch, & Fait, 2015; Hochberg, Degu, Cramer, Rachmilevitch, & Fait, 2015;

Hochberg et al., 2013). Most of these studies, however, focussed on the metabolic

profile of grapevine berries. However, as the abundance of various minor sugars and

sugar alcohols leads to improved tolerance towards different plant stress conditions,

e.g., water and cold stress (Gupta & Kaur, 2005; Rontein et al., 2002), and could play a

crucial role in plant metabolism (Valluru & Van den Ende, 2011), more work is needed

to improve the knowledge of an environmentally stress related change of minor sugar

abundance in different grapevine organs.

Sugar alcohols, i.e., non-reducing carbohydrates that are a class of polyols, include

sorbitol, mannitol, galactinol and myo-inositol, and are present in plants, commonly

synthesised as primary photosynthetic products (Moing, 2000). These compounds can

act as transport substances and storage compounds, sometimes appearing as minor

compounds in the roots of woody plants (Loescher et al., 1990). The abundance of these

compounds in plant tissues can increase when plants are exposed to abiotic constraints,

and could thereby promote resistance towards various abiotic plant stresses, including

low temperature, water constraints, and high salinity, as well as biotic stress (Gupta &


Kaur, 2005; Moing, 2000). In a variety of plant species, it is possible that hexoses and

other TNC (e.g. sucrose and starch) can be converted into sugar alcohols during dry

conditions (Wang, Quebedeaux, & Stutte, 1996). These compounds can then also act as

osmoprotectants (Rontein et al., 2002), and are therefore involved in the stabilisation of

cell membranes and enzymes (Krasensky & Jonak, 2012). The accumulation of sugar

alcohols can, furthermore, induce the importation of water into plant cells during

osmotic stress, by causing an adjustment in cell osmotic potential (Wang et al., 1996).

Myo-inositol is an important example of a plant sugar alcohol, and functions as a potent

osmoprotectant (Silva et al., 2011). It plays a role in structuring cell membranes, and

can also act as a signalling molecule in plants (Valluru & Van den Ende, 2011). Myo-

inositol accumulation was previously found to correlate with salt tolerance in tomato

plants (Cuartero & Fernández-Muñoz, 1998), while genes involved in myo-inositol

synthesis were also previously found to be upregulated during water stress in various

other plant species (Ishitani et al., 1996). Furthermore, myo-inositol serves as a

precursor of various other metabolites in plants. It serves as a precursor in the synthesis

of galactinol, and subsequently also the synthesis of the raffinose family

oligosaccharides (Valluru & Van den Ende, 2011). It is also proposed that myo-inositol

could provide an alternative substrate for a pathway for ascorbic acid synthesis

(Lorence, Chevone, Mendes, & Nessler, 2004). Ascorbic acid, on the other hand, is a

major substrate for the synthesis of tartaric acid, threonic acid, glyceric acid and oxalic

acid (Loewus, 1999).

Raffinose family oligosaccharides (RFOs) consist of raffinose, stachyose

and verbascose, and are suggested to play a role in plant osmoprotection under water

21


stress (Taji et al., 2002). RFOs were therefore previously indicated to contribute to cell

membrane protection (Krasensky & Jonak, 2012). RFOs can also be used as

transport compounds in plants, accumulate in response to a range of abiotic stresses, and

also as reserve compounds (Sengupta, Mukherjee, Basak, & Majumder, 2015; Valluru

& Van den Ende, 2011). Raffinose is known to accumulate in plant tissues during

chilling conditions, and may thereby increase the tolerance against chilling due to its

role in membrane stabilisation via interactions with phospholipid headgroups (Morsy et

al., 2007). It was also found that drought, high salinity, and cold conditions could

induce the accumulation of high levels of raffinose, along with its precursor, galactinol,

but not of stachyose. Raffinose and galactinol are therefore suggested to play a role in

stress tolerance in plants (Taji et al., 2002), and were found to play roles as

osmoprotectants in the leaves of Arabidopsis plants (Nishizawa-Yokoi, Yabuta, &

Shigeoka, 2008). It is also suggested that raffinose and galactinol could act as signals to

mediate stress responses in plants (Valluru & Van den Ende, 2011). While raffinose is

therefore perhaps an important osmoprotector, the role of stachyose is suggested to

rather be more related to the storage and transport of TNC in various woody plants,

cucurbits, and legumes (Dey & Harborne, 1997).

Other minor sugars include trehalose, which only accumulates in certain plants, and

mainly in so-called resurrection plants (which can survive extreme dehydration for

extended periods), where it can lead to increased drought tolerance, improved

photosynthesis and dry matter accumulation under water stressed conditions (Gupta &

Kaur, 2005). Trehalose has also been proposed to act as an osmoprotectant during water

constraints (Penna, 2003; Rontein et al., 2002), as it can protect proteins and cell

membranes against denaturation (Silva et al., 2011). Trehalose can also be an important

22


23

signalling metabolite, involved in the regulation of plant growth and development in

response to C availability (O’Hara, Paul, & Wingler, 2013; Silva et al., 2011). The Vitis

vinifera genome contains genes encoding the enzymes responsible for trehalose

synthesis and degradation, and trehalose was found to be present in grapevine tissue,

after exposure to excessive chilling (Fernandez et al., 2012).

2.2.5. Factors influencing carbohydrate reserve distribution in grapevines

2.2.5.1. Seasonal development

Seasonal developmental stage or environmental conditions usually play a larger role in

the grapevine TNC reserve dynamics, than viticultural practices (Holzapfel & Smith,

2012). TNC allocation towards different vine parts fluctuates throughout the season,

mainly due to changes in the grapevine developmental stage, and the size and activity of

TNC sinks (Holzapfel & Smith, 2007). The berries, for example, become sinks for

carbohydrate assimilates at fruit set (Hale & Weaver, 1962), although the woody

tissues, e.g. the roots, also start to accumulate TNC around flowering (Holzapfel et al.,

2010). Furthermore, the berries are also strong TNC sinks between véraison and harvest

(Davies & Robinson, 1996). TNC reserve levels in the roots and trunks are usually the

highest during dormancy (at leaf fall), and the lowest after their utilisation for the

reestablishment of vegetative growth (just before flowering) (Zufferey et al., 2012).

TNC levels are then usually restored again and can be as high as at budbreak, around

two weeks after flowering (Mullins, Bouquet, & Williams, 1992).

The translocation of TNC, synthesised in the leaves, towards woody vine parts, usually

starts at the 10-leaf stage and becomes prominent four weeks after flowering (Yang &

Hori, 1979). However, the rates of leaf C assimilation can vary throughout the season,


24

with the peak rates of individual leaf photosynthesis attained about 40 days after

unfolding, with the rates declining gradually thereafter (Kriedemann, Kliewer, & Harris,

1970). Maximum canopy photosynthesis rates are often found at around véraison (Poni,

Intrieri, & Magnanini, 2000). Photosynthesis rates decline after harvest, but a

considerable amount of assimilates can still be accumulated during the post-harvest

period (Williams, 1996). After harvest, it is possible for the leaves to assimilate C for

two to three months prior to leaf fall, if conditions permit it (Scholefield et al., 1978).

The duration and conditions of the post-harvest period are therefore important for

reserve accumulation and for the next season’s early vegetative growth and reproductive

development (Holzapfel, Smith, Mandel, & Keller, 2006), and should be maintained by

good viticultural practices (e.g. irrigation management and disease spray programs to

retain the leaves).

2.2.5.2. Abiotic conditions

Any factor that affects leaf photoassimilation will impact on reserve TNC accumulation

(Holzapfel & Smith, 2012). The reserves are utilised whenever the source TNC supply

cannot meet the demand of the sinks. Abiotic conditions, such as water availability,

atmospheric temperature, and leaf CO2 supply, have a major influence on leaf (source)

photoassimilation.

The leaf photosynthesis of higher plants is strongly affected by atmospheric temperature

(Berry & Bjorkman, 1980), and fluctuations in the temperature therefore affect

grapevine leaf photosynthesis, and subsequently impact on TNC accumulation and

distribution. High atmospheric temperatures can inhibit starch accumulation in the

leaves, as this can also cause starch to convert to lipid-like material within the


25

chloroplasts (Buttrose & Hale, 1971). TNC reserve concentrations in grapevine organs

in warmer climate regions can generally be much higher than in the cooler regions

(Zufferey et al., 2012). In warmer regions, such as the Riverina in Australia

(Holzapfel et al., 2006), there is a much longer period between fruit maturity and leaf

fall than in cooler regions, such as Canterbury in New Zealand (Bennett et al., 2005),

leading to prolonged post-harvest TNC reserve accumulation in warmer climates.

The longer post-harvest period in warmer regions is also important to sustain the higher

fruit yields that are generally associated with such regions (Holzapfel et al., 2006).

In cool climates, because there is usually no substantial accumulation period

after harvest, the maintenance of reserves before harvest should be prioritised (Bennett

et al., 2005). Soil temperature also affects the utilisation and depletion of reserve

TNC from the roots (Field, Smith, Holzapfel, Hardie, & Emery, 2009; Rogiers,

Hardie, & Smith, 2011). Warmer soil temperatures between budbreak and flowering

can, for example, lead to depleted root starch (Field et al., 2009), and soil

temperature can therefore modulate root TNC mobilisation.

Leaf stomatal conductance is sensitive to atmospheric CO2 conditions, and the CO2

abundance in the atmosphere therefore affects photosynthesis (Farquhar & Sharkey,

1982). Elevated atmospheric CO2 concentrations have been shown to be a cause of

increased assimilate supply and subsequent greater plant TNC concentrations (Schultz,

2000). A combination of high temperatures and CO2 in the atmosphere can increase

plant photoassimilation and, except for the subsequent increased shoot and root growth

rates, it also leads to increased leaf starch concentrations (Kriedemann, Sward, &

Downton, 1976). However, long term exposure to increased CO2 may result in

decreased photosynthesis due to the likely sink saturation by assimilates (Samarakoon

& Gifford, 1995).


26

Plants perceive and respond to water constraints through a rapid alteration in gene

expression in conjunction with physiological and biochemical changes, and this can

even start occurring under mild to moderate stress conditions (Chaves, Flexas, &

Pinheiro, 2009; Gambetta, Matthews, Shaghasi, McElrone, & Castellarin, 2010). Under

mild to moderate water constraints, one of the first plant responses is a reduction in

stomatal conductance, thereby reducing water loss through transpiration, also restricting

photosynthesis and C assimilation (Chaves, Maroco, & Pereira, 2003). Stomatal closure

due to water stress is associated with increased abscisic acid (ABA) in the petiole xylem

and leaf blades, and an increased xylem pH, while decreased plant hydraulic

conductance could also be involved (Lovisolo et al., 2010). Under water constraints,

chemical compounds such as ABA, synthesised in the roots, act as long-distance

signals, inducing stomatal closure and/or restricting leaf growth (Chaves et al., 2010).

Water constraints thereby limit photosynthesis, and can cause photo-oxidative damage

in grapevines, which reduces the photosynthetic rate and weakens plant growth

(Hochberg, Degu, Fait, & Rachmilevitch, 2012). Grapevine C assimilation is therefore

limited by water constraints due to restricted canopy photoassimilation (Escalona et al.,

1999) and restricted canopy size (Hochberg et al., 2012).

The inhibition of leaf stomatal conductance is often observed following deficit

irrigation, causing a reduction in root TNC concentrations and also, to a lesser extent,

the TNC concentrations in the trunks and shoots (Holzapfel et al., 2010). Furthermore,

as mentioned, water stress can induce the enzymatic degradation of starch in plant

tissues, as previously observed in the leaves (Jacobsen, Hanson, & Chandler, 1986; Li

& Li, 2005) and roots (Regier et al., 2009) of various plant species. This subsequently

leads to sugar accumulation in those tissues, and water constraints were therefore


27

previously linked to the accumulation of soluble sugars in grapevine roots and trunks,

while the starch concentrations in these organs are decreased (Dayer, Prieto, Galat, &

Perez Peña, 2013; Rogiers, Holzapfel, et al., 2011). A reduction in starch-to-sugar ratios

therefore regularly corresponds with water constraints, and this could therefore induce

reduced starch reserve availability at budburst.

2.2.5.3. TNC Source and sink organ relations

The translocation of TNC requires sugar export from a site of production/synthesis (the

source organ, e.g. the photoassimilating leaves or the starch abundant roots), phloem

transport via an osmotic gradient, and a site of sugar export where the sugar is further

utilised (the sink organ, e.g. the sugar accumulating fruit or the starch storing roots).

The canopy leaf-to-fruit ratio of grapevines is a frequently quantified measurement,

used when studying the effects of source-sink relations on grapevine physiological

development (Kliewer & Dokoozlian, 2005; Santesteban & Royo, 2006; Zufferey et al.,

2015). It was previously suggested, following a comparative study of grapevines with a

wide range of leaf-to-fruit ratios in a given climatic region, that a leaf area-to-fruit ratio

of 8-12 cm2

leaf area/g fruit fresh weight is required for field grown wine grapes to

reach the maximum level of soluble solids, fruit weight, and skin colour when cultivated

on a single canopy type trellis system (Kliewer & Dokoozlian, 2005). However,

environmental conditions during the berry maturation period impact on how these ratios

affect the accumulation of quality parameters in the berries (e.g., juice soluble solid

concentration and fruit yield) (Howell, 2001). Furthermore, the abundance of TNC

reserves, and their availability to be utilised towards fruit sugar accumulation, will also


28

help determine the required leaf-to-fruit ratio that would promote maximum fruit sugar

accumulation.

Alterations in the grapevine leaf-to-fruit ratio at different stages of the season differently

affect the TNC reserve distribution and partitioning within grapevines and,

subsequently, also impact on vine vegetative and reproductive development. Early

defruiting may, for example, increase the total TNC concentrations in the roots, and lead

to improved bud fruitfulness in subsequent seasons (J. P. Smith & Holzapfel, 2009).

However, defoliation at harvest may decrease the root TNC concentration, and can

cause reduced fruit yields in subsequent seasons (J. P. Smith & Holzapfel, 2009).

Although reduced leaf-to-fruit ratios can lead to stimulated leaf level photosynthesis

(Candolfi-Vasconcelos & Koblet, 1991), this can also cause a reduction in whole-vine

photoassimilation due to the subsequent restricted canopy leaf area. Furthermore, the

exportation of leaf TNC benefits from the presence of sink organs, such as the fruit, as

well as a low leaf-to-fruit ratio (Zufferey & Murisier, 2005). Cropped grapevines were

previously found to have higher or similar stomatal conductance and photassimilation

rates than vines that were defruited (Downton, Grant, & Loveys, 1987; Naor, Gal, &

Bravdo, 1997). However, it is also possible that a larger crop load could increase leaf

transpiration, leading to enhanced root signalling, and a subsequent reduction in leaf

stomatal conductance (Naor et al., 1997). These impacts of crop load on stomatal

conductance, can subsequently affect the TNC reserve accumulation.

Defoliation can increase the net leaf stomatal conductance and CO2 exchange rate per

leaf area basis (Poni et al., 2008). However, defoliation can also cause the

remobilisation of TNC reserves from the perennial structure towards the fruit during


29

berry maturation, as described earlier (Candolfi-Vasconcelos et al., 1994). Excessive

defoliation causes a reduction in canopy photosynthesis, causing lower TNC reserve

levels in grapevine tissues at the end of the growing season (Bennett et al., 2005;

Candolfi-Vasconcelos et al., 1994; Holzapfel et al., 2006). Root TNC abundance is

more sensitive to grapevine source-sink balances than the TNC levels in other woody

grapevine parts (e.g. the trunk) (J. P. Smith & Holzapfel, 2009). Starch concentrations

in woody grapevine parts usually decrease due to defoliation, while there can be an

increase in soluble sugar content in these tissues. However, sugar concentrations in the

roots can also be unresponsive towards defoliation and fruit removal (J. P. Smith &

Holzapfel, 2009), or can even reduce because of early defoliation (Bennett et al., 2005).

High grapevine crop loads may cause a delay in fruit maturation, and a subsequently

shorter post-harvest period, which can reduce reserve TNC accumulation (Holzapfel &

Smith, 2012). In fact, high crop loads reduce the starch reserve concentration in

grapevine trunks (Dayer et al., 2013). It was also previously found that the starch and

soluble solid concentrations in grapevine roots and wood were the highest by fruit

maturity in defruited vines, and that partially defruited vines have higher concentrations

than higher yielding vines (J. P. Smith & Holzapfel, 2009).

Defruiting of grapevines at the onset of fruit maturation increases TNC concentrations

in the roots and, to a lesser extent in the trunks, and this can result in fruit yield

increases the following season (J. P. Smith & Holzapfel, 2009). Crop load reductions

before véraison can induce increased starch and sugar reserve concentrations in roots in

the following winter, and the requirement of less TNC reserves to support fruit sugar

accumulation perhaps contributes to this (Holzapfel et al., 2010). Very low sink


30

demands following excessive early defruiting, might cause increased vegetative growth

because of reduced competition for TNC between vegetative and reproductive sinks,

and this frequently happens mainly through lateral shoot growth (Mattii & Orlandini,

2004). The duration of the storage and accumulation periods of TNC throughout the

season, and the length of the post-harvest period for reserve accumulation, can vary due

to climatic and canopy conditions, but are also influenced by grapevine crop load, and

the subsequent fruit yield (Holzapfel et al., 2006).

2.3. Grapevine nitrogen

The supply of grapevine nitrogen (N) plays an essential role in sustaining vine growth

and development (Cheng et al., 2004). The status of N reserve availability early in the

season could largely determine the fruit yield and the vegetative growth of grapevines

(Cheng et al., 2004).

N is taken up from the soil as nitrate ions and ammonium. However, nitrate is thought

to be the preferred form of N uptake by grapevine roots, and ammonium can be toxic

when taken up at high concentrations (Roubelakis-Angelakis & Kliewer, 1992). Nitrates

enter root cells, and are reduced and incorporated into organic molecules, stored, or

translocated to other vine parts for further utilisation (Roubelakis-Angelakis & Kliewer,

1992). The reduction of nitrates to ammonia results in a useable form of N, and nitrate

reductase (NR) catalyses the first step in nitrate reduction in higher plants (Roubelakis-

Angelakis & Kliewer, 1992). The Glutamine synthetase/glutamate synthase pathway

(GS/GOGAT) is the primary route of ammonia assimilation, while glutamate

dehydrogenase may also play a role. Amino acids are the first products of ammonia

assimilation, and are the building blocks of proteins, also playing roles in the regulation


of metabolism, N transport, and the storage of N (Roubelakis-Angelakis & Kliewer,

1992). Ammonium is first assimilated into glutamine and glutamic acid, and then,

through aminotransferase reactions, into other amino acids, such as aspartic acid and

asparagine.

N fertilisation, and the timing thereof, not only affects N reserve accumulation, but also

the partitioning and concentration of fruit N. Soil application of N at bloom, for

example, promotes the allocation of N towards annual grapevine tissues, while the

application after set could promote N accumulation in perennial vine parts (Holzapfel,

Watt, Smith, Suklje, & Rogiers, 2015). Furthermore, N application two weeks after

véraison could lead to increased berry juice yeast assimilable N (YAN) content,

which benefits the fermentation process during winemaking, and improves must

quality (Holzapfel et al., 2015).

The spring growth of grapevines depends on N reserve remobilisation from the roots,

as N uptake is usually still insufficient during this time of the season (Zapata et al.,

2004). This remobilisation during early spring growth accounts for most N

distribution until flowering (Zapata et al., 2004). The fruit, leaves and shoots are all

considerable N sinks between flowering and véraison (Conradie, 1991; Roubelakis-

Angelakis & Kliewer, 1992), and sufficient soil N availability during this period is

therefore important. Between véraison and harvest, when N uptake is reduced or absent,

redistribution of N from the roots, shoots and leaves towards the bunches take place

(Conradie, 1991). The assimilation of root N for the overwintering reserve N storage is

important late in the growing season, as these reserves are, as mentioned, crucial for

early vine development the next season, when N soil uptake is still insufficient (Cheng et

31


al., 2004). Root N reserve replenishment can start from about one month after

flowering (Bates, Dunst, & Joy, 2002).

2.3.1. Roles of N in grapevines

N plays a central role in plant metabolism, as it is a constituent of proteins, nucleic

acids, chlorophyll, co-enzymes, phytohormones, and secondary metabolites

(Hawkesford et al., 2012). The photosynthetic capacity of leaves is related to its N

content, as proteins involved in photosynthesis represent the majority of leaf N (Evans,

1989). N availability is required for plant growth, and sufficient N availability during

vine vegetative growth leads to increased leaf area, fruit yield, and overall shoot

development, while it also enhances leaf CO2 assimilation (Cheng et al., 2004). Higher

N availability can lead to increased vegetative growth (Smart & Robinson, 1991);

although it could also induce increased fruit yield (Benz, Bogdanoff, & Kliewer, 1991),

given that the N supply is not excessive (Ahmedullah & Roberts, 1991). Low N

availability around bloom causes reduced fruit set, and N availability at bloom is a

major determinant of fruit yield (Keller, Arnink, & Hrazdina, 1998).

N reserves are predominantly stored in the roots of grapevines, and consist of a range of

amino acids (mainly arginine), and proteins (Xia & Cheng, 2004; Zapata et al.,

2004). Proteins are used for structure, metabolism and N storage, and storage

proteins are therefore important for N metabolism, and play a role in the overwintering

N storage in deciduous plants (Roubelakis-Angelakis & Kliewer, 1992). N reserves are

essential for spring growth, as the early vegetative growth might even be more

responsive towards N reserve availability, than the availability of TNC reserves (Cheng

et al., 2004).

32


Shoot growth before flowering depends on N mobilisation from root reserves,

and the accumulation of N into the storage sinks of perennial organs prior to harvest is

therefore important for vine development the following season (Weyand & Schultz,

2006; Zapata et al., 2004). The vegetative and reproductive growth of young

Concord vines were found to be largely determined by the N reserve status, and N

supply sustains shoot and leaf growth, and contributes to fruit development from fruit

set (Cheng et al., 2004).

2.3.2. Fruit N accumulation

Fruit N content is influenced by scion and rootstock genetics, fruit maturity,

atmospheric temperature, mineral nutrition availability, fruit crop level, canopy

management practices, and disease prevalence (Roubelakis-Angelakis & Kliewer,

1992). However, it is suggested that fruit N accumulation peaks at two stages, one

pre-véraison (just before pea-size) and one just after véraison (Roubelakis-

Angelakis & Kliewer, 1992). The availability of N during the growing season can

therefore directly impact berry nitrogenous compounds, and can also

indirectly impact berry juice composition through the effects of N availability on

canopy vegetative growth and fruit yield (Smart & Robinson, 1991).

Fruit N accumulation is crucial for juice YAN concentrations, which

impact fermentation and wine composition. YAN in the must is composed of free

assimilable amino N (FAN) and ammonium. Low N supply reduces YAN, but

also leads to undesirable thiols and higher alcohols and lower concentrations of esters

and long chain volatile fatty acids in wine (Bell & Henschke, 2005).

33


34

However, excessively high fruit N content leads to increased ethyl acetate and acetic

acid, ethyl carbamate and biogenic amines in wine (Bell & Henschke, 2005). Arginine,

phenylalanine, histidine, valine, and glutamic acid in berry juice usually contribute the

towards berry juice amino N, useful towards fermentation by the yeasts

(Roubelakis-Angelakis & Kliewer, 1992). It is suggested that about a minimum of

130 mg YAN is required per litre of must, in order for the yeast to complete must

fermentation during winemaking (Agenbach, 1977). The composition of amino acids

in the must is also important for the development of aromatic compounds during

winemaking, as various amino acids provide C skeletons, utilised in the biosynthesis

of the aromatic compounds (Bell & Henschke, 2005).

2.3.3. Factors influencing N distribution

Any factor affecting N sink and source functioning, e.g. abiotic factors and management

practices (including crop load, leaf area, water availability, and disease prevalence), will

likely affect the N status of the grapevine, and subsequently the vine development the

following season (Cheng et al., 2004).

2.3.3.1. Seasonal grapevine development

No remarkable root N uptake from the soil takes place prior to budbreak, however,

significant uptake starts soon after budbreak, and peaks at four weeks post-flowering.

Soil N uptake can also take place soon after véraison (Löhnertz, 1991), and another

peak period of N uptake takes place shortly after harvest (Roubelakis-Angelakis &

Kliewer, 1992). Total N reserve content in permanent grapevine tissues (e.g. the roots)

decreases at budbreak, as it is utilised towards spring growth, and some reserve

accumulation starts again from around flowering (Zapata et al., 2004). When vegetative


35

growth slows down at the end of the season, the N taken up from the soil is primary

stored as reserves (Cheng et al., 2004), and the perennial vine parts therefore usually

start accumulating N reserves before berry maturation (Roubelakis-Angelakis &

Kliewer, 1992).

The highest concentrations and greatest fluctuations in N are thought to be found in the

roots, where N content stays constant early in the season and increases thereafter

(Roubelakis-Angelakis & Kliewer, 1992). Early in the season, N is remobilised from the

roots to support the vegetative growth in the shoots and leaves (Peacock, Christensen, &

Broadbent, 1989). In the shoots, N concentrations increase after budbreak and stay

constant towards the end of the vegetative growth period, and then increase again due to

retranslocation of N from senescing leaves (Roubelakis-Angelakis & Kliewer, 1992).

From budbreak, abundant N accumulation takes place in the leaves, and maximum leaf

N is found at full leaf expansion, while leaf N decreases towards the end of the growing

period (Roubelakis-Angelakis & Kliewer, 1992). Shoot and leaf N can be intermediate

N reservoirs between the roots and berries (Conradie, 1986).

2.3.3.2. Abiotic conditions

Water availability, and N fertilisation are the main drivers of N uptake, and water

constraints can limit N uptake by the roots (Keller, 2005). However, the metabolism and

distribution of nitrate and ammonium ions, and the partitioning of N in plants, are also

affected by other abiotic factors, such as light intensity and temperature, which may

particularly affect N partitioning related enzymes (Roubelakis-Angelakis & Kliewer,

1992).


36

Reduced water supply can result in the higher allocation of N to perennial structures,

and less to annual components of the vine (Holzapfel et al., 2015). Water stress also

reduces the activity of nitrate reductase in the leaves, due to a decreased nitrate flux

(Thomas & Stoddart, 1980), and this can have repercussions on N assimilation by the

plant. A number of N containing compounds accumulate in plants following abiotic

stress conditions (such as high salinity). These include amino acids, amids, imino acids,

proteins, quarternary ammonium compounds, and polyamines. The accumulation of

these compounds is suggested to be involved in cell osmotic adjustment, protection of

cellular macromolecules, storage of N, detoxification of cells, and the scavenging of

free radicals under stressful conditions (Mansour, 2000). The impact of abiotic

conditions on the abundance of certain amino acids within plants, is discussed later.

2.3.3.3. N Source and sink organ relations

The distribution of N requires a site of N storage (e.g. the leaves and roots) or the site of

soil N uptake (the roots) (i.e. the source organ), organic N transport, e.g., amino acid

xylem and phloem transport, and a site of N importation (e.g. N accumulating fruit or N

storing roots).

Vines developing a large vegetative vigour, depend less on N reserves and more on

current N supply (Cheng et al., 2004). However, early vine vegetative defoliation can

cause decreased total N content in grapevines at dormancy, and this happens as

mobilisation of N from leaves to storage tissues is interrupted, along with a decreased

root N uptake (Cheng et al., 2004).


37

The presence of fruit is also described to cause reduced N assimilation in grapevine

roots (Morinaga, Imai, Yakushiji, & Koshita, 2003), while higher crop loads cause

reduced N storage, at least as found in grapevine canes (Balasubrahmanyam, Eifert, &

Diofasi, 1978). The fruit of vines with low crop loads tends to have higher

concentrations of total N, arginine, proline, and total free amino acids (Kliewer & Ough,

1970).

Source-sink relations can greatly impact leaf N dynamics, and leaf senescence is often

associated with leaf N deficiency as a consequence of the remobilisation of leaf N

towards the development of reproductive organs (i.e., the fruit) (Thomas & Stoddart,

1980). N from the leaves that accumulated early in the season, is later used for

reproductive development, however, if the inflorescence or the developing fruit are

removed, leaf senescence can be delayed, or even reversed in some plant species

(Thomas & Stoddart, 1980). N remobilisation from the leaves of higher plants is related

to the biomasses of the source and sink organs (Diaz et al., 2008).

2.3.4. Amino acids

Major roles of amino acids in plants include their involvement in the regulation of N

metabolism, N transport, osmotic regulation, regulation of ion transport, and the storage

of N (Lam, Coschigano, Oliveira, Melo-Oliveira, & Coruzzi, 1996; Rai, 2002;

Roubelakis-Angelakis & Kliewer, 1992). Amino acids are also involved in modulating

stomatal opening, and the detoxification of heavy metals, while they also affect the

synthesis and activity of some enzymes, and impact on gene expression and redox

homeostasis (Rai, 2002). Amino acids can accumulate in high levels in the leaves and


38

roots of higher plants, and are transported in the xylem and phloem to sink organs

(Fischer et al., 1998).

One of the major pathways involved in amino acid metabolism in higher plants involves

α-ketoglutaric acid metabolism, and this pathway yields important plant amino acids

with well-defined functioning, i.e., glutamic acid, glutamine, arginine, and proline

(Verma, Zhang, & Singh, 1999). Glutamine and glutamic acid have known transport

roles (Coruzzi & Last, 2000) and are, in fact, major transporters of organic N from

source to sink tissues in many plants (Lam et al., 1996). Glutamic acid is also the

intermediate product of nitrate reduction and ammonium assimilation, and a key amino

donor in the synthesis of many other amino acids (Fritz, Mueller, Matt, Feil, & Stitt,

2006). In fact, glutamic acid is the substrate for the synthesis of glutamine from

ammonia and, in addition, the α-amino group of glutamic acid is transferred to all other

amino acids, and the C skeleton of glutamic acid forms the basis for the synthesis of γ-

aminobutyric acid (GABA), arginine and proline (Forde & Lea, 2007).

Arginine is one of the more abundant amino acids in grapevines, and is a major N-

storage compound, and participates in the biosynthesis of other amino acids

(Roubelakis-Angelakis & Kliewer, 1992). Arginine is actually the main form of storage

N in grapevines, and is the most abundant amino acid in both free amino acids and

protein in grapevine roots (Xia & Cheng, 2004). It is due to arginine’s high N:C ratio

(4:6) that it acts as a major N storage compound in higher plants. Besides the known N

transport role of glutamine in higher plants, it is also suggested that arginine and

asparagine could be involved as N transport compounds in various plants (Lea, Sodek,

Parry, Shewry, & Halford, 2007).


39

Proline accumulation in plants is suggested to frequently occur in response to biotic and

abiotic stresses. Proline is involved in cellular osmotic adjustment, the stabilisation of

subcellular structures, and the scavenging of free radicals (Hare & Cress, 1997). Free

proline reduces cellular water potential, and thereby serves as an osmotic regulator to

maintain turgor pressure in plant cells (Hare & Cress, 1997). Plants subjected to abiotic

stresses therefore typically accumulate certain amino acids, such as proline. Proline

accumulation is frequently described to coincide with plant water constraints in many

species, thereby promoting plant abiotic stress tolerance (Bertamini, Zulini,

Muthuchelian, & Nedunchezhian, 2006; Rai, 2002; Singh, Aspinall, & Paleg, 1972).

Arginine and proline (proline is not assimilated by yeast), are usually the predominant

amino acids in the must during winemaking (Bell & Henschke, 2005). Seasonal water

deficits elevate proline concentration, and also that of other amino N in the must, and

pre véraison water constraints can increase YAN in the berries (Hannam, Neilsen,

Forge, & Neilsen, 2013). Water constraints between flowering and harvest also increase

the YAN concentration of berry juice at harvest (Holzapfel et al., 2015). However,

partial rootzone drying (PRD) and regulated deficit irrigation can lower berry proline

and arginine concentrations (Wade, Holzapfel, Degaris, Williams, & Keller, 2002). The

composition and abundance of amino acids in the berries also affect flavour

development during winemaking (Bell & Henschke, 2005).

Another important pathway essential for amino acid biosynthesis in plants involves

shikimic acid metabolism (Haslam, 1993). This pathway yields the aromatic amino

acids, i.e., phenylalanine, tryptophan and tyrosine, which are essential compounds for

protein synthesis in plants, and serve as precursors for various secondary metabolites,


40

essential for plant growth (Maeda & Dudareva, 2012; Tzin & Galili, 2010).

Phenylalanine act as a precursor for phenylpropanoids, flavonoids, ligin, and

anthocyanins. Tyrosine is a precursor of various secondary metabolites, including

alkaloids, and several non-protein amino acids. Tryptophan is catabolised into indole-

containing secondary metabolites, such as auxin, and tryptamine derivatives (Tzin &

Galili, 2010). The metabolic pathway facilitating the synthesis of the aromatic amino

acids, is a major determinant of the phenolic content in the berries, affecting crucial

quality parameters (e.g., colour intensity and flavour). In fact, phenylalanine ammonia-

lyase enzyme activity plays a major role in this pathway, and is involved in the

catabolism of phenylalanine, thereby ultimately contributing to the biosynthesis of

anthocyanins in the berries (Boss, Davies, & Robinson, 1996).

Other amino acids that are present in different tissues of grapevines include alanine, β-

alanine, aspartic acid, asparagine, glycine, serine, threonine, valine, histidine,

methionine and lysine (Cuadros-Inostroza et al., 2016; Hochberg, Batushansky, et al.,

2015; Holzapfel et al., 2015).

2.4. Concluding remarks

The berry maturation period (between véraison and fruit maturity) is an important stage

in the annual grapevine developmental cycle, a period when strong sink organ

competition for plant metabolites and nutrients exists. The maturing fruit are strong

sinks for TNC and N, and the roots and leaves are potential sources of both these

compounds during berry maturation. However, the roots are also sinks for TNC and N

before dormancy, as these compounds are redistributed from the roots early in the next

season, to be utilised for spring vegetative and reproductive development.


41

Water availability and grapevine leaf-to-fruit relations are major determinants of the

availability, distribution, and partitioning of TNC and N reserves in the grapevine.

These factors could contribute to determining the abundance of TNC and N in both the

fruit and the storage organs, throughout berry maturation.

The existing literature in regards to grapevine TNC and N dynamics, is centred around

the concentrations of TNC and N at distinct stages of the annual growth cycle, in

specific organs. In addition to the existing literature, more work is required to

understand the kinetics of the allocation and distribution of TNC and N between

different grapevine organs, and the partitioning thereof within the organs, during the

berry maturation period. Such work will be essential in explaining the links between the

availability of reserves of TNC (especially in the roots) and N (especially in the leaves

and roots), and the accumulation of these compounds in the fruit. As major drivers of

TNC and N metabolism in higher plants, the water status, crop load, and leaf area

variability of grapevines during the berry maturation period, could supply additional

value in understanding the allocation of TNC and N between storage and temporary

grapevine sinks.

2.5. Literature cited

Agenbach, W. A. (1977, November). A study of must nitrogen content in relation to

incomplete fermentations, yeast production and fermentation activity.

Proceedings of the South African Society for Enology and Viticulture (pp. 66-

88). South African Society of Enology and Viticulture: Stellenbosch, South

Africa.

Ahmedullah, M., & Roberts, S. (1991, June). Effect of soil-applied nitrogen on the yield

and quality of Concord grapevines. Proceedings of the International Symposium

on Nitrogen in Grapes and Wine (pp. 200-201). American Society for

Enology and Viticulture: Seattle, WA.

Araujo, F., Williams, L. E., & Matthews, M. A. (1995). A comparative study of young

‘Thompson Seedless’ grapevines (Vitis vinifera L.) under drip and furrow


42

irrigation. II. Growth, water use efficiency and nitrogen partitioning. Scientia

Horticulturae, 60(3–4), 251-265.

Balasubrahmanyam, V., Eifert, J., & Diofasi, L. (1978). Nutrient reserves in grapevine

canes as influenced by cropping levels. Vitis, 17(1), 23-29.

Bates, T. R., Dunst, R. M., & Joy, P. (2002). Seasonal dry matter, starch, and nutrient

distribution in 'Concord' grapevine roots. HortScience, 37(2), 313-316.

Bell, S.-J., & Henschke, P. A. (2005). Implications of nitrogen nutrition for grapes,

fermentation and wine. Australian Journal of Grape and Wine Research, 11(3),

242-295.

Bennett, J., Javis, P., Creasy, G. L., & Throught, M. C. T. (2005). Influence of

defoliation on overwintering carbohydrate reserves, return bloom, and yield of

mature Chardonnay grapevines. American Journal of Enology and Viticulture,

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Benz, M., Bogdanoff, C., & Kliewer, W. M. (1991, June). Responses of Thompson

seedless grapevines trained to single and divided canopy trellis systems to

nitrogen fertilization. Proceedings of the International Symposium on Nitrogen

in Grapes and Wine (pp. 282-289). American Society for Enology and

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Berry, J., & Bjorkman, O. (1980). Photosynthetic response and adaptation to

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Bertamini, M., Zulini, L., Muthuchelian, K., & Nedunchezhian, N. (2006). Effect of

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Boss, P. K., Davies, C., & Robinson, S. P. (1996). Analysis of the expression of

anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape

berries and the implications for pathway regulation. Plant Physiology, 111(4),

1059-1066.

Bray, E. A. (1997). Plant responses to water deficit. Trends in Plant Science, 2(2), 48-

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Buttrose, M., & Hale, C. (1971). Effects of temperature on accumulation of starch or

lipid in chloroplasts of grapevine. Planta, 101(2), 166-170.

Çakir, B., Agasse, A., Gaillard, C., Saumonneau, A., Delrot, S., & Atanassova, R.

(2003). A grape ASR protein involved in sugar and abscisic acid signaling. The

Plant Cell, 15(9), 2165-2180.

Candolfi-Vasconcelos, M. C., Candolfi, M. P., & Koblet, W. (1994). Retranslocation of

carbon reserves from the woody storage tissues into the fruit as a response to

defoliation stress during the ripening period in Vitis vinifera L. Planta, 192(4),

567-573.

Candolfi-Vasconcelos, M. C., & Koblet, W. (1991). Influence of partial defoliation on

gas exchange parameters and chlorophyll content of field-grown grapevines-

Mechanisms and limitations of the compensation capacity. Vitis, 30, 129-141.

Canny, M. (1998). Transporting Water in Plants Evaporation from the leaves pulls

water to the top of a tree, but living cells make that possible by protecting the

stretched water and repairing it when it breaks. American Scientist, 152-159.

Chaves, M. M., Flexas, J., & Pinheiro, C. (2009). Photosynthesis under drought and salt

stress: regulation mechanisms from whole plant to cell. Annals of Botany,

103(4), 551-560.


43

Chaves, M. M., Maroco, J. P., & Pereira, J. S. (2003). Understanding plant responses to

drought - from genes to the whole plant. Functional Plant Biology, 30(3), 239-

264.

Chaves, M. M., Zarrouk, O., Francisco, R., Costa, J., Santos, T., Regalado, A., . . .

Lopes, C. (2010). Grapevine under deficit irrigation: Hints from physiological

and molecular data. Annals of Botany, 105(5), 661-676.

Cheng, L., Xia, G., & Bates, T. (2004). Growth and fruiting of young 'Concord'

grapevines in relation to reserve nitrogen and carbohydrates. Journal of the

American Society for Horticultural Science, 129(5), 660-666.

Conradie, W. (1980). Seasonal uptake of nutrients by Chenin blanc in sand culture: I.

Nitrogen. South African Journal of Enology and Viticulture, 1(1), 59-65.

Conradie, W. (1986). Utilization of nitrogen by the grape-vine as affected by time of

application and soil type. South African Journal of Enology and Viticulture,

7(2), 76-83.

Conradie, W. (1991). Distribution and translocation of nitrogen absorbed during early

summer by two-year-old grapevines grown in sand culture. American Journal of


Coruzzi, G., & Last, R. (2000). Amino acids. In B. Bunchanan, W. Gruissem, & R.

Jones (Eds.), Biochemistry & molecular biology of plants. (pp. 358-411).

Rockville, MD: American Society of Plant Physiologists.

Cuadros-Inostroza, A., Ruíz-Lara, S., González, E., Eckardt, A., Willmitzer, L., & Peña-

Cortés, H. (2016). GC–MS metabolic profiling of Cabernet Sauvignon and

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in 'Delaware' grapevines I. Retranslocation of 14

C-assimilates in the following spring after 14C feeding in summer and autumn. Tohoku Journal of Agricultural Research, 30(2), 43-56.


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Zapata, C., Deléens, E., Chaillou, S., & Magné, C. (2004). Mobilisation and distribution

of starch and total N in two grapevine cultivars differing in their susceptibility to

shedding. Functional Plant Biology, 31, 1127-1135.

Zufferey, V., & Murisier, F. (2005, August). Leaf to fruit ratio and photosynthetic

capacity of foliage in grapevines (cv. Chasselas). Proceedings of

XIV International GESCO Viticulture Congress (pp. 559-566). Groupe

d'Etude des Systemes de Conduite de la vigne (GESCO): Geisenheim,

Germany. Zufferey, V., Murisier, F., Belcher, S., Lorenzini, F., Vivin, P., Spring, J.-L., & Viret,

O. (2015). Nitrogen reserves in the grapevine (Vitis vinifera L. cv. Chasselas):

the influence of the leaf to fruit ratio. Vitis, 54(4), 183-188.

Zufferey, V., Murisier, F., Vivin, P., Belcher, S., Lorenzini, F., Spring, J.-L., & Viret,

O. (2012). Carbohydrate reserves in grapevine (Vitis vinifera L. ‘Chasselas’):

the influence of the leaf to fruit ratio. Vitis, 51(3), 103-110.

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Chapter 3: Paper 1

Carbohydrate distribution during berry ripening of potted

grapevines: Impact of water availability and leaf-to-fruit ratio

(Paper 1 has been published in Scientia Horticulturae as in the format below.)

3.1. Main objective for paper 1

To investigate the interactive effects of the leaf-to-fruit ratio and grapevine water status

during two phases of berry sugar accumulation (rapid and slow) on the carbohydrate

distribution between the different grapevine organs.

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Scientia Horticulturae 216 (2017) 215–225

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

Carbohydrate distribution during berry ripening of potted grapevines:Impact of water availability and leaf-to-fruit ratio

Gerhard C. Rossouwa,b,∗, Jason P. Smitha,1, Celia Barril a,b, Alain Deloirea,2,Bruno P. Holzapfela,c

a National Wine and Grape Industry Centre, Wagga Wagga 2678, New South Wales, Australiab School of Agriculture and Wine Sciences, Charles Sturt University, Wagga Wagga 2678, New South Wales, Australiac New South Wales Department of Primary Industries, Wagga Wagga 2678, New South Wales, Australia

a r t i c l e i n f o

Article history:Received 24 May 2016Received in revised form 3 January 2017Accepted 6 January 2017Available online 16 January 2017

Keywords:Carbon translocationWater stressLeaf areaCrop loadStarch reserveFruit maturation

a b s t r a c t

Insufficient leaf photoassimilation could allow mobilized carbohydrate reserves to contribute to berrysugar accumulation. However, the extent of this contribution during rapid and slow berry sugar accumu-lation is undefined. The potential effect of leaf-to-fruit ratio and water availability on carbohydrate reservedistribution in potted Tempranillo grapevines was examined during berry maturation. Within each leaf-to-fruit ratio treatment (full and 50% leaves), vines were grown under full or 50% reduced irrigationregimes. Dry biomass development, and the starch and soluble sugar concentrations were determinedin the roots, trunks, stems and leaves. Berry sugar and anthocyanin accumulation were also assessed.Under full irrigation, no starch remobilization from roots was observed, regardless of the leaf-to-fruitratio. Under reduced water supply, starch remobilization from roots was concurrent with rapid berrysugar accumulation, especially in grapevines with low leaf-to-fruit ratio. Soluble sugar accumulationcoincided with starch depletion in the roots of grapevines under reduced water availability. When berrysugar accumulation slowed, an increase in carbohydrates was observed in the roots. Sustained waterconstraints during rapid berry sugar accumulation resulted in a forced reliance on stored carbohydratesto support berry sugar accumulation, but did not significantly alter the tempo of berry sugar and antho-cyanin accumulation. A reduced leaf-to-fruit ratio intensified the reliance of fruit sugar accumulation onstored carbohydrates. Besides the importance of post-harvest carbohydrate reserve replenishment whenroot carbohydrate reserves are depleted during berry maturation, the reserves are also refilled duringmaturation when berry sugar accumulation slows. This study showed distinctly that root carbohydratereplenishment could already start a few weeks before harvest, and this replenishment could be importantwhen the post-harvest carbon assimilation period is ineffective.

© 2017 Elsevier B.V. All rights reserved.

1. Introduction

Leaf-to-fruit ratio and vine water status are parameters likelyto influence vine carbon balance during berry maturation (i.e. theberry sugar accumulation phase). Abiotic factors such as tempera-ture, light intensity, and water could limit vine carbon assimilationby restricting leaf photoassimilation (Escalona et al., 1999), whilereduced leaf-to-fruit ratios, up to a point, could result in an increase

∗ Corresponding author at: National Wine and Grape Industry Centre, WaggaWagga 2678, New South Wales, Australia.

E-mail address: [email protected] (G.C. Rossouw).1 Present address: Institut für Allgemeinen und ökologischen Weinbau,

Hochschule Geisenheim University, Geisenheim 65366, Germany.2 Present address: Montpellier SupAgro, Montpellier 34060, France.

of leaf photosynthetic activity (Candolfi-Vasconcelos and Koblet,1991). However, the importance of the contribution of root car-bohydrate reserves to support berry sugar accumulation underdiffering leaf-to-fruit ratios and grapevine water status is still aresearch question.

Carbohydrates are synthesized by plants through leaf photosyn-thesis and the effect of the abiotic factors in association with thevine internal competition for carbon, can affect the dynamics ofnon-structural carbohydrate reserve storage within the grapevine(Holzapfel and Smith 2012). These reserves are distributed tothe different plant organs, and the concentration and partitioningwithin different organs vary throughout the growing season. Thedistribution of carbohydrates could be affected by soil water avail-ability (soil depth, root implementation and functioning) and soil

http://dx.doi.org/10.1016/j.scienta.2017.01.0080304-4238/© 2017 Elsevier B.V. All rights reserved.

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216 G.C. Rossouw et al. / Scientia Horticulturae 216 (2017) 215–225

temperature (Dayer et al., 2013; Field et al., 2009; Rogiers et al.,2011a).

Studies have shown that grapevines with higher crop load, andthose subjected to elevated water constraint, exhibit reduced car-bohydrate reserve concentrations at budburst the following season.Water constraints during the growing season (pre-dawn leaf waterpotential values below −0.6 MPa), and high crop loads (leaf-to-fruitratios below 8 cm2 leaf area per gram of fruit), have been reportedto cause reduced starch concentrations in grapevine trunks duringthe dormancy period (Dayer et al., 2013). Furthermore, defruiting atthe onset of fruit ripening increases total non-structural carbohy-drate (TNC) concentration in the roots in subsequent seasons, whilea complete defoliation at harvest reduces it (Smith and Holzapfel2009). No previous studies, to the best of our knowledge, have how-ever investigated the potential effect of the interaction betweenwater availability and leaf-to-fruit ratio on the distribution of non-structural carbohydrate content between the different grapevineorgans, during the berry sugar accumulation phase.

Non-structural carbohydrates provide energy and carbon forgrapevine growth, and/or are stored as reserves in perennial plantorgans. The stored carbohydrates are used for early season veg-etative growth until leaf photosynthesis becomes the primarysource of carbon, generally around flowering for the grapevine(Zapata et al., 2004). Carbohydrate reserves are also utilizedtowards the reproductive development, including supporting berrysugar accumulation, as confirmed by 14C tracing studies (Candolfi-Vasconcelos et al., 1994). Woody tissues, especially perennial roots,also start to accumulate carbohydrates from anthesis, and the cropload influences the continuation of the perennial reserve accumu-lation during grape maturation (Holzapfel et al., 2010). Due to theinvolvement of carbohydrate reserves in these various functions,strong competition is expected to exist between the different sinksfrom véraison (berry softening) and the end of berry sugar accu-mulation (Davies and Robinson 1996; Wang et al., 2003a).

Carbohydrates are mainly stored as starch in grapevine roots,and this starch can be hydrolyzed to form soluble sugars. Duringdry conditions, the activity of starch-degrading enzymes, such as�-amylase, is often found to increase in plant tissues, resulting instarch breakdown, and an increase in soluble sugar concentrations(Jacobsen et al., 1986; Li and Li 2005). The ratio of starch to solublesugars has been reported to decrease in grapevine perennial organsduring water constraints (Rogiers et al., 2011b), as well as follow-ing early (Bennett et al., 2005) or late season (Smith and Holzapfel2009) defoliation.

Similar to sugar accumulation, fruit anthocyanin accumulationalso commences at véraison, and normally continues throughoutberry maturation (Boss et al., 1996). The accumulation of antho-cyanins in the berries is an important contributor to the qualityof the fruit from a wine quality perspective. The grapevine waterstatus is one of the major factors known to affect sugar (Wanget al., 2003b) and anthocyanin (Ojeda et al., 2002) accumulationin ripening berries.

The aim of this study was to investigate the interactive effectsof leaf-to-fruit ratio and vine water status during the berry sugaraccumulation stage on carbohydrate partitioning in perennial andannual grapevine organs. Although starch and/or soluble sugar con-centrations at certain stages of the annual grapevine growth cycle(mainly at dormancy, budburst or harvest) have been predomi-nantly reported for the roots and trunks, the kinetic of whole-vineTNC content distribution during the berry sugar accumulationphase is still a research question. The first goal was to determine thecombined effect of water constraint and limited leaf-to-fruit ratioon the TNC allocation to perennial and vegetative organs duringthe berry sugar accumulation phase. The second goal was to quan-tify the contribution of remobilized starch reserves towards berrysugar content when whole vine leaf photoassimilation becomes

insufficient for sink demands during berry maturation. The last goalwas to investigate how the accumulation of fruit sugar and antho-cyanins responds when a greater reliance is placed on the starchreserves to support berry sugar accumulation. Experiments wereconducted on grapevines grown in large pots, allowing the analy-sis of whole grapevines and individual organ biomass, including thewhole root systems, where carbohydrate distribution was deter-mined as affected by the different treatments (leaf-to-fruit ratioand water availability).

2. Materials and methods

2.1. Experimental design and treatments

Forty own-rooted Vitis vinifera L. cv Tempranillo (clone D8V12)grapevines were used in the 2013/2014 growing season, plantedin commercial potting mix soil in 50 L pots. The grapevines weregrown in an outside bird proof cage in the warm to very warmclimate of the Riverina region (Wagga Wagga, New South Wales,Australia). The three-year-old grapevines were spur pruned to fourtwo-bud spurs in the winter, left with eight primary shoots each,and distributed in four rows of ten vines each, with a three-wiretrellis system installed to support the vegetative growth. At fruitset, the total amount of bunches and berries per vine were counted,and vines were crop thinned just after fruit set so that all grapevineswere left with six to seven bunches, totaling 400 berries per vine.Prior to the application of the treatments at the onset of véraison(very first sign of berry softening), four randomly selected vines,one per row, were destructively harvested in order to representT0 for the population of grapevines. After removal of the four ini-tial vines through destructive harvesting, the nine remaining vinesper row were evenly spaced out in the row, resulting in a fourrow by nine column array, containing three treatment replicates.Two irrigation treatments, two defoliation treatments, and threedestructive harvest dates were randomized in the block design.Pressure compensated drip emitters (4 L/hr each) were used for irri-gation during the experiment. Rainfall, atmospheric temperature,and relative humidity were recorded and collected from an on-siteweather station and vapor pressure deficit (VPD) was calculated.Environmental conditions were summarized for three fortnightlyintervals during the experiment, referred to as intervals 1, 2 and 3.

In order to study the interaction between either low or high leaf-to-fruit ratios, and either low or high water availability throughoutthe berry sugar accumulation phase, four distinct treatments wereapplied, i.e., low leaf-to-fruit ratio and low water availability(LowL/F:50%); low leaf-to-fruit ratio and high water availability(LowL/F:100%); high leaf-to-fruit ratio and low water availability(HighL/F:50%); high leaf-to-fruit ratio and high water availability(HighL/F:100%). Vines with a low leaf-to-fruit ratio were left with50% less leaves than those with a high ratio (40 vs 80 leaves). Everysecond leaf from the base of each shoot was removed until each vinehad the desirable amount of leaves. Vines were irrigated three timesa day (0730, 1400 and 1800 h), with equal water volume appliedeach time of the day, ranging between 12 and 20 min of irrigationapplication time per irrigation event. The higher water availabilitytreatment was conducted with the aim of watering pots each dayjust to the point of first visual free draining during the midday irri-gation, via two irrigation emitters located to the left and right ofa vine near the middle of each pot. In the lower water availabilitytreatment, 50% of the water was delivered over the same period,through one irrigation emitter in the middle of the pot. Vines ofboth leaf-to-fruit ratio treatments received the same water volumewithin each irrigation treatment. Secondary shoots (laterals), andany newly formed leaves from primary shoots during the course of

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the experiment, were removed daily as soon as the regrowth wasobserved.

At the fortnightly destructive harvest dates, i.e., véraison (27 Dec2013, i.e., V), V + 14 (10 Jan 2014), V + 27 (23 Jan 2014) and V + 40(5 Feb 2014), the pre-selected grapevines were dismantled. Wholeroot systems, trunks, spurs, stems, petioles, and leaves were sep-arated, collected and washed with phosphate-free detergent andrinsed with deionized water. Leaves were collected in the morn-ing between 0800 and 1000 h on each of the destructive harvestdates. The fresh weights of these organs were determined, and thesamples were oven dried at 60 ◦C until a constant dry weight wasreached.

2.2. Leaf-to-fruit ratio and berry composition

The total leaf area of all the leaves that were sampled from eachindividual vine at the respective destructive harvest dates was mea-sured using a leaf area meter (LLI-3100C, LI-COR Biosciences Inc.,Lincoln, Nebraska, USA). The total fruit weight of each grapevinewas also recorded, and the leaf-to-fruit ratios determined. A 50berry subsample per vine was oven dried at 60 ◦C until constantweight, and total vine fruit dry weight determined. The averagesoluble solid content per berry per vine was determined in a sub-sample of 50 representative berries, on the basis of berry freshweight and juice soluble solid concentration (◦Brix).

Berry anthocyanin concentration was analyzed from a 50 berrysubsample per vine. The whole berries were homogenized (Ultra-Turrax T25, IKA, Selangor, Malaysia), and the phenolic compoundsextracted from 1 g homogenate in 10 mL 50% ethanol (pH 2)for two hours. The samples were centrifuged at 3000 rpm for10 min, and 1 mL supernatant was added to 9 mL HCl and leftfor 3 h at room temperature. The absorbance was measured at520 nm (�Quant universal spectrophotometer MQX200, Bio-Tek,Winooski, VT, USA), to determine berry total anthocyanin concen-trations (Iland et al., 2000).

2.3. Grapevine water status and leaf gas exchange

Weekly measurements of soil water content were made directlyunder an irrigation emitter in the midday period (between 1200and 1400 h), using a time-domain reflectometry (TDR) probe(Trime

®-FM3, Imko GmbH, Ettlingen, Germany). Measurements of

stem water potential (SWP) were started one week after the treat-ments were initiated, and undertaken once a week, with a singleleaf removed per vine per measurement to minimize the impacton vine leaf-to-fruit ratio. Following the method outlined by Chonéet al. (2001), one leaf from each vine on a main shoot was enclosed ina zip-lock plastic bag covered with aluminum foil, and left coveredfor 30 min in the midday period (between 1200 and 1400 h) to allowstomatal closure. The leaves were then removed by a single cut ofthe petiole using a sharp blade, the bags removed, and the leaveswere immediately placed in a pressure chamber for measurement(Model 1000, PMS instruments, Albany, Oregon, USA).

A portable photosynthesis system instrument (LCA-4, ADC Bio-scientific Ltd., Hoddesdon, Hertfordshire, UK) was used to measurecomparison values of leaf temperature, stomatal conductance (gs)and photosynthesis rates (A). Two fully intact leaves were chosenweekly on each vine between the 4th and 7th shoot node positionfrom the base, to be used for measurements during midday periods(between 1200 and 1400 h) on clear, non-cloudy days.

2.4. Non-structural carbohydrates

Whole, dried plant material, collected during the destructiveharvest dates (roots, trunks, spurs, stems, petioles and leaves),were ground through a heavy duty cutting mill (Retsch SM2000,

Hann, NRW, Germany) to 5 mm and a subsample was then groundthrough a 0.12 mm sieve using an ultracentrifugal mill (RetschZM200, Hann, NRW, Germany).

Following the method outlined in Smith and Holzapfel (2009),the starch concentration was determined in a 20 mg subsample bya commercial enzymatic assay (K-TSTA, Megazyme International,Bray, Ireland). In short, soluble solids were first extracted usingthree 1 mL portions of 80% (v/v) aqueous ethanol, two at 80 ◦C andone at room temperature for ten minutes. The extracts were cen-trifuged after each wash, the supernatants collected together, andthen used for later soluble sugar analysis. The remaining dried plantmaterial was suspended in 200 �L dimethyl sulfoxide and heatedat 98 ◦C for 10 min. Starch was then hydrolyzed with �-amylase(30 units) and amyloglucosidase (33 units), and the starch contentwas calculated from the concentration of released glucose in thesample.

For the soluble sugar analyses, the combined extracts preparedduring the starch analyses were diluted to 10 mL with deion-ized water, and used for determination of the concentrations ofsucrose, D-glucose, D-fructose and total sugars, with a commercialenzymatic assay (K-SUFRG, Megazyme International, Bray, Ireland),as outlined in Smith and Holzapfel (2009). In this method, eachsugar was converted into glucose-6-phosphate (G6P), and quan-tification of reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was performed, following oxidation in the presence ofnicotinamide-adenine dinucleotide phosphate (NADP+) and G6P-dehydrogenase.

2.5. Statistical analysis

Data were analyzed using Statistica 12 (Statsoft Inc., Tulsa, OK,USA), with the analysis of variance (ANOVA) used to test the sig-nificance of each variable. Fisher’s least significant difference (LSD)test was used to identify significant differences between means(P < 0.05). Significant differences in table columns and rows areindicated by upper case and lower case letters, respectively.

3. Results

The fortnightly periods between the four destructive harvestdates are referred to as intervals 1, 2 and 3. Intervals 1 and 2 rep-resent the rapid berry sugar accumulation period, while interval 3represents the slow berry sugar accumulation period.

3.1. Environmental conditions

The average daily temperature and vapor pressure deficit (VPD),and the total rainfall data collected during each interval of theexperiment are shown in Table 1.

3.2. Overview of non-structural carbohydrate assimilation andallocation

For all treatments, the soluble sugar content (SSC) per berryinitially accumulated significantly between V and V + 27 (rapidaccumulation), and slowed down between V + 27 and V + 40, whenthere were no significant changes in berry SSC (slow accumulation)(Fig. 1). The tempo (mg/berry/day) of berry sugar accumulation didnot differ significantly between the treatments during both, therapid and slow berry sugar accumulation periods (Fig. 1).

The leaf stomatal conductance (gs) of all treatments decreasedsignificantly between intervals 1 (V to V + 14) and 2 (V + 14 toV + 27) for all treatments, while leaf photoassimilation rates (A)also significantly decreased during this stage, except for treatmentLowL/F:100% (Fig. 1B). Further reduction in gs took place duringinterval 3 (slow berry sugar accumulation) for grapevines under

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Table 1Periodic rainfall, daily mean, minimum and maximum atmospheric temperature and vapor pressure deficit (VPD) averages, and berry juice soluble solid concentration (◦Brix)during the experimental period.

V to V + 14 (Interval 1) V + 14 to V + 27 (Interval 2) V + 27 to V + 40 (Interval 3)

Daily mean 22.9 29.6 28.0Mean min 15.6 21.8 20.1Mean max 29.3 36.7 35.0

Daily mean 2.1 3.3 2.9Mean min 0.8 1.5 1.1Mean max 3.4 5.4 4.9Total 1.9 1.2 14.8

Temperature (◦C)

VPD (kPa)

Rainfall (mm)

Total soluble solids (◦Brix) Mean change 7.3–15.4 15.4–21.6 21.6–23.2

Fig. 1. Effects of the different irrigation and leaf-to-fruit ratio treatments (A: LowL/F:50%, B: LowL/F:100%, C: HighL/F:50% and D: HighL/F:100%) on the soluble solid contentaccumulation per berry, leaf stomatal conductance (gs) and photosynthesis (A), and root starch, soluble sugar and total non-structural carbohydrate (TNC) content evolutionper vine (n = 3).

higher water availability, while A also reduced significantly for alltreatments except LowL/F:50% (Fig. 1A), during slow fruit sugaraccumulation.

Under higher water supply, the root starch content per vinewas not significantly affected during rapid berry sugar accumu-lation (Fig. 1B + D). However, under reduced irrigation, significantdepletion in root starch content occurred during rapid berry sugar

accumulation (Fig. 1A + C). During slow berry sugar accumulation,the starch content in these roots increased significantly, and backto their initial levels at V. The root soluble sugar content of treat-ment HighL/F:50% increased significantly during rapid berry sugaraccumulation, and was significantly higher than that of vines withhigh water availability at V + 27 (Fig. 1C). The root sugar content

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of treatment LowL/F:50% showed a similar increasing trend duringrapid berry sugar accumulation (Fig. 1A) (P = 0.07).

Reduced irrigation induced significant root TNC content deple-tion during rapid berry sugar accumulation (Fig. 1A + C). These TNCcontents then increased significantly during slow berry sugar accu-mulation.

3.3. Soil water content and vine water status

Reduced water supply induced significantly lower soil watercontent (Table 2). However, the leaf-to-fruit ratio treatments alsoaffected the soil water content of vines that received more irri-gation, where the high leaf-to-fruit ratio significantly caused 30%lower average soil water content compared to the treatment withlower leaf-to-fruit ratio, despite receiving the same water volume.Under reduced irrigation, the leaf-to-fruit ratio did not significantlyimpact the soil water content.

The stem water potential (SWP) was significantly affected bythe irrigation regime and the defoliation treatments (Table 2).Higher water supply resulted in less negative SWP values, andcorresponded to a moderate to weak overall grapevine waterconstraint according to published classification thresholds (VanLeeuwen et al., 2009); however, higher leaf-to-fruit ratio treat-ments showed more negative SWP values. During interval 2 (V + 14to V + 27), SWP values became significantly more negative for treat-ment lowL/F:100%. Reduced water supply resulted in a moderateto severe grapevine water constraint, according to previously sug-gested thresholds (Van Leeuwen et al., 2009).

3.4. Leaf and fruit structural development

Vines with a high leaf-to-fruit ratio lost 19 and 24% of their leafarea between V and V + 40 under higher and lower irrigation supply,respectively (Fig. 2A). No significant differences in leaf area amongthe different destructive harvests were observed for grapevineswith low leaf-to-fruit ratio, irrespective of the irrigation regime.

Total fresh fruit weight per vine increased significantly from Vto a maximum at V + 27 for all treatments, except HighL/F:100%,where the maximum fresh weight was observed at the end of inter-val 1 (V + 14) (Fig. 2B). After the initial increase in fresh weight, totalfresh fruit weight decreased for all treatments to varying degrees(significantly for treatments with high leaf-to-fruit ratio), withgrapevines from treatment HighL/F:50% showing the largest totalfresh fruit weight loss towards the end of interval 3 (25% of overalldecrease). There was no significant difference in leaf-to-fruit ratiosat the final destructive harvest date (V + 40) (Fig. 2C).

3.5. Leaf gas exchange

Leaf stomatal conductance (gs) and photosynthesis rates(A) were significantly affected by both water availability, andgrapevine leaf-to-fruit ratio (Table 2). Grapevines generallyexposed to the highest seasonal water constraints, according tothe SWP values (treatment HighL/F:50%), showed the lowest gs

and A. Under both irrigation regimes, the low leaf-to-fruit ratiotreatments showed significantly higher gs and A values thanthe corresponding high leaf-to-fruit ratio treatments. TreatmentLowL/F:100% resulted, on average, in 34% higher A values thantreatment HighL/F:100%, while treatment LowL/F:50% resultedin 43% higher A values than treatment HighL/F:50%. Vines fromtreatment LowL/F:100% showed the highest A, and treatmentHighL/F:50% the lowest A (P < 0.05).

The average mid-day leaf temperature per interval was con-stantly measured above 35 ◦C during the experiment (Table 2).

Fig. 2. Effects of the different irrigation and leaf-to-fruit ratio treatments on totalgrapevine leaf area (A), total berry fresh weight per vine (B), and leaf-to-fresh fruitweight ratio per vine (C) (mean ± SE; n = 3).

3.6. Berry composition

Treatment HighL/F:50% induced significantly lower berry sol-uble solid content (SSC) at V + 40 than the grapevines underhigh water availability, furthermore, the berries of treat-ment LowL/F:50% had significantly lower SSC than treatmentHighL/F:100% at V + 40 (Fig. 3A). The berry juice soluble solidconcentrations at V + 40 ranged from 22.3◦Brix for treatmentLowL/F:100% to 24.6◦Brix for treatment HighL/F:100%, but did notsignificantly differ between any of the treatments at this stage (datanot shown).

Seasonal berry dry weight evolution followed a similar patternthan that observed with berry sugar accumulation (Fig. 3A). Rapidberry dry weight increase was observed during rapid berry sugaraccumulation (V to V + 27), with a slower berry dry weight increaseduring slow fruit sugar accumulation (V + 27 to V + 40). The averagefresh weight per berry at V + 40 ranged from 1.62 g for treatmentHighL/F:50% to 1.93 g for treatment HighL/F:100%, and the differ-ences were significant between these treatments. There were noother significant differences in fresh weights per berry betweenany treatments (data not shown).

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Table 2Average leaf temperatures during the different intervals of the experiment, and the influence of different irrigation and leaf-to-fruit ratio treatments on soil water content,stem water potential, and leaf stomatal conductance and photosynthesis rates.

Treatment V to V + 14 V + 14 to V + 27 V + 27 to V + 40 Treatment main effect

Rapid berry sugar accumulation Slow berry sugar accumulation

Average 35.1 36.0 35.8 –Leaf temperature (◦C)Soil water content (%) LowL/F:50% C a 9.0 a b B 9.2 a B 9.0 a C 9.08

LowL/F:100% A 14.7 a A 16.6 a A 14.6 a A 15.32HighL/F:50% C 7.9 a B 8.9 a B 8.8 a C 8.40HighL/F:100% B 10.8 a B 10.7 a B 10.3 a B 10.68

Stem water potential (-MPa) LowL/F:50% C 1.21 a BC 1.34 a B 1.24 a C 1.26LowL/F:100% A 0.66 a A 0.97 b A 0.87 a A 0.80HighL/F:50% D 1.38 ab C 1.57 b 1.32B aHighL/F:100% B 1.00 a 1.15AB a A 0.93 a B 1.04

Stomatal conductance (mol/m2/s) LowL/F:50% B 0.03 a B 0.02 b B 0.02 b B 0.02LowL/F:100% A 0.05 a A 0.03 b A 0.02 c A 0.04HighL/F:50% C 0.02 a C 0.01 b C 0.01 b C 0.01HighL/F:100% B 0.03 a B 0.02 b BC 0.01 c B 0.02

Photosynthesis rate (�mol/m2/s) LowL/F:50% B 4.90 a B 4.18 b B 3.40 b B 4.39LowL/F:100% A 6.42 a A 6.09 a A 4.43 b A 5.94HighL/F:50% C 3.73 a C 2.86 b C 1.77 c C 3.06HighL/F:100% B 5.52 a 3.89B b 2.68 cB B 4.44

a Means separated within columns using Fisher’s LSD test, significant differences are indicated at P < 0.05. Where the same capital letter appears in a column, values donot differ significantly.

b Means separated within rows using Fisher’s LSD test, significant differences are indicated at P < 0.05. Where the same lower case letter appears in a row, values do notdiffer significantly.

Fig. 3. Impact of irrigation and leaf-to-fruit ratio on A: soluble solid content (SSC, left axis) and dry weight (DW, right axis) per berry, and B: anthocyanin content per berry(left axis) and berry anthocyanin concentration (right axis) during the experimental period (mean ± SE; n = 3).

The berry anthocyanin concentration increased significantlyfor all treatments during intervals 1 and 2, when rapid berrysugar accumulation also took place (Fig. 3B), and continued toincrease significantly during interval 3 for all treatments, exceptHighL/F:100%. At V + 27, the fruit of treatment HighL/F:100%had significantly higher anthocyanin concentration than that ofall other treatments. The total anthocyanin content per berryincreased significantly during rapid berry sugar accumulation,and especially accumulated rapidly for treatment HighL/F:100%during interval 2 (Fig. 3B). The anthocyanin content per berry con-tinued to increase significantly during interval 3 for treatmentLowL/F:100%. At V + 40, the anthocyanin content per berry did notdiffer significantly between the treatments, although the berries oftreatment HighL/F:100% tended to have higher anthocyanin con-tent than that of treatments HighL/F:50% (P = 0.06) and LowL/F:50%(P = 0.08).

3.7. Non-structural carbohydrates

Whole vine (excluding the fruit) and perennial tissue TNC con-tent evolution is represented in Fig. 4. In Table 3, organ-specificstarch and soluble sugar concentrations are reported, while organ-

specific total dry biomass and TNC content development is reportedin Table 4.

3.7.1. Whole-vine and perennial TNC contentThe TNC content for the combined perennial and vegetative

seasonal organs (total vine TNC) (Fig. 4A) was reflective of theoverall perennial tissue TNC content (Fig. 4B). Under higher wateravailability, the total vine and perennial tissue TNC content didnot significantly change during rapid berry sugar accumulation(V to V + 27), but increased significantly during slow berry sugaraccumulation (V + 27 to V + 40) when vines also had a high leaf-to-fruit ratio. Under lower water availability, total vine and perennialTNC contents reduced significantly during rapid berry sugar accu-mulation (Fig. 4). This was especially obvious in vines with lowleaf-to-fruit ratio, where both TNC contents decreased by 30%.The decrease was most pronounced during interval 2 (V + 14to V + 27), and especially for vines with low leaf-to-fruit ratio.During slow berry sugar accumulation, significant TNC replen-ishment took place, regaining the initial TNC contents observedat V.

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Table 3Influence of different irrigation and leaf-to-fruit ratio treatments on the starch and sugar concentrations in the roots, trunks, stems and leaves (% DW) at the various destructiveharvest dates (n = 3).

Rapid berry sugaraccumulation

Slow berry sugaraccumulation

Rapid berry sugaraccumulation

Slow berry sugaraccumulation

Treatment V V + 14 V + 27 V + 40 V V + 14 V + 27 V + 40

Starch% Roots LowL/F:50% 25.3 a Ba 20.7 bb B 18.4 b B 25.1 a Trunk 7.1 ab A 9.3 a B 6.2 b A 8.2 abLowL/F:100% 25.3 a A 26.8 a A 25.3 a AB 29.3 a 7.1 a A 8.6 a A 9.3 a A 9.5 aHighL/F:50% 25.3 a AB 22.7 ab B 18.9 b B 24.7 a 7.1 a A 8.0 a B 6.7 a A 8.1 aHighL/F:100% 25.3 b AB 25.5 b AB 20.3 b A 34.0 a 7.1 b A 7.4 b A 10.2 ab A 11.9 a

Sugar% LowL/F:50% 2.5 b B 2.4 b AB 2.9 ab A 3.8 a 1.5 a BC 1.3 a A 1.6 a A 2.3 aLowL/F:100% 2.5 b 1.7B c 2.3B bc A 3.1 a 1.5 a 0.8C c B 0.9 bc A 1.4 abHighL/F:50% 2.5 b A 3.1 ab A 4.0 a A 4.0 a 1.5 b A 2.1 a A 2.0 ab A 1.7 abHighL/F:100% 2.5 b B 2.4 b B 2.4 b A 3.4 a 1.5 a AB 1.6 a AB 1.4 a A 1.4 a

Starch% Stems LowL/F:50% 3.4 b A 4.8 a B 3.8 ab B 4.7 a Leaves 0.14 a C 0.06 b B 0.06 b B 0.15 aLowL/F:100% 3.4 b A 4.7 a B 4.7 a B 4.8 a 0.14 a AB 0.10 a B 0.14 a B 0.22 aHighL/F:50% 3.4 b A 4.6 ab B 3.5 b B 5.4 a 0.14 a BC 0.05 b B 0.06 b B 0.14 aHighL/F:100% 3.4 c A 5.5 b A 6.7 ab A 8.0 a 0.14 b A 0.12 b A 1.32 a A 0.55 ab

Sugar% LowL/F:50% 2.1 a A 1.9 a A 2.2 a B 1.9 a 4.9 a A 2.6 b B 2.4 b A 3.6 abLowL/F:100% 2.1 ab B 1.2 b B 1.2 b AB 2.8 a 4.9 a A 2.6 b AB 2.9 b A 3.3 bHighL/F:50% 2.1 b 2.5A a 2.5A a 2.4AB ab 4.9 a A 2.7 b AB 3.0 b A 3.2 bHighL/F:100% 2.1 b A 2.0 b A 2.2 b A 3.1 a 4.9 a A 3.4 b A 4.4 ab A 3.6 b



Table 4Influence of different irrigation and leaf-to-fruit ratio treatments on the dry biomass of total fruit per vine, and the dry biomass and total non-structural carbohydrate (TNC)content per organ.

Rapid berry sugar accumulation Slow berry sugar accumulation

Treatment V V + 14 V + 27 V + 40

Biomass (g) Fruit LowL/F:50% 55.0 c Ba 106.2 bb B 170.4 a AB 179.7 aLowL/F:100% 55.0 c B 118.4 b AB 185.6 a AB 180.7 aHighL/F:50% 55.0 d B 118.9 c AB 182.5 a B 161.0 bHighL/F:100% 55.0 c A 157.0 b A 196.3 a A 194.0 a

Biomass (g) Roots LowL/F:50% 191.5 a A 197.6 a B 171.0 b A 181.8 abLowL/F:100% 191.5 a A 168.7 a B 182.0 a A 182.6 aHighL/F:50% 191.5 a A 190.5 a AB 192.7 a A 183.2 aHighL/F:100% 191.5 b 163.3A b 236.1 aA A 207.2 ab

TNC content (g) LowL/F:50% 49.8 a A 42.2 a A 33.2 b B 47.9 aLowL/F:100% 49.8 a A 44.2 a A 48.1 a B 55.2 aHighL/F:50% 49.8 a A 45.1 a A 38.7 b B 48.9 aHighL/F:100% 49.8 b A 42.9 b A 49.3 b A 71.6 a

Biomass (g) Stems LowL/F:50% 54.7 a AB 69.2 a A 58.2 a A 63.6 aLowL/F:100% 54.7 a B 55.8 a A 70.8 a A 67.9 aHighL/F:50% 54.7 a AB 61.1 a A 55.2 a A 57.4 aHighL/F:100% 54.7 b A 77.9 a A 74.8 ab A 77.4 a

TNC content (g) LowL/F:50% 3.3 a AB 4.6 a B 3.5 a B 4.2 aLowL/F:100% 3.3 a B 3.3 a B 4.2 a B 5.0 aHighL/F:50% 3.3 b B 4.2 ab B 3.3 b B 4.5 aHighL/F:100% 3.3 c A 5.9 b A 6.6 ab A 8.6 a

Biomass (g) Trunk LowL/F:50% 84.7 a A 106.7 a A 81.8 a A 93.3 aLowL/F:100% 84.7 a AB 94.5 a A 87.8 a A 99.6 aHighL/F:50% 84.7 a B 78.7 a A 86.3 a A 85.4 aHighL/F:100% 84.7 a AB 97.9 a A 94.5 a A 95.9 a

TNC content (g) LowL/F:50% 7.3 b BC 11.4 a A 6.4 b A 9.8 abLowL/F:100% 7.3 b C 9.0 ab B 8.8 ab A 10.8 aHighL/F:50% 7.3 a A 7.9 a A 7.5 a A 8.4 aHighL/F:100% 7.3 c AB 8.8 bc AB 11.0 ab A 12.6 a

Biomass (g) Leaves LowL/F:50% 0.7 bc AB 0.9 a A 1.0 a A 1.0 aLowL/F:100% 0.7 b AB 0.9 a A 0.9 a A 1.0 aHighL/F:50% 0.7 b B 0.8 a A 0.8 a B 0.8 aHighL/F:100% 0.7 b A 1.0 a A 0.9 a AB 0.9 a

TNC content (g) LowL/F:50% 0.03 ac A 0.02 a B 0.02 a A 0.04 aLowL/F:100% 0.03 a A 0.02 a AB 0.03 a A 0.04 aHighL/F:50% 0.03 a A 0.02 a AB 0.02 a A 0.03 aHighL/F:100% 0.03 b A 0.04 ab A 0.05 a A 0.04 ab



c Leaf biomass and TNC content indicated as per leaf per vine.

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Fig. 4. Total non-structural carbohydrate (TNC) content per vine in combined grapevine roots, trunks, spurs, shoots, leaves and petioles (A) and in total perennial tissues (B),as affected by the irrigation and leaf-to-fruit ratio treatments (mean ± SE; n = 3).

3.7.2. Starch and soluble sugar concentration distributionUnder higher water supply, the total root starch concentration

was not significantly affected during rapid berry sugar accumula-tion (V to V + 27) (Table 3). However, the root starch concentrationdid significantly increase by V + 40 for the high leaf-to-fruit ratiotreatment. Under reduced irrigation, the root starch concentra-tions of treatments with low and high leaf-to-fruit ratio reducedsignificantly by 27 and 25% respectively, during rapid berry sugaraccumulation. During slow berry sugar accumulation (V + 27 toV + 40), the starch concentrations in these roots increased sig-nificantly. The root soluble sugar concentration of treatmentHighL/F:50% increased significantly during rapid berry sugar accu-mulation, and were significantly higher than that of vines with highwater availability at V + 27 (Table 3).

The trunk starch concentration of treatment HighL/F:100% wassignificantly higher at V + 40 than at V (Table 3). The trunk solu-ble sugar concentrations reduced significantly during rapid berrysugar accumulation for treatment LowL/F:100%, and was signifi-cantly lower than that of the reduced irrigated grapevines at V + 27(Table 3).

In the stems, vines with a higher water availability exhibiteda significant increase in starch concentration during rapid berrysugar accumulation, and this resulted in significantly higher stemstarch concentration for treatment HighL/F:100%, compared to theother treatments at V + 27 (Table 3). All treatments showed signif-icantly higher stem starch concentrations at V + 40 than at V. Stemsoluble sugar concentration increased significantly for treatmentHighL/F:50% during rapid berry sugar accumulation (Table 3).

The leaf starch concentrations for treatment HighL/F:100%increased significantly during rapid berry sugar accumulation(Table 3), and this treatment also induced significantly higher leafstarch concentrations at V + 40 than any of the other treatments.The leaf starch concentration of vines receiving reduced irriga-tion depleted significantly during rapid berry sugar accumulation.The leaf soluble sugar concentration also reduced significantly forall treatments, except treatment HighL/F:100% during rapid berrysugar accumulation (Table 3).

3.7.3. Organ dry biomass and TNC contentThe total fruit dry weight per vine increased significantly for

all treatments during rapid berry sugar accumulation (V to V + 27)(Table 4). The total fruit dry weight per vine remained constant dur-ing slow berry sugar accumulation for all treatments, apart from

treatment HighL/F:50% where it significantly reduced. At V + 40,treatment HighL/F:100% had significantly higher total fruit dryweight than treatment HighL/F50%.

The total root dry weight decreased significantly during rapidberry sugar accumulation for vines of treatment LowL/F:50%,while treatment HighL/F:100% induced significantly larger root dryweights at V + 27 than both of the low leaf-to-fruit ratio treatments(Table 4). Reduced irrigation induced significant root TNC con-tent depletion during rapid berry sugar accumulation (Table 4). AtV + 40, treatment HighL/F:100% exhibited significantly higher rootTNC content than any other treatments.

The trunk total dry weights did not significantly change duringthe experiment for any of the treatments (Table 4). The trunk TNCcontent of treatment HighL/F:100% increased significantly duringrapid berry sugar accumulation (Table 4).

The total stem dry weight of treatment HighL/F:100% was sig-nificantly higher at V + 40 than at V, but stem dry weights did notchange significantly during the experiment for any of the othertreatments (Table 4). Stem TNC contents increased significantlyfor treatment HighL/F:100% during rapid berry sugar accumulation(Table 4).

The dry weight per leaf increased significantly between Vand V + 14 for all treatments, and remained constant thereafter(Table 4). Both low leaf-to-fruit ratio treatments had significantlyhigher dry weight per leaf than treatment HighL/F:50% at V + 40.The TNC content per leaf of treatment HighL/F:100% increased sig-nificantly during rapid berry sugar accumulation.

4. Discussion

To study the contribution of non-structural carbohydrate (TNC)reserves towards berry dry matter accumulation, two distinct treat-ments of leaf-to-fruit ratio in combination with two vine watersupply regimes were implemented at véraison (onset of berry soft-ening). The reduced water supply and/or leaf area treatments wereaimed to reduce canopy photoassimilation enough so as for berrysugar accumulation to rely on remobilized stored carbohydratesfrom the perennial structure.

The relationship between the tempo of berry sugar accumu-lation and root TNC content, in potted grapevines subjected tomoderate to severe water constraints, has been illustrated in thepresent study. Although it has previously been confirmed by 14Ctracing studies that root carbohydrates can be relocated towards

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the berries during berry sugar accumulation (Candolfi-Vasconceloset al., 1994), this is to the best of our knowledge, the first studyto indicate an inverse relationship between the contents of rootTNC and berry sugar, when leaf photoassimilation is limited dur-ing rapid fruit sugar accumulation. The clear replenishment of rootTNC as the berry sugar accumulation tempo slowed down, is anadditional original result. The roots, therefore, became a comple-mentary source of TNC to supply towards the sink TNC demand ofmaturing fruit.

When comparing the TNC contents and concentrations betweenthe roots, trunks, stems and leaves: the roots had the highestvalues, and the root TNC content represented, on average, 73%of the total TNC content in these organs. The net loss of com-bined root, trunk, stem and leaf TNC content (Fig. 4A) during rapidberry sugar accumulation, in grapevines under reduced water sup-ply, can be attributed to root starch depletion. In fact, root starchremobilization accounted for 89% of the whole-vine (excluding thefruit) TNC loss during rapid berry sugar accumulation in treatmentLowL/F:50%. An apparent hydrolysis of root starch took place duringrapid fruit sugar accumulation in the reduced irrigated grapevines,corresponding to the soluble sugar accumulation in these roots. Thestarch hydrolysis was induced by a sugar deficit, prompted by thefruit (temporary TNC sink) sugar demand outweighing the leaf (TNCsource) photoassimilate supply (Eveland and Jackson, 2012). Solu-ble sugars are transported from the roots in the phloem, and TNCcan thereby be mobilized from the roots to the berries, contributingto the sink TNC demand. Further investigation is, however, neededto quantify the absolute amount of sugars relocated from the rootstoward the fruit during rapid berry sugar accumulation.

The net loss of TNC in the roots, trunks, stems and leaves duringrapid berry sugar accumulation, and in grapevines under moder-ate to severe water constraints, potentially contributed up to 18and 10% to total fruit dry biomass accumulation per vine for treat-ments LowL/F:50% and HighL/F:50%, respectively. As there wereno significant biomass increases for any other organs in grapevinesfrom these treatments during this period, it implies that significantamounts of stored TNC were unlikely used towards the struc-tural development of other organs. While not quantified in thepresent study, it must however also be noted that some of the TNCcould have been lost through respiration, although the whole-vinerespiration rate is likely reduced under limited water availabil-ity (Escalona et al., 2012). In addition, the amount of carbon lostthrough respiration in relation to the total pool of carbohydratesis also thought to be very limited, as previously illustrated ingrapevine berries (Romieu et al., 1992). It, therefore, seems likelythat root reserve TNC made a significant contribution to the berrysugar content for the grapevines that received reduced irrigation,to compensate the limited leaf assimilation (Candolfi-Vasconceloset al., 1994). To further clarify the relative contribution of rootrespiration to the change in TNC content during berry ripening,future studies could include the determination of root respirationrates.

When berry sugar accumulation slowed down, starch accumu-lated in the roots as the berry carbohydrate sink strength wasreduced. The content in TNC and especially starch, at the end ofberry maturation is an indicator of the starch reserve availability atbudburst for the following season (Smith and Holzapfel 2009). Thereserve TNC at budburst is utilized for early season vegetative andreproductive growth and development. Previous studies suggestthat low carbohydrate reserve content at budburst is detrimen-tal towards vegetative growth (Loescher et al., 1990), inflorescenceand flower initiation and development, fruit set, and overall fruityield (Bennett et al., 2005; Smith and Holzapfel 2009). It is, there-fore, probable that the vegetative and reproductive developmentof grapevines from treatments with low water availability couldbe affected in the following season, especially if further reserve

accumulation is impaired due to a short post-harvest period. Morework is however needed to quantify the post-harvest recovery ofroot TNC content following the depletion thereof during berry sugaraccumulation.

The leaf-to-fruit ratio at the final destructive harvest date(V + 40) of grapevines with low leaf-to-fruit ratios, was found to bewithin a range (8–12 cm2 leaf area per gram of fruit) estimated to,in a comparison of grapevines with a wide range of leaf-to-fruitratios in a given climatic region, allow the maximum accumu-lation of berry soluble solids, as well as maximum developmentof berry fresh weight and skin anthocyanins, on a single canopytrellis-system (Kliewer and Dokoozlian, 2005). However, whenusing potted vines, the present study indicates that the water sta-tus of grapevines with the same leaf-to-fruit ratios can significantlyimpact on the berry soluble solid content (SSC) of mature fruit, asgrapevines under reduced water supply and a high leaf-to-fruitratio had inferior berry SSC than those under higher water sup-ply, and the same leaf-to-fruit ratio. Nevertheless, the consistentpattern and tempo of berry sugar content accumulation betweenthe treatments, and the lack of significant treatment differences inberry anthocyanin content and soluble solid concentration (◦Brix)at V + 40, suggests that no treatment caused an inhibition of berrysugar import or skin anthocyanin biosynthesis. Water stress cancause alterations in the expression of genes involved in regulat-ing plant sugar transportation between source and sink organs(Williams et al., 2000), as well as those involved in berry skin antho-cyanin biosynthesis (Castellarin et al., 2007). A sustained waterstress, for example, causes an up- or down-regulation of the genesencoding hexose transporters in grapevines (Medici et al., 2014),and may thereby affect the tempo of berry sugar accumulation.Likewise, water stress can inhibit or promote anthocyanin biosyn-thesis in grapevine berries (Ojeda et al., 2002). Because there wereno significant alterations in the tempo of berry sugar accumula-tion and no significant differences in the berry anthocyanin contentof mature berries in the present study, it can be assumed thatthe reduced water supply treatments (treatments lowL/F:50% andHighL/F:50%) induced a sustained water constraint rather than astress. The water constraints, therefore, affected leaf photosynthe-sis (A), although not altering berry ripening in terms of sugar andanthocyanin accumulation.

Increased water availability and decreased leaf-to-fruit ratioimproved mid-day leaf gas exchange rates during the present study.Although leaf stomatal conductance (gs) and A declined as theexperiment progressed, the gas exchange rates in the present studywere determined by the treatments, rather than the variation inatmospheric temperatures and vapor pressure deficits (VPD) dur-ing the different intervals of the experiment. The gas exchangerates recorded in the present study, especially for grapevines undermoderate to weak water constraints, were however, relativelylow in comparison to values reported for field-grown Tempranillograpevines (Medrano et al., 2003). Constantly high atmosphericVPD, and air and leaf temperatures during the midday periods whenthese measurements were conducted could attribute to these lowvalues. Another contributor to the low gas exchange values is thefact that the scheduling of the irrigation events caused the soil mois-ture content in the pots to presumably reach its lowest volumes atthe time of the day when these measurements were conducted,and thereby promoted stomatal closure. Furthermore, the mea-surements were conducted late in the growing season, and on olderleaves towards the basal parts of the shoot, when leaf aging likelyalready impaired maximum leaf gas exchange rates (Poni et al.,1994).

Based on the observations from the present study, it is, however,important to note that limitations in whole-vine photoassimilationduring berry ripening is thought to be overcome through TNC remo-bilization from storage tissues (Candolfi-Vasconcelos et al., 1994).

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Under moderate to weak water constraints, no net whole-vine TNCremobilization was observed throughout the experiment (Fig. 4A),indicating sufficient canopy photoassimilation. However, undermoderate to severe water constraints, reserve TNC remobilizationtook place during rapid berry sugar accumulation, suggesting thatthe net photoassimilation of these grapevines was insufficient tomeet sink demands during berry maturation. The reduced leaf areaof the low leaf-to-fruit ratio grapevines likely suppressed canopytranspiration, causing lower water constraints compared to thatof the grapevines with full leaf area. However, the combinationof reduced leaf area and low water supply induced the greatestsuppression of canopy photoassimilation, resulting in the high-est relative remobilization of TNC from the roots of treatmentLowL/F:50%. This indicates that under limited water supply duringrapid berry sugar accumulation, a higher leaf-to-fruit ratio couldbe beneficial towards maintaining root carbohydrate reserves priorto harvest. This would be important when a post-harvest reserveaccumulation period is absent or insufficient, as often observed, forexample, in cooler climate areas.

5. Conclusion

The effects of water availability and grapevine sink-source rela-tions on carbohydrate reserve storage by dormancy, expressed ona concentration basis, have been studied previously. However, anovel approach was undertaken in the present study to inves-tigate carbohydrate content distribution, specifically during theactive berry sugar accumulation phase, and to quantify the contri-bution of carbohydrate reserves towards berry sugar accumulation.Moderate to severe water constraints resulted in less carbohydrateallocation to the perennial grapevine organs, although not alteringthe evolution of berry sugar and anthocyanin accumulation. Carbo-hydrate reserves were remobilized in reduced-irrigated grapevinesto contribute to the berry sugar content. When berry carbohydratesink demand decreased, carbohydrates were redirected towardsthe roots, and root starch accumulated. The largest relative con-tribution (up to 18%) of total perennial and vegetative seasonalorgan carbohydrate mobilization towards berry dry matter accu-mulation, occurred for vines with low leaf-to-fruit ratios and underreduced irrigation. In these grapevines, root starch mobilizationaccounted for up to 89% of the loss of total perennial and vegeta-tive seasonal organ carbohydrate content during rapid berry sugaraccumulation. Moderate to severe water constraints can cause agreater reliance on TNC reserves to support berry dry matter accu-mulation, although seemingly not impacting on the effectiveness ofberry sugar import or anthocyanin biosynthesis. Although reservecarbohydrate replenishment starts as soon as the berry sugar accu-mulation tempo slows (possibly even a few weeks prior to harvest),restricted water availability during berry maturation can causelower carbohydrate reserve content in the roots by fruit matu-rity. In vineyards where no, or an ineffective post-harvest periodoccurs, the impact of this prevention of root carbohydrate reserveaccumulation by fruit maturity is more severe.

Acknowledgements

This work was supported by the National Wine and GrapeIndustry Centre, and the Australian grapegrowers and winemak-ers through their investment body, Wine Australia, with matchingfunds from the Australian Government. The authors thank RobertLamont and David Foster for technical assistance, and BeverleyOrchard for statistical advice.

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Chapter 4: Paper 2

Implications of the presence of maturing fruit on

carbohydrate and nitrogen distribution in grapevines under

post-veraison water constraints

(Paper 2 has been published in the Journal of the American Society for

Horticultural Science as in the format below.)


To determine how the presence or absence of fruit during sustained post-véraison water

constraints influences the allocation of carbohydrates and N between the different

grapevine organs.

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J. AMER. SOC. HORT. SCI. 142(2):71–84. 2017. doi: 10.21273/JASHS03982-16

Implications of the Presence of Maturing Fruiton Carbohydrate and Nitrogen Distributionin Grapevines under Postveraison Water ConstraintsGerhard C. Rossouw3

National Wine and Grape Industry Centre, School of Agricultural and Wine Sciences, Charles SturtUniversity, Wagga Wagga, New South Wales 2678, Australia

Jason P. Smith1

National Wine and Grape Industry Centre, Charles Sturt University, WaggaWagga, New SouthWales2678, Australia

Celia BarrilNational Wine and Grape Industry Centre, School of Agricultural and Wine Sciences, Charles SturtUniversity, Wagga Wagga, New South Wales 2678, Australia

Alain Deloire2

National Wine and Grape Industry Centre, Charles Sturt University, WaggaWagga, New SouthWales2678, Australia

Bruno P. HolzapfelNational Wine and Grape Industry Centre, New South Wales Department of Primary Industries,Wagga Wagga, New South Wales 2678, Australia

ADDITIONAL INDEX WORDS. starch mobilization, nitrogen allocation, amino acids, water deficit, sucrose translocation

ABSTRACT. Grapevine (Vitis vinifera) berries are sugar and nitrogen (N) sinks between veraison and fruit maturity.Limited photoassimilation, often caused by water constraints, induces reserve total nonstructural carbohydrate (TNC)remobilization, contributing to berry sugar accumulation, while fruit N accumulation can be affected by vine watersupply. Although postveraison root carbohydrate remobilization toward the fruit has been identified through 14C tracingstudies, it is still unclear when this remobilization occurs during the two phases of berry sugar accumulation (rapid andslow). Similarly, although postveraison N reserve mobilization toward the fruit has been reported, the impact of waterconstraints during berry N accumulation on its translocation from the different grapevine organs requires clarification.Potted grapevines were grown with or without fruit from the onset of veraison. Vines were irrigated to sustain waterconstraints, and fortnightly root, trunk, shoot, and leaf structural biomass, starch, soluble sugar, total N, and amino Nconcentrations were determined. The fruit sugar and N accumulation was also assessed. Root starch depletion coincidedwith root sucrose and hexose accumulation during peak berry sugar accumulation. Defruiting at veraison resulted incontinuous root growth, earlier starch storage, and root hexose accumulation. Leaf N depletion coincided with fruitN accumulation, while the roots of defruited vines accumulated N reserves. Root growth, starch, and N reserveaccumulation were affected by maturing fruit during water constraints. Root starch is an alternative source to supportfruit sugar accumulation, resulting in reserve starch depletion during rapid fruit sugar accumulation, while root starchrefills during slow berry sugar accumulation. On the other hand, leaf N is a source toward postveraison fruit Naccumulation, and the fruit N accumulation prevents root N storage.

Grapevine berries are sinks for the incorporation of bothcarbohydrates (Davies and Robinson, 1996) and N (Roubelakis-Angelakis and Kliewer, 1992) between veraison and fruitmaturity. Restricted TNC availability, induced by limited leafphotoassimilation, can cause starch redistribution from the rootsduring berry sugar accumulation (Candolfi-Vasconcelos et al.,1994), while N concentrations in the berries and roots are

affected by abiotic conditions, such as vine water availability,during the growing season (Araujo et al., 1995). Furthermore,apart from also being affected by vine N supply, the N reserveaccumulation in the roots is restricted by the presence of fruitbefore and after veraison (Rodriguez-Lovelle and Gaudillere,2002). It is still unclear how the postveraison distribution ofTNC and N reserves among the different organs, are affectedby a combination of fruit presence and sustained water con-straints. A further question remains on how this distributioncontributes to, or inhibits, TNC and N reserve storage, or thecontents of TNC and N in the fruit during berry maturation.

In plant roots, TNC are mainly stored as starch, which can behydrolyzed, yielding osmotic active soluble sugars (Regieret al., 2009). Apart from the possible remobilization of sugarsvia phloem sucrose transportation (Ruan et al., 2010) between

Received for publication 23 Nov. 2016. Accepted for publication 9 Jan. 2017.This work was supported by the National Wine and Grape Industry Centre, andthe Australian grapegrowers and winemakers through their investment body,Wine Australia, with matching funds from the Australian Government.1Current address: Institut f€ur Allgemeinen und €okologischen Weinbau,Hochschule Geisenheim University, Geisenheim, 65366, Germany2Current address: Montpellier SupAgro, Montpellier, 34060, France3Corresponding author. E-mail: [email protected].

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the perennial vine parts (the roots and trunk) and the ripeningberries, thereby contributing to berry sugar accumulation(Candolfi-Vasconcelos et al., 1994), sugars also accumulate invarious tissues of water stressed plants, to aid in osmotic regu-lation (Regier et al., 2009). Therefore, upon water constraintsduring berry sugar accumulation, the accumulation of solublesugars in different vine parts could theoretically contribute tovarious functions; e.g., to facilitate TNC remobilization, and toimprove abiotic stress tolerance. As grapevines are perennialplants, the storage of starch reserves at the end of the season isessential for reserve TNC utilization the following season,required for vegetative and reproductive development frombudburst (Holzapfel et al., 2010). Depleted root starch concen-tration can then lead to limited initiation and development of theinflorescence, and decreased fruit set and fruit yield the followingseason (Smith and Holzapfel, 2009).

The N allocation to grapevine berries, and subsequent accu-mulation during berry maturation is, from a wine qualityperspective, essential as it determines the juice yeast assimilableN content, influencing fermentation and wine composition.However, root N accumulation late in the growing season isimportant for its overwintering storage (Cheng et al., 2004).Similar to TNC, N reserves are used toward the initiation of earlyseason vegetative growth, where their mobilization regulatespring growth and account for most of the N distribution untilaround flowering, as N soil uptake is usually still insufficient atthis stage (Zapata et al., 2004). Nitrogen accumulation in theperennial vine parts usually initiates before berry maturity, andthe reserves continue to increase until leaf fall (Roubelakis-Angelakis and Kliewer, 1992). The presence of fruit reduces Nassimilation in grapevine roots (Morinaga et al., 2003). Nitrogenis mostly stored in the roots, and these reserves consists of a rangeof amino acids and proteins (Zapata et al., 2004). Amino acids inplants are involved in the regulation of N metabolism, and playessential roles in N transport and storage (Roubelakis-Angelakisand Kliewer, 1992). The metabolic pathway related to thea-ketoglutarate family of amino acids, yields glutamic acid,glutamine, arginine, and proline. These amino acids are abundantin plants, and have distinct roles in N metabolism (Verma et al.,1999). Glutamic acid is the intermediate product of nitrate andammonium assimilation, and a precursor for the synthesis ofglutamine, arginine, and proline (Berg et al., 2002). Arginine isconsidered the main N-storage amino acid in grapevines (Xiaand Cheng, 2004), while glutamine is an essential N transporter(Coruzzi and Last, 2000). Proline accumulation is linked toosmotic adjustment following abiotic plant stresses (Hare andCress, 1997). The metabolism of the a-ketoglutarate-derivedamino acids is therefore essential to regulate plant N partitioningand distribution, particularly during abiotic constraints.

The aim of this experiment was to determine the effect offruit presence during sustained postveraison water constraints,on the TNC and N distribution within grapevines. The first goalwas to investigate the response in the structural development ofthe leaves, shoots, trunk, and roots, based on the presence offruit, during sustained water constraints. The second goal wasto determine how fruit presence affects TNC accumulation inthe different organs, during the sustained water constraints, andto assess which individual sugars accumulate in the grapevineroots during the two phases of berry maturation (rapid and slowsugar accumulation). The final goal was to determine how thepresence of maturing fruit affects the allocation of N betweenthe grapevine organs, and to identify potential contributions of

amino N, and especially the amino acids yielded from a-keto-glutarate metabolism, toward N storage or translocation.

Materials and Methods

EXPERIMENTAL DESIGN. Own-rooted ‘Shiraz’ grapevines,grown in 50-L pots containing commercial potting mix, wereused for this experiment in the 2014–15 growing season. Thegrapevines were grown in an outside bird-proof cage in thewarm to very warm climate Riverina region of New SouthWales, Australia. The 3-year-old grapevines were winterpruned to 10 spurs with two buds each, and arranged in fourrows of nine vines each. From just after budburst, thegrapevines were fertilized every 3 weeks with 250 mL of50:1 diluted complete liquid fertilizer (MEGAMIX Plus;Rutec, Tamworth, Australia). In total, �2.6 g N was appliedto each vine through fertilization, and the fertilization eventswere ceased 1 month before the start of the experiment, aimingto limit soil N uptake during the experiment. The grapevineswere well watered between budburst and veraison, whenirrigation was supplied three times a day to the point of visualfree drainage from the pots.

Vines were shoot thinned so as to leave 17 shoots per vinefrom fruit set, and at the onset of veraison, 2 d after the first signof berry softening was observed, the treatments were initiated.Four randomly selected vines, one from each row, weredestructively harvested on the day when then the treatmentswere initiated, to represent the population of grapevines beforethe implementation of the treatments. After removal of the fourinitial vines, the eight remaining vines per row were evenlyspaced out in the row. All bunches were removed on half thevines, to have 16 vines with, and 16 vines without fruit. Twovines in each row (one with fruit and one without fruit) wereused as a visual reference to control irrigation scheduling, andreceived double the irrigation volume than the other vines.Irrigation was scheduled three times per day (0800, 1400, and1800 HR) for all vines, and the vines receiving double theirrigation, were rewatered each day just to the point of visualfree drainage from the pots during the 1400 HR irrigation event,through two irrigation emitters per pot. The remaining 24 vineswere irrigated through one irrigation emitter, aiming to inducesustained water constraints. Selected vines were destructivelyharvested fortnightly from after the start of veraison, over fourdistinct harvest dates, as described later. Three vines with andwithout fruit each, and that received reduced irrigation, wereharvested during each of these dates. Vines were distributedin triplicate for each treatment and harvest date. Pressure-compensated drip emitters (4 L�h–1 each) were used to supplythe irrigation during the experiment, and the irrigation timeranged between 15 and 22min per irrigation event (the same foreach irrigation event per day) to reach free drainage from thepots receiving double irrigation, as described above. Beforeforecasted rainfall events, the top of the pots, around the trunksof the grapevines, were covered with plastic to avoid rain waterfrom entering the soil.

At the destructive harvest dates; i.e., veraison [V (22 Dec.2014)], V + 14 (5 Jan. 2015), V + 29 (20 Jan. 2014), V + 42(2 Feb. 2015), and V + 56 (16 Feb. 2014), the preselectedgrapevines were dismantled. Whole root systems, trunks,shoots, and leaves were separated, collected, and washed withphosphate-free detergent and rinsed with deionized water.Leaves were collected between 0800 and 1000 HR on each of

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modified from Reed et al. (2004), and used to calculate the roottotal sugar concentrations. Extractions were conducted, once in2 mL of a 200 mg�L–1 mannitol (internal standard) solution,followed by twomore extractions in 2mL ultrapure water. Extractsolutions were homogenized with the root tissue, before incuba-tion in an 80 �Cwater bath for 15 min, before being centrifuged at3000 gn for 5 min. The three supernatants were collected together,and purified through a solid-phase extraction cartridge, containingreverse phase C18 packing. The cartridges were first preequili-brated with 4 mL methanol followed by 8 mL ultrapure water,and the sample was finally eluted with 1.5 mL ultrapure water. Acentrifugal evaporator (CentriVap 7812014; Labconco, KansasCity, MO) was used to evaporate the purified supernatants todryness, before being resuspended in 1 mL ultrapure water. Thesuspensions were placed in an ultrasonic waterbath (FX14;Unisonics, Sydney, Australia) for 30 min, and filtered througha 0.2-mm cellulose acetate syringe filter. Final samples of 60 mLwere injected into a high-performance liquid chromatography(HPLC) system (600 series; Waters, Milford, MA), with ultrapurewater used as mobile phase, pumped at a flow rate of 0.4mL�min–1. Sugars in the samples were separated with a mono-saccharide column [300 · 7.8 mm, 8 mm (REZEX 8% Pb2+;Phenomenex, Torrance, CA)]. The column was heated to 75 �C,and sugars were detected with a refractive index detector (model2414; Waters). Standard solutions of sucrose, glucose, fructose,and mannitol were used to determine the retention times and toestablish calibration curves.

TOTAL N CONCENTRATION. N concentrations were determinedin the finely ground, dried samples of roots, trunks, shoots, andcombined leaf blades and petioles. For the fruit, 50 frozen berrieswere ground to a fine powder under liquid N with an analyticalmill (A11; IKA, Selangor, Malaysia), freeze-dried (Gamma 1-16LSC), and used for the determination of fruit N concentration. Nconcentration in 200 mg of a representative sample were deter-mined by the LECO method (Standard methods of Rayment andLyons, Soil chemical methods, Australasia, Dumas CombustionMethod 6B2b), using an elemental analyzer (CNS TruMAC;LECO Corp., St. Joseph, MI).

AMINO N CONCENTRATION. Subsamples of the fruit, roots,trunks, shoots, leaf blades, and petioles were taken from–80 �C storage and ground to a powder under liquid N, usingan analytical mill (A11; IKA). Free amino acids wereextracted from a 100 mg subsample of the ground tissue,using 100 mL of an 80% (v/v) methanol solution. Thesamples were vortexed for 1 min, and sonicated for 15 minat room temperature, before centrifugation at 12,000 gn for10 min. The supernatant (20 mL) was mixed with 475 mL 0.25 M

borate buffer (pH 8.5) and 5 mL internal standard (10 mM Lhydroxyproline), and 100 mL of the mixture used in thederivatization of the amino groups, according to Hayneset al. (1991), using 9-fluoreonylethyl chloroformate. Aminoacids were analyzed by a HPLC system (600 series; Waters),and were separated with a C18 column [4.6 · 150 mm, 5 mm(Zorbax Eclipse plus; Agilent, Santa Clara, CA)], and quan-tified with a fluorescence detector (model 2475; Waters)according to Haynes et al. (1991). The N concentration offree amino acids was determined in relation to the amino Natoms of each amino acid. Total free amino N concentrationswere determined from the amino N atoms of 17 free aminoacids.

STATISTICAL ANALYSIS. Data were analyzed using Statistica12 (Statsoft, Tulsa, OK), with the analysis of variance used to

the harvest dates. The fresh weights of these organs weredetermined, and the samples were oven-dried at 60 �C untilconstant dry weight. During the destructive harvests, root,trunk, shoot, leaf blade, and petiole subsamples were collected.The root subsamples consisted of full length roots taken within10 cm from the basal part of the trunk, always between 2 and6 mm in diameter, with at least 50 g in total fresh weight. Soilparticles were shaken off and the roots rapidly rinsed withdeionized water, before the samples being frozen in liquid N.Trunk subsamples, 10 cm in length, were taken from 20 cmabove soil level. One whole shoot per vine represented the shootsubsamples, whereas 20 leaves from the base of one shoot pervine represented the leaf blade and petiole subsamples. Berries(50 per vine) were also collected between 0800 and 1000 HR eachday, and all subsamples were immediately frozen in liquid N andstored at –80 �C. Rainfall, atmospheric temperature, and relativehumidity were recorded and collected from an on-site weatherstation, and the vapor pressure deficit was calculated (Castellvı�et al., 1996).

WATER STATUS AND LEAF GAS EXCHANGE. Measurements ofstem water potential (SWP) were conducted weekly accordingto Chon�e et al. (2001), selecting one healthy leaf from eachvine on a main shoot and enclosing it with aluminum foil bagsfor 30 min between 1200 and 1400 HR. The leaves were thenplaced in a pressure chamber to measure SWP (model 1000;PMS instruments, Albany, OR). A portable photosynthesis systeminstrument (LCA-4; ADC Bioscientific, Hoddesdon, UK) wasused to measure leaf surface temperature, stomatal conductance(gS), and photosynthesis (Pn). Two healthy, fully intact leaveswere chosen weekly on each vine between the fourth and seventhshoot node position, to be used for these measurements between1200 and 1400 HR on clear, noncloudy days.

VEGETATIVE AND REPRODUCTIVE DEVELOPMENT. The total fruitfresh weight of each grapevine was recorded at each destructiveharvest. Subsamples of 50 berries per vine were used to deter-mine the fresh weight per berry, and were juiced to measure thejuice total soluble solid (TSS) concentration with a digital benchrefractometer (PR-101; Atago, Tokyo, Japan). Berry solublesolid content (SSC) was calculated on the basis of berry freshweight and TSS concentration.

The total tissue dry weight of whole root systems, trunks,shoots, and leaves and petioles were calculated for each vine, bycombining the weights measured from the dried samples andthe estimated dry weights of the subsamples. The subsampledry weights were estimated through the percentage weight lossduring drying of the main samples. The structural biomass ofthese tissues was determined by subtracting the TNC contentfrom the total biomass. Ground berry powder (5 g) from eachvine was freeze-dried (Gamma 1-16 LSC; Christ, Osterode amHarz, Germany), and the berry dry weight estimated from theweight loss during drying.

TNC. Whole, dried plant parts, collected during the de-structive harvest dates (roots, trunks, shoots, and combined leafpetioles and blades), were ground through a heavy duty cuttingmill (SM2000;Retsch, Haan, Germany) to 5mmand a subsamplewas then ground to 0.12 mm by using a ultracentrifugal mill(ZM200; Retsch). Starch (in the roots, trunks, shoots, and leaves),and total sugar (in the trunk, shoots, and leaves) concentrations ina 20 mg subsample were determined following the methodsoutlined in Smith and Holzapfel (2009).

The concentrations of sucrose, glucose, and fructose in theground root tissue were measured from a 200 mg subsample, as


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test the significance of each variable. Fisher’s least significantdifference test was used to identify significant differencesbetween means (P < 0.05).

Results

Atmospheric conditions, leaf gas exchange, and SWPThe average daily temperature and VPD data collected

during each interval of the experiment are shown in Table 1.The leaf surface temperatures were significantly the lowest

during Interval 3, and significantly higher for the defruitedvines during Interval 4 (Table 2). Fruit absence or presence didnot induce significant differences in leaf gS and Pn at any stageof the experiment (Table 2). Stomatal conductance and Pn,however, reduced significantly between Intervals 1 and 2, andgS of the fruited vines reduced further between Intervals 2 and4. The fruited vines had significantly more negative SWPvalues than the defruited vines, albeit receiving the sameamount of irrigation water. The SWP values generally becamemore negative as the experiment progressed, and weresignificantly more negative than during Interval 1 by Interval2 for the fruited vines, and by Interval 3 for the defruited vines.Defruited vine SWP values also reduced significantly betweenIntervals 3 and 4.

Dry weight, TNC, and N accumulationThe fruit SSC per vine increased significantly during In-

tervals 1 and 2 (Table 3) at rates of 9.2 and 9.7 g�d–1respectively, and rapid berry sugar accumulation therefore tookplace between V and V + 29. The fruit SSC accumulation pervine slowed down during Interval 3 (P > 0.05) at a rate of 7 g�d–1,and no accumulation took place during Interval 4. The fruit Ncontent per vine increased significantly during Interval 1, andbetween V + 14 and V + 42 (Table 3) at rates of 111 and 32mg�d–1, respectively. The fruit dry weight per vine increasedsignificantly between V and V + 29 (Table 3). The fresh weightper berry increased significantly between V and V + 29, andthen decreased during Interval 4 (Table 3).

Combined roots, trunk, shoots, and leaves per vine exhibiteda significant increase in the TNC content of both treatmentsduring Interval 4 (Table 3). These TNC contents also increasedsignificantly in the defruited vines during Interval 3. Further-more, the defruited vines had higher TNC content than thefruited vines as showed by the treatment main effect. Root TNCcontent accounted for most (53% on average) of the total vine(excluding the fruit) TNC content (Table 3), and decreasedsignificantly between V and V + 29 in the fruited vines, beforeincreasing to the initial level by V + 56. The root TNC contentin the defruited vines increased significantly between V + 14

Table 1. Summary of the periodic atmospheric temperature, vapor pressure deficit (VPD) and the total irrigation volume applied per grapevineduring the different experimental intervals. Intervals 1, 2, 3, or 4 refer to the periods of V (veraison) to V + 14 (14 d after veraison), V + 14 toV + 29 (29 d after veraison), V + 29 to V + 42 (42 d after veraison), or V + 42 to V + 56 (56 d after veraison), respectively.

Treatment

Time after veraison (d)

V to V + 14(Interval 1)

V + 14 to V + 29(Interval 2)



Atmospheric temp (�C) Daily mean 25.4 24.1 23.7 26.2Mean minimum 18.1 18.3 17.3 18.9Mean maximum 33.3 31.6 31.0 34.4

VPD (kPa) Daily mean 2.3 1.6 1.7 2.2Mean minimum 0.8 0.5 0.6 0.7Mean maximum 4.3 3.5 3.4 4.4

Irrigation (L/vine) Periodic total 50.4 54 46.8 50.4

Table 2. The influence of grapevine fruiting during sustained water constraints on leaf surface temperature, stomatal conductance (gS), netphotosynthetic rate (Pn), and stem water potential (SWP) averaged during the different experimental intervals. Intervals 1, 2, 3, or 4 refer to theperiods of V (veraison) to V + 14 (14 d after V), V + 14 to V + 29 (29 d after V), V + 29 to V + 42 (42 d after V), or V + 42 to V + 56 (56 d after V),respectively. Themain effect indicates themeanmeasured values betweenV + 14 andV + 56 for each treatment. The fruited treatment consisted ofvines with their fruit intact between V and V + 56, whereas the defruited treatment consisted of vines without fruit during the same period.

Treatment


V to V + 14(Interval 1)



V + 42 to V + 56(Interval 4) Main effect

Fruited 33.6 a 33.8 a 30.2 b 33.0Defruited 33.7 a 33.7 a 31.0 b 33.3Fruited 27.7 a 8.9 b 7.1 b 7.5Defruited 29.3 a 12.2 b 6.3 bc 8.8Fruited 4.71 a 3.01 b 3.80 ab 3.71Defruited 4.91 a 3.44 b 3.59 b 3.81

Leaf surface temp (�C)

gS (mmol�m–2�s–1)

Pn (mmol�m–2�s–1)

SWP (–MPa) FruitedDefruited

*z1.47 ay

*1.37 a*1.71 b*1.41 ab

*1.65 b*1.48 b

*33.3 a*35.3 a

5.8 b1.7 c4.19 ab3.18 b1.68 b1.64 c

*1.62*1.43

zMeans separated within columns using Fisher’s least significant difference (LSD) test, significant differences between the treatments (*) areindicated at P < 0.05.yMeans separated within rows using Fisher’s LSD test, significant differences are indicated at P < 0.05.Where the same lower case letter appears ina row, values do not differ significantly.


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Furthermore, the leaf N concentra-tions (Table 3) in vines of bothtreatments were, at veraison, withina range considered to indicate ade-quate vine N supply, and the Nstatus of these vines could thereforebe considered sufficient for ‘Shiraz’vines after veraison (Holzapfel andTreeby, 2007). By estimation, andalthough not significantly affectingthe total N content in a combinationof the fruit, leaves, shoots, trunk,and roots per vine (P > 0.05), thefruited vines absorbed 0.6 and 0.4 gsoil N per vine during respectively,Intervals 1 and 2. However, andwhile also not significantly contrib-uting to the total vine N content (P >0.05), the defruited vines absorbedan estimated 0.8 and 0.5 g N pervine from the soil during Intervals 2and 3, respectively.

The total dry weight of com-bined roots, trunk, shoots, andleaves per vine did not changesignificantly in the fruited vines,whereas the defruited vines exhibiteda significant increase during Interval 3(Table 3). The defruited vines also hadsignificantly higher dry weights pervine than the fruited vines, as a treat-ment main effect.

Organ structural developmentThe leaf and shoot structural

dry weight per vine did not signifi-cantly differ among the treatments,and did not change significantly(Fig. 1A and B). There was nosignificant difference in trunk struc-tural dry weight for both treatments,apart from the defruited vinesexhibiting a larger trunk dry weightat V + 42 (Fig. 1C). The rootstructural dry weight did not signif-icantly change in the fruited vines,

whereas that of the defruited vines significantly increased fromV + 42 (Fig. 1D).

TNCSTARCH CONCENTRATION PER ORGAN. The leaf starch concen-

tration was never significantly different between the treatments,and was similar to that at V until Interval 4 where it significantlyincreased (Fig. 2A). The shoot starch concentration in the bothtreatments increased significantly during Intervals 3 and 4 (Fig.2B), and was significantly higher at V + 56 than at V. The shootstarch concentration was significantly higher by V + 42 in thedefruited vines.

The trunk starch concentration in the fruited vines wassignificantly higher at V than by V + 42, whereas that in thedefruited vines was significantly higher by V + 29 (Fig. 2C).The root starch concentration in the fruited vines decreased

Table 3. Influence of sustained grapevine water constraints on the fruit total soluble solidconcentration (TSS), total soluble solid content (SSC), N content, total fruit dry weight (DW)per vine, fresh weight (FW) per berry and the impact of grapevine fruiting on the nonstructuralcarbohydrate (TNC) content, N content, and DW of vegetative annual and perennial tissues pervine at the different destructive harvests. Intervals 1, 2, 3, or 4 refer to the periods of V (veraison)to V + 14 (14 d after V), V + 14 to V + 29 (29 d after V), V + 29 to V + 42 (42 d after V), or V + 42to V + 56 (56 d after V), respectively. The main effect indicates the mean measured valuesbetween V + 14 and V + 56 for each treatment. The fruited treatment consisted of vines with theirfruit intact between V and V + 56, whereas the defruited treatment consisted of vines without fruitduring the same period.

Treatment


Maineffect

Interval 1 Interval 2 Interval 3 Interval 4

V V + 14 V + 29 V + 42 V + 56

Fruit4.9 d 9.5 c 16.4 b 18.2 b 22.6 a —58.5 c 187.9 b 333.1 a 423.7 a 409.5 a —2.42 c 3.98 b 4.03 ab 4.87 a 4.54 a —

161.0 c 315.6 bc 435.9 ab 562.1 a 581.7 a —

TSS (%) FruitedSSC (g/vine) FruitedN content Fruited

(g/vine)DW (g/vine) FruitedFW (g/berry) Fruited 0.8 c 0.9 bc 1.3 a 1.3 a 1.0 b —

Rest of vineTNC

Total content(g/vine)x

Fruited 65.5 b 52.6 b *78.2 b 129.4 a *75.2Defruited 65.5 c 64.3 c 202.3 a *111.6

Root content(g/vine)

Fruited 41.4 ab 36.1 bcDefruited 41.4 c 37.9 c

*z53.4 by

*93.7 c*28.7 c*54.7 bc

*59.8 a*108.9 a

NTotal content

(g/vine)xFruited 10.3 a 9.3 a 9.7 a 8.7 aDefruited 10.3 a 9.9 a 10.7 a 10.6 a

Leaf content(g/vine)

Fruited 6.3 a 5.0 ab 5.5 ab 4.5 bDefruited 6.3 a 5.3 a 5.5 a 5.2 a

Leaf concn(% DW)

Fruit 2.64 a 2.50 a 2.51 a

*147.6 b*34.3 bc*79.6 b

*8.6 a*11.2 a

4.8 b5.8 a2.26 b 1.99 c

No fruit 2.64 a 2.47 ab 2.41 ab 2.38 b 2.03 c

*39.7*70.3

*9.1*10.6*4.9*5.52.322.32

DWTotalx

(g/vine)Fruit 767.3 a 759.2 a *751.8 a 864.7 a *785.0No fruit 767.3 b 804.6 b

*765.3 a*865.6 b *1,022.8 a 1,055.0 a *937.0

zMeans separated within columns using Fisher’s least significant difference (LSD) test, significantdifferences between the treatments (*) are indicated at P < 0.05.yMeans separated within rows using Fisher’s LSD test, significant differences are indicated atP < 0.05. Where the same lower case letter appears in a row, values do not differ significantly.xIndicate the combination of roots, trunks, shoots, and leaves per vine.

and V + 42, and also during Interval 4. The root TNC content indefruited vines was significantly higher than that in the fruitedvines from V + 29 onward.

The total N content in combined roots, trunks, shoots, andleaves per vine (vine N content) did not change significantly invines of both treatments (Table 3). However, the defruited vineshad significantly higher N content in combined roots, trunks,shoots, and leaves per vine, than the fruited vines as showed bythe treatment main effect (Table 3). Most of the vine N contentwas present in the leaves (61% of total at veraison), and the leafN content per vine in the fruited vines tended to decrease duringInterval 1 (P = 0.07) (Table 3). The leaf N content of those vinesdecreased significantly between V and V + 42, whereas that ofthe defruited vines did not change significantly. The defruitedvines had significantly higher leaf N content per vine than thefruited vines as demonstrated by the treatment main effect.


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significantly between V and V + 29, followed by a signifi-cant increase during Interval 4 back to the initial concentration(Fig. 2D). The root starch concentration in the defruited vineswas significantly higher at V + 56 than at V, and wassignificantly higher than that of the fruited vines at V + 29.

Total soluble sugar concentration per organThe leaf sugar concentration in the fruited vines signifi-

cantly increased between V + 14 and V + 42, whereas that inthe defruited vines significantly increased between V and V +42 (Fig. 2E). The shoot sugar concentration in the defruitedvines remained stable throughout the experiment (Fig. 2F).Although the shoot sugar concentration of the fruited vinessignificantly decreased during Interval 1 and then signifi-cantly increased until V + 42, the concentration at V + 56 wasnot different to that at V.

The trunk sugar concentration significantly increased duringInterval 2 and remained higher than at V until V + 56 in thefruited vines, whereas that in the defruited vines was signifi-cantly higher than at V at all stages (except V + 29) (Fig. 2G).The root total sugar concentration significantly decreasedduring Interval 1, and then significantly increased duringInterval 2 in the fruited vines (Fig. 2H). At V + 29, the rootsugar concentration in the fruited vines was significantly higherthan at V. The fruited vine root sugar concentration thenreduced significantly during Interval 4. There were no signif-icant changes in root sugar concentration in the defruited vines;however, the fruited vines had significantly higher root sugarconcentration at V + 29 and V + 42, than the defruited vines.

Individual root sugar concentrationsThe root sucrose concentration in the fruited vines signifi-

cantly decreased during Interval 1, and significantly increasedduring Interval 2 (Fig. 3A). By V + 42, these root sucroseconcentrations were significantly higher than at V, beforedecreasing to the concentration observed at V + 14 duringInterval 4. The root sucrose concentration in the defruited vineswas significantly lower than at V by V + 42. The sucroseconcentration of the roots was the same for the fruited anddefruited vines except at V + 29 and V + 42, where it wassignificantly higher in the fruited vines.

Total hexose concentration; i.e., combined fructose andglucose concentrations, increased significantly during Interval2 in the roots of the fruited vines (Fig. 3D). Glucose (Fig. 3C),and the total hexose concentrations, decreased significantlyduring Interval 4 in the roots. At V + 56, both the total hexoseand the fructose (Fig. 3B) concentrations in the roots of thefruited vines, were significantly higher than at V. The roothexose concentration in the defruited vines increased graduallyduring the experiment, and from V + 42, both the total hexoseand fructose concentrations were significantly higher than at V.At V + 42, the fruited vines had significantly higher root hexose,fructose and glucose concentrations per vine, than thosewithout fruit.

NitrogenTOTAL N CONTENT PER ORGAN. The development of total leaf

N content per vine between V and V + 56 (Table 3) wasdescribed earlier. The shoot N content in vines of bothtreatments did not alter significantly (Fig. 4A), although theshoot N content in defruited vines was significantly higher thanthat in fruited vines, as determined by the treatment main effect.

Fig. 1. Effects of grapevine fruiting during sustained water constraints on thetotal (A) leaf (combination of leaf blades and petioles), (B) shoot, (C) trunk,and (D) root structural dry weight accumulation per vine. Time after veraisonrefers the different destructive harvest dates [V (veraison), V + 14 (14 d afterV), V + 29 (29 d after V), V + 42 (42 d after V), and V + 56 (56 d after V)]. Thefruited treatment consisted of vines with their fruit intact between V and V +56, whereas the defruited treatment consisted of vines without fruit during thesame period [mean ± SE (n = 3)].


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harvests (except V + 42) than at V (Fig. 4B). In the defruitedvines, the trunk N content was significantly higher during therest of the experiment than at V. Furthermore, the defruited

Fig. 2. Effects of grapevine fruiting during sustained water constraints on the leaf (combination of leaf blades and petioles), shoot, trunk, and root starch (A, B, C,andD, respectively) and total sugar (E,F,G, andH, respectively) concentration dryweight) per vine. Time after veraison refers to the different destructive harvestdates [V (veraison), V + 14 (14 d after V), V + 29 (29 d after V), V + 42 (42 d after V), and V + 56 (56 d after V)]. The fruited treatment consisted of vines with theirfruit intact between V and V + 56, whereas the defruited treatment consisted of vines without fruit during the same period [mean ± SE (n = 3)].

The N content in vine trunks of both treatments increasedsignificantly during Interval 1. The trunk N content in thefruited vines was significantly higher at all the destructive


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vines had significantly higher trunk N content than those withfruit, as determined by the treatment main effect. The root Ncontent in the fruited vines did not change significantly

Fig. 3. Effects of grapevine fruiting during sustained water constraints on theroot (A) sucrose, (B) fructose, (C) glucose, and (D) total hexose concen-tration dry weight per vine. Time after veraison refers to the differentdestructive harvest dates [V (veraison), V + 14 (14 d after V), V + 29 (29 d afterV), V + 42 (42 d after V), and V + 56 (56 d after V)]. The fruited treatmentconsisted of vines with their fruit intact between V and V + 56, whereas thedefruited treatment consisted of vines without fruit during the same period[mean ± SE (n = 3)].

Fig. 4. Effects of grapevine fruiting during sustained water constraints on thenitrogen content per vine in the (A) shoots, (B) trunks, and (C) roots. Time afterveraison refers to the different destructive harvest dates [V (veraison), V + 14(14 d after V), V + 29 (29 d after V), V + 42 (42 d after V), andV + 56 (56 d afterV)]. The fruited treatment consisted of vines with their fruit intact betweenV and V + 56, whereas the defruited treatment consisted of vines without fruitduring the same period [mean ± SE (n = 3)].


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(Fig. 4C); however, the root N content in the defruited vineswas significantly higher than at V by V + 29. The defruitedvines also had significantly higher root N content than thosewith fruit, as determined by the treatment main effect.

TOTAL AMINO N CONCENTRATION PER ORGAN. In the fruitedvines, the fruit amino N concentration was significantly higherby V + 29 than at V (Fig. 5A). The fruit amino N concentrationalso increased significantly during Interval 4.

The leaf blade total amino N concentration in vines of bothtreatments increased significantly during Interval 1, but de-creased significantly during Interval 2, and did not differbetween the treatments (Fig. 5B). In the petioles of the fruitedvines, the total amino N concentration increased significantly

during Interval 1 (Fig. 5C), and was significantly higher thanthat in the defruited fruited vines at V + 14 and V + 29.Furthermore, during Interval 3, the petiole amino N concen-tration in the fruited vines decreased significantly. The totalshoot amino N concentration increased significantly in thefruited vines during Interval 1 (Fig. 5D), and was significantlyhigher than that in the defruited vines at V + 14. The shootamino N concentration of the fruited vines decreased signif-icantly during Interval 2.

In the trunks of both treatments, the total amino N concentra-tion increased significantly during Interval 1 (Fig. 5E). The trunkamino N concentration in the defruited vines was significantlylower than at V + 14 by V + 42, whereas that in the fruited vines

Fig. 5. Effects of grapevine fruiting during sustained water constraints on total amino nitrogen concentration fresh weight in the (A) fruit, (B) leaf blades, (C) leafpetioles, (D) shoots, (E) trunks, and (F) roots. Time after veraison (d) refers to the different destructive harvest dates [V (veraison), V + 14 (14 d after V), V + 29(29 d after V), V + 42 (42 d after V), and V + 56 (56 d after V)]. The fruited treatment consisted of vines with their fruit intact between V and V + 56, whereas thedefruited treatment consisted of vines without fruit during the same period [mean ± SE (n = 3)].


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decreased significantly during Interval 4. In the roots, the totalaminoN concentration did not change significantly (Fig. 5F), anddid not significantly differ between the treatments.

INDIVIDUAL AMINO ACID CONCENTRATIONS. The concentrationsof arginine, glutamic acid, glutamine and proline, and aminoacids derived from a-ketoglutarate metabolism, are presentedfor the different organs in Fig. 6.

Arginine was the most abundant amino acid in the fruit, whereits concentration increased significantly between V and V + 29.The fruited vines had higher leaf blade arginine concentration thanthe defruited vines at V + 14 and V + 29. Likewise, in the petioles,the arginine concentration in the fruited vines was significantlyhigher at V + 14 than that in the defruited vines. The shootarginine concentration in the fruited vines increased significantlyduring Interval 1, and was significantly higher than that in thedefruited vines at V + 14. In the defruited vines, the shoot arginineconcentration was significantly higher at V + 56 than at V. Therewere no significant differences in trunk and root arginineconcentrations between the treatments, although between all ofthe perennial and vegetative annual organs, the roots had thehighest arginine concentrations.

The fruit glutamic acid concentration was higher by V + 42than at V, whereas that in the leaf blades, petioles, and shootsnever differed between the treatments. The glutamic acid con-centration in leaf blades and petioles increased significantlyduring Interval 1 for both treatments. The trunk glutamic acidconcentration in the defruited vines was significantly higher thanthat in the fruited vines at V + 14; however, it was significantlyhigher in the trunks of fruited vines at V + 42. There were nosignificant differences in root glutamic acid concentration.

The fruit glutamine concentration did not change signifi-cantly, whereas that in the leaf blades, petioles, shoot, and trunkincreased significantly for both treatments during Interval 1.The glutamine concentration then decreased significantly in theleaf blades and shoots of the vines from both treatments duringInterval 2, and also in the petioles and trunk in the defruitedvines, whereas that in the petioles and trunk of the fruited vinesdecreased by V + 42. The glutamine concentration in thepetioles of fruited vines was significantly higher than that of thedefruited vines at V + 29, and at V + 14 in the shoots. The trunkand root glutamine concentrations never significantly differedbetween the treatments.

The fruit proline concentration increased significantly dur-ing Interval 4. There was no significant difference in the prolineconcentration between the treatments in the leaf blades (exceptat V + 56 where the defruited vines had higher concentration)and petioles, although these concentrations increased signifi-cantly during Interval 1, and also during Interval 4 in the leafblades. Likewise, the proline concentration in both, shoots andtrunks, did not differ significantly between the treatments,although it increased significantly during Interval 1 in thetrunks, and then decreased during Interval 2. The root prolineconcentration in the fruited vines increased significantly duringInterval 1 before decreasing significantly during Interval 2, butwas significantly higher in the roots of defruited vines at V + 29.

Discussion

Grapevines were grown with and without fruit betweenveraison and fruit maturity, and irrigation limited to createa sustained postveraison water constraint. The aim was to reduceleaf photoassimilation, and force greater reliance on stored

carbohydrates for berry sugar accumulation in the fruiting vines.For the nonfruiting vines, the absence of the strong reproductivesink allowed vegetative growth and partitioning responses towater constraints to be examined in more detail. The TNCcontent in different organs, and the concentration changes of themajor root sugars were investigated. The water constraints alsoaimed to alter the contribution of the different N sources towardfruit N accumulation, and the concentrations of amino N wasdetermined in the different organs.

Themore negative SWP values seenwith the presence of fruitis not uncommon in deciduous fruit species. Berman and DeJong(1996), for example, described the higher crop load of peach(Prunus persica) trees under reduced irrigation to induce morenegative SWP values. The crop load induced SWP differences inthat study were attributed to either increased leaf transpiration ofthe higher cropping trees, or the reduced root growth of thosetrees and a subsequent inferior soil water uptake. In the presentstudy, leaf gS and Pn were unaffected by fruit presence. However,if higher leaf transpiration rates of the fruited vines induced themore negative SWP values, it is probable that the leaf transpi-ration rate differences between the treatments occurred due to gSvariations during periods other than the middle of the day; e.g.,midafternoon (Downton et al., 1987). The low midday Pn valuesin the present study are, however, consistent with of the impact ofthe imposed grapevine water constraints on net photoassimila-tion (Medrano et al., 2003). In addition to the water constraints,and as illustrated in apple (Malus domestica) trees, the highmidday leaf surface temperatures likely also contributed to thecorresponding low gS values (Greer, 2015). Classification of themidday SWP values, according to published thresholds, confirmsthat water constraints were sustained from veraison to berrymaturity (Van Leeuwen et al., 2009).

A depletion of root TNC coincided with rapid fruit sugaraccumulation (Table 3), and root TNC reserves were seeminglynot used toward structural development in the fruited vines (Fig.1). In the absence of fruit, the vines stored the available photo-assimilate in the roots as starch, and as implied by the gain in rootstructural biomass, also used carbohydrates toward root devel-opment. When there was no more fruit sugar accumulation, thefruited vines also stored starch. The reduced demand from thefruit therefore caused surplus carbohydrates to be stored as starch,predominantly in the roots. The absence of fruit as a carbohydratesink induced the earlier storage of starch reserves in the roots,trunks, and shoots. Although the reduction in TNC content duringrapid fruit sugar accumulation was only observed in the roots, theTNC content in the leaves, shoots, and trunks only accumulatedonce fruit sugar accumulation slowed.

Root TNC depletion in the fruited vines was caused bystarch reduction, but coincided with increased total sugarconcentrations in these roots during the maximum rates of fruitsugar accumulation. This suggests that starch was hydrolyzed,resulting in the root sugar accumulation (Regier et al., 2009).The root TNC depletion during the phase of rapid berry sugaraccumulation resulted from TNC remobilization and/or rootrespiration. Candolfi-Vasconcelos et al. (1994) illustratedthrough 14C that perennial TNC reserves are directed towardthe sugar-accumulating berries when a C source limitation wasinduced by grapevine defoliation.

The water constraints of the present study restricted mid-day leaf Pn, with values comparable to field-grown grapevinessubjected to severe water constraints (Medrano et al., 2003).Restricted leaf-level Pn may limit canopy photoassimilation


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2012). As the demand from the sugar-accumulating berries inthe present study was apparently greater than could be met bycurrent photosynthesis, the abundant root starch reserveslikely provided an alternative carbohydrate source. The roots

Fig. 6. Effects of grapevine fruiting during sustained water constraints on the concentration fresh weight of arginine, glutamic acid, glutamine, and proline in the(A) fruit, (B) leaf blades, (C) petioles, (D) shoots, (E) trunks, and (F) roots. Time after veraison (d) refers to the different destructive harvest dates [V (veraison),V + 14 (14 d after V), V + 29 (29 d after V), V + 42 (42 d after V), and V + 56 (56 d after V)]. The fruited treatment consisted of vines with their fruit intact betweenV and V + 56, whereas the defruited treatment consisted of vines without fruit during the same period [mean ± SE (n = 3)].

enough to induce a C deficiency during a period of intensefruit C demand. When a plant sugar deficiency occurs, geneexpression related to TNC remobilization, and its exportationfrom source tissues is upregulated (Eveland and Jackson,


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had the highest TNC concentration, possibly explaining whysignificant starch depletion during rapid fruit sugar accumu-lation was only observed in the roots. In fact, root TNC isusually more affected by abiotic constraints and grapevineseasonal development, than the TNC in other vine parts(Holzapfel et al., 2010). As mentioned, respiration lossesmay also partly account for the root starch depletion duringrapid fruit sugar accumulation. However, the root respirationrates of fruited pot-grown grapevines have been found to beless than defruited vines during berry maturation (Morinagaet al., 2003). Furthermore, water constraints may reducerespiration in comparison with well-watered potted vines(Escalona et al., 2012). Therefore, although root respirationmay account for some of the depleted root TNC during rapidberry sugar accumulation, it also remains probable that theroots served as an alternative TNC source. Future studies mayinclude measuring root respiration rates to quantify therelated C expense.

The concentration of root total sugars initially decreased atthe start of berry sugar accumulation. The root sugar con-centration reduction, caused by sucrose depletion, suggeststhat existing free sugars were initially translocated out of theroot system. In the subsequent sampling root sugar concen-tration increased resulting from the apparent starch hydroly-sis (Fig. 2). The breakdown of starch through enzymaticdegradation, yields C-containing intermediates such as glu-cans, which is subsequently restructured as sugars (Smithet al., 2005). It was during the period of maximum fruit sugaraccumulation, when the root sugar concentration also rapidlyincreased. Sugars are osmotically active, and facilitatesource-to-sink C translocation via the pressure flow of thephloem. Through 14C labeling, Yang et al. (2002) observedrice (Oryza sativa) stem TNC reserves to be remobilized tothe grains during grain filling, when these plants weresubjected to water constraints. Furthermore, they describedstarch remobilization to coincide with stem sucrose accumu-lation, which was suggested to sustain the C flux to the grainswhen leaf photoassimilation was restricted. Therefore, thebiosynthesis of root sugars resulting from the starch hydro-lysis in the present study, suggests that these sugars becameavailable for translocation to the ripening berries underconditions of limited C availability. Concurrent with theslowdown of berry sugar loading, root sugar accumulationstopped, and total root sugar concentration then reduced whenberry sugar accumulation ceased. The lack of a change in thetotal sugar concentrations in the roots of the defruited vines isfurther evidence that the rate of berry sugar accumulationimpacted on the root sugar abundance.

The root sucrose depletion during Interval 1 suggests thatTNC translocation from the roots mainly took place throughsucrose export. Grapevines, like most other plants, transportcarbohydrates as sucrose (Ruan et al., 2010). The rapid rootsucrose accumulation during peak berry sugar accumulationsuggests that sucrose synthesis resulted from substratesprovided by starch hydrolysis (Smith et al., 2005). Thissucrose accumulation could create a concentration gradientbetween the root tissues and upper vine parts, as the berrysugar demand outweighed canopy photoassimilation, drivingsucrose availability for phloem translocation to the berries,where it is hydrolyzed into glucose and fructose (Davies andRobinson, 1996). In the roots of the defruited vines, thegradual sucrose decline coincided with increased hexoses,

suggesting sucrose cleavage. Furthermore, an increase inroot structural biomass was observed in these vines, inagreement with Wang et al. (2010), who concluded that roothexose accumulation contributes to root growth. In fact, theaccumulation of glucose and fructose in growing plant organssupports the generation of an osmotic gradient to regulatecell expansion, especially, during abiotic stresses, such asdrought (Roitsch and Gonz�alez, 2004). As significant rootsucrose was presumably exported during rapid berry sugaraccumulation, it is further possible that the hexose accumu-lation in the roots of the fruited vines occurred to maintaincell osmotic potential (Sturm, 1999).

Similar to vine TNC, defruiting also induced higher reserveN accumulation, although the abundance of fruit N was muchlower than that of its sugar. Leaves exhibited the highest Ncontent at veraison, and also the only significant postveraisonN depletion. The trend in leaf N reduction during Interval 1and the significant leaf N depletion by V + 42 coincided withperiods of fruit N accumulation, suggesting that leaf N re-distribution took place toward the fruit (Verdenal et al., 2016).The accumulation of root N in the defruited vines suggests, inagreement with Morinaga et al. (2003), that the roots were analternative N sink due to fruit absence. The lack of root Ndepletion indicates that the roots could not be considereda considerable N source. Root, shoot, and leaf N mobilizationis thought to take place toward the bunches during berrymaturation (Conradie, 1991). However, the water constraintsof the present study likely inhibited root N reserve mobili-zation, as preharvest water constraints are thought to promoteN allocation toward the roots (Holzapfel et al., 2015). Thereason for the lack of root N translocation under waterconstraint during fruit maturation, while root TNC utilizationwas apparently not limited, needs further exploration.

One possibility for the differing TNC and N mobilizationresponses is that the fruit was not a strong enough N sink tocause a substantial root N loss. Furthermore, the leaves were thelargest N source, and with almost three times more leaf thanroot N available at veraison, may have been sufficient to meetthe fruit demand.Water constraints, as well as reducing the flowof water to the shoot, can induce a loss of xylem transportfunctioning in grapevines, by restricting xylem hydraulic con-ductivity (Lovisolo and Schubert, 1998). As N translocation fromthe roots during the period of berry maturation can take placethrough both the xylem and phloem (Roubelakis-Angelakisand Kliewer, 1992), the question is raised whether root Nmobilization could have been limited due to a potential waterconstraint induced restricted xylem functioning, an aspect thatrequires further investigation. Nevertheless, despite the sug-gested leaf N contribution toward fruit N, and although Nfertilization was ceased 1 month before the experiment, thefruited vines seemingly absorbed N from the soil during thefirst two intervals. Grapevine soil N absorption is not unusualduring the period soon after the start of veraison (L€ohnertz,1991). This newly absorbed N did not significantly affect tothe total vine N content, but potentially contributed, togetherwith the exported leaf N, toward fruit N. With the absence offruit, the implied soil N uptake likely contributed to root Naccumulation.

The elevated concentrations of amino N in the petioles andshoots of the fruited grapevines within Intervals 1 and 2, arepossibly related to N translocation from the vegetative tissuestoward the fruit (Conradie, 1991). Elevated petiole and shoot


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glutamine concentrations in the fruited vines were majorcontributors toward these amino N increases, and coincided(during Interval 1) with rapid fruit N accumulation. Glutamineis a known N transporter from source to sink organs in plants(Coruzzi and Last, 2000). On the other hand, the highest overallamino N concentrations were found in the roots of bothtreatments, present as arginine. Arginine is the major N storagecompound in grapevines (Xia and Cheng, 2004), and most ofthe amino N in the perennial structures of both treatments wastherefore likely related to N storage.

In summary, this study was performed to understand therelationship, during sustained postveraison water con-straints, between the distributions of TNC and N, and therespective accumulation of sugar and N in the fruit. Fruitinginhibited root structural development, whereas defruiting atveraison prompted continuous root growth. During theperiod of rapid fruit sugar accumulation, root TNC remobi-lization occurred, where starch depleted, and sugars accu-mulated. The results suggest that root sucrose accumulationcreated a concentration gradient to drive sucrose transporttoward the fruit. In the absence of fruit, starch accumulated,and the sucrose-to-hexose ratio decreased, indicating a po-tential role for hexoses as important osmotic regulators bypromoting a gradient to attract water into expanding rootcells. Leaf N depletion in the fruited vines coincided withfruit N accumulation, suggesting the leaves to be an impor-tant N source. In the absence of fruit, the roots became analternative N sink. Amino N accumulation in the leaf petiolesand shoots, largely attributed to glutamine accumulation,peaked during the first half of the experiment in the fruitedvines, suggesting a role in N transport from the vegetativetissues toward the fruit.

Therefore, during sustained water constraints, TNC can besourced from the roots during the rapid berry sugar accumula-tion phase. This has repercussions on starch storage, and mayaffect the vegetative and reproductive development of grape-vines the following season if starch reserve replenishment isunsatisfactory. On the other hand, leaf N translocation cansupport berry N accumulation during sustained water con-straints, causing reduced root N accumulation. This too couldhave repercussions on spring growth the following season,especially if postharvest root N replenishment is insufficient.

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the impact of foliar-applied nitrogen on N accumulation in the grapemust. J. Intl. Sci. Vigne Vin 50:23–33.

Verma, D.P.S., C. Zhang, and B. Singh. 1999. Regulation of prolineand arginine biosynthesis in plants, p. 249–265. In: B.K. Singh (ed.).Plant amino acids: Biochemistry and biotechnology. Marcel Dekker,New York, NY.

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Xia, G. and L. Cheng. 2004. Foliar urea application in the fall affectsboth nitrogen and carbon storage in young ‘Concord’ grapevinesgrown under a wide range of nitrogen supply. J. Amer. Soc. Hort. Sci.129:653–659.

Yang, J., J. Zhang, Z. Wang, Q. Zhu, and L. Liu. 2002. Abscisic acidand cytokinins in the root exudates and leaves and their relationshipto senescence and remobilization of carbon reserves in rice subjectedto water stress during grain filling. Planta 215:645–652.

Zapata, C., E. Del�eens, S. Chaillou, and C. Magn�e. 2004. Mobilisationand distribution of starch and total N in two grapevine cultivarsdiffering in their susceptibility to shedding. Funct. Plant Biol.31:1127–1135.


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Chapter 5: Paper 3

Vitis vinifera root and leaf metabolic composition during fruit

ripening: Implications of defoliation

(Paper 3 has been accepted for publication in Physiologia Plantarum, subject to a

minor revision. The revised manuscript was submitted to the journal in the format

below. The table and figures are shown after the main manuscript text.)


To assess the implications of defoliation on fruit sugar and N accumulation in

conjunction with the carbohydrate, N and primary metabolite composition of the major

grapevine source organs (roots and leaves).

5.2. Supporting information

Supporting information, as referred to throughout Paper 3 (Tables S1 and S2, and

Figures S1, S2 and S3), is included in appendix A.

Chapter 5: Paper 3

Vitis vinifera root and leaf metabolic composition during fruit maturation: Implications of defoliation

Gerhard C. Rossouwa,b,*, Beverley A. Orchardc, Katja Šukljea,†, Jason P. Smitha,‡, Celia

Barrila,b, Alain Deloirea,§ and Bruno P. Holzapfela,c

aNational Wine and Grape Industry Centre, Wagga Wagga 2678, New South Wales, Australia. bSchool of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga 2678, New South

Wales, Australia. cNew South Wales Department of Primary Industries, Wagga Wagga 2650, New South Wales,

Australia.

*Corresponding author email: [email protected]†Present address: Wine Research Centre, University of Nova Gorica, Lanthieri Palace, Glavni trg 8,

5271 Vipava, Slovenia.‡Present address: Department of General and Organic Viticulture, Hochschule Geisenheim University,

Geisenheim 65366, Germany.§Present address: Montpellier SupAgro, Montpellier 34060, France.

Abstract

Grapevine (Vitis vinifera) roots and leaves represent major carbohydrate and nitrogen

(N) sources, either as recent assimilates, or mobilised from labile or storage pools. This

study examined the response of root and leaf primary metabolism following

defoliation treatments applied to fruiting vines during ripening. The objective was to

link alterations in root and leaf metabolism to carbohydrate and N source functioning

under conditions of increased fruit sink demand. Potted grapevine leaf area was

adjusted near the start of véraison to 25 primary leaves per vine compared to 100 leaves

for the control. An additional group of vines were completely defoliated. Fruit sugar

and N content development was assessed, and root and leaf starch and N

concentrations determined. An untargeted GC/MS approach was undertaken to

evaluate root and leaf primary metabolite concentrations. Partial and full defoliation

increased root carbohydrate source contribution towards berry sugar accumulation,

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evident through starch remobilisation. Furthermore, root myo-inositol metabolism

played a distinct role during carbohydrate remobilisation. Full defoliation induced

shikimate pathway derived aromatic amino acid accumulation in roots, while arginine

accumulated after full and partial defoliation. Likewise, various leaf amino acids

accumulated after partial defoliation. These results suggest elevated root and leaf

amino N source activity when leaf N availability is restricted during fruit ripening.

Overall, this study provides novel information regarding the impact of leaf source

restriction, on metabolic compositions of major carbohydrate and N sources during

berry maturation. These results enhance the understanding of source organ carbon and

N metabolism during fruit maturation.

Abbreviations

25L, 25 leaves treatment; ANOVA, analysis of variance; C, carbon; FL, full leaf area

treatment; GABA, γ-amino-n-butyric acid; GC/MS, gas chromatography/mass

spectrometry; LSD, least significant difference; N, nitrogen; NL, no leaf treatment;

PCA, principal component analysis; SCC, soluble solid content; TCA, tricarboxylic

acid; TSS, total soluble solids; V, véraison; V+9, 9 days after véraison; V+18, 18 days

after véraison; V+27, 27 days after véraison; V+37, 37 days after véraison; V+46, 46

days after véraison

Introduction

Maturing post-véraison grapevine (Vitis vinifera) berries are sinks for non-structural

carbohydrates and N (Davies and Robinson 1996, Roubelakis-Angelakis and Kliewer

1992). Carbohydrates are ultimately derived from leaf photoassimilation, but reserve

carbohydrate remobilisation from perennial tissues can provide an alternative carbon

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(C) source if photosynthetic supply is limited (Candolfi-Vasconcelos et al. 1994).

Among the different grapevine organs, root starch is often the largest pool of storage

carbohydrates, and this rapidly depletes if the period of high berry sugar demand after

véraison coincides with restricted canopy photoassimilation (Rossouw et al. 2017a,

2017b). After the slowing of fruit sugar accumulation in the later ripening period, the

roots instead become a C sink, and root starch storage initiates (Rossouw et al. 2017a).

Soil N uptake is limited during the berry maturation period, and redistribution of N

from roots, shoots, and leaves is thought to contribute to fruit N (Conradie 1991).

Mature leaves and the roots are, additionally, the major sources of amino N in higher

plants (Rentsch et al. 2007). Grapevine roots and leaves are, therefore, important

sources of C and/or N during fruit sugar and N accumulation. The extent of the

contribution of primary compound metabolism towards root and leaf C and N source

activity requires further research.

While starch is often the predominant non-structural carbohydrate, sucrose, glucose,

fructose, and less abundant (minor) sugars and sugar alcohols (e.g. raffinose and myo-

inositol) also contribute to the carbohydrate pool of higher plants (Noiraud et al. 2001,

Valluru and Van den Ende 2011). Conditions of high carbohydrate demand, such as

those created by reducing the leaf area of fruiting grapevines, induce enzymatic starch

breakdown, and subsequent carbohydrate exportation from reserve storage (Eveland

and Jackson 2012). Breakdown of starch reserves is therefore expected to alter the

relative composition of root non-structural carbohydrates, such as the different sugars,

and influence the metabolism of other C containing compounds (e.g. organic acids).

Organic acids, such as malic and citric acid, are important intermediates during C

metabolism (López-Bucio et al. 2000). The C skeletons of these tricarboxylic acid

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(TCA) cycle intermediates, are also utilised during N assimilation and amino acid

biosynthesis (Popova and Pinheiro de Carvalho 1998). Restricting the leaf C source

may cause altered root source activity by impacting on C flux through different

pathways of primary root metabolism. The C flux through the shikimate pathway,

which can represent up to 20% of the available C in plants (Haslam 1993), is for

example, likely affected by limited C availability. As the origin of many amino acids

and secondary metabolites (e.g. phenolic compounds), changes in C flux through the

shikimate pathway could have crucial consequences on plant C and N source organ

metabolic composition.

Grapevine N reserves are stored as proteins and amino acids (mainly arginine), with

the largest proportion located in the root system and mature leaves (Roubelakis-

Angelakis and Kliewer 1992). Amino acids provide a soluble source of organic N

which can be transported between sources (leaves and roots) and sinks (the fruit) (Lam

et al. 1996). As the fruit normally accumulates N during the post-véraison period

(Roubelakis-Angelakis and Kliewer 1992), the metabolism of N containing

compounds (e.g. amino acids) in N source organs are potentially altered by limited

post-véraison N availability. By subsequently restricting the availability of organic N,

defoliation may induce protein degradation in remaining N sources such as the roots

(Volenec et al. 1996). Protein degradation in the roots will subsequently promote root

amino N accumulation, which becomes available for further N translocation to sinks.

Monitoring changes in the abundance of primary metabolites in grapevines may

provide an insight into metabolic pathway responses to abiotic conditions, and to

canopy or crop load manipulations that are commonly used in commercial viticulture.

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84

Methods such as gas chromatography/mass spectrometry (GC/MS) allow the profiling

of a wide range of plant metabolites, including soluble sugars, sugar alcohols, organic

acids and amino acids from a single sample preparation (Lisec et al. 2006). In relation

to C and N containing storage compounds and their associated metabolism, the

responses of vegetative tissues is of particular interest. However, recent literature

concerning grapevines has focused more on comparative studies between genotypes.

Where the implications of abiotic conditions such as water or heat constraints have

been examined, most of the related analyses were conducted in fruit samples (Cuadros-

Inostroza et al. 2016, Hochberg et al. 2013, 2015a, 2015b). To the best of our

knowledge, no previous studies have conducted a detailed profiling of grapevine root

and leaf metabolite composition during the post-véraison period.

This study assessed the effects of post-véraison defoliation on the contents of non-

structural carbohydrates and N in the fruit and in major C and N sources (roots and

leaves), in conjunction with the source organ abundance of primary metabolites. By

changing leaf C and N source availability, the main objective was to profile the

primary metabolic composition (including soluble sugars, amino acids and organic

acids) of remaining leaves and/or the roots. A further objective was to link specific

alterations in source organ primary metabolite concentrations to carbohydrate or N

distribution between the source and sink organs, as influenced by leaf source

availability.

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Material and methods

Experimental design and sample collection

Forty own-rooted V. vinifera L. cv. Shiraz (clone EVOVS12) grapevines, grown in 30

litre pots containing commercial potting mix, were used for the study during the

2015/16 growing season. The grapevines were enclosed in a bird-proof cage in the hot

climate Riverina grape growing region of New South Wales, Australia. The three-year-

old grapevines were spur-pruned to five two-bud spurs in the winter and distributed in

four rows with ten vines each. Shortly after budburst, the grapevines were fertilised

every three weeks with 250 ml of 1:50 diluted complete liquid fertiliser (MEGAMIX

Plus, Rutec, Tamworth, Australia). In total, approximately 3.2 g N was applied to each

vine from after budburst, and the fertilisation events were ceased one month prior to

the start of the experiment, aiming to avoid excessive soil N uptake during the

experiment. After budburst the vines were trained where possible to 10 shoots, and the

number of bunches per vine was counted at fruit set. All vines naturally contained

between 13 and 19 bunches, and individual vines were subsequently classified as either

containing low (13-15), medium (15-16) or high (16-19) number of bunches. This

classification was later only used to minimise vine cropping variability among

treatments and harvest dates. Nine days after the first sight of berry softening (i.e.

véraison + 9 days; V+9), four vines, one out of each row, and from each of the bunch

classes (two from the medium class) to ensure the collection of vines to be as unbiased

as possible, were destructively harvested to represent the population of grapevines

prior to the start of the experiment. The remaining nine vines per row were separated

into three replicates, each representing a bunch number class, and randomly allocated

a specific treatment and harvest date. This resulted in three replicates, spread over a

four row, nine column randomised block design.

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The three experimental treatments: full leaf (i.e., control, 100 primary shoot leaves per

vine and all laterals, FL), 25% leaf (25 primary shoot leaves, with no lateral leaves,

25L), and no leaves (NL), were established at a stage (V+9) when berry sugar

accumulation was expected to occur rapidly. All the leaves were removed on NL vines,

while the 25L vines were left with 25 primary leaves each, adjacent to a bunch, and

additionally on one node above or below a bunch when required. When more than 100

primary shoot leaves were present, the leaves on FL vines were reduced to 100 per

vine. The leaf-to-fruit ratio of FL vines was adjusted to approximately 8 cm2 leaf area

per g fresh fruit weight, while that of 25L vines was adjusted to 2 cm2 g-1. The adjusted

FL ratio is on the lower end of a range (8-12 cm2 g-1) suggested to, in a given climatic

region, contribute towards maximum grapevine fruit sugar accumulation capacity

(Kliewer and Dokoozlian 2005). For NL and 25L vines, any new vegetative growth

was removed as soon as the regrowth of leaves and lateral shoots was observed.

A pressure compensated drip emitter (4 l h-1) was installed in the middle of each pot,

close to the vine trunk, and irrigation events were scheduled four times daily (08:00,

11:30, 14:30 and 18:00 h). All vines received the same amount of water during each

irrigation event. The irrigation event duration was the same at each application per

day, and ranged between 13 and 20 min per event throughout the experiment

(depending on daily atmospheric conditions), aiming to always allow visual free water

drainage from all pots during each irrigation event. Three vines from each treatment

were destructively harvested every 9-10 days after the start of the experiment. At the

destructive harvest dates, i.e., 28 Dec 2015 (V+9), 6 Jan 2016 (V+18), 15 Jan 2016

(V+27), 25 Jan 2016 (V+37), and 3 Feb 2016 (V+46), the pre-selected grapevines were

dismantled. Whole root systems, leaf blades and all fruit were collected from each

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vine, collected and washed with phosphate-free detergent and rinsed with deionised

water. The fresh weights of these organs were determined, and the root and leaf

samples were oven-dried at 60 °C until a constant dry weight was reached. During the

destructive harvests, subsamples of the roots, leaf blades and berries were collected.

The root subsamples consisted of full-length root parts taken from within 10 cm from

the basal part of the trunk, always between 2 and 6 mm in diameter, with at least 50 g

in total tissue fresh weight. Soil particles were shaken off and the roots rapidly rinsed

with deionised water, prior to freezing in liquid N. Leaf subsamples consisted of 20

leaves, taken adjacent to a bunch, or from the shoot node directly above or below a

bunch when required, and frozen in liquid N. Berry subsamples consisted of 100

berries per vine, immediately frozen in liquid N after their removal from the vine. The

snap-frozen subsamples were stored at -80 °C until further processing. The periods

between the different destructive harvest dates are referred to as Intervals 1, 2, 3 and

4, respectively.

Vegetative and reproductive development

The total area of the leaves collected from each individual vine at the respective

destructive harvest dates was measured using a leaf area meter (LI-3100C, LI-COR

Biosciences Inc., Lincoln, NE, USA). The leaf subsample areas were measured

immediately after removal of the leaves, prior to the snap-freezing of these leaves.

Total fruit weight of each grapevine was recorded, and subsamples of 50 berries per

vine were used to determine the fresh weight per berry and juice total soluble solid

(TSS) concentration. Berry soluble solid content (SSC) was calculated on the basis of

berry fresh weight and TSS. The total fruit sugar content per vine basis was

subsequently calculated and is henceforth referred to as the fruit sugar content.

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Total tissue dry weight of whole root systems and leaves were calculated for each vine

by combining the weights measured from the dried main samples with estimated dry

weights of the sub-samples. A subsample of 50 frozen berries per vine was ground to

a fine powder under liquid N using an analytical mill (A11 basic analytical mill, IKA,

Selangor, Malaysia), and freeze-dried (Gamma 1-16 LSC, Christ, Osterade am Harz,

Germany) until a constant weight was reached. Total fruit weight per vine was

estimated from the weight loss during drying. Root and leaf structural biomass per vine

were estimated by subtracting the non-structural carbohydrate content (total starch and

soluble sugar content) of these tissues from their total dry weight.

Non-structural carbohydrate determination

The root and leaf subsamples for each vine were taken from -80 °C storage and ground

to a fine powder under liquid N, using an analytical mill (A11 basic analytical mill,

IKA, Selangor, Malaysia). Frozen ground tissues of each sample were then freeze-

dried (Gamma 1-16 LSC, Christ, Osterade am Harz, Germany) until a constant weight

was reached. Starch and total soluble sugar concentrations in a 20 mg freeze-dried

sample of ground tissue were determined following the methods outlined in Smith and

Holzapfel (2009).

Total Nitrogen (N) determination

Nitrogen concentrations were determined in finely ground, freeze-dried samples of

roots, leaves and fruit. N concentration in 200 mg of a representative sample was

determined by the LECO method (Standard methods of Rayment and Lyons, Soil

chemical methods, Australasia, Dumas Combustion Method 6B2b), using a LECO

CNS TruMAC analyser (LECO corporation, St. Joseph, MI, USA).

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Metabolite extraction and analysis

Extraction and derivatisation of untargeted metabolites in freeze-dried root and leaf

subsamples were performed using the method outlined in Lisec et al. (2006) with some

modifications. Firstly, 100 mg ground tissue were homogenised with 1.4 ml 100%

(v/v) methanol and 30 µl internal standard solution (1 g l-1 of each, adonitol, L-

hydroxyproline, and adipic acid, dissolved in 50% v/v methanol). The homogenate

was shaken at 70 ºC for 10 min (Thermomixer 5436, Eppendorf, North Ryde, NSW,

Australia), before being centrifuged at 11 000 g for 10 min. The supernatant was

transferred to a glass vial, and mixed with 0.75 ml chloroform and 1.4 ml ultrapure

water. The mixture was centrifuged at 2200 g for 15 min, and 150 µl of supernatant

(polar phase) collected and dried under a constant stream of pure N2 gas.

Derivatisation of the extracted metabolites was initiated by adding 40 µl of 20 mg ml-

1 methoxyamin hydrochloride in pure pyridine, to the dried extracts. Samples were

then shaken at 37 ºC for 2 h, before being centrifuged at 5000 g for 2 min. N-Methyl-

N-(trimethylsilyl) trifluoroacetamide (MSTFA, 70 µl) was added, and samples were

shaken again for 30 min at 37 ºC, before being centrifuged at 5000 g for 2 min.

Solutions of analytical grade standards (10 µg l-1 in 50% v/v methanol), obtained from

Sigma-Aldrich (Sigma, St. Louis, MO, USA), consisted of soluble sugars: Sucrose,

D(+)-glucose, D(-)-fructose, D(+)-mannose, L-rhamnose, D(-)-mannitol, galactinol

dehydrate, D(+)-raffinose, melibiose, D(+)-turanose, D(+)-melezitose, D(+)-

cellobiose, D(-)-ribose, D(-)-arabinose, D(+)-trehalose, maltose monohydrate, D(+)-

galactose, D(+)-xylose, dulcitol (galactitol), L-fucose, and myo-inositol; amino acids:

L-glutamic acid, L-arginine, L-proline, L-glutamine, γ-amino-n-butyric acid (GABA),

L-threonine, L-methionine, β-alanine, L-lysine, L-asparagine, L-aspartic acid, L-

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leucine, L-isoleucine, L-valine, L-alanine, L-serine, glycine, L-tyrosine, L-

phenylalanine, L-tryptophan, L-histidine, L-cysteine and L-cysteine), and

miscellaneous compounds (L-ascorbic acid and protocathechuic acid), and were

prepared in order to assist in the retention index and spectra identification of these

compounds.

Extraction and analysis of samples were randomised, and after every tenth sample, a

quality control root sample was injected. GC/MS analyses were conducted by injecting

1 µl into the GC column (30 m ×0.25 mm, 0.25 µm HP-5MS, Agilent, Santa Clara,

CA, USA), in both split-less and split mode (300:1, to allow the measurement of more

abundant metabolites). The GC/MS system consisted of a 7683B series autosampler,

7890A gas chromatograph and 5975C mass spectrometer with an electron impact

ionisation source and a quadruple analyser (all from Agilent, Santa Clara, CA, USA).

The injection port was set at 250 ºC, the transfer line at 280 ºC, the ionisation source

at 230 ºC, and the quadrupole at 150 ºC. The helium carrier gas was set at a constant

flow rate of 1.3 ml min-1. The column temperature program was set at 65 ºC for 2 min,

followed by a 6 ºC min-1 ramp to 300 ºC, where it was held for 25 min. The ionisation

energy was set at 70 eV. Mass spectra were recorded in full mode at 2.66 scans s-1 with

a mass-to-charge ratio of 50 to 600 amu. Spectral deconvolution (signal-to-noise ratio

threshold = 10; mass absolute height ≥ 2000; compound absolute area ≥ 10000)

allowed the identification of co-eluting chromatographic peaks, and was conducted

through the MassHunter Workstation software (Qualitative Analysis, version B.07.00,

Agilent, Santa Clara, CA, USA). Acquired MS spectra were searched for, and

identified by using the National Institute of Standards and Technology algorithm

(NIST, Gaithersburg, MD, USA). The retention index for each compound in the

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analysed samples was calculated by using the retention times of a series of alkanes

(C8-C28) in an injected retention index solution (Fluka, Buchs, Switzerland).

Statistical analysis

Datasets regarding grapevine vegetative and reproductive development (Table 1), non-

structural carbohydrate (Fig. 1) and total N distribution (Fig. 2), and primary

metabolite concentration (Figs. 3 and 4, with Supporting Information in Tables S1 and

S2) were analysed using Statistica 13 (Dell Inc., Tulsa, OK, USA). For each variable,

both treatment differences at a single time and how a treatment changed over time

were of interest. It was also recognised that residual variance at each time may differ

and that the interventionist nature of the treatments may also lead to reduced residual

variance for some treatments. To facilitate these comparisons, univariate analysis of

variance (ANOVA) at each date (all treatments) and for each treatment (all dates) were

conducted. An average Fisher’s least significant difference (LSD) test was used to

identify significant differences between means (P < 0.05). Significant differences in

table and heat map columns and rows are indicated by upper case letters (between

treatments) and lower case letters (between dates), respectively.

For each of the grapevine organs (roots and leaf blades), at each harvest date after the

initial harvest (V+18, V+27, V+37 and V+46), a linear mixed model was fitted for

every unequivocally identified primary metabolite (78 for roots and 75 for leaves)

using ASReml-R (Butler et al. 2007). Each model included Treatment as a fixed effect

and Replicate as a random effect. For the roots, Treatment included FL, 25L and NL,

while for the leaves Treatments included FL and 25L. The significance of treatment

effects was assessed using approximate F-tests using the techniques of Kenward and

Chapter 5: Paper 3

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Roger (1997). For each vine organ, primary metabolites which had significant

treatment effects (P < 0.05) for any of V+18, V+27, V+37 and V+46 were retained for

a Principal Component Analysis (PCA). The PCA was only conducted as an initial

data analysis step to interpret the interactions between metabolites, treatment and

harvest dates. Predicted means from the ASReml-R analysis were used in a PCA based

on the correlation matrix, conducted in Genstat 17th Edition (VSN International Ltd.,

Hemel Hempstead, Hertfordshire, UK). Biplots were drawn using R statistical

software plus the add-in package ‘shape’ (developed by Karline Soetaert, Royal

Netherlands Institute of Sea Research Yerseke, The Netherlands). These biplots and

further PCA information are provided in the Supplementary Information (Figs. S1 and

S2).

To test the relationship between the concentration of root starch and myo-inositol, a

cubic smoothing spline was fitted using the linear mixed model methods of Verbyla et

al. (1999). The fixed effects included intercept effects for the overall mean and

treatments, and effects for linear trend including overall linear trend and trend due to

treatment. The random effects included overall spline curvature and curvature due to

treatment as well as effects due to replicates at each time of measurement. The

significance of fixed treatment effects was assessed by the approximate F-tests using

the techniques of Kenward and Roger (1997) and the significance of spline curvature

was assessed by examining 0.5(1-Pr( χ 2 ≤ d)) where d refers to models which differ in

a single spline curvature term. This linear model was fitted using ASReml 3.0

(Gilmour et al. 2009).

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Results

Vegetative and reproductive development

The total leaf area (data not shown) and corresponding leaf-to-fruit ratio per control

(FL) vine were initially (at V+9) adjusted to 1.4 m2 and 8 cm2 leaf area g-1 fresh fruit

(Table 1) respectively, and these values did not change significantly during the

experiment. The leaf area and leaf-to-fruit ratio of the 25 leaf treatment (25L) was

adjusted to 0.6 m2 and 1.9 cm2 g-1 respectively, with the latter being significantly lower

than those of FL from V+18.

The root structural biomass per FL vine increased significantly during Interval 1 (Table

1), while that of 25L and no leaf (NL) did not alter significantly. FL had significantly

larger root structural biomass at V+46 than 25L or NL. The FL leaf structural biomass

per vine increased significantly between V+18 and V+46 (Table 1), while that of 25L

decreased during Interval 1 due to the defoliation. After treatment implementations,

FL leaf structural biomass was significantly larger than that of 25L at all harvest dates.

The FL and 25L total fruit dry weight per vine increased significantly during Interval

1 (Table 1). The FL total fruit dry weight continued to increase between V+18 and

V+37, while that of 25L increased significantly between V+18 and V+46. The NL

total fruit dry weight per vine increased significantly between V+9 and V+27. FL

significantly induced the largest fruit dry weight per vine from V+27.

Carbohydrate distribution

Fruit sugar accumulation: FL total fruit sugar content per vine increased rapidly during

Intervals 1 (12 g d-1) and 2 (14 g d-1) (Fig 1A). It continued to increase significantly

Chapter 5: Paper 3

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during Interval 3 although at a reduced rate (7 g d-1), and did not change significantly

during Interval 4 (3 g d-1). Although at rates lower than that of the control, 25L fruit

sugar content per vine increased significantly during Intervals 1 (7 g d-1) and 2 (8 g d-

1). The NL total fruit sugar content per vine increased at a slow but significant rate

during Interval 1 (4 g d-1) and between V+18 and V+37 (3 g d-1). Among treatments,

FL fruit had significantly higher sugar content from V+27, and 25L fruit contained

significantly more sugar per vine than those of NL at V+27.

Root carbohydrate abundance: Starch concentration in FL roots decreased

significantly between V+9 and V+27, and then increased back to its original

concentration during Interval 4 (Fig. 1B). The 25L and NL root starch concentrations

reduced significantly during Intervals 1 and 3. Among the treatments, FL root starch

concentration was highest at V+18, V+37 and V+46. The FL root total sugar

concentration was significantly higher at V+46 than at V+9 and V+27, and was

significantly higher than that of 25L at V+46 (Fig. 1B). The NL root total sugar

concentration was significantly higher from V+37 than at V+18.

Leaf carbohydrate abundance: FL leaf starch concentration decreased significantly

during Interval 2, and increased significantly during Intervals 3 and 4 (Fig. 1C). The

25L leaf starch concentration reduced significantly during Intervals 1 and 2. Among

treatments, FL significantly induced the highest leaf starch concentration from V+37.

The only significant leaf sugar concentration changes occurred where the FL

concentration increased during Intervals 3 and 4 (Fig. 1C). No leaf sugar concentration

treatment differences occurred.

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Nitrogen (N) distribution

Fruit N accumulation: FL total fruit N content per vine increased significantly between

V+9 and V+46 (0.02 g d-1) (Fig. 2A). The 25L and NL total fruit N contents per vine

increased significantly between V+9 and V+27 (0.04 g d-1). No significant treatment

differences occurred in total fruit N content.

Root N abundance: The only significant root N concentration change occurred where

25L N increased during Interval 3 (Fig. 2B). Among treatments, root N concentrations

of 25L and NL was significantly higher than that of FL at V+46.

Leaf N abundance: FL leaf N concentration reduced significantly during Interval 3,

and was significantly lower at V+46 than before V+37 (Fig. 2C). The 25L leaf N

concentration increased significantly during Interval 1, and reduced significantly

during Intervals 2 and 3. Among the treatments, 25L leaf N concentration was the

highest at V+18.

Metabolic adjustments

Primary metabolites from the roots and leaves were categorised as sugars, sugar

alcohols, amino acids, miscellaneous acids, or others (including flavonoids and

stilbenoids). Further information regarding the metabolite abundance and MS spectra

are indicated in Tables S1 and S2 (Supporting Information). Simplified listings of all

metabolites which had significant treatment effects for any of the destructive harvest

dates, and the associated biosynthetic pathway of each metabolite, are indicated for

roots (Table 2) and leaves (Table 3).

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Figures 3 and 4 illustrate the effects of the defoliation treatments on root and leaf

metabolite concentrations respectively. The concentrations of metabolites that

exhibited significant treatment differences, and are involved in major C and N

metabolic pathways, are indicated in the figures. For ease of interpretation, treatment

effects and notable metabolite responses are described in further detail below. This

description is structured in accordance to the simplified metabolic pathways for roots

(myo-inositol metabolism, amino acid metabolism including shikimate pathway

derived amino acids, and the TCA cycle) and leaves (sugar alcohol and further myo-

inositol metabolism, the shikimate pathway including aromatic amino acids, the TCA

cycle, and amino acid metabolism other than those related to the shikimate pathway).

Root metabolism

Myo-inositol metabolism: The FL root myo-inositol concentration decreased between

V+9 and V+27, and then increased between V+27 and V+46. For the 25L and NL

treatments root myo-inositol concentrations decreased during both the first two

intervals. FL roots subsequently contained more myo-inositol than those of 25L and

NL at V+18, and again from V+37 (Fig. 3). While FL root galactinol decreased

between V+9 and V+46, that in 25L and NL roots reduced during Interval 1. Among

treatments, FL roots contained the most galactinol at V+37, and additionally more

galactinol than those of 25L and NL at V+18 and V+46, respectively. FL root raffinose

increased during Interval 3, while that of 25L and NL reduced between V+9 and V+27.

Among treatments, FL roots exhibited the highest raffinose concentration from V+27.

FL root melibiose increased during Interval 2 before decreasing during Interval 3,

while that in 25L and NL roots increased between V+9 and V+37. Melibiose was

Chapter 5: Paper 3

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subsequently more abundant in 25L and NL roots than those of FL at V+37, while NL

roots additionally contained more melibiose than those of FL at V+46.

FL root ascorbic acid increased between V+18 and V+46, while that in 25L and NL

roots reduced during Interval 1. Among treatments, FL roots contained the most

ascorbic acid at V+27 and V+46. While FL root tartaric acid did not change

significantly, that in 25L and NL roots increased between V+9 and V+37. 25L and NL

roots subsequently contained more tartaric acid than FL roots from V+37 and at V+46,

respectively.

Amino acid metabolism: The FL root glutamic acid concentration never changed

significantly, however, that in 25L and NL roots decreased during Interval 1. FL roots

subsequently contained more glutamic acid than those of NL and 25L from V+27 and

at V+37, respectively. Furthermore, 25L roots contained more glutamic acid than those

of NL at V+46. The FL root arginine concentration also did not change during the

experiment, however, that in 25L and NL roots accumulated during Interval 3 and

between V+18 and V+37, respectively. Among treatments, 25L contained most

arginine at V+46, when NL roots contained more arginine than FL roots.

For shikimate pathway derived amino acids, FL and 25L root tryptophan

concentrations did not change during the experiment, however, that in NL roots

accumulated during Interval 3. NL roots subsequently exhibited more tryptophan than

those of FL at V+37. Like with tryptophan, FL and 25L root tyrosine concentrations

did not change significantly, however, NL roots accumulated tyrosine between V+9

and V+27. Among treatments, NL roots contained most tyrosine at V+27. FL root

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phenylalanine increased between V+18 and V+37, before decreasing during Interval

4. While 25L root phenylalanine did not change significantly, that in NL roots

increased between V+9 and V+27. NL roots subsequently contained more

phenylalanine than those of FL and 25L at V+27.

Aspartic acid reduced in FL roots between V+9 and V+27, before increasing towards

V+46. 25L aspartic acid reduced between V+9 and V+27, while NL root aspartic acid

concentration did not alter significantly. FL roots contained more aspartic acid than

those of NL at V+46. FL root lysine did not change significantly, while that of 25L

and NL increased during Interval 3. At V+46, 25L roots contained the most lysine,

while NL roots exhibited higher lysine concentration than FL roots.

TCA cycle intermediate metabolism: FL root citric acid increased during Interval 4,

while that in 25L and NL roots accumulated during Interval 3, the NL citric acid then

decreased during Interval 4. Among treatments, 25L and NL roots contained more

citric acid than those of FL from V+37, while 25L roots also contained more citric acid

than NL roots at V+46. No significant maleic acid concentration changes occurred,

however, 25L roots contained more maleic acid than those of FL at V+37.

Leaf metabolism

Sugar alcohol and further myo-inositol metabolism: While the FL leaf mannitol

concentration did not change significantly, that of 25L leaves decreased during Interval

1. As a result, FL leaves contained more mannitol than those of 25L at V+46 (Fig. 4).

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FL myo-inositol decreased between V+9 and V+27, while that in 25L leaves gradually

decreased from V+9 to V+46. Among treatments, myo-inositol concentration was

lower in 25L than FL leaves, from V+27. FL leaf ascorbic acid concentration did not

change significantly, while that in 25L leaves decreased between V+9 and V+27. The

25L leaf ascorbic acid concentration was subsequently higher than that of FL leaves

from V+37. Tartaric acid decreased in FL leaves during Interval 2 and between V+27

and V+46. The 25L leaf tartaric acid decreased during Intervals 1 and 2, and among

treatments, FL leaves contained more tartaric acid at V+27 and V+37. While the FL

leaf threonic acid concentration did not change significantly, that in 25L leaves

decreased during Interval 1. 25L leaves subsequently exhibited more threonic acid

from V+27. FL leaf glyceric acid increased between V+9 and V+27, before decreasing

towards V+46. 25L leaf glyceric acid decreased during Interval 1, and among

treatments, FL leaves contained more glyceric acid from V+27.

Shikimate pathway derivatives: For amino acids, leaf phenylalanine accumulated

during Interval 2 regardless of the treatments, before decreasing during Interval 3.

However, 25L leaves contained more phenylalanine than those of FL at V+37. Leaf

tyrosine also accumulated during Interval 2, before depleting with no significant

concentration differences among treatments. Likewise, tryptophan accumulated during

Interval 2 before depletion in 25L leaves at V+37, while increasing between V+9 and

V+27 in FL leaves. In FL leaves, 5-hydroxytryptophan decreased between V+9 and

V+37, while reducing during Interval 1 and between V+18 and V+37 in 25L leaves.

Among treatments, FL leaves contained more 5-hydroxytryptophan at V+27 and

V+37.

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Caffeic acid depleted in the leaves of both treatments between V+9 and V+46, without

significant treatment differences. On the other hand, gallic acid was significantly less

abundant at V+27 than at V+9 in FL leaves, while 25L leaf gallic acid reduced during

Interval 1. FL leaf gallic acid was less abundant than that of 25L leaves from V+37.

Arbutin accumulated in leaves of both treatments between V+9 and V+37, and

additionally during Interval 4 in those of 25L. 25L leaves subsequently contained most

arbutin at V+46. While FL (+)-Catechin did not change significantly, it decreased

between V+9 and V+37 in 25L leaves, before increasing during Interval 4. The 25L

leaves contained more (+)-catechin than those of FL at V+46.

TCA cycle intermediates: Citric acid reduced in leaves of both treatments during

Interval 1 and between V+18 and V+37. However, among treatments, FL leaves

contained more citric acid from V+18.

Amino acid metabolism: FL and 25L leaf glutamic acid decreased during Interval 1,

before increasing in FL leaves during interval 2. However, no leaf glutamic acid

treatment differences were observed. While FL leaf GABA concentration did not

change significantly, that in 25L leaves accumulated during Interval 3. 25L leaves

subsequently exhibited more GABA at V+18, and from V+37.

FL leaf aspartic acid decreased between V+9 and V+37, while that in 25L leaves

decreased both, during Interval 1, and between V+18 and V+37. However, no leaf

aspartic acid treatment differences were observed. Threonine decreased in leaves of

both treatments during Interval 1, before accumulating in those of 25L during Interval

3. Among treatments, 25L leaves contained most threonine from V+18. While FL leaf

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isoleucine concentration did not change significantly, that of 25L leaves accumulated

during Interval 4. 25L leaves subsequently contained most isoleucine at V+27 and

V+46. FL leaf leucine concentration did not alter significantly, however, 25L leaf

leucine accumulated between V+9 and V+46. Among treatments, 25L leaves

contained most leucine at V+46. Although no significant valine concentration changes

occurred for both treatments, 25L leaves contained more valine at V+27 and V+46.

Serine decreased in FL leaves between V+9 and V+27, while increasing in 25L leaves

during Intervals 1 and 3. The 25L leaves contained most serine among treatments from

V+18. Cysteine accumulated in FL leaves during Interval 2, while it accumulated in

25L leaves between V+18 and V+37. The 25L leaves contained more cysteine than

those of FL at V+37.

Relationship between root starch and myo-inositol

Changes in root starch and myo-inositol concentrations were similar over time (Fig.

5). In FL roots, both starch and myo-inositol significantly declined from V+9 to V+37

but recovered to the abundance at V+9, by V+46. For 25L and NL, root starch and

myo-inositol declined significantly between V+9 and V+37, and plateaued during

Interval 4. The relationship between starch and myo-inositol is additionally illustrated

in the Supplementary Information (Fig. S3).

Discussion

The current study evaluated implications of reduced carbohydrate and N source-sink

biomass ratios during berry maturation for metabolite concentrations of the major

source organs. Specific defoliation induced changes in root and leaf carbohydrate and

Chapter 5: Paper 3

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N utilisation, and linkages to primary metabolism, are explained below. Leading into

the discussion, it is particularly noteworthy that decreasing source-sink ratios during

berry ripening distinctly increased root carbohydrate source activity. Conversely,

removing the leaf N source only had minor effects on total N re-distribution from

leaves and roots. Defoliation did, however, alter N composition of roots and leaves,

suggesting increased amino N source activity when the total vine N source-sink

biomass ratio was reduced.

Root carbohydrate reserve remobilisation

Following the removal of all leaves in the full defoliation treatment, root starch

concentrations rapidly declined (Fig 1B). However, the continued accumulation of

berry sugar in the corresponding period, albeit at a lower rate than control vines,

indicates a clear contribution of reserve carbohydrates to berry ripening (Fig 1A). Such

contributions from stored carbohydrates has previously been demonstrated by 14C

labelling, when carbohydrates from perennial tissues were translocated to fruit after

defoliation during fruit sugar accumulation (Candolfi-Vasconcelos et al. 1994). The

retention of some leaves on partially defoliated vines did not alter the rate of root

carbohydrate mobilization relative to the fully defoliated vines, but the availability of

extra carbohydrates from concurrent photosynthesis did allow an increased rate of

berry sugar accumulation. Under full leaf area, root starch concentration reduced only

during the phase of rapid fruit sugar accumulation, and then increased when fruit sugar

accumulation slowed. This starch reduction may imply reserve remobilisation towards

the sugar-accumulating berries as these vines carried a substantial crop load. However,

root reserves could also be utilised for respiration and structural development

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(Holzapfel et al. 2010), of which latter was observed during Interval 1 under full leaf

area.

Total N distribution was unaffected by defoliation

Under full leaf area, fruit sugar content per vine increased by 252% in the 37-day

period following the start of the experiment (Fig. 1A). During the same period fruit N

content only increased by 39% (Fig. 2A), implying a proportionally greater importance

of the véraison to harvest period for berry carbohydrate than N accumulation. The leaf

N concentrations of 2.1% (Fig 2C) were adequate according to published levels

(Holzapfel and Treeby 2007), and suggest that the lower N accumulation reflected

lower fruit sink demand rather than reduced availability of N from the vegetative parts

of the vine.

Although fruit N accumulation rates varied somewhat across the five sampling dates,

the fruit did not exhibit any significant N content treatment differences by the final

harvest. Therefore, despite the reduction or complete removal of leaves as an N source

(Rossouw et al. 2017b), fruit N accumulation was maintained. The lack of change in

root N concentration after partial or full defoliation (Fig. 2B), suggests the roots did

not become a significant net source during fruit N accumulation (Conradie 1991).

Although N fertilisation was ceased a month prior the experiment, it is likely that soil

N uptake contributed to maintaining fruit N accumulation. Soil N uptake is not unusual

shortly after véraison (Löhnertz 1991), and the limitation or absence of the leaf N

source did not interfere with N allocation towards the fruit by the final harvest.

Chapter 5: Paper 3

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A central role for myo-inositol during root carbohydrate remobilisation

The parallel changes of root starch and myo-inositol concentrations with treatment and

time (Fig. 5), suggests a role for myo-inositol during root carbohydrate remobilisation.

The roles of myo-inositol in plants include signalling, involvement in the synthesis of

cell wall polysaccharides, and precursor for other metabolites including galactinol,

raffinose, and ascorbic acid (Valluru and Van den Ende 2011). However, while myo-

inositol has been found in the phloem sap of various plants, suggesting a potential

long-distance transport role (Noiraud et al. 2001), myo-inositol is not generally

considered a major C transport compound. The close similarities between starch and

myo-inositol concentration profiles in the grapevine roots of the present study is an

original result, and further investigation is needed to determine the underlying

connection. The impact of defoliation on the decreased root concentrations of myo-

inositol and its derivatives, i.e., galactinol and raffinose (Loewus and Murphy 2000)

(Fig. 3), suggests that these metabolites play a role during carbohydrate reserve

remobilisation.

Similar to myo-inositol, root ascorbic acid depleted shortly after partial or full

defoliation (Fig. 3). It has been established that myo-inositol metabolism provides an

alternative pathway for ascorbic acid biosynthesis (Lorence et al. 2004). The

similarities in root myo-inositol and ascorbic acid concentrations over time, in terms

of both, for example, exhibiting higher concentration in FL roots by V+46, is therefore

potentially related to myo-inositol providing the initial substrate towards an ascorbic

acid biosynthetic route (Lorence et al. 2004, Valpuesta and Botella 2004).

Furthermore, ascorbic acid is a precursor for tartaric acid, and defoliation led to a

depletion of root ascorbic acid, while tartaric acid accumulated (Fig. 3). Therefore,

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root ascorbic acid catabolism potentially resulted in tartaric acid accumulation (DeBolt

et al. 2006). Additionally, root citric acid also accumulated under reduced leaf area

(Fig. 3). Intermediates of the TCA cycle, such as citric acid, play an essential role

during C metabolism, supplying C skeletons for the biosynthesis of various

metabolites, such as phenolic compounds and amino acids (Popova and Pinheiro de

Carvalho 1998).

Defoliation induced root amino acid accumulation

Glutamic acid was the only root amino acid that depleted under partial and full

defoliation (Fig. 3). The biosynthesis of amino acids in higher plants mainly occurs in

roots and mature leaves, from where it is transportable to sinks, thereby facilitating the

distribution of organic N between plant organs (Rentsch et al. 2007). Although

defoliation did not affect the root total N concentration, it did impact on root amino N

composition. As an essential amino-group donor during the synthesis of many other

amino acids, glutamic acid plays a crucial role during N partitioning (Forde and Lea

2007). The depletion of root glutamic acid after partial and full defoliation may

indicate its involvement in amino N repartitioning within these roots.

Various other amino acids accumulated in the roots of partially or fully defoliated

vines, including arginine, lysine, phenylalanine, tryptophan, and tyrosine (Fig. 3). It

can thus be proposed that glutamic acid metabolism was involved in the accumulation

of these amino acids in the roots, either directly by providing a C skeleton for arginine

synthesis (Berg et al. 2002), or as an amino donor towards the synthesis of the others.

The accumulation of arginine in roots, only after partial or full defoliation may relate

to a N transport role. In fact, arginine is characterised by a high N:C ratio, and is known

Chapter 5: Paper 3

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to be transported in the xylem and phloem between source and sink organs, facilitating

organic N distribution (Lea et al. 2007). When the leaves as a N source was restricted

or limited, root amino acid accumulation presumably contributed to the root amino N

source activity.

The aromatic amino acids (i.e., phenylalanine, tryptophan and tyrosine) only

accumulated in the roots after full defoliation (Fig. 3). These amino acids originate

from the shikimate pathway, and are precursors for many secondary metabolites,

including phenolic compounds (Maeda and Dudareva 2012). Various secondary

metabolites derived from the aromatic amino acids, including anthocyanins (Boss et

al. 1996), accumulate in post-véraison grapevine berries. The removal of leaves as an

amino acid source seemingly induced the biosynthesis of root amino acids through the

shikimate pathway. Genes related to the aromatic amino acids are expressed in post-

véraison grapevine berries (Berdeja et al. 2015). However, many amino acids,

including phenylalanine, are also present in the vascular tissues of higher plants (as for

example, indicated in Trifolium repens and Lupinus albus), where they accrete after

defoliation (Hartwig and Trommler 2001). The possibility is, therefore, raised that

grapevine leaves are important aromatic amino acid sources, from where they may be

phloem translocated to the fruit to contribute to secondary metabolism. The roots may

become an alternative aromatic amino acid source after exclusion of the leaf source.

Leaf sugar alcohols and organic acids depleted rapidly after partial defoliation

Myo-inositol and mannitol, some of the most prevalent sugar alcohols in higher plants

(Noiraud et al. 2001), depleted rapidly in remaining leaves after partial defoliation

(Fig. 4). Apart from sucrose as the principal plant transported sugar, some sugar

Chapter 5: Paper 3

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alcohols and raffinose family oligosaccharides can also be important C transporters

(Noiraud et al. 2001). Mannitol, unlike myo-inositol, is a known primary

photosynthetic product in mature leaves, and a recognised transport compound

(Noiraud et al. 2001). During the present study, mannitol was, therefore, presumably

synthesised in leaves during photosynthesis, and its rapid depletion in leaves after

partial defoliation suggests an important C transport role during limited canopy

photoassimilation. Although there has been ambiguity around the transport role of

myo-inositol (Noiraud et al. 2001), the current study suggests that root myo-inositol

plays a central role during carbohydrate remobilisation, and perhaps similarly in

leaves.

In addition to the sugar alcohols mentioned above, various leaf organic acids rapidly

depleted after partial defoliation (Fig. 4). Citric acid was among the organic acids

depleted under reduced leaf area, and like other TCA intermediates, it is a vital

metabolic branch point as its conversion provides C skeletons for N assimilation, in

addition to playing an important role in plant energy and C metabolism (Popova and

Pinheiro de Carvalho 1998). Leaf ascorbic acid concentration, like that of myo-inositol,

was negatively impacted by partial defoliation (Fig. 4). As a potential precursor, leaf

myo-inositol metabolism may have affected the ascorbic acid concentration. Likewise,

leaf tartaric acid, threonic acid and glyceric acid also depleted after partial defoliation

(Fig. 4). These organic acids are derived from ascorbic acid (Loewus 1999), further

indicating a change in ascorbic acid metabolism in remaining source leaves. The

phenolic acids, caffeic acid and gallic acid, depleted towards the end of berry ripening

in remaining leaves after partial defoliation, while arbutin and (+)-catechin increased

(Fig. 4). These compounds are products of the shikimate pathway (Balasundram et al.

Chapter 5: Paper 3

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2006, Siegler 1998) and, therefore, like in roots, leaf metabolites yielded from the

shikimate pathway were affected by post-véraison leaf source limitation.

Partial defoliation induced leaf amino acid accumulation

Various amino acids accumulated in remaining leaves after partial defoliation (Fig. 4).

Amino acid synthesis in plants, as mentioned above, mainly occurs in roots and mature

leaves, where they are utilised or stored, or exported to sinks to contribute to growth

and secondary metabolism (Rentsch et al. 2007). Leaf proteins (e.g. Rubisco and

chloroplast proteins) are extensively degraded during leaf ageing, thereby producing

free amino acids, outsourceable to sinks (Masclaux-Daubresse et al. 2010). In the

present study, GABA, leucine, isoleucine, as well as cysteine and serine were among

the amino acids that accumulated in the remaining leaves after partial defoliation (Fig.

4). After defoliation in the present study, the ratio of the leaf N source to the fruit N

sink was drastically reduced. It could, therefore, be argued that the increased source

requirement placed upon the remaining leaves of treatment 25L advanced its ageing

process, prompting amino acid accumulation and its subsequent exportation.

Conclusion

A study was conducted to determine the implications of reduced leaf carbohydrate and

N source availability, during a period of considerable fruit C and N sink demand, on

remaining leaf or root source activity. A focus was placed upon primary metabolite

abundance responses in the source organs, ultimately with the goal of identifying

metabolites that contribute to carbohydrate and N source functioning in roots and

leaves. In terms of carbohydrate distribution, post-véraison leaf source absence

slowed, but did not completely stop fruit sugar accumulation. In the absence of leaf C

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assimilation, root starch provided an alternative C source for berry ripening. Root myo-

inositol concentrations were directly related to starch concentration, suggesting an

important, if yet to be elucidated role, in starch metabolism. Furthermore, the depletion

of myo-inositol metabolism derived metabolites (galactinol, raffinose and ascorbic

acid) after defoliation, illustrates the contribution of this proposed pathway towards

root carbohydrate source functioning. Compared to carbohydrates, defoliation did not

have a considerable effect on fruit N content. In fact, vegetative N pools did not

contribute to post-véraison fruit N requirements, which instead appear to have been

met by root uptake from the soil and/or regulation of amino N composition in the

leaves and/or roots. However, arginine and shikimate pathway derived root aromatic

amino acids did accumulate after full defoliation, indicating there is a least a response

in this pool in roots during leaf N absence. The remaining leaves also accumulated

various amino acids (including GABA, leucine, isoleucine and serine) after partial

defoliation, suggesting protein degradation could make a small N contribution to the

fruit. Overall, this study has shown that myo-inositol metabolism and the flux through

the shikimate pathway play central roles in grapevine carbohydrate and N source

organs during fruit ripening. The findings of this study contribute to understanding leaf

and root C and N metabolism and utilisation during fruit maturation.

Author contributions

GCR conducted the experiment, wrote the body of the paper, and carried out sample

preparations, and laboratory and data analyses. KŠ contributed to sample preparation

and led the GC/MS analysis. BAO contributed to the experimental layout and

conducted statistical analyses. BPH coordinated the project and supported the

experimental planning. AD contributed to treatment planning and experimental design.

Chapter 5: Paper 3

110

JPS and CB reviewed the methods and results. All authors reviewed, edited and

approved the final version of the manuscript.

Acknowledgements - This work was supported by the National Wine and Grape

Industry Centre, and the Australian grapegrowers and winemakers through their

investment body, Wine Australia, with matching funds from the Australian

Government. The authors thank Robert Lamont and Peter Carey for technical

assistance.

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Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1. Root metabolite concentration and GC/MS information.

Table S2. Leaf metabolite concentration and GC/MS information.

Fig. S1. Root primary metabolite PCA.

Chapter 5: Paper 3

113

Fig. S2. Leaf primary metabolite PCA.

Fig. S3. Linear and curvilinear trends of root starch concentration and myo-inositol

concentration.

Tab

le 1

Eff

ect o

f def

olia

tion

treat

men

ts (f

ull l

eaf –

FL,

25%

leav

es –

25L

and

no

leaf

– N

L) o

n gr

apev

ine l

eaf-

to-fr

esh

frui

t wei

ght r

atio

, roo

t and

leaf

stru

ctur

al

biom

ass

per v

ine,

and

tota

l fru

it dr

y w

eigh

t per

vin

e at

the

dest

ruct

ive

harv

ests

(V+9

, V+1

8, V

+27,

V+3

7 an

d V

+46 ;

mea

n ±

SE, n

=3).

Mea

ns a

re s

epar

ated

w

ithin

row

s an

d co

lum

ns u

sing

Fish

er’s

LSD

test

, sig

nific

ant d

iffer

ence

s ar

e in

dica

ted

at P

< 0

.05.

Whe

re d

iffer

ent l

ower

cas

e le

tter a

ppea

rs in

a ro

w, v

alue

s di

ffer

sign

ifica

ntly

bet

wee

n da

tes.

Whe

re d

iffer

ent u

pper

cas

e le

tter a

ppea

rs in

a c

olum

n, v

alue

s diff

er si

gnifi

cant

ly b

etw

een

treat

men

ts.

Inte

rval

1

Inte

rval

2

Inte

rval

3

Inte

rval

4

Tre

atm

ent

V+9

V

+18

V+2

7 V

+37

V+4

6

Lea

f-to-

fres

h fr

uit

wei

ght r

atio

(c

m2 /g

)

FL

11 ±

2

a A

8

± 1

ab

A

6.9

± 0.

6 b

A

7.0

± 0.

8 b

A

7.5

± 0.

4 b

25L

11 ±

2

a B

1.

9 ±

0.2

b B

1.

9 ±

0.2

b B

1.

6 ±

0.1

b B

1.

5 ±

0.1

b N

L 11

± 2

-

- -

-

Roo

t st

ruct

ural

bi

omas

s (g

/vin

e)

FL

172

± 6

b A

22

6 ±

19

a A

22

6 ±

19

a A

22

4 ±

19

a A

20

1 ±

5 a

25L

172

± 6

a A

18

2 ±

17

a A

18

2 ±

17

a A

16

8 ±

16

a B

14

7 ±

13

a N

L 17

2 ±

6 a

A

197

± 19

a

A

197

± 19

a

A

199

± 24

a

B

155

± 13

a

Lea

f st

ruct

ural

bi

omas

s (g

/vin

e)

FL

76 ±

3

b A

76

± 2

b

A

82 ±

3

ab

A

82 ±

4

ab

A

91 ±

3

a 25

L 76

± 3

a

B

19 ±

1

b B

19

± 3

b

B

22.8

± 0

.4

b B

22

± 2

b

NL

76 ±

3

- -

- -

Tot

al fr

uit

dry

wei

ght

(g/v

ine)

FL

203

± 20

c

A

338

± 53

b

A

457

± 25

ab

A

54

4 ±

46

a A

57

5 ±

51

a 25

L 20

3 ±

20

c A

29

2 ±

19

b B

35

6 ±

11

ab

B

365

± 7

ab

B

408

± 49

a

NL

203

± 20

b

A

272

± 37

ab

B

30

1 ±

24

a B

31

6 ±

17

a B

32

2 ±

8 a

Chapter 5: Paper 3

114

Chapter 5: Paper 3

115

Fig. 1. Impact of defoliation (full leaf – FL, 25% leaves – 25L and no leaf – NL) on total fruit sugar content per vine (A), root starch and total sugar (total non-structural carbohydrates, TNC) concentrations (B), and leaf starch and total sugar (TNC) concentrations (C) during the experimental period (mean ± SE; n=3). Significant differences (P < 0.05) between harvest dates for each treatment are indicated by different lower case letters. Significant differences (P < 0.05) between treatments at each harvest date are indicated by different numerals [1: FL > (25L and NL) and 2: FL > 25L > NL]. To allow clarity of the most important results, these significant differences are indicated for fruit sugar content (A), and root (B) and leaf (C) starch concentrations only.

Chapter 5: Paper 3

116

Fig. 2. Impact of defoliation (full leaf – FL, 25% leaves – 25L and no leaf – NL) on total fruit nitrogen (N) content per vine (A), root N concentration (B), leaf N concentration (C) during the experimentalperiod (mean ± SE; n=3). Significant differences (P < 0.05) between harvest dates for each treatmentare indicated by different lower case letters. Significant differences (P < 0.05) between treatments ateach harvest date are indicated by different numerals [1: FL < (25L and NL) and 2: FL < 25L].

Chapter 5: Paper 3

117

Table 2 Proposed metabolic pathways related to the biosynthesis of significantly treatment affected root metabolites. All metabolites significantly differing among the defoliation treatments (full leaf, 25% leaves and no leaf) for any of the destructive harvest dates after treatment implementation (V+18, V+27, V+37 and V+46) are listed. The metabolites are categorised based on their compound properties (sugars, sugar alcohols, amino acids or miscellaneous acids).

Classification Metabolite Proposed primary pathway

Sugars

Sucrose Raffinose Melibiose Arabinose

Sugar alcohols

Myo-inositol Galactinol Mannitol Arabitol Glycerol

Primary carbohydrate metabolism Myo-inositol metabolism Myo-inositol metabolism Glucose-6-phosphate

Glucose-6-phosphate Myo-inositol metabolism Fructose metabolism Glucose-6-phosphate Glycerate

Amino acids

α-Ketoglutarate α-Ketoglutarate α-Ketoglutarate Shikimate Shikimate Shikimate

Miscellaneous acids

Glutamic acid Arginine Glutamine Tryptophan Phenylalanine Tyrosine Glycine Lysine Threonine Valine

Ascorbic acid Tartaric acid Citric acid Maleic acid 3-Hydroxyanthranilic acidProtocatechuic acid2-Keto-gluconic acid

3-PhosphoglycerateOxaloacetateOxaloacetatePyruvate

Myo-inositol metabolism Myo-inositol metabolism Tricarboxylic acid cycle Tricarboxylic acid cycle Shikimate Shikimate Gluconate

Chapter 5: Paper 3

118

Table 3 Proposed metabolic pathways related to the biosynthesis of significantly treatment affected leaf metabolites. All metabolites significantly differing among the defoliation treatments (full leaf and 25%) for any of the destructive harvest dates after treatment implementation (V+18, V+27, V+37 and V+46) are listed. The metabolites are categorised based on their compound properties (sugars, sugar alcohols, amino acids, miscellaneous acids and others).

Classification Metabolite Proposed principle pathway

Sugars

Glucose Raffinose Melibiose Rhamnose Melezitose Ribose

Primary carbohydrate metabolism Myo-inositol metabolism Myo-inositol metabolism Fructose metabolism Sucrose metabolism Glucose-6-phosphate

Sugar alcohols Mannitol

Amino acids

Fructose metabolism Glucose-6-phosphate

α-Ketoglutarate α-Ketoglutarate 3-Phosphoglycerate3-PhosphoglyceratePyruvatePyruvatePyruvateShikimateShikimateShikimate

Miscellaneous acids

Other compounds

Myo-inositol

Arginine GABA Serine Cysteine Valine Leucine Isoleucine Phenylalanine Tryptophan 5-HydroxytryptophanThreonine

Ascorbic acid Tartaric acid Threonic acid Glyceric acid Caffeic acid Gallic acid Lactic acid Citric acid Fumaric acid 2-Keto-glutaric acidPhosphoric acidGluconic acidRibonic acidNonanoic acidPalmitic acid

Arbutin Catechin Glycerol monostearate

Oxaloacetate

Myo-inositol metabolism Myo-inositol metabolism Myo-inositol metabolism Myo-inositol metabolism Shikimate Shikimate Pyruvate Tricarboxylic acid cycle Tricarboxylic acid cycle Tricarboxylic acid cycle Tricarboxylic acid cycle Glucose metabolism Glucose-6-phosphate Glycerol metabolism Glycerol metabolism

Shikimate Shikimate Glycerol metabolism

Chapter 5: Paper 3

119

Fig.

3. S

impl

ified

pat

hway

resp

onse

of t

he ro

ot p

rimar

y m

etab

olite

s si

gnifi

cant

ly a

ffec

ted

by th

e tre

atm

ents

(ful

l lea

f: FL

; 25%

leav

es: 2

5L; n

o le

af: N

L) a

nd

othe

r met

abol

ites

dire

ctly

invo

lved

in th

e pa

thw

ays

(1).

Sign

ifica

nt d

iffer

ence

s ar

e in

dica

ted

at P

< 0

.05,

hea

tmap

col

umns

indi

cate

the

thre

e tre

atm

ents

, whi

le

heat

map

row

s in

dica

te th

e ha

rves

t dat

es (V

+9, V

+18,

V+2

7, V

+37

and

V+4

6). W

here

diff

eren

t upp

er c

ase

lette

rs a

ppea

r in

heat

map

col

umns

(2),

valu

es d

iffer

sig

nific

antly

bet

wee

n tre

atm

ents

. Whe

re d

iffer

ent l

ower

cas

e le

tters

app

ear

in a

row

, val

ues

diff

er s

igni

fican

tly b

etw

een

harv

est d

ates

. Ave

rage

met

abol

ite

abun

danc

e is

colo

ur c

oded

acc

ordi

ng to

the

scal

e on

the

left

(3).

3-PG

A: 3

-pho

spho

glyc

eric

aci

d; P

EP: p

hosp

hoen

olpy

ruvi

c ac

id; T

CA

: tric

arbo

xylic

aci

d.

Chapter 5: Paper 3

120

Fig.

4. S

impl

ified

pat

hway

resp

onse

of t

he le

af p

rimar

y m

etab

olite

s sig

nific

antly

aff

ecte

d by

the

treat

men

ts (f

ull l

eaf:

FL; 2

5% le

aves

: 25L

) and

oth

er

met

abol

ites

dire

ctly

invo

lved

in th

e pa

thw

ays (

1). S

igni

fican

t diff

eren

ces a

re in

dica

ted

at P

< 0

.05,

hea

tmap

col

umns

indi

cate

the

thre

e tre

atm

ents

, whi

le

heat

map

row

s ind

icat

e th

e ha

rves

t dat

es (V

+9, V

+18,

V+2

7, V

+37

and

V+4

6). W

here

diff

eren

t upp

er c

ase

lette

rs a

ppea

r in

heat

map

col

umns

(2),

valu

es d

iffer

sig

nific

antly

bet

wee

n tre

atm

ents

. Whe

re d

iffer

ent l

ower

cas

e le

tters

app

ear i

n a

row

, val

ues

diff

er si

gnifi

cant

ly b

etw

een

harv

est d

ates

. Ave

rage

met

abol

ite

abun

danc

e is

colo

ur c

oded

acc

ordi

ng to

the

scal

e on

the

left

(3).

3-PG

A: 3

-pho

spho

glyc

eric

aci

d; P

EP: p

hosp

hoen

olpy

ruvi

c ac

id; T

CA

: tric

arbo

xylic

aci

d.

Chapter 5: Paper 3

121

Fig. 5. Impact of defoliation (full leaf – FL, 25% leaves – 25L and no leaf – NL) on root starch (A) and myo-inositol (B) concentration during the experimental period (mean ± SE; n=3). Significant differences (P < 0.05) between harvest dates for each treatment are indicated by different lower case letters. Significant differences (P < 0.05) between treatments at each harvest date are indicated by a numeral [1: FL > (25L and NL)].

Chapter 6: Paper 4

122

Chapter 6: Paper 4

Impact of post-véraison leaf source limitation on the

metabolic profile of Vitis vinifera cv. Shiraz berries

(Paper 4 has been submitted for publication in Plant Physiology and Biochemistry.

The tables and figures are shown after the main manuscript text.)


To study the implications of defoliation on the post-véraison metabolic composition of

grapevine berries.

6.2. Supplementary material

Supplementary material, as referred to in Paper 4 (Supplementary table S1 and figure

S1), is included in appendix B.

Chapter 6: Paper 4

123

Impact of post-véraison leaf source limitation on the metabolic profile of Vitis vinifera cv. Shiraz berries

Gerhard C. Rossouw a,b,*, Katja Šuklje a,1, Beverley A. Orchard c, Jason P. Smith a,2, Celia Barril a,b, Alain Deloire a,3, Bruno P. Holzapfel a,c

a National Wine and Grape Industry Centre, Wagga Wagga 2678, New South Wales, Australia. b School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga 2678, New South Wales, Australia. c New South Wales Department of Primary Industries, Wagga Wagga 2650, New South Wales, Australia. 1 Present address: Wine Research Centre, University of Nova Gorica, Lanthieri Palace, Glavni trg 8, 5271 Vipava, Slovenia. 2 Present address: Institut für Allgemeinen und ökologischen Weinbau, Hochschule Geisenheim University, Geisenheim 65366, Germany. 3 Present address: Montpellier SupAgro, Montpellier 34060, France.

*Corresponding author (email [email protected])

Keywords: Amino acids, carbon allocation, defoliation, fruit ripening, grape metabolism, nitrogen partitioning, sugar

Abstract

Leaves are an important contributor toward berry sugar and nitrogen (N)

accumulation during fruit maturation. The post-véraison grapevine (Vitis vinifera L.)

leaf area may, therefore, affect the development of fruit composition. The aim of this

study was to investigate the impact of leaf source limitation or absence on key berry

quality attributes in conjunction with the accumulation of primary berry metabolites.

Shortly after the start of véraison, potted grapevines were defoliated (total defoliation

and 25% of the control), and the accumulation of berry soluble solids, N and

anthocyanins were compared to that of a full leaf area control. An untargeted approach

was undertaken to measure the content in primary metabolites by Gas

Chromatography/Mass Spectrometry. Defoliation resulted in reduced berry sugar and

anthocyanin accumulation, while total berry N content was unaffected. The juice yeast

assimilable N (YAN), however, increased upon partial and full defoliation.

Remobilized carbohydrate reserves allowed accumulation of the major berry sugars

during source limitation. Berry anthocyanin biosynthesis was strongly inhibited by

Chapter 6: Paper 4

defoliation, which could relate to the carbon source limitation and/or increased bunch

exposure. Arginine accumulation, likely resulting from reserve translocation,

contributed to increased YAN upon defoliation. Furthermore, assessing the

implications on various products of the shikimate pathway, suggests the carbon flux

through this pathway to be largely affected by leaf source limitation during fruit

maturation. This study provides a novel investigation of impacts of carbon and N

source limitation during berry maturation on the development of key berry quality

parameters as underlined by alterations in primary metabolism.

1. Introduction

The post-véraison period is essential for the development of key grapevine berry

quality parameters. During this berry maturation period, the accumulation of large

quantities of sugars takes place (Davies and Robinson, 1996). Furthermore, berry

Nitrogen (N) (Roubelakis-Angelakis and Kliewer, 1992) and anthocyanin (Boss et al.,

1996) incorporation also occurs, while the organic acid content declines (Degu et al.,

2014). Leaf photoassimilation yields soluble sugars which are translocated to the fruit,

mostly as sucrose, where it is hydrolyzed into glucose and fructose (Davies and

Robinson, 1996). The grapevine leaves are also a major source of organic N toward

the fruit (Rossouw et al., 2017b), and amino acid export from mature leaves provides

a soluble form of N, transportable to sink organs (Rentsch et al., 2007). Canopy

defoliation during berry maturation, therefore, removes a potential source of both,

carbon (C) and N, which can induce a severe reduction in berry sugar and N

accumulation. To the best of our knowledge, the implications of a post-véraison C and

N source limitation, as induced by severe defoliation, on the kinetic development of

fruit C and N containing primary metabolites are yet to be determined. 124

Chapter 6: Paper 4

Developments in gas chromatography/mass spectrometry (GC/MS) allow the

detection and measurement of complex mixtures of plant metabolites (Lisec et al.,

2006). These metabolites include organic acids, amino acids, sugars, sugar alcohols,

phosphorylated intermediates and lipophilic compounds. Although glucose and

fructose predominate, some minor sugars and sugar alcohols, in addition to sucrose,

are also accumulated in the grapevine berries (Cuadros-Inostroza et al., 2016). Apart

from being essential for alcoholic fermentation during winemaking, berry sugars are,

amongst other things, utilized as structural components and cell nutrients (Çakir et al.,

2003). In addition to sucrose, raffinose family oligosaccharides (e.g. raffinose) and

sugar alcohols (e.g. mannitol) can also play important roles in transporting C from

source to sink organs in higher plants (Noiraud et al., 2001). Sink organs, such as the

fruit, have little to no capacity to synthesize sugar alcohols, which are generally

produced as primary photosynthetic products (Loescher and Everard, 1996). In Olea

europaea fruit, sugar alcohols have been found to act as storage compounds and are

important during metabolic transformation (Marsilio et al., 2001). Some minor sugars,

such as trehalose, can play a role in signaling of the plant C status or as a cell

membrane constituent (O’Hara et al., 2013).

The content and composition of berry amino acids are essential from a wine

quality perspective, as it determines the juice yeast assimilable N (YAN)

concentration, influencing fermentation and wine aroma potential (Bell and Henschke,

2005). The major amino acids in berries are usually arginine and proline, the latter not

assimilable by yeasts (Bell and Henschke, 2005). Furthermore, the C skeletons of

different amino acids are utilized during the biosynthesis of secondary flavor

125

Chapter 6: Paper 4

compounds in berries (Bell and Henschke, 2005). Phenolic compounds in berries, such

as anthocyanins, are also derived from C skeletons provided by amino acid metabolism

(Boss et al., 1996). One of the major organic acids in the grapevine berries, malic acid,

is catabolized after véraison and provides a vital source of C, utilized during secondary

metabolism (Sweetman et al., 2009). Other organic acids, such as citric acid, do not

accumulate in large quantities in grapevine berries, while tartaric acid can be abundant

in the berries, but has ambiguous roles during plant metabolism (Sweetman et al.,

2009). Observing changes in the content of different berry primary metabolites during

maturation, as influenced by excessive defoliation, can provide a novel indication of

the impact of source limitation on the accumulation of key berry quality attributes

(e.g., soluble solids, N, anthocyanins and YAN).

Leaves are, however, not the only source of C and N during berry maturation.

When grapevine leaf area is limited, carbohydrate reserves, mostly stored in the roots,

are remobilized towards the fruit to support the fruit sugar content (Candolfi-

Vasconcelos et al., 1994). In addition to leaves, grapevine roots, trunks and shoots are

also transitional reservoirs of soil-absorbed N, from where it is translocated to the fruit

during maturation as soil N uptake is usually limited or absent during this period

(Roubelakis-Angelakis and Kliewer, 1992). The removal of leaf C and N source,

therefore limits, but not necessarily inhibits, fruit sugar and N accumulation during

berry maturation. A complete defoliation would also be detrimental towards sap flow

through the xylem, thereby potentially limiting root N exportation. Profiling berry

primary metabolite accumulation between véraison and fruit maturity can, however,

indicate which metabolites are likely leaf-derived and synthesized in the berries, or

derived from source organs other than the leaves.

126

Chapter 6: Paper 4

127

The aim of this experiment was to determine how defoliation near the onset of

fruit maturation affects key berry quality attributes (soluble solids, pH, TA, total N

and YAN), and especially how these parameters relate to the berry primary metabolite

composition development. The first goal was to better understand the leaf contribution

to fruit maturation and composition. It was, therefore, evaluated if a partial or complete

defoliation near véraison inhibits berry sugar, N and anthocyanin accumulation, and

the juice YAN concentration. The second goal was to conduct an untargeted profiling

of the primary metabolites in the berries during the berry maturation period. These

metabolites include sugars, sugar alcohols, organic acids, amino acids and fatty acids.

By profiling the metabolic changes in maturing grapevine berries due to defoliation,

information was gathered to better understand how primary berry metabolism is

affected by limited leaf area availability during fruit maturation.

2. Materials and methods

2.1. Experimental design and treatments

Three-year-old Vitis vinifera L. cv Shiraz (clone EVOVS12) grapevines, planted

in 30 L pots containing commercial potting mix, were grown in a bird-proofed

enclosure and used for an experiment during the 2015-16 growing season. The

experiment was conducted in the warm to very warm Riverina region, New South

Wales, Australia. Forty vines were distributed in four rows of ten vines each, and were

spur-pruned during the winter preceding the experiment to five, two-bud spurs each.

Vines were thinned to ten primary shoots after budburst. Between budburst and

approximately one month prior to the start of véraison, the grapevines were fertilized

once every three weeks with 250 mL 1:50 diluted liquid fertilizer (MEGAMIX Plus,

Chapter 6: Paper 4

Rutec, Tamworth, Australia), and an approximate total of 3.2 g N was subsequently

applied per vine. Vines were well-watered throughout the experiment to avoid any

water constraints.

At fruit-set, the bunch amount per vine was counted, and each vine was classified

as naturally containing low (13 – 15), medium (15 – 16) or high (16 – 19) bunch

numbers. This classification was later only used to minimise natural vine cropping

variability among treatments and harvest dates, and vines from the different bunch

number classes were thus equally distributed among the treatments and harvest dates.

The experiment was initiated on 28 Dec 2015 (V+9), nine days after the very first

indication of berry softening (véraison). On V+9, all the fruit from four vines, one out

of each row, were harvested in order to represent the population of grapevines prior to

the start of the experiment. To ensure an unbiased selection of vines, these vines

consisted of one vine from each of the low and high bunch number classes and two

from the medium class. Additionally, a 50 berry subsample from each of the above

mentioned vines was collected and immediately frozen in liquid N and stored at -80

ºC. To assess the vine N status, 20 petioles from each of the four vines were also

collected (from adjacent a bunch or from the shoot node directly above or below a

bunch when required). The petioles were frozen in liquid N and ground to a fine

powder (IKA A11, Selangor, Malaysia) before being freeze dried (Gamma 1-16 LSC,

Christ, Osterade am Harz, Germany). The petiole N concentration was subsequently

determined by the method also used to determine berry N, described in section 2.2.

The remaining nine vines per row were spread over a four row, nine column

randomized block design, consisting of three treatment replicates. The experimental

treatments were implemented on V+9, with the control treatment (FL) consisting of

128

Chapter 6: Paper 4

129

vines with 100 primary shoot leaves and all the intact laterals. The partial defoliation

treatment (25L) consisted of vines with 25 primary shoot leaves, and the full

defoliation treatment (NL) consisted of vines without leaves. The leaves of treatment

25L were left adjacent to a bunch or on a node directly above or below a bunch. To

eliminate any new vegetative growth, any newly developing buds were removed from

the shoots of treatments 25L and NL daily, as soon as the growth was observed. Every

9 - 10 days after the start of the experiment, all the fruit from three vine replicates per

treatment were harvested and 50 berries per vine were selected and immediately frozen

in liquid N and stored at -80 ºC to later be used for primary metabolite analysis. Fruit

was harvested on 6 Jan 2016 (V+18), 15 Jan 2016 (V+27), 25 Jan 2016 (V+37) and 3

Feb 2016 (V+46). The periods between each of the harvest dates are referred to as

Interval 1, 2, 3 and 4, respectively. All leaves per vine were removed at the time of

fruit harvest, and the leaf area determined with a leaf area meter (LI-3100C, LI-COR

Biosciences Inc., Lincoln, Nebraska, USA).

2.2. Berry weight and composition

At each harvest, the total fresh fruit weight per vine was recorded, and a

subsample of 50 fresh berries per vine was collected, weighed, and the fresh weight

per berry determined. These berries were juiced by hand in a plastic bag and the juice

total soluble solid concentration (TSS) measured using a bench refractometer (PR-

101, Atago, Tokyo, Japan). The soluble solid content per berry (SSC) was

subsequently calculated based on the berry fresh weight and TSS concentration. The

remaining fruit from each vine was stored at -20 ºC until further processing as

described below. A subsample of 50 frozen berries per vine was ground to a fine

powder under liquid N (IKA A11, Selangor, Malaysia) and freeze-dried until constant

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weight (Gamma 1-16 LSC, Christ, Osterade am Harz, Germany) to determine the

weight loss percentage during the drying process. The dry weight per berry was

subsequently calculated. To determine the berry total N concentration, a 200 mg

freeze-dried sample was analyzed using the LECO method (Standard methods of

Rayment and Lyons, Soil chemical methods, Australasia, Dumas Combustion Method

6B2b), through a LECO CNS TruMAC analyzer (LECO corporation, St. Joseph, MI,

USA).

Another subsample of 50 frozen berries per vine was thawed in a shaking water-

bath at 30 ºC for 30 min, juiced and vortexed, again left at 30 ºC for 30 min and then

centrifuged for 5 min at 3000 × g. The juice titratable acidity (TA) and pH was

determined by sodium hydroxide (0.1 M) titration using an automatic titrator

(Metrohm Fully Automated 59 Place Titrando System, Metrohm AG, Herisau,

Switzerland) to an end point of pH 8.2. The ammonium and α-amino acid

concentrations of the juice were determined by using a commercially available

enzymatic assay kit, designed for an Arena discrete analyzer (Thermofisher, Scoresby,

Australia). The yeast assimilable N (YAN) concentration was subsequently calculated

from the ammonium and free amino N (FAN) (Iland et al., 2004). Fruit total

anthocyanin concentration was analyzed from a third 50 berry subsample per vine.

Whole berries were thawed at 4 °C and homogenized (Ultra-Turrax T25, IKA,

Selangor, Malaysia), and the total anthocyanin concentration determined as described

in Iland et al., (2004).

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2.3. Primary metabolite analysis

The subsample of 50 berries per vine, stored at -80 ºC, was ground to a fine

powder under liquid N and freeze-dried as described above. Sample extraction

and derivatization from a 100 mg freeze-fried sample was conducted according to

the method of Lisec et al. (2006) with some modifications. In summary, the berry

tissue of each vine was homogenized with 1.4 mL methanol and 30 µL of an internal

standard solution (1 g.L-1 of each, adonitol, L-hydroxyproline, and adipic acid, in

50% v/v methanol). The homogenate was shaken at 70 ºC for 10 min

(Thermomixer 5436, Eppendorf, North Ryde, NSW, Australia), and centrifuged at

11000 × g for 10 min. The supernatant was transferred to a glass vial, and mixed

with 0.75 mL chloroform and 1.4 mL ultrapure water. The mixture was

centrifuged at 2200 × g for 15 min, before the polar phase supernatant was

collected. In order to avoid saturation of samples with highly abundant metabolites,

the supernatant was diluted with 50% (v/v) methanol. To measure less abundant

metabolites, the dilution factor (DF) was 10, and to measure more abundant metabolites

(e.g., glucose, fructose and malic acid), the DF was 100. The diluted supernatant

(150 µL) was subsequently dried under a constant flow of pure N gas.

Methoxyamin hydrochloride (40 µL of 20 mg.mL-1) in pure pyridine, was added

to the dried extracts. The samples were then shaken at 37 ºC for 2 h, before being

centrifuged at 5000 × g for 2 min. N-Methyl-N-(trimethylsilyl) trifluoroacetamide

(70 µL) was added, and the samples were shaken at 37 ºC for 30 min, before being

centrifuged at 5000 × g for 2 min, after which the supernatant was collected. The

sample extraction, derivatization and measurement order was randomized.

Gas chromatography/mass spectrometry (GC/MS) analyses of the samples were

conducted by injecting 1 µL into the GC column (30 m × 0.25 mm, 0.25 µm HP-5MS,

Agilent, Santa Clara, CA, USA). The GC/MS system consisted of a

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7683B series autosampler, 7890A gas chromatograph, and 5975C mass spectrometer

with an electron impact ionization source and a quadrupole analyser (all from Agilent,

Santa Clara, CA, USA). The injection port was set at 250 ºC, the transfer line at 280

ºC, the ionization source at 230 ºC, and the quadrupole at 150 ºC. Helium gas, set at a

constant flow rate of 1.3 mL.min-1, was the carrier gas. The column temperature

program was set at 65 ºC for 2 min, followed by a 6 ºC.min-1 ramp to 300 ºC, where it

was held for 25 min. The ionization energy was set at 70 eV. Mass spectra were

recorded in full scan mode at 2.66 scans per second with a mass-to-charge ratio of 50

to 600 amu. Spectral deconvolution (signal-to-noise ratio threshold = 10; mass

absolute height ≥ 2000; compound absolute area ≥ 10000) allowed the identification

of co-eluting chromatographic peaks, and was conducted using MassHunter

Workstation software (Qualitative Analysis, version B.07.00, Agilent, Santa Clara,

CA, USA). Acquired MS spectra were searched for, and compounds identified using

the National Institute of Standards and Technology algorithm (NIST, Gaithersburg,

USA). The retention index for each compound in the analyzed samples was calculated

by using the retention times of a series of alkanes (C8-C28) in an injected retention

index solution (Fluka, Buchs, Switzerland).

Analytical grade standards, obtained from Sigma-Aldrich (Sigma, St. Louis, MO,

USA), were prepared in order to assist in the identification of these compounds. These

standards (10 mg.L-1 in 50% v/v methanol) consisted of various soluble sugars, i.e.,

sucrose, D(+)-glucose, D(-)-fructose, D(+)-mannose, L-rhamnose, D(-)-mannitol,

galactinol dehydrate, D(+)-raffinose, melibiose, D(+)-cellobiose, D(-)-ribose, D(-)-

arabinose, D(+)-trehalose, maltose monohydrate, D(+)-galactose, D(+)-xylose,

dulcitol (galactitol), L-fucose, and myo-inositol; amino acids, i.e., L-glutamic acid, L-

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arginine, L-proline, L-glutamine, γ-amino-n-butyric acid (GABA), L-threonine, L-

methionine, β-alanine, L-lysine, L-asparagine, L-aspartic acid, L-leucine, L-

isoleucine, L-valine, L-alanine, L-serine, glycine, L-tyrosine, L-phenylalanine, L-

tryptophan, L-histidine, L-cysteine and L-cysteine; and the organic acids, L-ascorbic

acid and protocathechuic acid.

2.4. Statistical analysis

The datasets in regard to the grapevine leaf area and basic berry composition

(Table 1), berry size and sugar, N and anthocyanin content (Fig. 1), and primary

metabolite abundance (Fig. 2, Supplementary Table S1) were analyzed using Statistica

13 (Dell Inc., Tulsa, OK, USA). For each variable, both treatment differences at a

single time and how a treatment changed over time were of interest. It was also

recognized that residual variance at each time may differ and that the interventionist

nature of the treatments may also lead to reduced residual variance for some

treatments. To facilitate these comparisons univariate analysis of variance (ANOVA)

at each date (all treatments) and for each treatment (all dates) were conducted. An

average Fisher’s least significant difference (LSD) test was used to identify significant

differences between means (P < 0.05). Significant differences in table and heat map

columns and rows are indicated by upper case letters (between treatments) and lower

case letters (between dates), respectively.

For each harvest date (V+18, V+27, V+37 and V+46) after the initial pre-

treatment harvest (V+9), a linear mixed model was fitted for every identified primary

metabolite (79 metabolites) using ASReml-R (Butler et al., 2007). Each model

included Treatment as a fixed effect and Replicate as a random effect. The significance

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of treatment effects was assessed by approximate F-tests, using the techniques of

Kenward and Roger (1997). For each vine organ, primary metabolites which had

significant treatment effects (P < 0.05) for any of V+18, V+27, V+37 and V+46 were

retained for a Principal Component Analysis (PCA). The PCA was only conducted as

an initial data analysis step to interpret the interactions between metabolites, treatment

and harvest dates. Predicted means from the ASreml-R analysis were used in a PCA

based on the correlation matrix, conducted in Genstat 17th Edition (VSN International

Ltd., Hemel Hempstead, Hertfordshire, UK). Biplots were drawn using R statistical

software plus the add in package ‘shape’ (developed by Karline Soetaert, Royal

Netherlands Institute of Sea Research Yerseke, The Netherlands). These biplots and

further PCA information (latent vector loadings and contributions of the measured

metabolites to principal components 1 and 2) are provided in Supplementary Fig. S1.

3. Results

3.1. Leaf area, petiole N and basic berry juice composition

The defoliation of 25L and NL caused FL vines to exhibit significantly larger leaf

area from V+18 (Table 1). While partial or full defoliation did not cause any

significant differences in the fruit fresh weight per vine, the FL fresh fruit weight

increased between V+9 and V+27, while that of 25L and NL increased during Interval

1 (Table 1). The petiole N concentration at the start of the experiment was 0.54% (±

0.02) (data not shown), and therefore, within a range indicating adequote N supply to

Shiraz vines by véraison (Holzapfel and Treeby, 2007).

Under full leaf area, an increase in berry juice TSS was noticed during Intervals

1 to 3 (Table 1). The reduced leaf area of 25L resulted in a slower TSS accumulation

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135

compared to FL, whereas a small increase in NL juice TSS was only noticed during

Interval 2. Therefore, the FL juice TSS was significantly higher than that of 25L and

NL at all sampling dates after V+9. In addition, 25L resulted in significantly higher

juice TSS than NL from V+37 onwards. Reduced leaf area had no significant effect

on the juice TA and pH (Table 1). The juice TA of all treatments decreased during

Intervals 1 to 3, while the juice pH increased during the same intervals, and that of FL

and NL further increased during Interval 4.

Defoliation resulted in significant alterations of the juice nitrogenous

composition. Under full leaf area, juice FAN concentrations were significantly lower

from V+18 compared to the defoliated treatments, while no significant differences in

juice FAN concentrations occurred between 25L and NL (Table 1). Generally, the

FAN concentration increased from the first to the last sampling date irrespective of

the treatment. The FL juice FAN concentration, however, decreased during Interval 1,

then increased during Interval 2, and was significantly higher at V+46 than at V+9

(Table 1). The 25L juice FAN increased during Intervals 2 and 3, while that of NL

increased during Interval 2 and between V+27 and V+46. The FL juice ammonium

concentration was significantly lower than that of 25L at V+18 and V+46. Similar to

the juice FAN, the YAN concentrations of FL were generally lower than those of 25L

and NL after V+9, with an exception at V+37 when there was no significant difference

amongst FL and NL (Table 1). Furthermore, no significant YAN differences occurred

between 25L and NL.

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3.2. Berry dry weight, SSC, N and anthocyanin content

The dry weight per FL berry was significantly larger than those of the defoliation

treatments from V+18 (Fig. 1A). Similarly, among the treatments, SSC per FL berry

was the highest from V+18, while the SSC per 25L berry was significantly higher than

that of NL from V+37 (Fig. 1B). The FL berry SSC increased significantly during

Intervals 1 to 3, while that of 25L increased more slowly, albeit significantly during

every interval. The SSC per NL berry increased significantly between V+9 and V+27,

and V+27 and V+46. Comparable to the SSC content per berry, the anthocyanin

content was generally superior in FL berries, where it was significantly the highest

from V+27 (Fig. 1C). The anthocyanin content per berry generally increased for all

treatments between V+9 and V+46, although at a much slower rate in the 25L and NL

berries.

Although no significant berry N content differences occurred at the final harvest

date, the N content per 25L berry was significantly superior to that of FL at V+37 (Fig.

1D). Furthermore, although the N content per berry increased between V+9 and V+46

irrespective of the leaf area, FL berry N content increased between V+18 and V+46,

while that of 25L increased during Interval 3. The N content per NL berry increased

between V+9 and V+27, and also between V+18 and V+37.

3.3. Metabolic adjustments

Inspection of the metabolite spectra, obtained from the GC/MS analyses, resulted

in the identification of 75 metabolites, categorized according to their chemical classes

(Table 2). In addition, three unidentified metabolites with sugar-like spectra were

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classified as unknown sugars 1, 2 and 3. Another metabolite with a spectrum similar

to that of raffinose was classified as unknown oligosaccharide 1.

Figure 2 illustrates the changes in the metabolite content per berry as induced by

partial or full defoliation. The contents of the metabolites that exhibited significant

treatment differences (listed in Supplementary Fig. S1), are indicated in the figure and

are briefly described below. In addition, metabolites that are directly involved in the

proposed metabolic pathways are also shown in the figure. Table 3 additionally lists a

simplified summary of the metabolites that accumulated and/or depleted during berry

maturation.

3.3.1. Accumulative metabolites

The majority of sugars accumulated in the berries between V+9 and V+46.

Glucose, fructose and sucrose accumulated irrespective of the treatments, and were

more abundant under full leaf area at V+37. A number of less abundant (minor) sugars

also accumulated as the berries matured, and mostly irrespective of the leaf area. These

include cellobiose, trehalose, tagatose, arabinofuranose, fucose, 3α-mannobiose and

unknown sugars 1, 2 and 3. These sugars were, however, generally more abundant

under full leaf area. In fact, at V+46 the FL fruit contained more cellobiose, trehalose,

arabinofuranose, 3α-mannobiose and unknown sugars 1, 2 and 3 than those of the

defoliated treatments (with the exception of unknown sugar 1, which did not

significantly differ between FL and 25L at V+46). The sugar alcohols myo-inositol

and dulcitol accumulated in FL berries, which had a superior myo-inositol content

compared to NL berries from V+27. Dulcitol also accumulated in the other treatments,

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138

however, FL berries contained the most dulcitol at V+46. Arabitol, on the other hand,

only accumulated after full defoliation.

A number of amino acids accumulated as the berries matured. Included among

these were glutamic acid, which accumulated irrespective of the leaf area, but was

more abundant in FL berries compared to those of NL from V+27. In addition, the 25L

berries contained more glutamic acid than those of NL at V+46. Similarly, berry

proline accumulated especially under full leaf area, where it was most abundant at

V+46. The 25L berry proline content was additionally superior to that of NL at V+46.

β-Alanine accumulated irrespective of the treatment, but was more abundant in FL

berries compared to those of NL from V+27. Arginine also accumulated irrespective

of the treatment, however, the NL berries contained more arginine than those of FL

from V+27. In addition, the 25L berries contained more arginine than those of FL from

V+37. Tyrosine accumulated in the berries of treatments FL between V+9 and V+46,

but the content did not differ among the treatments.

Unlike most other organic acids, pyruvic acid and lactic acid generally

accumulated in the berries. In fact, pyruvic acid was the most abundant in FL berries

at V+46, and additionally more abundant than in those of NL from V+18. Lactic acid

was more abundant in FL berries, compared to those of 25L at V+46. The phenolic

compounds, gallic acid and benzoic acid, also generally accumulated as the berries

matured. Gallic acid accumulated in FL and 25L berries, and was more abundant in

those of FL at V+37. Benzoic acid accumulated irrespective of leaf area, and was more

abundant in FL and 25L berries compared to those of NL from V+37 and at V+46,

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respectively. The other compounds that exhibited accumulating trends during berry

maturation were glycerol monostearate, 1-monopalmatin and hydroxylamine.

3.3.2. Metabolites exhibiting accumulative and depletive trends

Galactinol accumulated between V+9 and V+27, irrespective of the leaf area, and

then depleted between V+27 and V+46. However, 25L and NL berries contained more

galactinol than FL berries at V+27. Similarly, raffinose accumulated between V+9 and

V+27 before depleting. The FL berries contained more raffinose than those of 25L at

V+27 and more than those of NL at V+37. Unknown oligosaccharide 1 increased

between V+9 and V+27 in NL berries, and was significantly the most abundant in

these berries at V+37.

Aspartic acid accumulated until V+18 in NL berries and until V+27 in FL and

25L berries. The 25L berries contained the most aspartic acid from V+37.

Additionally, 25L and NL berries contained more aspartic acid at V+18. Asparagine

accumulated in NL berries during Interval 1, but depleted irrespective of the leaf area,

towards V+46. Asparagine was more abundant in the NL berries, than in the berries

of other treatments at V+37. Although the phenylalanine content never significantly

differed among the treatments, it accumulated in 25L berries during Interval 3, while

depleting during Interval 4. Likewise, the phenylalanine content in NL berries

increased during Interval 2.

3.3.3. Depletive metabolites

Rhamnose was the only sugar that depleted under full leaf area as the berries

matured. Among the amino acids, threonine depleted in the FL berries during Interval

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140

1, however, it accumulated in those of 25L during Interval 3. Tryptophan depleted in

the FL berries, and was lower than in 25L and NL at V+27, and lower than in 25L at

V+46.

Various organic acids depleted as the berries matured. Although malic acid

accumulated during Interval 1, the contents subsequently decreased in all treatments

towards V+46, and malic acid was less abundant in NL berries at V+37. Citric acid

also depleted over time, and at V+27, FL berries contained more citric acid than in

25L and NL, while NL berries contained less than those of the other treatments at

V+37. Fumaric acid depleted irrespective of treatments, and the FL berries contained

less fumaric acid than those of 25L and NL from, and at, V+37 respectively. Gluconic

acid only reduced upon partial and full defoliation, and the FL berries contained more

gluconic acid than those of 25L and NL from V+18 and V+27, respectively. Ascorbic

acid reduced in the berries, irrespective of leaf area, while tartaric acid only reduced

in 25L and NL berries, where it was significantly less abundant than in those of FL at

V+27. Threonic acid reduced irrespective of leaf area, and was more abundant in FL

berries from V+27. Glyceric acid reduced in the berries of all treatments, but was the

least abundant in those of FL from V+27. In terms of the phenolic acids, the berry

protocatechuic acid depleted irrespective of leaf area, but was less abundant in those

of FL at V+46, while caffeic acid only depleted upon partial or full defoliation. In

addition to the acids, (+)-catechin also reduced irrespective of leaf area, as the berries

matured.

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4. Discussion

Grapevines were partially (25L) or fully (NL) defoliated nine days after the start

of véraison, and the berry sugar, nitrogen (N) and anthocyanin accumulation, and juice

yeast assimilable N (YAN) concentration was compared to that of grapevines with full

leaf area (FL). The defoliation treatments were aimed to force a partial or full reliance

on carbohydrate and N reserves during restricted or no leaf availability towards canopy

photoassimilation. An untargeted investigation of the primary metabolite content in

the berries was conducted, aiming to emphasize the impact of C and N source

limitation on the different metabolites.

After treatment implementation, partially defoliated vines had a leaf-to-fresh fruit

ratio of around 0.2 m2.kg-1, while the ratio of the control treatment was 0.8 m2.kg-1.

According to published threshold levels (0.8 – 1.2 m2.kg-1), the leaf-to-fruit ratio of

the control vines should be sufficient to allow maximum accumulation of berry sugar

and color in a given climatic region (Kliewer and Dokoozlian, 2005). In contrast, the

partially and fully defoliated vines exhibited an insufficient leaf C source to support

its fruit sugar and color development. The 35 and 49% reduction in berry soluble solid

content (SSC) at the final harvest under partial and full defoliation, respectively, is

therefore not surprising. However, in the absence of leaf photosynthesis (full

defoliation, NL), there was still an 86% increase in SSC per berry at the last harvest.

The remobilization of carbohydrate reserves from the perennial structure (mostly the

roots) therefore contributed to the fruit sugar content after excessive defoliation

(Candolfi-Vasconcelos et al., 1994; Rossouw et al., 2017a). Furthermore, partial and

full defoliation caused a 71 and 79% reduction in berry anthocyanin content,

respectively by the final sampling date. Carbon limitation due to the defoliation

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treatments, may have caused a greater down regulation of berry anthocyanin

biosynthesis than sugar accumulation, however, an intensified sunlight exposure after

defoliation is likely to have inhibited anthocyanin metabolism (Haselgrove et al.,

2000). In their study, Bobeica et al. (2015) concluded that under C limitation, the

grapevine can manage the metabolic fate of C in such a way that sugar accumulation

is maintained at the expense of secondary metabolites. In fact, grapevine defoliation

may inhibit anthocyanin biosynthesis at protein (Wu et al., 2013) and transcription

(Pastore et al., 2013) levels.

While berry sugar and anthocyanin contents were highly affected by defoliation,

the total N content per berry was unaffected by the final harvest date. During berry

maturation, leaves provide a source of organic N towards fruit N accumulation

(Rossouw et al., 2017b), while soil N absorption could be restricted or absent.

However, other grapevine organs, such as roots and shoots, are also sources of N

during this period (Roubelakis-Angelakis and Kliewer, 1992), and soil N uptake by

the potted grapevines of the present study seems likely, even though N fertilization

was ceased a month prior the experiment. Redistribution of N from source organs other

than the leaves, and/or from soil N uptake, therefore, supported the accumulation of

berry N, irrespective of the leaf area. Nevertheless, the juice YAN concentration was

increased by partial and full defoliation, and was at a level (close to 130 mg.L-1)

thought to be just sufficient to allow the completion of must fermentation (Bell and

Henschke, 2005). Without must N supplementation, the lower juice YAN of the

control treatment could result in a sluggish fermentation, and inferior wine aroma

development (Bell and Henschke, 2005). The underlying aspects contributing to the

increased YAN after defoliation are discussed later, however, although defoliation had

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an insignificant influence on the total berry N content, the amino N content increased

after the defoliation treatments.

Most sugars, including many minor sugars, accumulated during berry maturation,

but the accumulation was reduced after partial and full defoliation. Accumulation of

the major berry sugars (glucose, fructose and sucrose) occurred faster under full leaf

area during the period of rapid berry SSC accumulation (until V+37), but as the berry

SSC accumulation slowed, accumulation of these sugars ceased. The resulting lack in

significant differences in the respective contents of berry glucose, fructose and sucrose

among the treatments at V+46, indicates the contribution of mobilized carbohydrate

reserves toward the fruit sugar content (Rossouw et al., 2017a), and in contrast to the

significantly higher content of most minor sugars (e.g. trehalose) under full leaf area

at V+46. Therefore, it seems that berry sugar metabolism, sourced from reserve starch

hydrolysis (Smith et al., 2005) rather than leaf photoassimilation, favored the major

sugars. In grapevines, like most other plants, sucrose is the major transport sugar

through the phloem (Ruan et al., 2010). Sucrose is translocated from the leaves or

reserve organs, such as the roots, towards the fruit where it is hydrolyzed into glucose

and fructose (Davies and Robinson, 1996). Carbon limitation, however, restricted the

accumulation of many minor sugars, although most still accumulated.

Galactinol, raffinose and unknown oligosaccharide 1 did not, like most other

sugars, accumulate progressively, but rather typically accumulated until midway

through the experiment, before depleting. Among their functions, the raffinose family

oligosaccharides (RFOs) can serve as transport compounds between plant sources and

sinks (Sengupta et al., 2015). Galactinol is derived from myo-inositol, and is a

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precursor for raffinose and its subsequent higher molecular weight RFOs (e.g.

stachyose and verbascose) (Sengupta et al., 2015). The accumulation pattern of

galactinol suggests that, especially after partial and full defoliation, it was transported

to the berries while the fruit was presumably a strong C sink. Although little is known

about their transport roles in grapevines, through studying Solanum tuberosum,

Hannah et al. (2006) found that both, galactinol and raffinose, are transported in the

phloem. It is therefore possible that either, galactinol and the RFOs were transported

to the berries, or that after translocation, galactinol was metabolized in the berries, and

the RFOs subsequently accumulated. The accumulation of galactinol, raffinose and

unknown oligosaccharide 1 after full defoliation, suggests that these compounds were

sourced from carbohydrate reserves during a period of strong berry C demand. On the

other hand, instead of accumulating during berry maturation like most other sugars,

rhamnose depleted under full leaf area. A similar observation was made by Cuadros-

Inostroza et al. (2016), who reported the post-véraison depletion of berry rhamnose.

Apart from being a cell wall pectic polysaccharide component, rhamnose is also

present in many secondary metabolites, such as anthocyanins and flavonoids (Watt et

al., 2004). As the only sugar that clearly depleted during berry maturation, further

work is needed to determine the functioning of rhamnose during berry maturation.

Unlike most sugars that progressively accumulated during berry maturation, the

different amino acids had diverse patterns of accumulation or depletion. Volenec et al.

(1996) described root and stem protein degradation to occur rapidly after forage plant

species, such as Medicago sativa, are defoliated, when vegetative regrowth occurs.

The degradation of storage proteins results in amino acid accumulation, which

becomes available to be translocated from the source to sink organs. As mentioned

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earlier, there was an increase in juice YAN after defoliation. Typically, arginine and

proline are the most abundant amino acids in mature grapevine berries (Bell and

Henschke, 2005). Proline, however, is not yeast assimilable, and arginine is therefore

a major contributor to juice free amino N (FAN). The increased accumulation of

arginine after partial and full defoliation explains the increased FAN and subsequent

YAN, in the berry juice of those treatments. Apart from being the major storage amino

acid in grapevine roots as a free amino acid or protein component (Xia and Cheng,

2004), arginine also plays an important role as a N transport compound (Lea et al.,

2007). The elimination or restriction of the leaf N source, therefore, presumably

induced N translocation as arginine from the storage tissues towards the berries,

contributing to berry N accumulation, and effectively raising juice YAN. Asparagine

only accumulated after a complete defoliation and has, like arginine, a high N:C ratio

making it an effective N storage compound, while also a major transport amino acid

in plants (Lea et al., 2007). Like arginine, the accumulation of berry asparagine in the

absence of leaves, therefore, implies its mobilization from N storage tissues (e.g. the

roots), subsequently also contributing to the increased YAN (Bell and Henschke,

2005). Furthermore, the defoliation treatments of the present study, especially full

defoliation, would force a greater reliance on phloem amino acid translocation, as the

xylem flow would likely be interrupted. Tromp and Ovaa (1971) described arginine

and asparagine as being major phloem translocated amino acids in Malus domestica,

and likely similarly so in the grapevines of the present study. The bulk phloem flow

solution, moving from source to sink organs contains sugars and amino N, and is

propelled by osmotically generated hydrostatic pressure differences (Lalonde et al.,

2003). In the present study, the elimination of the leaf C source enforced root

carbohydrate reserves to be become a sugar source towards the maturing berries, a

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strong C sink (Candolfi-Vasconcelos et al., 1994). If root N repartitioning and

subsequently, amino acid accumulation, occurred after removal of the leaf N source,

these amino acids could follow the sugar regulated osmotic phloem flow towards the

berries. The results of the present study, therefore, suggest amino N translocation from

reserve tissues to the berries.

Although the berry phenylalanine content never significantly differed among

treatments, it only accumulated after partial and full defoliation. The transport

mechanisms of phenylalanine in grapevines has not yet been studied, however,

phenylalanine accumulated in the xylem of Trifolium repens and the phloem of

Lupinus albus after defoliation (Hartwig and Trommler, 2001). Such accumulation

likely implies the possibility of phenylalanine transport between source and sink

organs in plants. Phenylalanine is an important precursor for anthocyanin biosynthesis

in grapevine berries (Boss et al., 1996), and the results of the present study suggest

that the grapevine regulates its berry phenylalanine accumulation under source (leaf)

limitation. Roots and mature leaves are the major sources of amino acids in higher

plants, from where they are translocated to sink organs (Rentsch et al., 2007). In the

present study, the accumulation of amino acids like arginine, asparagine and

phenylalanine after partial and full defoliation, suggests that they were sourced from

N reserve tissues (likely the roots) when the leaf source was limited or unavailable.

On the other hand, many amino acids, e.g., glutamic acid, proline and β-alanine

accumulated more in the berries under full leaf area, implying the leaves to be an

important source of these amino acids. More work is, however, needed to determine

the extent of amino acid biosynthesis within the grapevine berries. In Solanum

lycopersicum fruit, peptidases are very active during ripening, and they are able to

146

Chapter 6: Paper 4

release free amino acids from endogenous proteins (Sorrequieta et al., 2010).

Therefore, although the results of the present study suggest berry amino acids being

sourced from the leaves or alternatively, reserve tissues (e.g. the roots), the occurance

of amino acid biosynthesis within the berries needs further exploration.

Defoliation did not affect the juice titratable acidity (TA) and pH, implying that

organic acids are less responsive to C limitation, than sugars (Bobeica et al., 2015).

This lack of TA differences was thus expected to yield minimal differences in berry

malic acid, tartaric acid and citric acid content among the treatments. In fact, at the

final harvest, there were no significant differences in the contents of any of these major

berry acids. The intermediates of the tricarboxylic acid (TCA) cycle usually decline in

the berries after véraison, which likely matches the increased demand of precursors in

the synthesis of carbohydrates, amino acids and subsequent flavonoids (Degu et al.,

2014). The initial accumulation of malic acid, before its decline was thus surprising,

however, the other TCA cycle intermediates generally depleted with berry maturation.

These organic acids were therefore likely catabolized, and were useful precursors for

energy production and further C metabolism (López-Bucio et al., 2000).

The abundance of many berry metabolites derived from the shikimate pathway

were affected by defoliation. The implication of the defoliation treatments on the

aromatic amino acids (e.g. phenylalanine) has already been described above, however

arbutin, (+)-catechin and various phenolic acids (gallic acid, benzoic acid, caffeic acid

and protocatechuic acid) were also affected in different ways. Furthermore,

anthocyanins are also secondary products of the shikimate pathway, and the berry

anthocyanin content was, as mentioned, greatly decreased by defoliation. The flux

147

Chapter 6: Paper 4

through the shikimate pathway can represent up to 20% of the total C in plants

(Haslam, 1993), and the diverse reactions of products of this pathway illustrate how C

limitation greatly impacts this pathway in grapevine berries. In addition, the

intensified berry sunlight exposure as associated with the defoliation treatments, may

have affected the expression of genes involved in encoding enzymes that play key

roles in the shikimate pathway of grapevine berries (Zhang et al., 2012). Therefore,

further investigation is needed to separate the implications of C source availability and

sunlight exposure during the post-véraison period, on berry products of the shikimate

pathway.

In summary, the objective of this study was to illustrate the impact of an imposed

reliance on stored carbohydrate and N reserves during berry maturation on the

accumulation of berry soluble solids, N and anthocyanins in conjunction with the

content of primary berry metabolites. The study was additionally aimed to better

understand the role of the leaves towards berry maturation and composition. Carbon

source limitation, induced by defoliation from near the start of berry maturation,

limited fruit sugar accumulation and anthocyanin biosynthesis. The major berry

sugars, and most minor sugars and sugar alcohols, accumulated during maturation and

the accumulation was stimulated under full leaf area. Glucose, fructose and sucrose

accumulation was maintained under C source limitation at the expense of anthocyanin

biosynthesis. Defoliation did not influence the N content of mature berries, but

increased the juice YAN. Arginine, asparagine and phenylalanine accumulated after

defoliation, while various other amino acids accumulated under full leaf area. Arginine

accumulation contributed to the increased juice YAN after defoliation, and was likely

148

Chapter 6: Paper 4

149

sourced from reserve tissues. Anthocyanins and various other shikimate pathway

products, such as phenolic acids, were largely affected by defoliation.

Therefore, excessive limitation of canopy leaf area had severe implications on

sugar and anthocyanin accumulation during berry maturation, and also altered the

berry sugar profile. Defoliation, may however, increase juice YAN through enhanced

allocation of certain amino acids to the berries, supposedly originating from N

repartitioning in alternative N source organs. This study provides a novel indication

of the effects of leaf source limitation on development of key berry quality

characteristics in conjunction with underlying metabolic alterations.

Acknowledgements

This work was supported by the National Wine and Grape Industry Centre, and

the Australian grapegrowers and winemakers through their investment body, Wine

Australia, with matching funds from the Australian Government. The authors thank

Robert Lamont, Peter Carey and Viera Mendoza Huallanca for technical assistance.

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Chapter 6: Paper 4

153

Tabl

e 1 L

eaf a

rea

and

fresh

frui

t wei

ght p

er v

ine,

and

juic

e to

tal s

olub

le so

lid c

once

ntra

tion,

titra

tabl

e ac

idity

(TA

), pH

, fre

e am

ino

nitro

gen

(FA

N)

conc

entra

tion,

am

mon

ium

con

cent

ratio

n an

d ye

ast a

ssim

ilabl

e ni

troge

n (Y

AN

) co

ncen

tratio

n, a

t the

diff

eren

t har

vest

dat

es (V

+9, V

+18,

V+2

7,

V+3

7 an

d V

+46)

as a

ffect

ed b

y th

e de

folia

tion

treat

men

ts (f

ull l

eaf:

FL; 2

5% le

aves

: 25L

; no

leaf

: NL;

mea

n ±

SE, n

=3).

Inte

rval

1

Inte

rval

2

Inte

rval

3

Inte

rval

4

Trea

tmen

t V

+9

V+1

8 V

+27

V+3

7 V

+46

Leaf

are

a pe

r vi

ne (m

2 ) FL

1.

46 ±

0.0

6 A

1 1.

43 ±

0.0

6 A

1

.44

± 0.

04

A

1.47

± 0

.05

A

1.56

± 0

.03

25L

1.46

± 0

.06

a2 B

0.

35 ±

0.0

2 b

B

0.3

4 ±

0.04

b

B

0.36

± 0

.01

b B

0.

34 ±

0.0

3 b

NL

1.46

± 0

.06

- -

- -

Fres

h fr

uit

wei

ght p

er

vine

(kg)

FL

1.34

± 0

.1

b 1.

85 ±

0.3

ab

2.1

2 ±

0.1

a 2.

16 ±

0.2

a

2.08

± 0

.2

a 25

L 1.

34 ±

0.1

b

1.93

± 0

.2

a 2

.21

± 0.

1 a

2.10

± 0

.1

a 2.

17 ±

0.1

a

NL

1.34

± 0

.1

b 1.

90 ±

0.3

a

1.8

4 ±

0.1

a 2.

00 ±

0.1

a

1.92

± 0

.1

a

Juic

e TS

S (º

Bri

x)

FL

10.0

± 0

.4

d A

13

.5 ±

0.8

c

A

17.

5 ±

0.8

b A

20

.6 ±

0.8

a

A

22.4

± 0

.3

a 25

L 10

.0 ±

0.4

c

B

10.4

± 0

.3

c B

1

2.3

± 0.

2 b

B

13.7

± 0

.2

a B

14

.5 ±

0.5

a

NL

10.0

± 0

.4

bc

B

9.0

± 0

.3

c B

1

1.0

± 0.

3 ab

C

11

.3 ±

0.1

ab

C

12

.1 ±

0.7

a

Juic

e TA

(g

.L-1

) FL

24

.5 ±

0.9

a

11.9

± 0

.6

b 6

.7 ±

0.3

c

4.5

± 0

.1

d 4.

0 ±

0.1

d 25

L 24

.5 ±

0.9

a

11.6

± 0

.4

b 7

.0 ±

0.3

c

5.3

± 0

.1

d 4.

6 ±

0.1

d N

L 24

.5 ±

0.9

a

12.0

± 0

.2

b 6

.8 ±

0.2

c

5.1

± 0

.1

d 4.

3 ±

0.1

d

Juic

e pH

FL

2.

80 ±

0.0

2 e

3.09

± 0

.06

d

3.4

8 ±

0.03

c

3.8

4 ±

0.06

b

3.97

± 0

.03

a

25L

2.80

± 0

.02

d 3.

20 ±

0.0

3

c 3

.55

± 0.

02

b 3

.90

± 0.

02

a 3.

94 ±

0.0

1

a N

L 2.

80 ±

0.0

2 e

3.13

± 0

.02

d

3.5

9 ±

0.02

c

3.8

7 ±

0.02

b

3.98

± 0

.04

a

Juic

e FA

N

(mg.

L-1)

FL

36.3

± 2

.6

b B

22

.7 ±

3.8

c

B

45.

0 ±

1.7

ab

B

46.

3 ±

5.8

ab

B

48.

3 ±

1.3

a 25

L 36

.3 ±

2.6

c

A

47.7

± 5

.5

c A

7

5.7

± 3.

8 b

A

110.

7 ±1

1.9

a A

10

4.0

± 8.

2 a

NL

36.3

± 2

.6

c A

43

.3 ±

5.2

c

A

80.

3 ±

10.2

b

A

85.

3 ±

8.7

ab

A

103.

3 ±

6.8

a

Juic

e A

mm

oniu

m

(mg.

L-1)

FL

28.0

± 3

.6

B

8.7

± 2

.8

17.

7 ±

9.7

21.

3 ±

12.5

B

16

.7 ±

5.7

25

L 28

.0 ±

3.6

A

39

.7 ±

11.

5 3

1.0

± 0.

6 3

8.7

± 5.

2 A

39

.3 ±

7.8

N

L 28

.0 ±

3.6

A

B

25.7

± 3

.8

36.

0 ±

3.8

35.

7 ±

3.7

AB

36

.7 ±

4.5

Juic

e Y

AN

(m

g.L-1

) FL

57

.9 ±

4.6

a

B

27.3

± 5

.8

b B

5

4.7

± 11

.0

ab

B

59.

5 ±

17.1

a

B

56.

5 ±

5.1

ab

25L

57.9

± 4

.6

c A

78

.9 ±

8.6

bc

A

9

3.3

± 2.

8 b

A

129.

9 ±

13.7

a

A

125.

0 ±

2.1

a N

L 57

.9 ±

4.6

b

A

61.4

± 7

.6

b A

10

2.2

± 12

.1

a A

B

106.

0 ±

10.8

a

A

121.

8 ±

9.6

a

1 Mea

ns s

epar

ated

with

in c

olum

ns u

sing

Fis

her’

s LSD

test,

sign

ifica

nt d

iffer

ence

s are

indi

cate

d at

P <

0.0

5. W

here

diff

eren

t upp

er c

ase

lette

rs a

ppea

r in

a co

lum

n, v

alue

s diff

er si

gnifi

cant

ly b

etw

een

treat

men

ts.

2 Mea

ns s

epar

ated

with

in ro

ws u

sing

Fis

her’

s LSD

test,

sign

ifica

nt d

iffer

ence

s are

indi

cate

d at

P <

0.0

5. W

here

diff

eren

t low

er c

ase

lette

rs a

ppea

r in

a ro

w, v

alue

s diff

er si

gnifi

cant

ly b

etw

een

harv

est d

ates

.

Chapter 6: Paper 4

154

Fig. 1. Impact of leaf area (full leaf: FL; 25% leaves: 25L; no leaf: NL) during berry maturation on the development of the dry weight (A), soluble solid content (SSC) (B), anthocyanin content (C) and nitrogen (N) content per berry (D) (mean ± SE; n=3). Significant differences (P < 0.05) between harvest dates for each treatment are indicated by different lower case letters. Significant differences (P < 0.05) between treatments at each harvest date are indicated by different numerals [1: FL > (25L and NL), 2: FL > 25L > NL and 3: FL < 25L].

Chapter 6: Paper 4

155

Table 2 Classification of the different metabolites in the berries by GC/MS analysis.

Compound class Number of metabolites

20

9

20

4

4

10

Sugars1

Sugar alcohols (polyols)

Amino acids

Phenolic acids

TCA intermediates (acids)

Other acids2

Fatty acids 6

2

2

Flavonoids

Glycerides

Glycosides 1

1 Unclassified

Total 79

3 Includes four unidentified compounds with sugar-like spectra. 4 Includes sugar acids, dicarboxylic acids, keto acids and α-hydroxy acids.

Chapter 6: Paper 4

156Fig.

2. A

sim

plifi

ed p

athw

ay r

espo

nse

of th

e be

rry

prim

ary

met

abol

ites

signi

fican

tly a

ffect

ed b

y tre

atm

ents

(fu

ll le

af: F

L; 2

5% le

aves

: 25L

; no

leaf

: NL)

and

oth

er m

etab

olite

s dire

ctly

invo

lved

in th

e pa

thw

ays (

3). T

he si

mpl

ified

pat

hway

s inc

lude

suga

r and

suga

r alc

ohol

met

abol

ism

, am

ino

acid

met

abol

ism

, fat

ty a

cid

met

abol

ism

, phe

nolic

aci

d m

etab

olis

m, t

he tr

icar

boxy

lic a

cid

(TC

A)

cycl

e an

d th

e sh

ikim

ate

path

way

. Sig

nific

ant

diffe

renc

es a

re in

dica

ted

at P

< 0

.05,

hea

tmap

col

umns

indi

cate

the

thre

e tre

atm

ents

, whi

le h

eatm

ap ro

ws

indi

cate

the

harv

est d

ates

(V+9

, V+1

8,

V+2

7, V

+37

and

V+4

6). W

here

diff

eren

t upp

er c

ase

lette

rs a

ppea

r in

heat

map

col

umns

(1),

valu

es d

iffer

sig

nific

antly

bet

wee

n tre

atm

ents

. Whe

re

diffe

rent

low

er c

ase

lette

rs a

ppea

r in

a r

ow, v

alue

s di

ffer

sig

nific

antly

bet

wee

n ha

rves

t da

tes.

Ave

rage

met

abol

ite a

bund

ance

is c

olou

r co

ded

acco

rdin

g to

the

scal

e on

the

right

(2).

TCA

: tric

arbo

xylic

aci

d; G

-6-P

: glu

cose

-6-p

hosp

hate

; PEP

: pho

spho

enol

pyru

vic

acid

.

Chapter 6: Paper 4

157

Table 3 Summary of accumulation patterns of the different berry metabolites during the experiment.

Late accumulative (until V+37/V+46)

More abundant under FL

Sugars: Glucose, fructose, sucrose, cellobiose, trehalose, tagatose, arabinofuranose, fucose, 3α-mannobiose, unknown sugars 1, 2 and 3, ribose, arabinose. Sugar alcohols: Dulcitol, myo-inositol Amino acids: Glutamic acid, proline, pyroglutamic acid, β-alanine, tyrosine1 Organic acids: Pyruvic acid, lactic acid Phenolic acids: Gallic acid, benzoic acid Other: Glycerol monostearate, 1-monopalmitin, hydroxylamine

More abundant under 25L/NL Sugar alcohols: Arabitol Amino acids: Arginine

Early accumulative (until V+27), late depletive (after V+27)


More abundant under 25/NL

Sugars: Raffinose

Sugars: Unknown oligosaccharide 1 Sugar alcohols: Galactinol Amino acids: Aspartic acid, asparagine, phenylalanine1

Late depletive (until V+37/V+46)


More abundant under 25L/NL

Organic acids: Malic acid, citric acid, threonic acid, gluconic acid Phenolic acids: caffeic acid Sugars: Rhamnose Amino acids: Tryptophan Organic acids: Fumaric acid, glyceric acid Phenolic acids: Protocatechuic acid

Early depletive (until V+27)

More abundant under FL Organic acids: Tartaric acid Other: Catechin

More abundant under 25L/NL Sugar alcohols: cis-inosi tol

Amino acids: ThreonineOrganic acids: Ascorbic acid

5 No significant differences occurred between the treatments, although there were some changes over time.

Chapter 7: General conclusions and future work

158


Rapid berry sugar accumulation distinctly occurs during the post-véraison period, a

substantial sink demand for photoassimilates, therefore, exists during berry maturation.

In comparison, the extent of fruit nitrogen (N) incorporation often varies at distinct

stages of the growing season, the berries are nevertheless expected to be notable N sinks

after véraison. Grapevine water supply, and the relationship between the vegetative and

reproductive organ sizes, are likely major determinants of carbon (C) and N allocation

between the perennial and reproductive structures during the post-véraison period. Four

key research objectives were evaluated in order to better understand grapevine total

non-structural carbohydrate (TNC; starch and soluble sugars) and N (total N and amino

acids) partitioning and distribution during berry maturation. The effect of vine water

supply and/or the relationship between the leaf area and crop load on TNC and N was

also investigated.

Objective 1:

To investigate the interactive effects of the leaf-to-fruit ratio and grapevine water status

during two phases of berry sugar accumulation (rapid and slow) on the carbohydrate

distribution between the different grapevine organs (chapter 3).

The extent of TNC reserve contribution towards fruit sugar accumulation, when leaf

photoassimilation is restricted during the post-véraison period, was investigated in

chapter 3. Previous studies largely focussed on TNC concentrations in roots and trunks

at distinct stages of the season (predominantly budburst), on the other hand, the results


159

of this chapter are novel in demonstrating the changes of TNC content in whole

grapevine organs during berry ripening.

Sustained water constraints during the rapid berry sugar accumulation phase enforced a

reliance on root TNC reserves to support the berry sugar content. In fact, root starch

reserves accounted for 89% of the total perennial and vegetative organ TNC content

loss during rapid berry sugar accumulation. On the other hand, the perennial and

vegetative TNC content at véraison contributed to up to 18% of the berry dry matter

accumulation. The contribution from root starch reserves, as induced by water

constraints, may maintain the rate of berry sugar accumulation relative to that of well-

water vines. In addition, a reduced leaf-to-fruit ratio intensified the reliance of fruit

sugar accumulation on stored TNC. Besides the well documented replenishment of root

TNC reserves during the post-harvest period, the reserves were also stored before

harvest during the phase of slower berry sugar accumulation.

Root TNC reserve storage can significantly start occurring a few weeks prior to berry

maturity. In a practical sense, where grapegrowing regions only experience a short or no

substantial post-harvest period, the maintenance of a functional canopy leaf area during

the final few weeks before harvest could be essential in order to ensure sufficient

reserve TNC replenishment. Late season irrigation management could especially be

beneficial to allow reserve storage when post-véraison water constraints prevailed.


160

Objective 2:

To determine how the presence or absence of fruit during sustained post-véraison water

constraints influences the allocation of carbohydrates and N between the different

grapevine organs (chapter 4).

Chapter 4 evaluated the content development of both TNC and N in the different

grapevine organs during berry maturation. This study is novel in terms of demonstrating

the partitioning of both TNC and N within the different organs, thereby enhancing the

understanding of TNC and N reserve utilisation during post-véraison water constraints.

The study additionally focussed on root reserve utilisation and replenishment, and

investigatedthe implications of post-véraison water constraints on TNC and N storage

by berry maturity.

Root TNC reserves were remobilised throughout the phase of rapid berry sugar

accumulation during sustained post-véraison water constraints. However, root N

reserves, in terms of total content, were less affected during the corresponding fruit N

accumulation. Root starch hydrolysis, and subsequently root sucrose accumulation,

occurred when rapid fruit sugar accumulation coincided with sustained water

constraints. The accumulated root sucrose presumably became available for

translocation towards the fruit, as leaf photoassimilation was restricted. The

accumulation of root hexoses after defruiting may have played a role in regulating root

osmotic potential during structural root development, which also occurred after

defruiting. During sustained post-véraison water constraints, leaf N was likely exported

to the fruit, contributing to the N content. However, subsequent to defruiting, the roots

were an alternative N sink, prompting the storage of root N reserves.


When the post-véraison canopy photoassimilation is limited enough due to water

constraints, the fruit C requirement are sourced from the roots. On the other hand, leaf N

could support the post-véraison fruit N requirements. Root starch depletion during berry

sugar accumulation is subsequently detrimental towards reserve carbohydrate storage.

Furthermore, the presence of post-véraison fruit during sustained water constraints is

also detrimental towards root N storage by fruit maturity. Therefore, when water

constraints are sustained between véraison and fruit maturity, the post-harvest

replenishment of TNC and N reserves are especially crucial in order to ensure sufficient

reserve availability by budburst the next season.

Objective 3:

To assess the implications of defoliation on post-véraison fruit sugar and N

accumulation in conjunction with the carbohydrate, N and primary metabolite

composition of the major grapevine source organs (roots and leaves) (chapter 5).

In chapter 5, an untargeted approach was undertaken to determine the profiles of root

and leaf primary metabolites during berry maturation. These analyses were not intended

to describe full metabolic pathways, but were rather conducted to enable a description

of the compounds (sugars, sugar alcohols, amino acids, organic acids, etc.) involved in

source organ C and N metabolism. This study is thereby original in terms of

demonstrating the implications of C and N source limitations during berry maturation,

on subsequent C and N metabolism in the remaining source organs.

A complete defoliation shortly after the start of véraison enabled confirmation of the

post-véraison contribution of remobilised root TNC reserves towards fruit sugar

161


162

content. In fact, after the leaves, a C assimilation source, were completely removed,

some continuation of fruit sugar accumulation occurred, in conjunction with an intense

root starch depletion. The absence or limitation of the leaf source was detrimental

towards the rate of fruit sugar accumulation, but did not affect the total N content of the

mature fruit. Sugars and organic acids generally depleted in the roots and remaining

leaves after defoliation, while root and leaf amino acids accumulated.

Two metabolic pathways were identified as being largely affected in the roots by leaf

source limitation during berry maturation. Firstly, defoliation induced a rapid decrease

in root myo-inositol concentration, and likewise, the depletion of known myo-inositol

derivatives, including galactinol, raffinose and ascorbic acid. Changes in root starch and

myo-inositol concentrations were also closely related, and the results suggest myo-

inositol to play an underlying role during root starch remobilisation. Secondly,

defoliation induced the accumulation of shikimate pathway-derived aromatic amino

acids, i.e., phenylalanine, tyrosine and tryptophan, in the roots. The biosynthesis of

shikimate pathway products in the roots is, therefore, likely enhanced during a C source

limitation during berry ripening. Furthermore, as the only amino acid that clearly

depleted in the roots after defoliation and a known amino-group donor, glutamic acid is

likely involved in modulating the root amino acids composition when the leaf N source

is restricted or absent. The accumulated amino acids in the roots after defoliation, which

in addition to the aromatic amino acids also included arginine, may play a role in root to

fruit amino N translocation. As observed in the roots, amino acids accumulated in the

remaining leaves after partial defoliation, suggesting leaf protein degradation, and a

likely intensified amino N export from the remaining leaves to the fruit sink.


163

Myo-inositol metabolism and shikimate pathway derivatives are largely affected in

major TNC and N source organs (roots and leaves) during berry maturation, when a leaf

area restriction prevails. A qualitative untargeted evaluation of the primary root and leaf

metabolic composition was conducted during this study, and the results contribute to the

understanding of source organ TNC and N reserve utilisation during berry ripening.

Objective 4:

To study the implications of defoliation on the post-véraison metabolic composition of

grapevine berries (chapter 6).

Restricting the leaf area during fruit maturation limits C and likely N supply towards the

berries. Chapter 6 illustrates the implications of limiting canopy leaf area on berry

compositional parameters (soluble solids, total N, yeast assimilable N and

anthocyanins), as underlined by the contents in primary berry metabolites. The study

enhances the understanding of the contribution of primary berry metabolites towards

overall berry composition during fruit ripening.

Apart from restricting berry sugar accumulation, post-véraison leaf source limitation

strongly suppressed the berry anthocyanin accumulation rate. The inhibition of berry

anthocyanin accumulation was likely related to a C source limitation and/or an

intensified bunch exposure subsequent to defoliation. Although the total N content per

berry remained largely unaffected by the vine leaf area, the yeast assimilable N (YAN)

content increased when grapevines were partially or fully defoliated. Defoliation,

therefore, did not affect the total berry N content, but did influence N composition in the

berries through a preference towards certain amino acids.


164

Berry arginine accumulation largely explained the increased YAN after defoliation, and

was likely sourced from the roots. Glucose, fructose and sucrose content in mature

berries were less affected by post-véraison leaf source limitation, however, the

accumulation of many minor sugars and sugar alcohols, such as trehalose and myo-

inositol, were inhibited by defoliation. Galactinol and its sugar derivatives (e.g.

raffinose) accumulated early on during berry maturation, before subsequently depleting,

suggesting important transport roles for these compounds during a period of intense

fruit sugar demand, especially after defoliation. The C flux through the shikimate

pathway, as implied by the impacts on its products in the berries (e.g. anthocyanins and

phenolic acids), was largely affected by leaf source restriction during fruit maturation.

Excessive limitation of canopy leaf area had severe implications on sugar and

anthocyanin accumulation during berry maturation, and additionally altered the berry

sugar composition. Defoliation may, however, increase the juice YAN through a

subsequent increased amino acid allocation to the berries.

Synopsis of the general conclusions and future work:

This research provides an original perspective on the post-véraison utilisation of TNC

reserves towards the fruit when canopy photoassimilation is restricted by water

constraints and/or an insufficient leaf area. In comparison to TNC, the effects of vine

water supply or vegetative and reproductive organ size relations on the post-véraison

total N distribution between the different grapevine organs is less obvious. The

difference is likely attributable to the post-véraison period being the predominant stage

of fruit C demand, while fruit N incorporation may be more variable over time.

Nevertheless, the study still contributes to the understanding of post-véraison N


165

partitioning in the different grapevine organs, as demonstrated by the N compositional

changes occuring in source and sink organs during berry N accumulation.

In order to quantify TNC reserve recovery during the post-harvest period, future studies

involving post-véraison water constraints and/or restricted leaf area could include the

determination of root TNC reserve content at budburst the following season. Thereby,

the implications of restricted vine photoassimilation during berry ripening on reserve

TNC availability at the start of the next season could be revealed to a greater extent.

Furthermore, as root respiration is an additional carbohydrate expense, future studies

could include the quantification of post-véraison root respiration rate to determine its

contribution towards root TNC depletion. Carbon isotopic labelling could also be

considered in future studies in order to accurately trace TNC translocation between the

roots and fruit during the post-véraison period. Likewise, N isotopic labelling could be

utilised in order to better illustrate N distribution between the different grapevine organs

during berry ripening.

In terms of metabolic studies, future work could include the targeted monitoring of

certain metabolites in source organs and fruit during berry ripening. In fact, a particular

focus on myo-inositol and its derivatives, and compounds yielded from the shikimate

pathway, would enhance the understanding of the roles these metabolites play in

grapevine source organs during berry ripening. The relationship between myo-inositol

metabolism and root starch remobilisation especially requires further investigation.

Appendix

166

Appendix

Appendix A: Supporting information (Chapter 5 - Paper 3)

Tables S1 and S2; Figures S1, S2 and S3.

Appendix B: Supplementary material (Chapter 6 - Paper 4)

Supplementary table S1 and figure S1.

Appendix A – Table S1

167 Days

aft

er v

erai

son

Root

met

abol

ite a

bund

ance

Trea

tmen

tva

lue

SDEV

valu

eSD

EVva

lue

SDEV

valu

eSD

EVva

lue

SDEV

RT (m

in)

m/z

Kegg

IDst

anda

rd id

entif

icat

ion

NIS

T lib

rary

com

poun

dRe

tent

ion

inde

x

FL0.

009

0.01

90.

060

0.00

60.

064

0.05

60.

055

0.02

30.

032

0.05

625

L0.

009

0.01

90.

376

0.57

90.

110

0.03

80.

165

0.01

40.

196

0.02

8N

L0.

009

0.01

90.

000

0.00

00.

084

0.08

80.

378

0.10

50.

225

0.03

9FL

0.06

10.

041

0.07

30.

024

0.06

10.

021

0.06

50.

020

0.07

70.

067

25L

0.06

10.

041

0.14

90.

028

0.10

50.

023

0.09

40.

085

0.09

50.

026

NL

0.06

10.

041

0.10

20.

013

0.14

30.

060

0.06

00.

050

0.10

80.

011

FL0.

377

0.05

80.

465

0.03

00.

624

0.26

50.

580

0.26

90.

506

0.08

825

L0.

377

0.05

80.

469

0.16

40.

511

0.14

71.

045

0.29

30.

794

0.18

5N

L0.

377

0.05

80.

537

0.10

10.

399

0.21

61.

097

0.25

90.

703

0.21

1FL

31.7

196.

219

41.6

9915

.954

27.3

671.

237

47.0

206.

867

68.4

4526

.144

25L

31.7

196.

219

33.7

1012

.955

42.7

528.

251

34.5

617.

973

45.6

215.

719

NL

31.7

196.

219

25.2

6512

.532

41.4

6721

.694

41.5

0517

.838

44.8

3721

.004

FL1.

645

0.73

10.

972

0.40

60.

757

0.77

31.

043

0.60

70.

555

0.07

525

L1.

645

0.73

10.

273

0.17

50.

004

0.00

60.

160

0.15

60.

225

0.28

7N

L1.

645

0.73

10.

336

0.33

90.

076

0.13

20.

171

0.18

00.

027

0.02

6FL

0.67

60.

130

1.04

90.

739

0.73

00.

187

1.30

60.

164

1.10

10.

397

25L

0.67

60.

130

1.12

40.

862

0.74

10.

088

1.09

60.

100

1.36

60.

264

NL

0.67

60.

130

0.64

20.

246

0.51

00.

141

1.29

80.

315

1.29

10.

200

FL21

.246

6.18

831

.543

16.8

0918

.364

1.16

835

.554

8.42

037

.702

13.5

6325

L21

.246

6.18

826

.564

12.3

9131

.565

6.63

626

.294

3.56

533

.711

2.49

6N

L21

.246

6.18

820

.883

9.93

626

.837

10.7

3435

.589

7.10

140

.119

6.67

3FL

0.70

80.

552

0.46

40.

121

0.30

50.

255

0.26

70.

154

2.38

52.

675

25L

0.70

80.

552

1.65

90.

597

0.47

50.

199

0.24

90.

431

0.65

60.

967

NL

0.70

80.

552

0.82

90.

646

1.72

42.

700

0.20

20.

349

0.12

00.

141

FL8.

287

1.26

56.

322

0.82

34.

346

1.79

84.

652

0.68

26.

725

1.36

325

L8.

287

1.26

54.

198

1.05

42.

124

0.77

21.

511

0.30

02.

132

0.38

5N

L8.

287

1.26

54.

205

0.56

22.

381

0.75

61.

709

0.24

71.

860

0.14

9FL

0.96

90.

347

0.86

50.

075

0.99

80.

269

1.23

10.

168

1.22

90.

430

25L

0.96

90.

347

0.90

10.

161

0.94

50.

094

1.26

60.

654

0.79

40.

512

NL

0.96

90.

347

1.05

00.

152

0.84

90.

271

0.59

80.

336

0.83

40.

265

FL0.

542

0.46

41.

005

1.45

31.

141

0.68

60.

593

0.14

80.

580

0.43

525

L0.

542

0.46

40.

738

0.17

50.

521

0.24

30.

278

0.09

10.

587

0.28

7N

L0.

542

0.46

40.

461

0.22

90.

349

0.07

60.

770

0.05

60.

357

0.08

6FL

0.44

20.

119

0.41

60.

361

0.36

90.

322

0.37

60.

483

0.91

80.

614

25L

0.44

20.

119

0.44

60.

207

0.58

90.

067

0.68

80.

698

0.23

30.

191

NL

0.44

20.

119

0.51

60.

220

0.47

60.

240

0.31

90.

172

0.81

71.

228

FL0.

004

0.00

90.

008

0.00

70.

012

0.02

00.

035

0.04

20.

000

0.00

025

L0.

004

0.00

90.

000

0.00

00.

000

0.00

00.

013

0.02

30.

012

0.01

2N

L0.

004

0.00

90.

008

0.01

40.

002

0.00

20.

030

0.01

80.

001

0.00

3FL

0.14

70.

157

0.12

10.

105

0.51

30.

293

0.11

50.

137

0.04

30.

074

25L

0.14

70.

157

0.35

80.

066

0.26

00.

232

0.74

10.

182

0.36

10.

317

NL

0.14

70.

157

0.21

40.

198

0.30

80.

273

0.75

40.

302

0.60

60.

214

FL1.

695

1.13

41.

024

0.13

51.

530

0.61

43.

410

1.58

41.

993

0.46

625

L1.

695

1.13

40.

663

0.59

60.

032

0.00

80.

082

0.07

80.

044

0.03

9N

L1.

695

1.13

40.

795

0.64

70.

021

0.02

40.

125

0.12

30.

005

0.00

8FL

0.12

20.

075

0.05

60.

049

0.05

50.

009

0.06

10.

006

0.29

50.

248

25L

0.12

20.

075

0.13

30.

044

0.06

80.

036

0.13

70.

066

0.08

90.

038

NL

0.12

20.

075

0.10

60.

057

0.19

50.

212

0.15

60.

088

0.08

60.

009

FL34

6.33

248

.470

364.

187

129.

338

187.

393

18.3

9039

8.26

593

.810

671.

744

194.

295

25L

346.

332

48.4

7018

4.68

254

.999

278.

863

77.6

1527

1.10

517

.301

239.

451

57.6

19N

L34

6.33

248

.470

244.

579

48.4

8129

5.98

165

.031

368.

200

93.4

9145

4.56

521

3.55

0FL

1.00

10.

216

1.11

30.

144

0.88

20.

356

1.17

20.

625

1.62

00.

625

25L

1.00

10.

216

1.19

70.

347

1.52

70.

373

1.48

11.

021

1.27

00.

328

NL

1.00

10.

216

1.20

50.

176

1.43

00.

299

0.72

40.

538

1.16

00.

044

FL3.

964

0.88

55.

670

3.36

18.

444

3.16

57.

922

1.29

14.

596

1.64

325

L3.

964

0.88

54.

652

3.15

04.

544

0.58

28.

208

0.79

56.

508

1.58

8N

L3.

964

0.88

55.

633

2.54

05.

122

1.76

99.

371

0.52

56.

780

1.57

527

98

1792

2705

1292

1749

V+9

V+18

V+27

V+37

V+46

3647

2944

3834

1933

1968

2060

2123

1939

1927

3066

1915

2769

1800

1814

Suga

rs (i

nclu

ding

suga

r alc

ohol

s):

ARAB

ITO

L22

.210

3; 2

17; 3

07C0

0532

Ar

abito

l (5T

MS)

ARAB

INO

SE21

.610

3; 1

47; 2

17C0

0216

*

DL-A

rabi

nose

, tet

raki

s(tr

imet

hyls

ilyl)

ethe

r, tr

imet

hyls

ilylo

xim

e (is

omer

2)

FRUC

TOSE

25.6

; 25.

814

7; 1

03C1

0906

*Fr

ucto

se, (

5TM

S)

CELL

OBI

OSE

38.1

204;

217

; 361

C001

85

*D-

(+)-

Cello

bios

e, o

ctak

is(t

rimet

hyls

ilyl)

ethe

r (is

omer

2)

GALA

CTO

SE25

.831

9; 2

05; 2

17C0

0124

*D-

Gala

ctos

e, 2

,3,4

,5,6

-pen

taki

s-O

-(tr

imet

hyls

ilyl)-

GALA

CTIN

OL

41.5

103;

305

; 361

C012

35*

Gala

ctin

ol, n

onak

is(t

rimet

hyls

ilyl)

ethe

r

myo

-INO

SITO

L29

.314

7; 2

17; 3

05C0

0137

*m

yo-In

osito

l (6T

MS)

GLUC

OSE

26.0

; 26.

314

7; 3

19C0

0031

*D-

Gluc

ose,

2,3

,4,5

,6-p

enta

kis-

O-(

trim

ethy

lsily

l)-

MAN

NIT

OL

26.5

319;

205

C003

92*

Man

nito

l, (6

TMS)

-

scyl

lo-IN

OSI

TOL

28.2

318

C061

53In

osito

l, 1,

2,3,

4,5,

6-he

xaki

s-O

-(tr

imet

hyls

ilyl)-

, scy

llo-

MEL

EZIT

OSE

50.6

361

C082

43*

Mel

ezito

se, 1

1TM

S

MAN

NO

SE25

.920

4C0

0159

*D-

(+)-

Man

nose

, pen

taki

s(tr

imet

hyls

ilyl)

ethe

r, tr

imet

hyls

ilylo

xim

e (is

omer

1)

RAFF

INO

SE48

.436

1C0

0492

*Ra

ffin

ose,

11T

MS

MEL

IBIO

SE40

.120

4C0

5402

*M

elib

iose

, oct

akis

(trim

ethy

lsily

l)-

SUCR

OSE

37.2

361;

217

C000

89*

Sucr

ose,

oct

akis

-O-(

trim

ethy

lsily

l)-

RHAM

NO

SE22

.611

7C0

0507

*D-

(-)-

Rham

nose

, tet

raki

s(tr

imet

hyls

ilyl)

ethe

r, m

ethy

loxi

me

(ant

i)

TREH

ALO

SE38

.536

1; 2

04; 1

91C0

1083

*D-

(+)-

Treh

alos

e, o

ctak

is(t

rimet

hyls

ilyl)

ethe

r

TAGA

TOSE

23.4

217

C007

95D-

(-)-

Taga

tose

, pen

taki

s(tr

imet

hyls

ilyl)

ethe

r

GLYC

ERO

L12

.914

7; 2

05; 1

17C0

0116

Glyc

erol

, tris

-TM

S

Tabl

e S1

: Im

pact

of d

efol

iatio

n tr

eatm

ents

(ful

l lea

f: FL

; 25

leav

es: 2

5L; n

o le

af: N

L) o

n m

easu

red

prim

ary

root

met

abol

ite a

bund

ance

dur

ing

the

expe

rimen

tal p

erio

d. T

he G

C/M

S re

tent

ion

time,

maj

or fr

agm

ent m

/z, r

eten

tion

inde

x, a

nd th

e N

IST

libra

ry co

rres

pond

ing

com

poun

d in

form

atio

n is

incl

uded

.


168

FL0.

238

0.07

30.

191

0.07

90.

241

0.11

30.

334

0.09

20.

362

0.21

025

L0.

238

0.07

30.

321

0.11

90.

270

0.09

30.

475

0.11

80.

337

0.09

4N

L0.

238

0.07

30.

372

0.13

30.

357

0.17

50.

421

0.06

90.

492

0.27

7FL

0.00

70.

014

0.00

00.

000

0.00

60.

007

0.00

30.

006

0.00

00.

000

25L

0.00

70.

014

0.00

00.

000

0.00

00.

000

0.01

70.

028

0.01

00.

017

NL

0.00

70.

014

0.00

00.

000

0.01

40.

021

0.02

10.

004

0.02

40.

021

FL1.

406

0.32

91.

246

0.28

10.

876

0.52

11.

374

0.31

32.

598

1.44

825

L1.

406

0.32

91.

447

0.75

41.

490

0.36

41.

487

0.49

51.

145

0.44

9N

L1.

406

0.32

91.

182

0.13

92.

925

2.69

61.

046

0.71

11.

138

0.69

6FL

0.91

30.

779

0.52

00.

797

0.43

60.

206

1.19

31.

147

0.64

70.

354

25L

0.91

30.

779

2.47

23.

799

1.57

51.

325

16.9

4316

.480

20.1

850.

901

NL

0.91

30.

779

0.45

30.

279

3.78

73.

670

14.7

1013

.139

9.00

86.

854

FL0.

000

0.00

00.

000

0.00

00.

000

0.00

00.

000

0.00

00.

000

0.00

025

L0.

000

0.00

00.

000

0.00

00.

000

0.00

00.

000

0.00

00.

050

0.04

4N

L0.

000

0.00

00.

000

0.00

00.

017

0.02

90.

072

0.06

90.

043

0.03

7FL

0.28

10.

098

0.16

00.

121

0.11

50.

040

0.14

80.

022

0.27

90.

109

25L

0.28

10.

098

0.16

00.

165

0.07

60.

012

0.16

90.

097

0.22

10.

042

NL

0.28

10.

098

0.16

50.

152

0.23

40.

169

0.16

00.

090

0.13

80.

010

FL1.

561

0.77

20.

973

0.24

60.

507

0.17

10.

936

0.31

82.

911

2.52

225

L1.

561

0.77

21.

654

0.75

30.

974

0.28

00.

943

0.84

01.

190

0.46

3N

L1.

561

0.77

21.

330

0.42

72.

423

2.76

10.

949

0.70

30.

986

0.24

6FL

0.42

70.

128

0.30

50.

162

0.30

70.

106

0.35

50.

068

0.32

60.

084

25L

0.42

70.

128

0.14

40.

034

0.18

70.

040

0.19

90.

092

0.26

30.

015

NL

0.42

70.

128

0.23

70.

153

0.06

80.

065

0.11

70.

042

0.10

50.

005

FL0.

280

0.07

40.

274

0.16

00.

271

0.14

70.

216

0.01

70.

494

0.45

025

L0.

280

0.07

40.

290

0.19

90.

166

0.04

10.

240

0.09

10.

220

0.06

9N

L0.

280

0.07

40.

312

0.20

50.

331

0.20

60.

603

0.18

80.

338

0.12

2FL

0.07

70.

101

0.02

80.

048

0.07

80.

136

0.03

40.

059

0.00

00.

000

25L

0.07

70.

101

0.00

00.

000

0.05

70.

099

0.00

00.

000

0.00

00.

000

NL

0.07

70.

101

0.23

40.

083

0.14

70.

131

0.00

00.

000

0.00

00.

000

FL0.

004

0.00

70.

000

0.00

00.

000

0.00

00.

000

0.00

00.

000

0.00

025

L0.

004

0.00

70.

006

0.01

10.

000

0.00

00.

000

0.00

00.

000

0.00

0N

L0.

004

0.00

70.

036

0.03

50.

019

0.03

30.

000

0.00

00.

000

0.00

0FL

0.00

20.

004

0.00

20.

004

0.00

00.

000

0.00

00.

000

0.00

00.

000

25L

0.00

20.

004

0.00

00.

000

0.00

00.

000

0.00

00.

000

0.00

00.

000

NL

0.00

20.

004

0.01

20.

011

0.00

50.

008

0.00

80.

014

0.00

00.

000

FL0.

028

0.03

80.

018

0.03

10.

000

0.00

00.

016

0.02

80.

017

0.03

025

L0.

028

0.03

80.

074

0.12

80.

016

0.02

80.

527

0.60

60.

642

0.08

5N

L0.

028

0.03

80.

023

0.04

00.

101

0.10

60.

441

0.36

00.

214

0.12

3FL

0.24

70.

123

0.17

60.

039

0.00

00.

000

0.16

10.

142

0.54

10.

409

25L

0.24

70.

123

0.36

30.

084

0.09

10.

158

0.19

50.

146

0.19

10.

170

NL

0.24

70.

123

0.17

30.

150

0.40

20.

591

0.13

60.

236

0.13

60.

147

FL0.

079

0.01

60.

062

0.04

20.

067

0.02

50.

110

0.01

70.

013

0.02

325

L0.

079

0.01

60.

114

0.15

90.

058

0.05

70.

225

0.17

90.

136

0.09

9N

L0.

079

0.01

60.

157

0.04

90.

418

0.18

60.

321

0.26

20.

318

0.12

0FL

0.32

30.

195

0.22

10.

043

0.10

60.

048

0.23

00.

085

0.84

50.

752

25L

0.32

30.

195

0.45

30.

253

0.25

30.

163

0.35

20.

146

0.31

90.

172

NL

0.32

30.

195

0.32

00.

154

0.65

40.

868

0.24

00.

246

0.18

60.

107

FL1.

329

0.38

21.

161

0.88

10.

872

0.10

21.

312

0.34

70.

996

0.11

025

L1.

329

0.38

20.

661

0.48

50.

603

0.12

70.

833

0.14

20.

862

0.12

4N

L1.

329

0.38

21.

239

0.83

90.

870

0.23

20.

929

0.40

91.

025

0.30

2FL

0.12

00.

023

0.11

50.

060

0.05

90.

036

0.13

20.

045

0.14

70.

029

25L

0.12

00.

023

0.12

40.

055

0.07

80.

024

0.13

30.

032

0.10

70.

071

NL

0.12

00.

023

0.34

30.

386

0.12

10.

071

0.10

60.

089

0.05

20.

014

FL0.

093

0.04

50.

082

0.07

30.

062

0.00

70.

060

0.02

70.

045

0.04

125

L0.

093

0.04

50.

076

0.05

40.

063

0.01

40.

068

0.06

10.

076

0.02

8N

L0.

093

0.04

50.

188

0.10

40.

096

0.01

90.

157

0.07

00.

145

0.06

3FL

0.35

50.

235

0.21

60.

284

0.19

00.

044

0.27

50.

153

0.24

40.

207

25L

0.35

50.

235

0.32

00.

333

0.16

40.

143

0.67

80.

541

0.54

50.

195

NL

0.35

50.

235

0.29

50.

145

0.54

70.

299

1.25

60.

581

0.80

80.

403

2798

2230

1336

1787

GLYC

INE

13.6

174

C000

37

GLUT

AMIN

E23

.315

6C0

0064

LEUC

INE

12.8

158

C001

23

ISO

LEUC

INE

13.3

158

1433

ASPA

RTIC

ACI

D18

.523

2; 1

47; 2

18C0

0049

*

L-As

part

ic a

cid,

(3TM

S)-

ASPA

RAGI

NE

21.6

116;

231

C001

52

*As

para

gine

, O,O

',N-t

ris(t

rimet

hyls

ilyl)-

1836

1130

1674

Amin

o ac

ids:

XYLO

SE21

.221

7; 3

07C0

0181

*D-

(+)-

Xylo

se, t

etra

kis(

trim

ethy

lsily

l) et

her,

met

hylo

xim

e (a

nti)

1414

1532

1313

1635

1532

1944

1288

1313

*Gl

y, O

,N,N

-tris

-TM

S

*L-

Glut

amin

e, N

,N2-

bis(

trim

ethy

lsily

l)-, t

rimet

hyls

ilyl e

ster

*L-

Leuc

ine,

N-(

trim

ethy

lsily

l)-, t

rimet

hyls

ilyl e

ster

1635

1542

1542

1696

TURA

NO

SE38

.820

4; 3

61; 3

07C1

9636

*D-

(+)-

Tura

nose

, oct

akis

(trim

ethy

lsily

l) et

her,

met

hylo

xim

e (is

omer

1)

ARGI

NIN

E 2

0.3;

24.

2;

142

C000

62

*Ar

gini

ne, (

3TM

S)

ALAN

INE

8.9

147

C000

41

*l-A

lani

ne, N

-(tr

imet

hyls

ilyl)-

, trim

ethy

lsily

l est

er

GLUT

AMIC

ACI

D20

.424

6C0

0025

*Gl

utam

ic a

cid

(3TM

S)

GABA

(γ-A

min

obut

yric

aci

d)18

.517

4C0

0334

*

GABA

3TM

S

C004

07*

Isol

euci

ne, d

i-TM

S

MET

HIO

NIN

E18

.312

8C0

0073

*M

et, (

2TM

S)

LYSI

NE

26.1

156

C000

47*

l-Lys

ine,

N2,

N6-

bis(

trim

ethy

lsily

l)-, t

rimet

hyls

ilyl e

ster

PRO

LIN

E13

.321

6; 1

47; 1

58C0

0148

*Pr

olin

e, d

i-TM

S

PHEN

YLAL

ANIN

E20

.421

8C0

0079

*Ph

enyl

alan

ine

(N,O

-TM

S)

SERI

NE

14.9

204;

218

C000

65*

Serin

e, (3

TMS)

PYRO

GLUT

AMIC

ACI

D (5

-oxo

-Pro

line)

18.3

156

C018

79Py

rogl

utam

ic a

cid,

(N,O

-TM

S)

Tryp

toph

an, N

,N,O

-triT

MS

THRE

ON

INE

15.5

218

C001

88*

Thre

onin

e (N

,O,O

-TM

S)

TRYP

TOPH

AN30

.829

1; 2

02C0

0078

*


169

FL0.

076

0.04

20.

068

0.02

70.

105

0.09

70.

064

0.05

60.

060

0.01

925

L0.

076

0.04

20.

071

0.03

20.

027

0.02

30.

117

0.00

50.

127

0.03

2N

L0.

076

0.04

20.

115

0.00

70.

042

0.04

70.

087

0.08

50.

054

0.04

0FL

0.10

10.

042

0.09

30.

080

0.10

10.

031

0.11

50.

024

0.12

40.

215

25L

0.10

10.

042

0.08

50.

147

0.07

70.

026

0.17

70.

060

0.09

70.

041

NL

0.10

10.

042

0.17

80.

066

0.23

30.

052

0.27

40.

138

0.16

70.

065

FL0.

022

0.00

90.

013

0.02

20.

007

0.01

30.

011

0.01

00.

003

0.00

525

L0.

022

0.00

90.

000

0.00

00.

007

0.01

20.

011

0.01

90.

000

0.00

0N

L0.

022

0.00

90.

071

0.04

50.

066

0.02

80.

013

0.01

10.

029

0.02

8

FL0.

270

0.09

00.

127

0.10

80.

222

0.07

20.

263

0.10

80.

342

0.05

225

L0.

270

0.09

00.

116

0.05

90.

076

0.04

70.

134

0.05

80.

100

0.03

9N

L0.

270

0.09

00.

133

0.04

30.

099

0.02

50.

144

0.06

10.

091

0.04

6FL

12.2

037.

095

12.7

283.

233

1.31

51.

930

9.89

54.

236

28.0

7925

.404

25L

12.2

037.

095

10.6

902.

991

7.88

66.

181

12.1

9711

.198

12.6

947.

372

NL

12.2

037.

095

11.4

343.

879

24.2

5029

.199

10.2

1311

.017

8.90

15.

862

FL0.

054

0.02

20.

064

0.02

40.

031

0.02

90.

085

0.02

40.

095

0.03

625

L0.

054

0.02

20.

101

0.04

30.

055

0.01

10.

129

0.07

60.

140

0.07

7N

L0.

054

0.02

20.

080

0.02

90.

122

0.12

20.

078

0.11

40.

066

0.02

4FL

0.17

90.

109

0.20

10.

154

0.28

50.

131

0.06

10.

023

0.27

90.

470

25L

0.17

90.

109

0.27

90.

408

0.63

20.

584

0.21

20.

274

0.19

10.

224

NL

0.17

90.

109

0.09

80.

062

0.20

50.

243

0.02

40.

016

0.30

40.

105

FL0.

039

0.00

70.

022

0.01

10.

032

0.01

50.

021

0.01

80.

018

0.03

125

L0.

039

0.00

70.

035

0.03

10.

045

0.03

10.

046

0.04

30.

042

0.02

3N

L0.

039

0.00

70.

020

0.01

90.

025

0.02

20.

022

0.01

90.

050

0.00

4FL

1.57

20.

344

1.11

80.

332

1.12

80.

604

1.32

40.

464

2.21

20.

490

25L

1.57

20.

344

2.86

73.

696

1.40

60.

278

7.07

64.

451

8.53

21.

370

NL

1.57

20.

344

0.91

80.

056

2.34

62.

446

6.70

31.

122

4.36

31.

067

FL0.

121

0.04

90.

088

0.00

30.

036

0.02

20.

056

0.05

10.

307

0.27

525

L0.

121

0.04

90.

173

0.04

90.

126

0.09

10.

156

0.01

50.

131

0.03

1N

L0.

121

0.04

90.

129

0.06

90.

222

0.27

20.

065

0.08

30.

096

0.03

0FL

2.23

40.

861

2.27

00.

517

1.53

50.

425

2.92

50.

467

3.69

61.

533

25L

2.23

40.

861

3.04

01.

310

2.61

40.

433

3.21

11.

758

3.29

41.

126

NL

2.23

40.

861

2.83

80.

503

3.48

11.

796

3.03

81.

674

3.05

00.

311

FL0.

453

0.10

50.

189

0.11

40.

104

0.05

00.

150

0.03

00.

531

0.39

725

L0.

453

0.10

50.

134

0.12

10.

067

0.06

00.

099

0.09

00.

178

0.04

5N

L0.

453

0.10

50.

115

0.04

80.

000

0.00

00.

060

0.05

90.

125

0.01

4FL

0.10

20.

044

0.07

90.

009

0.02

50.

022

0.03

30.

040

0.13

00.

096

25L

0.10

20.

044

0.10

50.

025

0.00

70.

012

0.00

30.

006

0.05

60.

049

NL

0.10

20.

044

0.10

10.

031

0.01

80.

032

0.08

50.

025

0.06

50.

025

FL0.

143

0.17

00.

108

0.02

40.

000

0.00

00.

058

0.10

10.

538

0.58

525

L0.

143

0.17

00.

257

0.17

70.

098

0.08

80.

112

0.13

00.

000

0.00

0N

L0.

143

0.17

00.

179

0.15

60.

330

0.57

10.

093

0.12

60.

060

0.06

1FL

0.06

30.

051

0.05

80.

013

0.05

10.

016

0.04

00.

026

0.41

80.

534

25L

0.06

30.

051

0.05

20.

032

0.06

00.

025

0.06

50.

019

0.06

60.

013

NL

0.06

30.

051

0.03

20.

009

0.30

30.

428

0.12

10.

021

0.05

90.

010

FL1.

365

0.46

11.

532

0.66

80.

734

0.09

71.

643

0.96

42.

164

1.85

425

L1.

365

0.46

11.

282

0.58

01.

184

0.65

21.

566

0.70

11.

146

0.52

1N

L1.

365

0.46

11.

347

0.59

92.

489

2.76

20.

960

0.77

61.

047

0.50

3FL

0.12

50.

118

0.13

10.

037

0.11

60.

011

0.06

30.

023

0.11

10.

040

25L

0.12

50.

118

0.14

50.

079

0.14

00.

063

0.18

10.

035

0.15

10.

069

NL

0.12

50.

118

0.05

80.

024

0.17

00.

043

0.12

60.

091

0.18

60.

067

FL2.

643

0.72

42.

598

0.57

71.

768

0.65

32.

098

1.01

31.

738

0.24

325

L2.

643

0.72

42.

186

0.78

61.

947

0.63

34.

467

1.34

74.

828

1.27

3N

L2.

643

0.72

42.

294

1.06

92.

051

0.52

14.

167

0.89

93.

083

1.38

5FL

3.93

61.

389

2.96

90.

072

2.46

01.

305

3.45

50.

677

7.35

94.

632

25L

3.93

61.

389

4.57

02.

263

3.54

60.

566

5.36

02.

385

3.85

91.

925

NL

3.93

61.

389

3.61

50.

825

7.22

45.

475

3.23

43.

187

3.06

23.

313

FL8.

448

0.70

45.

592

1.70

05.

513

0.82

05.

967

0.85

97.

724

1.01

125

L8.

448

0.70

48.

096

2.42

79.

370

1.42

816

.193

4.92

113

.188

4.55

7N

L8.

448

0.70

49.

063

1.13

68.

161

1.34

313

.483

0.55

112

.080

1.52

1

1175

1508

PHO

SPHO

RIC

ACID

12.9

299

C000

09Ph

osph

oric

aci

d, tr

iTM

S12

92

MAL

IC A

CID

17.8

233

C001

49M

alic

aci

d (3

TMS)

1847

1328

1088

1991

1242

1956

2323

1382

1898

2049

1980

1397

1944

2130

1635

1260

1870

Mis

cella

neou

s aci

ds:

TYRO

SIN

E26

.321

8; 2

80C0

0082

*Ty

rosi

ne, (

3TM

S)

5-hy

drox

y-TR

YPTO

PHAN

32.1

290;

218

; 146

C010

17L-

5-Hy

drox

ytry

ptop

han,

trim

ethy

lsily

l eth

er, t

rimet

hyls

ilyl e

ster

ASCO

RBIC

ACI

D24

.814

7C0

0072

*As

corb

ic a

cid

(4TM

S)

VALI

NE

11.5

144;

218

C001

83*

Valin

e, d

i-TM

S

4-HY

DRO

XY-B

ENZO

IC A

CID

20.4

267

C001

564-

Hydr

oxyb

enzo

ic a

cid

(2T

MS)

BEN

ZOIC

ACI

D12

.017

9C0

0180

Benz

oic a

cid,

TM

S

P-CO

UMAR

IC A

CID

26.1

219

C008

11p-

Coum

aric

aci

d (T

MS)

CAFF

EIC

ACID

29.4

396

C011

97Ca

ffei

c aci

d (3

TMS)

FUM

ARIC

ACI

D14

.424

5C0

0122

Fum

aric

aci

d (2

TMS)

CITR

IC A

CID

24.4

273

C001

58Ci

tric

aci

d, (4

TMS)

GLUC

ON

IC A

CID

28.0

292;

333

C002

57Gl

ucon

ic a

cid,

(6TM

S)

GALL

IC A

CID

26.7

281;

458

C014

24Ga

llic a

cid,

tetr

aTM

S

GLYC

ERIC

ACI

D14

.214

7; 1

89; 2

92C0

0258

Glyc

eric

aci

d, (3

TMS)

2-KE

TO-G

LUCO

NIC

ACI

D23

.729

2; 1

03C0

6473

2-Ke

to-l-

gluc

onic

aci

d, p

enta

(O-t

rimet

hyls

ilyl)-

LACT

IC A

CID

8.1

147;

117

C001

86La

ctic

aci

d, (2

TMS)

3-HY

DRO

XYAN

THRA

NIL

IC A

CID

26.9

354

C006

323-

Hydr

oxya

nthr

anill

ic a

cid,

(3TM

S)

MAL

EIC

ACID

13.5

147;

245

C01

384

Mal

eic a

cid,

(2TM

S)

OXA

LIC

ACID

9.8

147;

133

C002

09O

xalic

aci

d (2

TMS)


170

FL0.

711

0.36

10.

607

0.08

20.

362

0.26

20.

501

0.11

10.

878

0.11

425

L0.

711

0.36

10.

744

0.17

00.

905

0.56

60.

517

0.35

50.

474

0.19

8N

L0.

711

0.36

10.

759

0.05

30.

866

0.67

40.

320

0.33

60.

462

0.20

8FL

0.04

70.

035

0.03

20.

056

0.33

70.

512

0.05

10.

046

0.04

60.

079

25L

0.04

70.

035

0.17

10.

187

0.06

90.

060

0.00

00.

000

0.04

80.

043

NL

0.04

70.

035

0.10

00.

012

0.37

40.

531

0.02

30.

040

0.11

60.

061

FL4.

028

0.30

23.

415

0.75

512

.095

16.5

562.

748

1.41

15.

257

2.70

325

L4.

028

0.30

24.

840

4.87

76.

575

2.40

520

.539

7.41

224

.883

7.85

2N

L4.

028

0.30

23.

651

0.87

48.

062

5.46

814

.699

5.65

114

.777

5.23

5FL

1.14

50.

936

0.29

30.

271

0.07

60.

030

0.08

50.

027

0.11

70.

107

25L

1.14

50.

936

0.33

90.

351

0.08

00.

078

0.01

00.

018

0.05

80.

051

NL

1.14

50.

936

0.55

80.

316

0.00

00.

000

0.03

90.

051

0.38

10.

615

FL0.

064

0.06

10.

031

0.01

70.

039

0.03

10.

056

0.03

30.

270

0.36

925

L0.

064

0.06

10.

115

0.13

00.

019

0.00

50.

080

0.06

80.

054

0.03

0N

L0.

064

0.06

10.

097

0.07

70.

190

0.29

90.

076

0.04

90.

027

0.01

7FL

0.44

80.

319

0.34

30.

066

0.20

10.

066

0.32

50.

087

1.24

11.

297

25L

0.44

80.

319

0.59

20.

373

0.29

00.

109

0.47

50.

325

0.51

80.

233

NL

0.44

80.

319

0.52

90.

289

1.01

61.

303

0.35

90.

285

0.26

20.

132

FL0.

089

0.02

40.

136

0.04

60.

042

0.01

40.

087

0.04

20.

167

0.13

725

L0.

089

0.02

40.

125

0.06

20.

128

0.07

10.

112

0.00

80.

086

0.03

2N

L0.

089

0.02

40.

122

0.08

00.

128

0.08

40.

073

0.07

90.

103

0.07

2FL

0.06

10.

068

0.00

90.

014

0.00

20.

003

0.03

90.

013

0.22

70.

265

25L

0.06

10.

068

0.22

80.

278

0.03

30.

050

0.03

50.

060

0.04

00.

042

NL

0.06

10.

068

0.33

80.

482

0.10

60.

175

0.05

10.

088

0.00

20.

002

FL2.

294

1.46

81.

636

0.53

10.

742

0.25

41.

924

0.48

35.

661

5.11

425

L2.

294

1.46

83.

873

2.99

61.

789

1.11

42.

706

1.01

72.

461

1.26

7N

L2.

294

1.46

82.

829

1.94

25.

212

6.78

31.

942

2.38

11.

329

0.71

8FL

0.00

30.

006

0.00

00.

000

0.00

00.

000

0.00

00.

000

0.02

40.

042

25L

0.00

30.

006

0.01

50.

026

0.00

00.

000

0.00

00.

000

0.00

00.

000

NL

0.00

30.

006

0.05

70.

071

0.02

40.

042

0.00

00.

000

0.00

00.

000

FL3.

949

3.03

82.

823

0.84

11.

037

0.20

43.

167

1.20

110

.874

10.5

5825

L3.

949

3.03

86.

832

5.27

42.

485

1.54

84.

064

2.55

94.

501

2.55

9N

L3.

949

3.03

84.

339

2.70

79.

160

12.0

543.

237

3.99

71.

961

1.06

8

FL0.

264

0.06

80.

302

0.04

00.

405

0.08

30.

462

0.03

60.

445

0.12

225

L0.

264

0.06

80.

352

0.24

60.

250

0.09

30.

712

0.19

50.

477

0.05

3N

L0.

264

0.06

80.

272

0.07

10.

508

0.27

60.

656

0.05

30.

463

0.08

0FL

5.77

22.

439

5.63

20.

372

6.11

22.

538

6.47

90.

452

6.00

90.

211

25L

5.77

22.

439

5.24

13.

321

4.88

70.

667

9.48

43.

401

7.62

10.

917

NL

5.77

22.

439

7.33

50.

926

7.37

22.

506

7.91

54.

534

6.55

81.

118

FL17

.075

11.0

1213

.641

3.19

19.

191

5.16

615

.227

2.24

441

.513

36.8

2725

L17

.075

11.0

1223

.170

14.8

9312

.503

2.49

518

.053

12.4

8617

.881

7.31

1N

L17

.075

11.0

1218

.522

7.88

239

.290

47.8

0215

.556

10.7

8710

.065

5.94

5FL

0.41

20.

097

0.35

00.

053

0.24

00.

104

0.36

80.

054

0.77

90.

453

25L

0.41

20.

097

0.36

90.

076

0.50

90.

145

0.44

00.

132

0.39

20.

120

NL

0.41

20.

097

0.33

80.

033

0.59

20.

367

0.21

20.

286

0.26

70.

218

FL0.

007

0.00

50.

009

0.00

80.

008

0.00

60.

006

0.00

50.

004

0.00

625

L0.

007

0.00

50.

009

0.01

60.

013

0.01

10.

005

0.00

80.

006

0.01

0N

L0.

007

0.00

50.

002

0.00

30.

025

0.02

40.

002

0.00

30.

008

0.00

3FL

8.17

45.

032

6.79

51.

447

4.16

22.

297

6.97

50.

991

22.3

6921

.796

25L

8.17

45.

032

11.1

496.

728

6.49

91.

708

8.59

76.

101

9.16

73.

262

NL

8.17

45.

032

8.72

73.

693

19.1

5822

.971

6.80

05.

225

5.16

82.

967

FL0.

220

0.31

60.

193

0.02

20.

685

0.59

60.

688

0.27

00.

081

0.12

425

L0.

220

0.31

60.

319

0.39

40.

180

0.08

80.

509

0.23

20.

331

0.14

5N

L0.

220

0.31

60.

419

0.38

10.

353

0.29

51.

642

0.93

30.

238

0.18

3FL

0.13

00.

104

0.10

60.

033

0.08

20.

053

0.12

10.

050

0.06

80.

041

25L

0.13

00.

104

0.23

60.

076

0.24

20.

251

0.13

10.

090

0.13

70.

117

NL

0.13

00.

104

0.13

30.

074

0.14

90.

153

0.19

90.

103

0.12

80.

100

Adip

ic a

cid

1814

7; 2

75C0

6104

*Ad

ipic

aci

d, (2

TMS)

1528

Adon

itol (

Ribi

tol)

22.8

147;

217

; 103

C00474

*Ri

bito

l, 5T

MS

1760

L-hy

drox

ypro

line

18.5

230

C011

57*

3-Hy

drox

ypro

line,

N,O

,O'-t

ris(t

rimet

hyls

ilyl)-

1542

C000

42

1831

Succ

inic

aci

d (2

TMS)

2237

1940

2039

2209

1405

2407

2990

2734

4300

2602

3108

1135

2791

2889

2626

Mis

calle

neou

s com

poun

ds:

NO

NAN

OIC

ACI

D14

.611

7; 2

15C0

1601

Non

anoi

c aci

d, T

MS

este

r

MYR

ISTI

C AC

ID

L-Th

reon

ic a

cid,

tris

(trim

ethy

lsily

l) et

her,

trim

ethy

lsily

l est

er

Fatt

y ac

ids:

1586

PRO

TOCA

TECH

UIC

ACID

24.1

193;

370

C002

30*

Prot

ocat

echu

ic a

cid

(tm

s)

SUCC

INIC

ACI

D13

.714

7; 2

47

1666

1344

TART

ARIC

ACI

D21

.029

2C0

0898

Tart

aric

aci

d, T

MS

33.3

343

C064

24M

yris

tic a

cid,

2,3

-bis

(trim

ethy

lsilo

xy)p

ropy

l est

er

EICO

SAN

OIC

ACI

D40

.642

7C0

6425

Eico

sano

ic a

cid,

2,3

-bis

[(tr

imet

hyls

ilyl)o

xy]p

ropy

l est

er

THRE

ON

IC A

CID

19.4

147;

292

; 220

C016

20

PALM

ITIC

ACI

D27

.811

7; 3

13C0

0249

Palm

itic a

cid,

TM

S

OLE

IC A

CID

30.5

339

C007

12O

leic

aci

d, tr

imet

hyls

ilyl e

ster

STEA

RIC

ACID

30.9

117;

341

C015

30St

earic

aci

d, tr

imet

hyls

ilyl e

ster

PEN

TADE

CAN

OIC

ACI

D26

.211

7; 2

99C1

6537

Pent

adec

anoi

c aci

d, T

MS

este

r

GLYC

ERO

L MO

NO

STEA

RATE

38.4

399

N/A

Glyc

erol

mon

oste

arat

e, 2

tms d

eriv

ativ

e

CATE

CHIN

39.5

; 39.

836

8C0

6562

Cate

chin

e, p

enta

-TM

S-et

her,

(2R-

cis)

-

ARBU

TIN

36.2

254

C061

86Hy

droq

uino

ne-β

-d-g

luco

pyra

nosi

de,p

enta

kis(

trim

ethy

lsily

l)-

KAEM

PFER

OL

42.0

559

C059

03Ka

empf

erol

, 4TM

S

HYDR

OXY

LAM

INE

9.0

133

C001

92Hy

drox

ylam

ine,

N,N

,O-t

ris-T

MS

CIS-

RESV

ERAT

ROL

37.6

444;

147

; 207

C035

82ci

s-Re

sver

atro

l, 3T

MS

Inte

rnal

stan

dard

s:

CIS-

PICE

ID56

.144

4; 3

61; 2

17C1

0275

cis-

Pice

id, 6

TMS

1-M

ON

OPA

LMIT

IN35

.937

1N

/A1-

Mon

opal

miti

n tr

imet

hyls

ilyl e

ther


171

Day

s af

ter

vera

iso

n

Ro

ot

me

tab

oli

te a

bu

nd

ance

Tre

atm

en

tva

lue

SDEV

valu

eSD

EVva

lue

SDEV

valu

eSD

EVva

lue

SDEV

RT

(min

)m

/zK

egg

IDst

and

ard

ide

nti

fica

tio

nN

IST

lib

rary

co

mp

ou

nd

Re

ten

tio

n in

de

x

FL0.

215

0.0

67

0.23

60.1

31

0.16

30.0

68

0.06

00.0

68

0.05

10.0

88

25L

0.21

50.0

67

0.15

60.0

98

0.12

00.0

53

0.13

40.0

09

0.20

20.1

18

FL0.

166

0.0

90

0.20

70.0

33

0.15

50.0

86

0.21

00.0

97

0.18

80.0

92

25L

0.16

60.0

90

0.16

30.0

61

0.22

40.0

79

0.32

50.0

27

0.09

30.0

21

FL18

.198

3.9

14

16.9

902.8

08

19.0

494.8

41

19.0

193.2

55

19.3

220.6

27

25L

18.1

983.9

14

17.6

991.5

60

18.3

192.3

54

20.2

532.5

40

23.2

047.0

08

FL0.

457

0.1

96

0.18

00.1

56

0.57

70.5

98

0.32

40.1

08

0.25

70.0

42

25L

0.45

70.1

96

0.00

00.0

00

0.15

60.1

66

0.10

30.1

79

0.05

30.0

92

FL53

.722

4.2

39

50.8

7230.5

98

29.5

357.2

26

74.0

103.7

46

76.0

0121.3

11

25L

53.7

224.2

39

38.5

0211.2

85

26.9

727.4

35

47.5

537.4

41

54.2

9619.5

74

FL3.

848

1.5

22

3.97

50.2

78

5.69

01.2

73

4.61

60.4

62

2.99

40.7

43

25L

3.84

81.5

22

3.17

90.9

65

5.17

11.1

90

3.68

00.6

26

4.20

41.6

05

FL1.

041

0.0

71

1.13

70.3

16

0.94

10.0

97

1.05

70.0

65

1.11

00.2

56

25L

1.04

10.0

71

0.77

20.0

20

0.89

80.2

24

1.01

10.1

79

1.55

90.7

33

FL40

.426

3.8

01

34.7

4918.0

70

22.9

354.8

26

53.6

605.3

61

45.4

089.7

09

25L

40.4

263.8

01

33.0

089.7

16

25.6

764.4

47

45.6

252.6

59

50.8

0123.1

69

FL1.

581

3.0

37

0.00

00.0

00

0.01

30.0

23

0.00

00.0

00

1.92

62.3

48

25L

1.58

13.0

37

0.00

00.0

00

0.42

30.7

33

0.43

10.7

47

0.00

00.0

00

FL24

7.90

614.2

94

232.

271

37.8

21

198.

083

9.7

08

183.

468

20.2

60

179.

005

13.8

46

25L

247.

906

14.2

94

186.

784

9.2

52

137.

310

22.1

76

116.

523

5.7

60

103.

425

13.8

78

FL1.

523

0.1

21

0.86

10.5

08

1.13

50.1

78

0.68

60.2

90

0.63

30.3

09

25L

1.52

30.1

21

0.93

50.0

83

1.00

20.1

96

0.48

00.0

47

0.32

50.1

56

FL2.

094

0.8

56

2.82

21.7

02

0.78

60.4

17

1.10

10.1

88

2.48

31.3

32

25L

2.09

40.8

56

4.16

01.0

21

1.25

70.5

55

1.63

30.7

58

4.13

20.2

86

FL8.

427

0.8

54

7.67

81.3

99

6.98

60.8

44

8.99

32.1

26

8.60

10.3

98

25L

8.42

70.8

54

6.07

20.9

51

5.80

91.2

67

6.40

01.2

35

6.02

00.8

34

FL0.

220

0.3

09

0.06

40.0

57

0.05

70.0

50

0.21

20.3

68

0.08

60.0

78

25L

0.22

00.3

09

0.07

70.0

69

0.25

40.3

89

0.11

80.0

53

0.12

40.0

83

FL0.

019

0.0

28

0.00

30.0

05

0.02

20.0

30

0.00

40.0

07

0.03

30.0

43

25L

0.01

90.0

28

0.01

40.0

18

0.01

20.0

20

0.04

10.0

18

0.13

90.0

41

FL3.

367

0.8

45

4.08

50.7

96

5.03

51.8

12

4.74

70.8

81

4.64

71.4

91

25L

3.36

70.8

45

4.89

61.6

45

5.99

60.3

02

5.85

51.1

28

6.90

80.9

29

FL1.

299

0.4

11

0.86

40.0

84

1.77

41.2

26

2.35

30.2

57

3.58

43.1

38

25L

1.29

90.4

11

1.03

00.6

51

0.98

30.6

32

2.30

00.6

86

7.20

33.6

08

FL0.

103

0.0

23

0.07

10.0

26

0.07

90.0

11

0.05

40.0

47

0.06

20.0

20

25L

0.10

30.0

23

0.07

70.0

20

0.13

70.0

68

0.20

90.0

35

0.62

30.4

31

FL1.

058

0.1

89

0.86

80.0

56

0.66

60.0

70

0.87

90.2

88

0.98

30.2

06

25L

1.05

80.1

89

0.54

00.1

45

0.47

80.0

95

0.54

30.1

35

0.51

00.0

98

FL74

2.82

866.5

34

807.

892

44.7

75

716.

296

70.5

17

764.

979

10.1

08

780.

693

52.2

92

25L

742.

828

66.5

34

784.

238

38.2

96

809.

772

36.6

14

723.

916

222.2

36

773.

685

49.1

73

FL3.

954

1.2

01

4.95

80.7

45

3.81

20.9

70

4.59

41.2

29

3.64

11.0

98

25L

3.95

41.2

01

4.01

11.4

63

4.50

02.1

58

5.65

61.1

20

3.34

10.1

97

FL7.

692

0.7

19

6.25

90.7

86

7.09

73.1

75

6.51

80.4

33

5.63

60.7

64

25L

7.69

20.7

19

5.58

90.8

05

6.37

80.3

34

5.94

81.2

37

6.52

91.1

64

FL0.

148

0.0

97

0.06

60.0

84

0.12

40.0

35

0.11

00.0

72

0.07

80.0

86

25L

0.14

80.0

97

0.07

60.0

44

0.14

20.0

21

0.09

70.0

15

0.06

00.0

30

FL0.

488

0.1

30

0.41

50.0

10

0.40

60.1

55

0.48

70.0

28

0.49

70.0

56

25L

0.48

80.1

30

0.51

50.0

49

0.56

90.1

17

0.53

70.0

78

0.51

00.1

40

Tab

le S

2: Im

pac

t o

f d

efo

liat

ion

tre

atm

en

ts (

full

leaf

: FL;

25

leav

es:

25L

) o

n m

eas

ure

d p

rim

ary

leaf

me

tab

oli

te a

bu

nd

ance

du

rin

g th

e e

xpe

rim

en

tal p

eri

od

. Th

e G

C/M

S re

ten

tio

n t

ime

, maj

or

frag

me

nt

m/z

, re

ten

tio

n in

de

x, a

nd

th

e N

IST

lib

rary

co

rre

spo

nd

ing

com

po

un

d in

form

atio

n is

incl

ud

ed

.

GLY

CER

OL

12.9

147;

205

; 117

C00

116

Gly

cero

l, t

ris-

TMS

ALA

NIN

E8.

914

7C

0004

1 *

l-A

lan

ine

, N-(

trim

eth

ylsi

lyl)

-, t

rim

eth

ylsi

lyl e

ste

r

XYL

OSE

21.2

217;

307

C00

181

* D

-(+)

-Xyl

ose

, te

trak

is(t

rim

eth

ylsi

lyl)

eth

er,

me

thyl

oxi

me

(an

ti)

Am

ino

aci

ds:

1130

TREH

ALO

SE38

.536

1; 2

04; 1

91C

0108

3*

D-(

+)-T

reh

alo

se, o

ctak

is(t

rim

eth

ylsi

lyl)

eth

er

1674

2798

TAG

ATO

SE23

.421

7C

0079

5D

-(-)

-Tag

ato

se, p

en

taki

s(tr

ime

thyl

sily

l) e

the

r

SUC

RO

SE37

.236

1; 2

17C

0008

9*

Sucr

ose

, oct

akis

-O-(

trim

eth

ylsi

lyl)

-

RIB

OSE

21.8

103;

217

; 307

C00

121

*d

-Rib

ose

, 2,3

,4,5

-te

trak

is-O

-(tr

ime

thyl

sily

l)-,

O-m

eth

ylo

xim

e

RH

AM

NO

SE22

.611

7C

0050

7*

D-(

-)-R

ham

no

se, t

etr

akis

(tri

me

thyl

sily

l) e

the

r, m

eth

ylo

xim

e (

anti

)

RA

FFIN

OSE

48.4

361

C00

492

*R

affi

no

se, 1

1TM

S

MEL

IBIO

SE40

.120

4C

0540

2*

Me

lib

iose

, oct

akis

(tri

me

thyl

sily

l)-

MEL

EZIT

OSE

50.6

361

C08

243

*M

ele

zito

se, 1

1TM

S

MA

NN

OSE

25.9

204

C00

159

*D

-(+)

-Man

no

se, p

en

taki

s(tr

ime

thyl

sily

l) e

the

r, t

rim

eth

ylsi

lylo

xim

e (

iso

me

r 1)

MA

NN

ITO

L26

.531

9; 2

05C

0039

2*

Man

nit

ol,

(6T

MS)

-

MA

LTO

SE38

.620

4; 2

17; 3

62; 5

98C

0020

8*

Mal

tose

, oct

akis

(tri

me

thyl

sily

l) e

the

r, m

eth

ylo

xim

e (

iso

me

r 2)

scyl

lo-I

NO

SITO

L28

.231

8C

0615

3In

osi

tol,

1,2

,3,4

,5,6

-he

xaki

s-O

-(tr

ime

thyl

sily

l)-,

scy

llo

-

myo

-IN

OSI

TOL

29.3

147;

217

; 305

C00

137

*m

yo-I

no

sito

l (6T

MS)

GLU

CO

SE26

.0; 2

6.3

147;

319

C00

031

*D

-Glu

cose

, 2,3

,4,5

,6-p

en

taki

s-O

-(tr

ime

thyl

sily

l)-

GA

LAC

TOSE

25.8

319;

205

; 217

C00

124

*D

-Gal

acto

se, 2

,3,4

,5,6

-pe

nta

kis-

O-(

trim

eth

ylsi

lyl)

-

GA

LAC

TIN

OL

41.5

103;

305

; 361

C01

235

*G

alac

tin

ol,

no

nak

is(t

rim

eth

ylsi

lyl)

eth

er

FRU

CTO

SE25

.6; 2

5.8

147;

103

C10

906

*Fr

uct

ose

, (5T

MS)

*D

L-A

rab

ino

se, t

etr

akis

(tri

me

thyl

sily

l) e

the

r, t

rim

eth

ylsi

lylo

xim

e (

iso

me

r 2)

DU

LCIT

OL

(gal

acti

tol)

26.7

217;

147

C01

697

*D

ulc

ito

l, (

6TM

S)

CEL

LOB

IOSE

38.1

204;

217

; 361

C00

185

*

D-(

+)-C

ell

ob

iose

, oct

akis

(tri

me

thyl

sily

l) e

the

r (i

som

er

2)

V+9

V+1

8V

+27

V+3

7V

+46

Suga

rs (

incl

ud

ing

suga

r al

coh

ols

):

1939

1927

3066

1915

1980

2769

1800

1814

AR

AB

ITO

L22

.210

3; 2

17; 3

07C

0053

2

Ara

bit

ol (

5TM

S)

AR

AB

INO

SE21

.610

3; 1

47; 2

17C

0021

6

2060

2123

1792

2705

1706

1749

3647

2944

3834

1933

1968

2806

1292


172

FL0.

018

0.0

36

0.07

50.0

80

0.06

10.0

57

0.05

60.0

52

0.08

10.0

14

25L

0.01

80.0

36

0.01

80.0

30

0.06

70.0

29

0.05

90.0

52

0.01

50.0

26

FL0.

000

0.0

00

0.00

00.0

00

0.00

00.0

00

0.00

00.0

00

0.00

00.0

00

25L

0.00

00.0

00

0.00

00.0

00

0.00

00.0

00

0.00

00.0

00

0.00

80.0

14

FL0.

630

0.1

98

0.43

30.2

19

0.43

00.1

21

0.22

50.0

85

0.22

00.0

22

25L

0.63

00.1

98

0.40

40.0

62

0.24

70.0

26

0.16

30.0

33

0.21

00.0

17

FL0.

037

0.0

26

0.04

10.0

37

0.10

20.0

24

0.09

20.0

26

0.10

30.0

14

25L

0.03

70.0

26

0.06

00.0

03

0.10

30.0

19

0.15

70.0

19

0.21

20.1

06

FL0.

596

0.1

66

0.52

50.0

90

0.74

10.1

47

0.78

30.1

07

0.57

90.1

78

25L

0.59

60.1

66

0.81

10.1

17

1.11

40.2

80

2.08

00.2

60

2.48

80.5

90

FL1.

292

0.2

21

0.75

60.2

06

1.18

10.2

44

0.85

50.2

16

0.90

00.0

60

25L

1.29

20.2

21

0.97

80.0

67

0.84

50.2

21

0.75

10.0

45

0.85

60.0

65

FL0.

603

0.0

67

0.53

70.0

65

0.52

30.0

25

0.60

80.1

25

0.71

80.1

08

25L

0.60

30.0

67

0.51

50.0

80

0.56

50.1

56

0.76

70.0

48

0.85

00.1

21

FL0.

000

0.0

00

0.00

00.0

00

0.00

00.0

00

0.00

00.0

00

0.00

00.0

00

25L

0.00

00.0

00

0.00

00.0

00

0.03

00.0

07

0.03

10.0

36

0.07

30.0

33

FL0.

000

0.0

00

0.00

00.0

00

0.00

00.0

00

0.00

00.0

00

0.00

00.0

00

25L

0.00

00.0

00

0.00

00.0

00

0.01

70.0

19

0.01

10.0

19

0.02

10.0

06

FL0.

000

0.0

00

0.00

00.0

00

0.26

60.1

10

0.00

00.0

00

0.00

00.0

00

25L

0.00

00.0

00

0.00

00.0

00

0.45

30.3

34

0.09

50.0

08

0.07

70.0

68

FL1.

736

0.1

96

1.23

00.3

13

1.55

10.1

57

1.39

40.2

21

1.14

60.2

17

25L

1.73

60.1

96

1.26

80.3

96

1.16

30.3

20

1.16

80.1

45

1.32

80.0

94

FL0.

280

0.1

83

0.12

30.1

19

0.06

30.0

24

0.00

50.0

09

0.08

30.0

21

25L

0.28

00.1

83

0.83

00.0

75

0.25

80.1

17

0.61

60.1

89

0.57

70.1

09

FL0.

438

0.0

57

0.15

10.0

27

0.17

30.0

02

0.13

50.0

72

0.15

10.0

09

25L

0.43

80.0

57

0.28

70.0

41

0.25

10.0

35

0.35

40.0

05

0.36

90.0

15

FL0.

048

0.0

96

0.17

10.1

78

0.35

10.1

27

0.24

20.2

04

0.23

60.1

36

25L

0.04

80.0

96

0.10

90.0

95

0.60

70.2

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0.18

90.0

08

0.41

40.2

47

FL0.

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0.0

59

0.28

10.1

49

0.29

30.0

62

0.24

30.0

17

0.18

70.0

33

25L

0.40

50.0

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0.21

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0.11

90.0

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0.08

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0.0

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0.00

00.0

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30.0

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11.5

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12.9

174.5

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9.34

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82

9.32

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46

FL3.

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1.5

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4.74

00.7

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4.50

61.1

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4.65

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3.95

32.3

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11.5

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2.57

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3.95

22.5

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14

4.54

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3.53

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0.09

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0.11

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0.5

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1.53

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0.24

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0.24

10.2

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0.26

90.1

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0.36

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0.14

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0.10

80.0

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0.4

60

2.00

10.4

06

1.65

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2.32

60.4

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2.59

90.5

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1.46

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1.31

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0.73

80.3

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0.51

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GLU

CO

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AC

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luco

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aci

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6TM

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GA

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4TM

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P-C

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TMS)

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29.4

396

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AC

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Asc

orb

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MS)

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LIN

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18C

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280

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082

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, N,N

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S

THR

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15.5

218

C00

188

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N,O

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C00

065

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3TM

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mic

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246

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id)

18.5

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A 3

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CYS

TEIN

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7; 2

04C

0009

7*

Cys

tein

e, N

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s-TB

DM

S

ASP

AR

TIC

AC

ID18

.523

2; 1

47; 2

18C

0004

9 *

L-A

spar

tic

acid

, (3T

MS)

-

ASP

AR

AG

INE

21.6

116;

231

C00

152

*

Asp

arag

ine

, O,O

',N-t

ris(

trim

eth

ylsi

lyl)

-

AR

GIN

INE

; 20.

3; 2

4.2;

14

2C

0006

2

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rgin

ine

, (3T

MS)

1313

1787

1635

1542

2316

1542

1696

1836

1635

1397

1847

1944

2130

1635

1260

1870

1288

Mis

cell

ane

ou

s ac

ids:

PH

ENYL

ALA

NIN

E20

.421

8C

0007

9*

Ph

en

ylal

anin

e (

N,O

-TM

S)

LEU

CIN

E12

.815

8C

0012

3*

L-Le

uci

ne

, N-(

trim

eth

ylsi

lyl)

-, t

rim

eth

ylsi

lyl e

ste

r

PYR

OG

LUTA

MIC

AC

ID (

5-o

xo-P

roli

ne

)

1956

2323

2230

1433

1414

1532

2049

1980

1242


173

FL2.

195

1.0

31

1.56

10.1

63

1.51

40.9

67

1.64

00.2

11

2.03

20.8

37

25L

2.19

51.0

31

1.60

41.0

43

1.03

50.4

51

0.61

60.1

89

1.50

00.4

07

FL1.

041

0.3

63

1.11

00.6

63

1.72

90.2

35

1.58

20.1

66

0.96

10.2

69

25L

1.04

10.3

63

0.55

20.1

74

0.82

00.2

23

0.38

90.2

21

0.29

90.0

88

FL0.

142

0.0

31

0.09

30.0

10

0.08

20.0

35

0.07

90.0

02

0.05

60.0

52

25L

0.14

20.0

31

0.10

30.0

54

0.09

30.0

07

0.09

20.0

13

0.15

90.1

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FL0.

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0.2

63

0.67

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95

0.94

40.1

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0.64

60.5

64

0.48

70.1

31

25L

0.78

60.2

63

0.42

20.2

64

0.61

10.3

27

0.80

50.4

01

0.50

20.0

41

FL0.

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0.3

76

1.05

90.3

45

0.76

70.3

64

0.89

50.2

45

0.96

40.5

64

25L

0.88

70.3

76

0.44

50.1

98

0.89

60.8

16

1.13

20.4

76

0.81

30.1

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3.5

34

17.9

002.5

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19.9

372.6

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19.4

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21.2

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25.7

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13.9

112.4

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18.0

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0.0

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1.35

20.1

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1.71

50.1

63

1.39

20.3

44

1.57

40.1

12

25L

1.43

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1.65

60.0

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1.82

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26

1.95

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68

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20.3

47

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21.5

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11.8

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11.5

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14.6

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14.7

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12.7

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10.7

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0.0

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0.17

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0.20

50.1

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0.28

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0.19

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0.15

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0.36

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0.16

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0.33

30.0

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0.28

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0.40

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0.33

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0.22

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0.16

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0.19

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0.45

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0.48

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0.75

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18

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0.39

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0.65

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0.81

60.2

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0.15

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0.22

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0.38

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0.02

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0.64

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0.47

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0.64

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0.45

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5.61

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4.59

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4.62

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5.02

30.9

94

25L

6.38

81.4

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5.35

40.5

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5.20

60.7

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4.52

30.9

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70.9

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2.2

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5.35

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5.89

81.6

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6.13

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5.00

30.4

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0.09

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40.0

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10.0

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0.06

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0.24

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0.20

50.0

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60.0

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25L

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00.0

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0.28

30.0

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50.0

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90.0

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90.1

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0.6

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15.0

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14.3

424.1

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12.6

621.8

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13.9

601.4

76

25L

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15.0

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13.6

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15.8

351.5

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16.1

543.4

44

FL0.

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0.1

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0.02

50.0

13

0.14

20.2

19

0.04

90.0

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0.15

30.2

35

25L

0.10

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0.17

20.2

59

0.22

60.3

63

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10.0

14

0.41

00.3

97

Ad

ipic

aci

d18

147;

275

C06

104

*A

dip

ic a

cid

, (2T

MS)

1528

Ad

on

ito

l (R

ibit

ol)

22.8

147;

217

; 103

C00474

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ol,

5TM

S17

60

L-h

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xyp

roli

ne

18.5

230

C01

157

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Hyd

roxy

pro

lin

e, N

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rim

eth

ylsi

lyl)

-15

42

QU

ERC

ETIN

57.3

575

C00

389

Qu

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eti

n, (

5TM

S)

Inte

rnal

sta

nd

ard

s:

1-M

ON

OP

ALM

ITIN

35.9

371

N/A

1-M

on

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alm

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tri

me

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l eth

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KA

EMP

FER

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42.0

559

C05

903

Kae

mp

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l, 4

TMS

HYD

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XYL

AM

INE

9.0

133

C00

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Hyd

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lam

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, N,N

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CER

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MO

NO

STEA

RA

TE38

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9N

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on

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ear

ate

, 2tm

s d

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vati

ve

CA

TEC

HIN

39.5

; 39.

836

8C

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2C

ate

chin

e, p

en

ta-T

MS-

eth

er,

(2R

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)-

AR

BU

TIN

36.2

254

C06

186

Hyd

roq

uin

on

e-β

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luco

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sid

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s(tr

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l)-

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AC

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IC A

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30.5

339

C00

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Ole

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me

thyl

sily

l est

er

2039

2209

1405

NO

NA

NO

IC A

CID

14.6

117;

215

C01

601

EIC

OSA

NO

IC A

CID

40.6

427

C06

425

Eico

san

oic

aci

d, 2

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is[(

trim

eth

ylsi

lyl)

oxy

]pro

pyl

est

er

THR

EON

IC A

CID

19.4

147;

292

; 220

C01

620

L-Th

reo

nic

aci

d, t

ris(

trim

eth

ylsi

lyl)

eth

er,

tri

me

thyl

sily

l est

er

Fatt

y ac

ids:

1586

OX

ALI

C A

CID

9.8

147;

133

C00

209

Oxa

lic

acid

(2T

MS)

MA

LIC

AC

ID17

.823

3C

0014

9M

alic

aci

d (

3TM

S)

MA

LEIC

AC

ID13

.514

7; 2

45 C

0138

4M

ale

ic a

cid

, (2T

MS)

LAC

TIC

AC

ID8.

114

7; 1

17C

0018

6La

ctic

aci

d, (

2TM

S)

3-H

YDR

OX

YAN

THR

AN

ILIC

AC

ID26

.935

4C

0063

23-

Hyd

roxy

anth

ran

illi

c ac

id, (

3TM

S)

GLY

CER

IC A

CID

14.2

147;

189

; 292

C00

258

Gly

ceri

c ac

id, (

3TM

S)

2-K

ETO

-GLU

TAR

IC A

CID

25.3

157;

173

C00

026

α-K

eto

glu

tari

c ac

id, t

rim

eth

ylsi

lyl e

ste

r

1175

1508

1328

1088

1991

1382

1898

2407

2990

4402

2602

3108

1135

2791

2889

2626

Mis

call

en

eo

us

com

po

un

ds:

2237

No

nan

oic

aci

d, T

MS

est

er

MYR

ISTI

C A

CID

33.3

343

C06

424

Myr

isti

c ac

id, 2

,3-b

is(t

rim

eth

ylsi

loxy

)pro

pyl

est

er

PA

LMIT

IC A

CID

27.8

117;

313

C00

249

Pal

mit

ic a

cid

, TM

S

RIB

ON

IC A

CID

23.7

292;

217

C01

685

Tart

aric

aci

d, T

MS

SUC

CIN

IC A

CID

13.7

147;

247

C00

042

Succ

inic

aci

d (

2TM

S)

TAR

TAR

IC A

CID

21.0

292

C00

898

Rib

on

ic a

cid

, pe

nta

kis-

TMS

PR

OTO

CA

TEC

HU

IC A

CID

24.1

193;

370

C00

230

*P

roto

cate

chu

ic a

cid

(tm

s)

1666

1344

1808

PH

OSP

HO

RIC

AC

IDP

ho

sph

ori

c ac

id, t

riTM

S12

92C

0000

929

912

.9

1831

Appendix A – Figure S1

174

Roots PCA Analysis (using only data where treatment was significant at 5% for at least one time)

Principal component analysis of root primary metabolites for the three defoliation treatments (full leaf –

FL, 25% leaves – 25L and no leaf – NL), at each destructive harvest after the implementation of the

treatments (V+18, V+27, V+37 and V+46). A minimum convex polygon or convex hull (red

polygon) is included to define the treatment score space.


175

1. Input: Analysis based on the Correlation Matrix.

2. Eigenvalues or latent Roots (non-zero to 3 decimal places):

Eigenvalues 12 to 26 not included

1 2 3 4 5 6 7 8 9 10 11

Eigenvalue 9.819 4.688 3.638 2.855 1.711 1.092 0.752 0.602 0.449 0.275 0.120

3. Percentage Variation accounted for

1 2 3 4 5 6 7 8 9 10 11

%Variation 37.76 18.03 13.99 10.98 6.58 4.20 2.89 2.31 1.73 1.06 0.46


176 4.Ei

genv

ecto

rs o

r Lo

adin

gs

Clas

sifica

tion:

M

etab

olite

: 1

2 3

4 5

6 7

8 9

10

11

Suga

rs

Sucr

ose

-0.0

906

-0.0

006

0.40

03

-0.1

044

0.19

63

0.09

43

-0.4

566

-0.3

248

0.10

98

0.03

74

0.17

13

Raffi

nose

-0

.254

7 0.

1348

0.

0959

0.

1119

0.

1345

0.

0956

-0

.161

9 0.

4835

0.

0551

0.

0893

0.

1760

Mel

ibio

se

0.26

98

0.11

27

0.01

06

0.11

43

-0.0

581

0.22

55

0.11

48

0.00

99

-0.2

365

0.50

63

-0.4

199

Arab

inos

e 0.

2123

0.

0927

0.

0911

-0

.090

7 -0

.465

6 0.

0703

-0

.112

8 0.

3416

-0

.093

3 -0

.111

7 0.

0583

Suga

r alco

hols

Myo

-inos

itol

-0.2

894

0.02

45

0.13

12

-0.0

140

-0.1

263

-0.0

784

0.10

05

-0.0

866

0.32

65

0.09

15

-0.3

612

Gala

ctin

ol

-0.2

442

0.20

22

0.02

20

0.17

09

0.02

57

0.08

20

0.07

83

0.14

87

0.46

81

-0.1

447

-0.2

462

Man

nito

l -0

.135

1 0.

2254

-0

.012

7 0.

1397

-0

.391

4 0.

1691

0.

4981

-0

.209

5 0.

0605

-0

.195

3 0.

2573

Arab

itol

0.08

53

-0.3

150

-0.1

519

-0.2

798

-0.1

770

-0.0

503

-0.0

183

0.33

15

0.22

03

0.20

18

0.04

87

Glyc

erol

-0

.113

3 -0

.232

1 0.

2153

-0

.332

6 -0

.063

8 -0

.130

4 0.

3208

0.

2318

0.

0161

0.

0745

0.

0319

Amin

o ac

ids

Glut

amic

_aci

d -0

.241

1 0.

2495

-0

.006

8 0.

0470

0.

1922

-0

.240

9 0.

0486

-0

.036

8 0.

0285

0.

0297

-0

.108

5

Argi

nine

0.

2659

0.

1787

0.

0577

-0

.064

6 0.

1530

-0

.270

0 0.

0468

0.

0571

0.

0838

-0

.142

2 -0

.056

2

Glut

amin

e 0.

0542

-0

.048

4 0.

4780

0.

1168

-0

.166

8 0.

0688

0.

1363

-0

.103

6 -0

.184

9 -0

.061

4 -0

.045

0

Tryp

toph

an

0.26

73

0.03

50

0.23

95

0.14

30

-0.0

264

0.08

12

-0.0

952

0.05

61

0.03

04

-0.1

704

0.00

35

Phen

ylal

anin

e 0.

2391

-0

.195

7 0.

0889

0.

1367

0.

1104

0.

2493

0.

0184

0.

1998

0.

3570

-0

.068

9 0.

2120

Tyro

sine

0.18

29

-0.1

711

0.25

95

0.25

76

0.13

81

0.14

46

0.11

96

0.11

46

-0.0

314

-0.1

927

-0.3

546

Glyc

ine

-0.0

695

-0.3

207

-0.1

492

0.29

13

0.13

30

-0.1

846

0.30

47

-0.0

054

-0.1

370

0.05

85

0.06

65

Lysin

e 0.

2554

0.

1815

0.

0576

-0

.066

9 0.

1517

-0

.322

2 0.

1065

0.

0791

0.

0896

-0

.180

2 -0

.157

9

Thre

onin

e 0.

1308

-0

.193

2 0.

0785

0.

3990

-0

.171

4 -0

.268

6 -0

.188

8 -0

.168

5 0.

0347

0.

1385

0.

1123

Valin

e 0.

0287

-0

.380

3 -0

.029

3 0.

2705

0.

1769

-0

.087

0 0.

0942

-0

.012

5 0.

2565

0.

1163

0.

0023

Misc

ella

neou

s acid

s

Asco

rbic

_acid

-0

.209

7 0.

1311

0.

2922

0.

0044

0.

1994

0.

0704

0.

0825

0.

2487

-0

.223

5 0.

3751

-0

.005

8

Tart

aric

_acid

0.

2458

0.

2022

-0

.010

2 -0

.083

1 0.

2007

-0

.183

6 0.

2450

-0

.116

9 0.

0247

0.

2888

0.

3292

Citr

ic_ac

id

0.25

80

0.18

11

0.10

61

-0.1

141

0.09

49

-0.2

797

0.05

87

0.10

18

0.06

73

-0.0

954

-0.0

401

Mal

eic_

acid

0.

2183

-0

.008

0 -0

.046

0 -0

.316

0 -0

.010

0 0.

2957

0.

0619

-0

.309

7 0.

3747

0.

2472

-0

.156

4

3 Hy

drox

yant

hran

ilic a

cid

-0.0

592

-0.1

704

0.36

02

-0.2

502

0.21

57

0.13

17

0.29

87

-0.0

871

-0.0

315

-0.1

132

0.19

56

Prot

ocat

echu

ic_a

cid

-0.1

242

-0.3

304

-0.0

365

-0.2

965

0.02

95

-0.0

986

-0.0

728

-0.0

867

-0.2

399

-0.2

881

-0.3

113

2-Ke

to G

luco

nic

acid

-0

.089

6 -0

.054

5 0.

3283

0.

0021

-0

.400

0 -0

.436

9 -0

.087

7 -0

.060

4 0.

1516

0.

2316

-0

.008

6


177

5. Scores

Time_Treatment 1 2 3 4 5 6 7 8 9 10 11

T1_NL -1.5765 -3.2855 -0.6909 2.9007 0.0141 -1.9999 -0.0482 -0.0671 -0.3186 0.308 -0.1563

T2_NL 1.5895 -4.5492 0.0581 -0.596 1.1963 1.1299 1.0445 0.4201 0.5977 -0.3814 0.1591

T3_NL 4.4383 1.1417 3.1937 2.1682 -1.1573 0.3971 0.1549 0.0337 -0.6242 -0.6453 -0.0239

T4_NL 3.4055 -0.5346 0.4077 0.2756 -0.2211 0.6286 -1.4933 -0.7066 0.6207 0.8464 0.3655

T1_25L -0.2412 -0.6291 -0.6867 -1.9752 -3.0871 -0.1197 -0.0054 1.2281 0.0618 0.2578 -0.1359

T2_25L -0.5221 -0.8472 -2.7482 -1.4665 0.0055 0.4973 -0.7676 -0.8286 -1.2867 -0.5589 0.1093

T3_25L 3.7753 1.4292 -0.7637 -0.8197 1.7096 0.0926 -0.0049 0.243 -0.117 0.3794 -0.8061

T4_25L 2.7684 2.4369 -0.8056 -1.3935 0.6289 -2.0584 0.5956 0.0225 0.3906 -0.2949 0.4814

T1_FL -3.1123 1.0608 -0.5584 0.4658 -0.9542 0.175 0.0367 -1.1674 1.2463 -0.5847 -0.3735

T2_FL -2.1867 2.2389 -1.3433 1.3851 -0.1576 1.1241 1.6085 -0.2292 -0.3933 0.7152 0.2327

T3_FL -3.8363 1.7876 -0.2734 1.3461 1.2999 0.4971 -1.1781 1.4512 0.1938 -0.3241 0.1625

T4_FL -4.5019 -0.2496 4.2106 -2.2905 0.723 -0.3637 0.0574 -0.3998 -0.371 0.2825 -0.0148


178

Leaves PCA Analysis (using only data where treatment was significant at 5% for at least one time)

Principal component analyses of leaf primary metabolites for the three defoliation treatments (full leaf – FL, 25% leaves – 25L and no leaf – NL), at each destructive harvest after the implementation of thetreatments (V+18, V+27, V+37 and V+46). A minimum convex polygon or convex hull (red polygon)is included to define the treatment score space.


179

1. Input: Analysis based on the Correlation Matrix.

2. Eigenvalues or latent Roots (non-zero to 3 decimal places): 3. Eigenvalues or latent Roots (non-zero to 3 decimal places):

Eigenvalues 8 to 37 not included

1 2 3 4 5 6 7

Eigenvalue 19.558 5.339 4.557 3.593 2.027 1.290 0.635

4. Percentage Variation accounted for

1 2 3 4 5 6 7

%Variation 52.86 14.43 12.32 9.71 5.48 3.49 1.72


180

5. Eigenvectors or Loadings

Classification Metabolite 1 2 3 4 5 6 7 8

Sugars

Glucose 0.05381 0.33079 -0.01594 -0.19866 0.2327 0.10283 -0.38217 0.00219

Raffinose 0.14445 0.31981 -0.00858 0.02357 -0.01447 -0.10421 0.21392 0.07497

Melibiose 0.21706 0.00865 0.10766 0.04541 -0.05201 -0.09406 -0.03922 0.32557

Rhamnose 0.20081 0.15728 -0.04225 0.12726 0.03481 0.03101 0.12026 0.225

Melezitose 0.18479 0.21014 -0.05118 0.07387 0.01329 -0.08501 0.29454 -0.16781

Ribose -0.1763 0.24392 0.02686 -0.08148 0.04943 0.14339 0.15772 -0.00666

Sugar alcohols

Mannitol -0.16594 0.26431 0.07983 -0.08949 0.09189 0.03578 -0.13668 0.01946

Myo-inositol -0.20782 -0.02238 -0.14147 0.11935 -0.02417 0.05843 0.08528 -0.03326

Amino acids

Arginine -0.14035 -0.02498 0.28823 -0.06419 0.08223 0.28606 0.39431 -0.12982

GABA 0.21236 0.05229 0.02996 0.00068 0.22011 -0.02834 -0.01031 -0.05467

Serine 0.15167 -0.14713 -0.2695 -0.15336 0.05333 -0.08247 -0.0839 0.07941

Cysteine 0.19223 0.15413 0.12181 -0.00158 0.11636 -0.18285 0.13889 -0.00422

Valine 0.19677 0.01756 0.11437 0.11965 -0.18852 0.21141 -0.02349 -0.28563

Leucine 0.20738 -0.01456 0.13461 0.06387 -0.06596 0.20018 -0.00815 -0.24495

Isoleucine 0.21527 0.08121 0.04996 0.08633 0.01458 0.11818 0.05443 -0.01778

Phenylalanine 0.05423 -0.23828 0.29301 0.17414 -0.25373 -0.05964 0.07974 0.11112

Tryptophan 0.09496 -0.04163 0.30051 0.20041 -0.35261 0.07059 0.00999 0.0784

5-Hydroxytryptophan -0.19364 0.02586 -0.10521 0.22118 -0.08687 -0.12867 -0.01621 0.09981

Threonine 0.21026 -0.0946 -0.09164 -0.07118 0.11621 -0.05281 0.01478 -0.00915

Miscellaneous acids

Ascorbic_acid -0.17796 0.18059 -0.13327 -0.0621 -0.23382 0.02032 0.02313 0.40895

Tartaric_acid -0.16398 -0.0643 -0.18205 0.24326 0.07487 0.23911 0.07188 -0.03773

Threonic_acid -0.17535 0.21123 0.15868 -0.02677 0.00161 -0.1807 -0.03965 -0.04779

Glyceric_acid -0.17847 0.04999 0.15241 0.21036 -0.01914 -0.24949 -0.16762 -0.44368

Caffeic_acid -0.17726 -0.16657 -0.07047 0.13546 0.14613 0.28517 -0.03017 0.07324

Gallic_acid -0.15784 0.28989 0.01702 -0.0878 -0.11277 0.07493 0.05566 -0.16274

Lactic_acid -0.04948 -0.12734 0.23024 0.21894 0.40448 -0.27656 0.18375 0.08809

Citric_acid -0.18736 -0.0085 -0.10568 0.24807 0.04173 0.16515 0.05987 0.04003

Fumaric_acid -0.08571 -0.04889 0.33218 -0.26462 0.11965 0.13496 0.24001 0.26634

2-Ketoglutaric acid -0.11325 0.26538 -0.16072 0.01885 -0.33159 -0.09349 0.17722 -0.06427

Phosphoric_acid 0.01702 -0.1137 0.08621 -0.36855 -0.39039 -0.26848 0.04802 -0.05084

Gluconic_acid -0.20098 0.12582 0.09876 0.08837 -0.03167 -0.1723 -0.14101 0.1549

Ribonic_acid -0.13394 0.09879 0.15856 0.31908 0.10198 -0.2721 0.00143 0.12845

Nonanoic_acid -0.11691 0.14679 0.28151 -0.24567 0.09127 0.13187 0.02173 -0.04756

Palmitic_acid 0.08669 0.10435 0.31532 0.15049 -0.10445 0.26767 -0.4792 0.19256

Other compounds

Arbutin 0.20182 0.169 -0.01144 0.05224 0.13144 0.00489 0.09239 0.11068

Catechin 0.15011 0.1835 -0.17142 0.18226 -0.15247 0.21928 0.16397 0.13006

Glycerol monostearate 0.16757 0.21801 -0.01884 0.22606 0.02691 -0.06087 -0.0933 -0.12568


181

6. Scores

Time_Treatment 1 2 3 4 5 6 7

T1_25L -0.1092 -2.1327 -4.1121 -1.2603 -0.9652 -0.6701 -0.4453

T2_25L 2.2436 -2.8131 2.7636 0.3146 -1.926 0.9644 -0.2882

T3_25L 3.5286 -1.7166 1.0721 -2.0486 2.659 -0.1031 0.1135

T4_25L 8.3158 2.9697 -0.9956 1.5239 -0.2611 0.0392 0.1574

T1_FL -4.4244 -0.2239 -1.5675 1.7802 1.0028 1.9414 0.418

T2_FL -2.868 -1.0011 1.0163 2.4751 0.1714 -1.9522 0.6254

T3_FL -3.5698 2.4148 1.0846 -0.0186 0.4157 -0.2368 -1.5929

T4_FL -3.1167 2.5029 0.7387 -2.7662 -1.0965 0.0172 1.0121


182

Figure S3: Linear and curvilinear relationship between root starch and myo-inositol concentrations as

estimated by the linear mixed model. While the model indicates that the spline curvature is not

statistically significant, when a linear trend is fitted, the residuals do not appear to be random with fitted

values in the range 0.884 to 1.587 consistently smaller than observed values, and fitted values in the

range 3.076 to 4.211 consistently larger than observed values. NL: no leaf treatment, 25L: 25 leaves

treatment, FL: full leaf treatment, V+9: 9 days after the onset of véraison.

Appendix B – Supplementary table S1

183

Day

s af

ter

vera

iso

n

Me

tab

oli

te a

bu

nd

ance

pe

r b

err

yTr

eat

me

nt

valu

eSD

EVva

lue

SDEV

valu

eSD

EVva

lue

SDEV

valu

eSD

EVR

T (m

in)

m/z

Ke

gg ID

stan

dar

d id

en

tifi

cati

on

NIS

T li

bra

ry c

om

po

un

dR

ete

nti

on

ind

ex

FL0.

313

0.0

54

0.5

55

0.08

00.6

41

0.08

10.

850

0.16

60.

982

0.1

69

25L

0.31

30.0

54

0.42

00.

064

0.45

70.

062

0.52

40.

013

0.65

50.0

80

NL

0.31

30.

054

0.40

30.

058

0.39

50.

075

0.51

70.

035

0.53

40.144

FL0.

336

0.0

49

0.6

23

0.11

80.

599

0.1

79

0.8

34

0.39

80.

922

0.2

34

25L

0.33

60.0

49

0.42

90.

032

0.32

70.2

88

0.51

90.

107

0.67

50.1

42

NL

0.33

60.0

49

0.42

60.

075

0.46

10.1

24

0.52

10.

031

0.56

20.1

06

FL0.

008

0.00

90.0

17

0.01

30.

005

0.00

50.0

23

0.02

80.

018

0.030

25L

0.00

80.0

09

0.00

90.

012

0.01

50.0

10

0.03

30.

030

0.02

50.0

40

NL

0.00

80.

009

0.02

50.

038

0.03

10.

022

0.01

80.

019

0.05

10.024

FL0.

146

0.02

60.

346

0.05

90.

473

0.20

40.

843

0.27

10.

977

0.4

17

25L

0.14

60.

026

0.10

20.

016

0.14

50.

036

0.29

60.

091

0.26

50.1

30

NL

0.14

60.

026

0.12

70.

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1288

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N,O

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35

1336

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7*

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13

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18.5

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914

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1 *

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trim

eth

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128

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237

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ito

l, t

etr

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thyl

sily

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the

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38.8

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437

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N/A

2798

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04; 1

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3

3834

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fura

no

se, p

en

taki

s(tr

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thyl

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l) e

the

r (i

som

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3.56

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45 C

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MS)

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1980

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6TM

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4TM

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1260

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2130

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cell

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ou

s ac

ids:

ASC

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0007

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Asc

orb

ic a

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MS)

1870

1956

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LIN

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ine

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TMS

1242

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280

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MS)

2230

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18; 1

46C

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7L-

5-H

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ph

an, t

rim

eth

ylsi

lyl e

the

r, t

rim

eth

ylsi

lyl e

ste

r23

23

TRYP

TOP

HA

N30

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1; 2

02C

0007

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Tryp

top

han

, N,N

,O-t

riTM

S

1414

THR

EON

INE

15.5

218

C00

188

*Th

reo

nin

e (

N,O

,O-T

MS)

1433

SER

INE

14.9

204;

218

C00

065

*Se

rin

e, (

3TM

S)

1313

PYR

OG

LUTA

MIC

AC

ID (

5-o

xo-P

roli

ne

)18

.315

6C

0187

9P

yro

glu

tam

ic a

cid

, (N

,O-T

MS)

1532

PR

OLI

NE

13.3

216;

147

; 158

C00

148

*P

roli

ne

, di-

TMS


186

FL2.

394

0.51

72.6

89

0.17

01.

589

0.33

61.0

57

0.36

20.

525

0.392

25L

2.39

40.5

17

2.40

90.

328

2.07

20.2

45

1.26

60.

204

1.10

40.0

70

NL

2.39

40.5

17

3.10

40.

637

2.17

00.1

44

1.45

10.

148

1.33

80.1

36

FL0.

094

0.01

30.1

41

0.00

80.

155

0.02

80.2

00

0.06

70.

272

0.071

25L

0.09

40.0

13

0.12

00.

015

0.13

10.0

14

0.14

20.

021

0.15

10.0

27

NL

0.09

40.

013

0.11

30.

016

0.11

50.

015

0.11

40.

013

0.15

30.032

FL0.

104

0.03

60.0

00

0.0

00

0.0

00

0.0

00

0.0

00

0.0

00

0.0

00

0.0

00

25L

0.10

40.

036

0.03

80.

037

0.00

00.

000

0.00

00.

000

0.00

00.000

NL

0.10

40.0

36

0.0

11

0.01

90.

000

0.0

00

0.0

00

0.00

00.

000

0.0

00

FL12

.147

2.29

912

.242

7.35

312

.794

5.68

318

.448

5.71

915

.865

10.8

85

25L

12.1

472.

299

4.26

91.

260

3.84

32.

229

11.4

142.

884

8.58

72.4

42

NL

12.1

472.

299

12.9

449.

394

2.20

20.

872

5.23

31.

134

7.49

53.4

76

FL1.

343

0.13

90.

364

0.23

00.

112

0.02

50.

154

0.04

70.

135

0.0

43

25L

1.34

30.

139

0.16

60.

049

0.02

30.

020

0.02

80.

048

0.02

20.0

19

NL

1.34

30.

139

0.14

80.

045

0.01

80.

016

0.01

30.

023

0.00

00.0

00

FL0.

025

0.00

60.0

36

0.01

00.

045

0.01

70.0

56

0.01

10.

046

0.008

25L

0.02

50.0

06

0.04

90.

032

0.04

60.0

28

0.05

60.

011

0.04

20.0

04

NL

0.02

50.

006

0.02

80.

008

0.04

20.

025

0.03

90.

012

0.03

70.007

FL0.

102

0.0

20

0.1

42

0.03

80.

154

0.0

78

0.2

41

0.11

30.

129

0.0

46

25L

0.10

20.0

20

0.17

50.

075

0.16

00.0

79

0.19

30.

053

0.13

20.0

26

NL

0.10

20.

020

0.1

01

0.01

30.

131

0.05

80.1

40

0.04

40.

093

0.021

FL0.

046

0.03

40.0

52

0.02

00.

028

0.03

00.0

25

0.00

60.

014

0.025

25L

0.04

60.0

34

0.10

30.

062

0.03

70.0

28

0.02

30.

026

0.03

60.0

13

NL

0.04

60.0

34

0.04

40.

034

0.03

90.0

18

0.05

00.

037

0.02

10.0

20

FL0.

023

0.02

60.0

09

0.00

90.

049

0.03

50.0

60

0.04

60.

033

0.021

25L

0.02

30.0

26

0.10

20.

072

0.01

00.0

13

0.02

80.

004

0.01

20.0

17

NL

0.02

30.

026

0.04

50.

041

0.02

20.

013

0.00

80.

009

0.01

80.019

FL1.

255

1.2

06

1.7

95

1.77

00.

895

0.3

58

1.8

31

0.48

91.

436

0.3

73

25L

1.25

51.2

06

2.27

61.

597

1.08

20.2

35

1.16

20.

096

1.01

20.2

61

NL

1.25

51.2

06

1.07

40.

460

0.90

70.3

00

1.27

00.

667

0.63

30.3

07

FL1.

430

0.9

80

1.9

13

1.42

41.

383

0.5

52

2.4

39

0.80

21.

723

0.3

93

25L

1.43

00.9

80

2.77

71.

753

1.63

10.7

42

1.74

70.

309

1.48

40.4

07

NL

1.43

00.9

80

1.45

60.

715

1.26

80.4

39

2.00

91.

388

0.89

40.3

65

FL0.

048

0.02

10.0

43

0.02

90.

092

0.01

80.0

91

0.10

60.

042

0.073

25L

0.04

80.0

21

0.01

40.

019

0.01

40.0

25

0.09

60.

011

0.09

10.0

32

NL

0.04

80.

021

0.00

90.

008

0.03

20.

021

0.03

00.

026

0.03

40.028

FL8.

041

1.3

72

6.3

17

1.05

33.

488

0.4

19

3.1

15

0.58

12.

889

1.2

19

25L

8.04

11.3

72

5.76

40.

552

2.65

80.4

15

2.21

30.

352

1.89

90.3

16

NL

8.04

11.

372

6.60

62.

026

2.96

50.

235

1.61

60.

272

2.05

80.238

FL7.

397

0.35

911.4

90

1.82

814

.009

2.11

716.7

81

1.26

318

.887

5.380

25L

7.39

70.3

59

10.2

692.

235

11.4

061.2

37

14.9

950.

936

13.6

231.8

57

NL

7.39

70.3

59

9.22

21.

683

9.49

31.7

41

9.19

90.

855

10.6

660.5

11

FL0.

186

0.06

80.2

81

0.06

60.

363

0.07

10.4

00

0.09

70.

518

0.293

25L

0.18

60.0

68

0.16

00.

057

0.19

00.0

26

0.32

40.

075

0.28

00.0

37

NL

0.18

60.0

68

0.28

60.

083

0.20

60.0

14

0.09

40.

126

0.28

60.0

70

FL0.

001

0.00

30.0

01

0.00

10.

001

0.00

10.0

00

0.00

10.

000

0.001

25L

0.00

10.0

03

0.00

00.

000

0.00

10.0

01

0.00

10.

000

0.00

80.0

13

NL

0.00

10.

003

0.00

00.

000

0.00

00.

000

0.00

10.

000

0.00

00.001

FL3.

729

0.2

40

5.5

81

0.97

36.

597

1.6

82

8.6

52

1.94

48.

219

1.8

16

25L

3.72

90.2

40

5.21

21.

520

5.51

80.8

80

6.93

60.

773

6.16

00.4

97

NL

3.72

90.2

40

4.27

90.

428

4.71

21.1

05

4.55

80.

683

4.64

60.2

25

Ad

ipic

aci

d18

147;

275

C06

104

*A

dip

ic a

cid

, (2T

MS)

1528

Ad

on

ito

l (R

ibit

ol)

22.8

147;

217

; 103

C00474

*R

ibit

ol,

5TM

S17

60

L-h

ydro

xyp

roli

ne

18.5

230

C01

157

*3-

Hyd

roxy

pro

lin

e, N

,O,O

'-tr

is(t

rim

eth

ylsi

lyl)

-15

42

Inte

rnal

sta

nd

ard

s:

3108

1-M

ON

OP

ALM

ITIN

35.9

371

N/A

1-M

on

op

alm

itin

tri

me

thyl

sily

l eth

er

2602

KA

EMP

FER

OL

42.0

559

C05

903

Kae

mp

fero

l, 4

TMS

2791

HYD

RO

XYL

AM

INE

9.0

133

C00

192

Hyd

roxy

lam

ine

, N,N

,O-t

ris-

TMS

1135

GLY

CER

OL

MO

NO

STEA

RA

TE38

.439

9N

/AG

lyce

rol m

on

ost

ear

ate

, 2tm

s d

eri

vati

ve

2889

CA

TEC

HIN

39.5

; 39.

8 a

368

C06

562

Cat

ech

ine

, pe

nta

-TM

S-e

the

r, (

2R-c

is)-

Mis

call

en

eo

us

com

po

un

ds:

AR

BU

TIN

36.2

254

C06

186

Hyd

roq

uin

on

e-β

-d-g

luco

pyr

ano

sid

e,p

en

taki

s(tr

ime

thyl

sily

l)-

2626

STEA

RIC

AC

ID30

.911

7; 3

41C

0153

0St

ear

ic a

cid

, tri

me

thyl

sily

l est

er

2237

2209

PA

LMIT

IC A

CID

27.8

117;

313

C00

249

Pal

mit

ic a

cid

, TM

S20

39

OLE

IC A

CID

30.5

339

C00

712

Ole

ic a

cid

, tri

me

thyl

sily

l est

er

2407

NO

NA

NO

IC A

CID

14.6

117;

215

C01

601

No

nan

oic

aci

d, T

MS

est

er

1405

MYR

ISTI

C A

CID

33.3

343

C06

424

Myr

isti

c ac

id, 2

,3-b

is(t

rim

eth

ylsi

loxy

)pro

pyl

est

er

Fatt

y ac

ids:

EIC

OSA

NO

IC A

CID

40.6

427

C06

425

Eico

san

oic

aci

d, 2

,3-b

is[(

trim

eth

ylsi

lyl)

oxy

]pro

pyl

est

er

2990

1666

THR

EON

IC A

CID

19.4

147;

292

; 220

C01

620

L-Th

reo

nic

aci

d, t

ris(

trim

eth

ylsi

lyl)

eth

er,

tri

me

thyl

sily

l est

er

1586

TAR

TAR

IC A

CID

21.0

292

C00

898

Tart

aric

aci

d, T

MS

1831

SUC

CIN

IC A

CID

13.7

147;

247

C00

042

Succ

inic

aci

d (

2TM

S)13

44

PR

OTO

CA

TEC

HU

IC A

CID

24.1

193;

370

C00

230

*P

roto

cate

chu

ic a

cid

(tm

s)

PYR

UV

IC A

CID

13.6

73; 1

47C

0002

2P

yru

vic

acid

oxi

me

, bis

(tri

me

thyl

sily

l)-

de

riv.

1335

a M

ore

th

an o

ne

pe

ak c

hro

mat

ogr

aph

ic p

eak

s e

lute

d f

or

eac

h t

he

se m

eta

bo

lite

s.Th

e c

om

bin

ed

pe

ak a

reas

we

re u

sed

to

cal

cult

e t

he

me

tab

oli

te a

bu

nd

ance

.

Figure S1 Principle component analysis of the significantly affected berry primary metabolites for the three defoliation treatments (full leaf – FL, 25% leaves – 25L and no leaf – NL), at each destructive harvest after the implementation of the treatments (V+18, V+27, V+37 and V+46). A minimum convex polygon or convex hull (red polygon) has been included to define the treatment score space.

Appendix B – Supplementary figure S1

187

Appendix B – Supplementary figure S1

188

Pri

nci

pal

co

mp

on

ent

anal

ysis

(P

CA

) la

ten

t ve

cto

r lo

adin

gs o

f th

e d

iffe

ren

t b

erry

met

abo

lites

sig

nif

ican

tly

affe

cted

by

the

trea

tmen

ts d

uri

ng

the

exp

erim

enta

l per

iod

.

PC

1P

C2

1-M

onop

alm

itin

0.18

178

-0.0

0041

3α-M

anno

bios

e0.

1871

0.02

784

Ara

bino

fura

nose

0.18

744

-0.0

0168

Ara

bito

l-0

.015

79-0

.179

52A

rbut

in0.

1142

-0.0

9782

Arg

inin

e-0

.016

73-0

.292

44A

spar

agin

e-0

.114

860.

1908

8A

spar

tic a

cid

-0.0

6385

-0.0

3286

Ben

zoic

aci

d0.

1761

3-0

.011

45C

affe

ic a

cid

0.03

690.

2813

5C

atec

hin

-0.0

5034

0.31

738

Cel

lobi

ose

0.18

795

0.02

869

cis

-Inos

itol

0.06

622

-0.1

1093

Citr

ic a

cid

0.07

354

0.25

846

Dul

cito

l0.

1507

6-0

.151

18Fr

ucto

se0.

1743

8-0

.058

95Fu

cose

0.19

126

0.01

827

Fum

aric

aci

d-0

.131

270.

2272

3G

alac

tinol

-0.0

6606

-0.1

4315

Gal

lic a

cid

0.17

321

0.12

873

Glu

coni

c ac

id0.

1118

10.

2484

1G

luco

se0.

1787

5-0

.056

74G

luta

mic

aci

d0.

1774

6-0

.076

72G

lyce

ric a

cid

-0.1

4901

0.12

735

Gly

cero

l mon

oste

reat

e0.

1846

1-0

.019

77H

ydro

xyla

min

e0.

1708

60.

0550

4La

ctic

aci

d0.

1641

10.

0275

8M

alic

aci

d-0

.051

0.28

287

myo

-Inos

itol

0.16

940.

1282

Pro

line

0.17

55-0

.058

95P

roto

cate

chui

c ac

id-0

.142

870.

2091

8P

yrog

luta

mic

aci

d0.

1409

9-0

.135

51P

yruv

ic a

cid

0.18

045

0.00

619

Raf

finos

e0.

0133

4-0

.053

3R

ham

nose

-0.1

5514

-0.0

231

Suc

rose

0.17

284

-0.0

4049

Taga

tose

0.19

032

0.02

443

Tarta

ric a

cid

0.15

558

0.13

678

Thre

onic

aci

d0.

0325

30.

3059

7Th

reon

ine

-0.0

5206

-0.2

1073

Treh

alos

e0.

1822

30.

0462

8Tr

ypto

phan

-0.1

594

-0.1

0485

Unk

now

n ol

igos

acch

arid

e 1

-0.0

382

-0.1

651

Unk

now

n su

gar 1

0.18

948

0.02

395

Unk

now

n su

gar 2

0.18

905

0.03

217

Unk

now

n su

gar 3

0.18

854

0.03

777

β-a

lani

ne0.

1890

30.

0018

4

grapevine carbohydrate and nitrogen allocation during ...€¦ · grapevine carbohydrate and...

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