comparative characterization of olive prolyl 4 hydroxylases and … · 2018-10-03 · aristotelis...

Aristotelis Azariadis1, Konstantinos Blazakis1, Mohamed Kouhen1, Khansa Mekkaoui1, Georgios Kostelenos2, and Panagiotis Kalaitzis1

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1Dept of Horticultural Genetics Biotechnology, Mediterranean Agronomic Institute of Chania (MAICh (CIHEAM))

2Kostelenos Olive Nurseries

Comparative characterization of olive prolyl 4hydroxylases and cell wall glycoproteins in Greekolive cultivars under climate change conditions

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Fig. 1The Mediterranean countries, their Mediterranean regions and their different limits(Source: Gaussen & De Philippis – FAO, 2003)

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140000

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Salinity in the Meditarrenean

Population(in thousands)

Availability(m3/inh.year)

Fig. 2Availability of water in the Mediterranean(Source: N.V. Paranychianakis, K.S. Chartzoulakis, 2005)

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2005

Fig. 4 Gravimetric assay of arabinogalactan proteins (AGPs) isolated fromgrowth medium of salt-adapted compared with non-adapted controls. This enablesa comparison of AGPs released by non-adapted and salt-adapted cells. Data fromsingle representative experiments. Solid columns, salt-adapted; hatched columns,non-adapted controls.

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Fig. 3 A, B. Localization of AGPs in cultured tobacco cells with 13-glucosylYariv reagent staining. Yellowish-red color is due to Yariv-reagent staining. A So cells10 d after subculture. Insert shows a plasmolyzed cell with Yariv-reagent stainingthe protoplast surface. B S25 cells 15 d after subculture.

Alterations in expression and/or suppression of expression of plant P4Hs

Alterations in frequency of occurrence of proline hydroxylation events in substrate proteins

MECHANISM OF ACTION

Direct or indirect alterations in abiotic stress responses such as salinity

STRUCTURAL ALTERATIONS √ - VARIATION IN PROTEIN CONTENT √ - DEGRADATION ?

TFs (?) Hormone Peptides (?) AGP/Ext proteins √

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Fig. 5Leaf K/Na ratios of nine indigenous olive cultivars grown with 0, 50, 100 and 200mM NaCl. 5

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Salinity (120 mM NaCl) and Heat stress

Barrels of 150 Lt and 90 cm height

Koroneiki

Gaidourolia

Lefkolia

Arvanitolia

The facility

Fig. 6The greenhouse facilities of MAICh (CIHEAM)

Fig. 7Layout of the experimentalgreenhouse facility in thepremises of MAICh(CIHEAM)

The facility

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Temperature and Relative Humidity

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Rel

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idit

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Max Temperature: 44.03 oCMin Temperature: 21.53 oCAv. Temperature: 31,28 oC

Max Rel. Hum.: 85,81 %Min Rel. Hum.: 16,68 %Av. Rel. Hum.: 49,22 %

Fig. 8 Temperature and Relative Humidity

June July August June July August

The facility

Data collected from period: 21.06.18 – 09.08.18

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a. 90D Control

The morphological analysis was performed by oliveId

b. 90D Salinity

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45DAYS 90DAYS

Leaf

are

a(cm

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KORONEIKI

control

salinity

oliveId

Fig. 10In 90 days the average leaf area of plants thatwere submitted to salinity stress, was reducedby almost 29% in relation to the control plants.

Phenomics

Fig. 9Canopy of olive trees: a. control plants;b. salinity treated for 90 days.

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Fig. 12Dendrogram prepared with the deduced AGP amino acidsequences from olive. The dendrogram was constructedusing MATLAB_R2014b.

Arabinogalactans (AGPs) & Prolyl-4-Hydroxylases (P4Hs)in Olive (O. Europea)

Fig. 11Dendrogram prepared by comparison of the deduced P4Hamino acid sequences from olive, arabidopsis and tomato.The dendrogram was constructed using MATLAB_R2014b.

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P4H4 gene expression in olive roots

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0D 45D 90D

Arvanitolia

control

salinity

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Gaidourolia

control

salinity

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0D 45D 90D

Koroneiki

control

salinity

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Lefkolia

control

salinity

P4H4Fig. 13P4H4 gene expression in olive roots

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0D 45D 90D

Arvanitolia

control

salinity

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Gaidourolia

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salinity

P4H7 gene expression in olive roots

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Koroneiki

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Lefkolia

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salinity

P4H7Fig. 14P4H7 gene expression in olive roots

Monoclonal Antibody

Plant of Origin

AntigenEpitope Structure for Carbohydrate Antigen

LM2 Rice

Arabinogalactan

beta-linked glucuronic acid

JIM13 Carrot (beta)GlcA1->3(alpha)GalA1->2Rha

MAC207 Pea (beta)GlcA1->3(alpha)GalA1->2Rha

The Antibodies

Fig. 15The antibodies that were used in order to bind, detect and quantify arabinogalactan proteins (AGPs)Source: E.A. Yates, J. F. Valdor, S.M. Haslam, H.R. Morris, A. Dell, W. Mackie, and J.P. Knox. (1996)

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0 45 90 0 45 90

CONTROL SALINITY

ARVANITOLIA

LM2

ACTIN

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MW

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36

0 45 90 0 45 90

CONTROL SALINITY

GAIDOUROLIA

0 45 90 0 45 90

CONTROL SALINITY

KORONEIKI

0 45 90 0 45 90

CONTROL SALINITY

LEFKOLIA

JIM13

ACTIN

250

130

95

72

MW

55

36

MAC207

ACTIN

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95

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MW

55

36

Fig. 16 Western Blots of AGPs’ content.

Conclusions

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• P4Hs activity is affected by abiotic stress

• The characterization of the molecular response to salinity and heat stress might lead to thedevelopment of new breeding strategies.

• AGPs content is affected by abiotic stress

• Direct or indirect alterations of plant’s responses

Thank YouChania Alsyllio Agrokepio, 1 Makedonias str Chania 73100 Crete GREECE, Greece

a.azariadis@maich.gr

0030 2821035000Aristotelis Azariadis

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