factors affecting isoflavone concentration in soybean glycine max l
TRANSCRIPT
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Factors Affecting Isoflavone Concentration
in Soybean Glycine max L.
By
Abdel Rahman Al-Tawaha
Department ofPlant Science
c
Gill University, Macdonald Campus
Ste-Anne-de-Bellevue, QC, Canada
June 2006
A thesis submitted to the Office of Graduate and Postdoctoral Studies in partial
fulfillment of the requirements for the degree
of
Doctor ofPhilosophy
© Abdel Rahman AI-Tawaha 2006)
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1 1
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Dedication
To
the
Soul
o
y
Father Mohamed
Said AI-
Tawaha
To the Soul o
y Aunt
Khdeja Said AI-Tawaha
To li
the Honest
and Hardworking
People
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Contribution
o
authors
This thesis has been written in the form
o
manuscripts which have been submitted to
scientific joumals. This format has been approved by the Office
o
Graduate and Post
doctoral Studies as outlined in Guidelines for Thesis Preparation .
This thesis contains four manuscripts prepared by myself and Prof. Philippe
Seguin. Contributions o co-authors are described in this section. The first author on each
o the manuscripts is myself. The second co-author on each
o
the four manuscripts is my
supervisor; Prof. Philippe Seguin (Department
o
Plant Science, McGill University), who
provided supervision, funding throughout this research, technical assistance, and valuable
suggestions throughout the work, and corrected the resulting manuscripts. Prof. D.
L
Smith (Department
o
Plant Science, McGill University) is a co-author on the
manuscripts contained in chapters
4
5 and 6; he provided valuable suggestions and
corrected the resulting manuscripts. Prof. C. Beaulieu (Department ofBiology, Université
Sherbrooke) is a co-author on the manuscripts contained in chapters 5 and 6; she provided
biological materials and corrected the resulting manuscripts. Prof. B. BonneIl
(Department o Bioresource Engineering, McGill University) is a co-author on the
manuscript contained in chapters 4; he provided valuable suggestions and corrected the
resulting manuscript.
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ummary
Soybean [ lycine max L.) Merr.] seeds contain isoflavones that have positive impacts on
human health. Field and greenhouse experiments were conducted in Québec Canada to
determine the effects of management and environmental factors [seeding date (late May
and mid June), row spacing (20-, 40- and 60-cm), weeds (presence or absence), irrigation
levels (low, moderate, and high) and genotypes (Proteina, Orford, and Golden)] and of
foliar applications of elicitor compounds (i.e., LCOs, chitosan, and actinomycetes spores),
on the isoflavone concentrations of mature soybean seeds, and other important seed
characteristics. Our results indicated that environmental and agronomical factors have a
great impact on soybean seed isoflavone concentrations of early maturity soybean
cultivars. Year, seeding date, and weeds affected total and individual isoflavone
concentrations, row spacing had no effect. Total isoflavone concentration was greater in
2003 than 2004. Seeding in mid June increased isoflavone concentration by 38 ,
compared to seeding in May. The presence
ofwee s
increased total isoflavone
concentrations by 9 . Isoflavone concentrations were significantly affected by cultivars
and irrigation levels. In both of two growing seasons, Proteina had significantly greater
isoflavone concentrations compared to Orford. Irrigation effects on isoflavone
concentrations differed between years and cultivars. However, most responses were
observed with the lower of the two irrigation levels, which increased isoflavone
concentrations by as much as 60 compared to a non-irrigated control. Our results
suggest that under greenhouse conditions most biotic elicitors tested increased the
concentration
of
individual and total isoflavones in soybean seeds when compared to
untreated control plants. LCOs proved to be the most effective in studies contrasting
IV
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various elicitors. Response o field grown plants was more variable than that
o
greenhouse grown plants.
v
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ésumé
La fève de soya [ lycine max
L.)
Merr.] contient des isoflavones qui ont un effet positif
sur la santé humaine. Des expériences en champs et en serre ont été menées au Québec,
Canada, pour déterminer les effets d'applications foliaires de composés éliciteurs (LCO,
chitosane, et spores d'actinomycètes) et de la régie [date de semis (fin mai et mi-juin),
espacement entre les rangs (20, 40 et 60 cm), présence ou absence de mauvaises herbes,
niveau d'irrigation (bas, moyen, et élevé) et du génotype ('Proteina', 'Orford', et
'Golden')] sur la concentration en isoflavone des graines matures de soya, ainsi que sur
d'autres caractéristiques importantes, dont le rendement. Nos résultats indiquent que la
régie et les conditions environnementales ont un impact majeur sur la concentration en
isoflavone des graines de soya de cultivars hâtifs. L'année de croissance, la date de semis
et la présence de mauvaises herbes affectent la concentration de certains isoflavones ainsi
que la concentration totale. L'espacement entre les rangs n a eu aucun effet. La
concentration totale en isoflavones était plus élevée en 2003
qu en
2004. La concentration
en isoflavone était supérieure de 38 pour le semis de mi-Juin, comparativement au semi
de fin-mai. La présence de mauvaises herbes a augmenté la concentration totale en
isoflavones de 9 . La concentration en isoflavone a été affectée de façon significative par
les différents cultivars et les niveaux d'irrigations. Lors des deux saisons e croissance, le
cultivar 'Proteina' a produit la concentration en isoflavone la plus élevée et le cultivar
'Orford' la plus basse. La réponse à l'irrigation a été plus fréquente avec
le
plus faible de
deux niveaux d'irrigation, qui a augmenté la concentration en isoflavones jusqu'à 60
lorsque comparé a un contrôle non-irrigué. Nos résultats suggèrent que sous les
conditions de serre, tous les éliciteurs biotiques testés ont causé une augmentation de la
concentration de daidzein, genistein, glycitein, ainsi que la concentration totale
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d isoflavone, lorsque comparé au contrôle non-traité. a réponse aux traitements en
champs était plus variable que celles des plantes en serre.
vu
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cknowledgments
l acknowledge and thank many people for their help with this project. In
particular l would like to give great thanks to my supervisor Dr. Philippe Seguin who
gave me the opportunity to work on this project and provided excellent support and
guidance through the duration o the work and especially when it came time to write up
the papers. l would like to thank the members o my supervisory committee Dr. Alan
Watson and Dr. Don Smith for their time and precious advice during the past 3 years. l
am very grateful to Drs. A Souleimanov and W Zheng Mr. R Smith Mr. Jim
Straughton and Ms. Amélie Désilets-Roy for their help. l am very grateful to Dr. B.
Bonnell and his lab members especially Dr. Taher Waheed for their help through the
duration o the work in the irrigation experiment. l would also like to thank Mr. Bruce
Gelinas for translation o the thesis abstract to French. l would like to thank the secretarial
staffo the Department o Plant science. And finally l would like to thank my family and
their support and encouragement.
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Table of Contents
SUMMARY
. . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . .
IV
RÉSUMÉ
................................................................................................................... VI
ACKNOWLEDGMENTS
.............................................................................................
VIII
TABLE
OF
CONTENTS
. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . .
IX
LIST OF
FIGURES
...................................................................................................
XIII
LIS
T 0 F
TABLES
XIV
LIST OF APPENDICES . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . .
XVIII
1 0 GENERAL INTRODUCTION 1
2 0
LITERATURE RE
VIEW 4
2 1
SOYBEAN AGRONOMY 4
2 2
NUTRACEUTICALS: DEFINITION
: 5
2 3 BENEFITS AND IMPORTANCE OF SOYBEAN ISOFLAVONE ........................................ 6
2 4
FUNCTIONS OF ISOFLAVONES
IN PLANTS 8
2 5 FACTORS
AFFECTING ISOFLAVONES CONCENTRATION
IN PLANTS 9
2 5 1 GENETIC FACTORS
. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . .
9
2 5 2
CORRELATION
BETWEEN ISOFLAVONES
CONCENTRATIONS
AND OTHER
IMPORTANT AGRONOMIC
F ATORS
.................. .............................................. 11
2 5 3
ENVIRONMENTAL AND MANAGEMENT
FACTORS
..................................................
12
2 5 3 1
TEMPERATURE . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . .
12
2 5 3 2 WATER STRESS
..................................................................................... 13
2 5 3 3 SOIL
FERTILITY
AND FERTILIZATION
..........................................................
14
2 5 3 4 LIGHT . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . ..
15
2 5 3 5 PEST
PRESSURE ...................................................................................
16
2 5 3 6
NATURAL
INDUCERS
. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . .
16
2 5 3 7
ROLE
OF
INDUCERS AND
FLAVONOIDS IN
PLANT
RESISTANCE
..................
17
2 5 3 8
PLANT MATURITY
. . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . .. .
19
PREFACE TO CHAPTER
3
..............................................................................................
20
CHAPTER3. EFFECTS OF
SEEDING
DATE ROW SPACING AND WEEDS ON SOYBEAN
ISOFLA
VONE
CONCENTRATIONS
.......................
'
............................................
21
3 1
SUMMARY. . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . .. . .
21
3 2 INTRODUCTION
.................................................................................................
22
IX
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3.3. MATERIAL AND METHODS 24
3.3.1. SITE DESCRIPTION AND MANAGEMENT 24
3.3.2. ISOFLAVONE EXTRACTION AND HPLC ANALySES
25
3.3.3. OTHER VARIABLES MEASURED 26
3.3.4. STATISTICAL ANALySES 26
3.4. RESULTS AND DISCUSSION 27
3.4.l . CLIMATE DATA 27
3.4.2. ISOFLAVONE CONCENTRATIONS 28
3.4.3. SEED YIELD 30
3.4.4. CRUDE PROTEIN AND OIL CONCENTRATIONS 31
3.4.5. ISOFLAVONES AND CRUDE PROTEIN YIELDS 32
3.4.6. CORRELATIONS BETWEEN ISOFLAVONE CONCENTRATIONS AND OTHER SEED
CHARACTERISTICS
33
3.4.7. CONCLUSIONS 34
PREFACE TO CHAPTER 4 42
CHAPTER
4
EFFECTS
OF
IRRIGATION ON ISOFLAVONE CONCENTRATIONS
OF
SOYBEAN GROWN IN SOUTHWESTERN QUÉBEC
43
4.1. SUMMARY :
43
4.2. INTRODUCTION 44
4.3. MATERIAL AND METHODS 46
4.3.1. SITE DESCRIPTION AND MANAGEMENT 46
4.3.2. IRRIGATION TREATMENTS 47
4.3.3. ISOFLAVONE EXTRACTION AND HPLC ANALySES .47
4.3.4. OTHER VARIABLES MEASURED .48
4.3.5. STATISTICAL ANALySES 49
4.4. RESULTS AND DISCUSSION 49
4.4.1. CLIMATE DATA 49
4.4.2. ISOFLAVONE CONCENTRATIONS 50
4.4.3. SEED YIELD AND RELATED VARIABLES
52
4.4.4. CRUDE PROTEIN AND OIL CONCENTRATIONS 54
4.4.5. ISOFLAVONE YIELDS 54
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4 4 6 CONCLUSIONS ....................................................................................... 55
PREFACE TO CHAPTER 5 .........................................................................................64
CHAPTER 5
BIO
TIC ELICITORS
AS A
MEANS
OF INCREASING ISO
FLAVONE
CONCENTRATION
OF SOYBEAN SEEDS ....................................................................
65
5 1
SUMMARY
...........................................................................................................
65
5 2
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
66
5 3 MATERIALS AND METHODS 69
5 3 1 GROWTH CONDITIONS ............................................................................69
5 3 2 PREPARATION OF ELICITORS................................................................... 69
5 3 3 ISOFLAVONE EXTRACTION AND HPLC ANALySES
......................................
70
5 3 4 EXPT 1 TREATMENT OF SOYBEAN WITH FOLIAR APPLICATIONS OF NATURAL
ELICITORS AT DIFFERENT CONCENTRATIONS .....................................................
71
5 3 5 EXPT 2 TREATMENT OF SOYBEAN WITH NATURAL ELICITORS AT DIFFERENT
STAGES OF DEVELOPMENT
..............................................................................
72
5 3 6 EXPT 3 TREATMENT OF TWO SOYBEAN CULTIVARS WITH DIFFERENT
CHITOSAN CONCENTRATIONS AT DIFFERENT GROWTH STAGES ......................... 72
5 3 7 EXPT 4 TREATMENT OF SOYBEAN WITH YEAST EXTRACT
.........................
73
5 3 8 STATISTICAL ANALySES .........................................................................
73
5 4 RESULTS ..........................................................................................................74
5 4 1 EXPT
1
TREATMENT OF SOYBEAN WITH FOLIAR APPLICATIONS OF BlOTIC
ELICITORS AT DIFFERENT CONCENTRATIONS ................................................... 74
5 4 2 EXPT 2 TREATMENT OF SOYBEAN WITH BIOTIC ELICITORS AT DIFFERENT
STAGES OF DEVELOPMENT ............................................................................. 75
5 4 3 EXPT 3 TREATMENT OF SOYBEAN CULTIVARS WITH DIFFERENT CHITOSAN
CONCENTRATIONS AT DIFFERENT GROWTH STAGES
..........................................
76
5 4 4 EXPT 4 TREATMENT OF SOYBEAN WITH DIFFERENT CONCENTRATIONS OF
YEAST EXTRACTS ..........................................................................................77
5 5 DISCUSSION ...................................................................................................... 77
PREFACE
TO CHAPTER 6.......................................................................................... 86
CHAPTER
6
FOLIAR
APPLICATION
OF ELICITORS
ALTERS ISO
FLA
VONE
CONCENTRATIONS AND OTHER SEED CHARACTERISTICS
OF FIELD GROWN
SOYBEAN ................................................................................................................87
6 1 SUMMARY ........................................................................................................ 87
6 2 INTRODUCTION ................................................................................................
88
6 3 MATERIAL AND METHODS ................................................................................. 90
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6 3 1 SITE DESCRIPTION AND MANAGEMENT 90
6 3 2 PREPARATION OF ELICITORS
91
6 3 3 ISOFLAVONE EXTRACTION AND HPLC ANALYSES
92
6 3 4
OTHERMEASURED
VARIABLES
93
6 3 5 STATISTICAL DESIGN AND ANALYSES
93
6 4 RESULTS AND DISCUSSION 94
6 4 1 ISOFLAVONE CONCENTRATIONS 94
6 4 2 YIELD AND YIELD COMPONENTS 97
6 4 3 CRUDE PROTEIN AND OIL CONCENTRATIONS 100
6 4 4 CONCLUSIONS 101
7 0 SUMMARY AND GENERAL CONCLUSION
106
8 0 REFERENCES 114
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List
o
figures
Fig 3 1
Precipitation mm) and average temperature
OC)
in Sainte-Anne-de-Bellevue,
QC from May to September 2003 and 2004 36
Fig 4 1 Precipitation mm) and average temperature OC) in Sainte-Anne-de-Bellevue,
QC from May to September 2003 and 2004 57
Fig 5 1
Isoflavone concentrations in mature seeds
of
soybean plants treated at the
early podding stage R3) with foHar applications ofyeast extract in different
concentrations
85
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List of Tables
Table
3.1. Analysis
of
variance ofisoflavone concentrations and other seed
characteristics of field-grown soybean as affected by date of seeding, row
spacing and weeds 37
Table 3.2. Isoflavone concentrations in seeds
of
field-grown soybean as affected by year,
date of seeding, row spacing, and weeds .38
Table
3.3. Seed yield, crude protein and oïl concentrations in seeds
of
field-grown
soybean as affected by year, date of seeding, row spacing, and
weeds 39
Table 3.4. Crude protein and isoflavone yields offield.:.grown soybean seeds as affected
by year, date
of
seeding, row spacing, and
weeds 40
Table 3.5. Correlation coefficients
of
isoflavone concentrations and other seed
characteristics offield-grown soybean grown in Sainte-Anne-de-Bellevue, QC
in different years, and subjected to different date of seeding, row spacing, and
weed control treatments 4
Table
4.1. Monthly precipitation and irrigation levels mm) in Montreal, QC from May
to September 58
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Table 4 2 Analysis
of
variance ofisoflavone concentrations and other seed and plant
characteristics ofthree soybean cultivars (AC Orford, AC Protein, and
Golden) grown at three irrigation levels (none, low, and
high) .....................................................................................59
Table 4 3
Isoflavone concentrations
g l
DM)
ofthree
soybean cultivars grown at
three irrigation levels. Results represent main treatments effects and their
interaction for plants grown at Sainte-Anne-de-Bellevue, QC in 2003 and
2004
......................................................................................
60
Table 4 4 Yield, yield components, and phonological traits
of
three soybean cultivars
grown at three irrigation levels. Results represent main treatments effects for
plants grown at Sainte-Anne-de-Bellevue, QC in 2003 and
2004 ......................................................................................
61
Table 4 5 Oil and crude protein concentrations (g kil ofthree soybean cultivars grown
at three irrigation levels. Results represent main treatments effects for plants
grown at Sainte-Anne-de-Bellevue, QC in 2003 and
2004 ............................................... .................................... 62
Table 4 6
Isoflavone yields (kg
ha
l
DM) ofthree soybean cultivars grown at three
irrigation levels. Results represent main treatments effects and their
interaction for plants grown at Sainte-Anne-de-Bellevue, QC in 2003 and
2004 ................................................................................... 63
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Table 5 1
Isoflavone concentrations in mature seeds of soybean plants treated at the
early podding stage (R3) with foliar applications of natural elicitors in
different concentrations
82
Table 5 2
Isoflavone concentrations in mature seeds of soybean plants treated with foliar
applications
of
natural elicitors at different stages of
development. 83
Table 5 3
Isoflavone concentrations in mature seeds
of
soybean cultivars Orford and
Proteina submitted to seed and/or foliar treatments with chitosan in different
concentrations 84
Table 6 1 Monthly precipitation and average temperature in Sainte-Anne-de-Bellevue,
QC from April to September and the 30-year average 102
Table 6 2
Analysis
of
variance
of
isoflavone concentrations and other seed
characteristics
of
field-grown (Sainte-Anne-de-Bellevue 2003-2004) soybean
cultivars (AC Orford and AC Proteina) submitted to various foliar applied
elicitor treatments 1 3
Table 6 3 Isoflavone concentrations in mature seeds of field-grown (Sainte-Anne-de
Bellevue 2003 and 2004) soybean cultivars (AC Orford and AC Proteina)
submitted to various foliar applied elicitor treatments 104
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Table 6 4 Yield and yield components
of
field-grown Sainte-Anne-de-Bellevue 2003
and 2004) soybean cultivars AC Orford and AC Proteina) submitted to
various foliar applied elicitor treatments 105
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List
o
Appendices
Appendix 1
Description of development stages
of
soybean 3
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Chapter
General introduction
Soybean Glycine max L. is one of the oldest foods known to
human
kind. Chine se
have grown it for five thousand years Hymowitz and Harlan, 1983; Hymowitz and
Shurtleff, 2005). Soybean was introduced
in
North America about 200 years ago
Hymowitz and Harlan, 1983; Hymowitz and Shurtleff, 2005). Soybean grain is used
as feed, food and
in
industrial products because
of
its unique chemical composition
Williams, 1897; Amy 1926; Salunkhe et al., 1983).
Yield of soybean is influenced by numerous factors including genotype,
growing season, geographical site, and agronomic practices Nelson and Weaver,
1980; Boerma
and
Ashley, 1982; Board et al., 1992;
Chen
et al., 1992; Frederick et
al., 1998; Ashlock et al., 2000;
Yin
and Vyn, 2002;
Yin
and Vyn, 2003). Soybean
contains isoflavones, which
may
have numerous valuable health effects including
antiatherosclerotic, antioxidative, antitumorial, and antiestrogenic activities Messina
and Messina, 1994; SetcheU and Cole, 2003). Isoflavones also have been reported to
have beneficial effects
on
diabetes and renal diseases Ranich et al., 2001).
Isoflavones, which are found mainly
in
legumes, are involved
in
the communication
process between legumes and rhizobia that lead to nodulation and
N
fixation, disease
resistance mechanisms, and plant fertility Stafford, 1977; Long, 1989;
Mo
et al.,
1992; Yistra, 1992; Ols son et al., 1998;
Mohr and
CahiU, 2001).
Early studies have shown that genetic factors as well as environmental and
management factors such as temperature, water stress, soil fertility, light,
pest
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· pressure, and plant maturity are important factors affecting isoflavone concentrations
in soybean seeds Tsukamoto et al., 1995; Pratt and Birac, 1997; Carrao-Panizzi et al.,
1998; Vyn et al., 2002; Seguin
et
al., 2003; Bennett et al., 2004; Seguin
et
al., 2004
a,b; Swanson et al., 2004; AI-Tawaha
et
al., 2005).
Objectives:
1 General objective:
To identify strategies that will allow the production of soybean seed with high
isoflavone concentrations, with the goal of developing a new value-added niche
market for agricultural producers of eastem Canada.
2 Specifie objectives:
a Determine the effects ofmanagement i.e., irrigation, row spacing, date of seeding,
and weed control) on isoflavone concentrations and other seed characteristics
of
soybean.
b Evaluate the potential of using natural elicitors as a means of increasing isoflavone
concentration
of
soybean.
3 Hypotheses:
a The isoflavone content of soybean seeds can be affected
y
management and
environmental factors including seeding date, row spacing, weeds, irrigation levels
and cultivar selection.
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b Biotic elicitor compounds including Leos chitosan actinomycetes spores and
yeast extract can be used to increase the isoflavone content of soybean seeds.
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Chapter 2
2 0 Literature review
2 1 Soybean agronomy
Soybean [ lycine max
L.)
Merr.] is one of the most important crop plants cultivated
in eastern Canada, with an annual production over l million ha (Zhang et al., 2002).
Soybean production in eastern Canada is rapidly growing, with most production in
Ontario, followed
by
Quebec and the Maritimes (Cober, 2003). Soybean was
produced
on
3000
ha
in 1985 and 199000 ha in 2004 (Riley, 2004). The top soybean
producing countries are the United States, Brazil, China, Argentina, India, Canada,
and Paraguay (Wrather
et
al., 2001). Canada produced 1.8
of
the world total
soybean crop in 1998 (Wrather et al., 2001). The cultivated soybean, which was
originally domesticated in central China in the I l h Century C was first introduced
into North America
by
Samuel Bowen in 1765 as a hay crop (Hymowitz and Harlan,
1983; Hymowitz and Shurtleff, 2005).
Cultivated soybean is included in the family Leguminosae, the subfamily
Papilionoideae, the tribe Phaseoleae, the genus lycine Willd. and the subgenus Soja
(Moench). Soybean cultivars are seperated into three types of growth habit including
determinate, semideterminate and indeterminate (Bernard and Weiss, 1973). Soybean
in Asia
s
used mainly as food crop. However, in Canada soybean is often not
consumed directly by humans but is rather processed to produce vegetable oils and
protein meals. The average composition of cultivated soybean is 40 protein and
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20 oil on a dry matter basis (Hartwig and Kilen, 1991). Recently, soybean has
proved to be
ofv lue
in the nutraceutical industry, because it contains chemical
compounds, mainly isoflavones that are thought to have health promoting, disease
preventing or medicinal properties (Messina and Messina, 1994).
Soybean has the ability to convert atmospheric nitrogen gas (N2) into a form
utilizable by the plant through their relationship with Bradyrhizobium japonicum
Biological nitrogen fixation has been documented to mitigate environmental impacts
of agriculture by decreasing the level of ground water pollution by nitrate and
reducing greenhouse gas production (Watanabe, 1992; Wani
et
al., 1995; Vance,
1997; Van Kammen, 1997). Soybean can fix more than 100 - 200 kg/ha
per
year
of
atmospheric nitrogen (Smith and Hume, 1987). Agronomically, soybean may also
play an important role with the intensification
of
crop rotations as an alternative to
fallow in sorne traditional rotations (Clegg, 1992; Mohammed and Clegg, 1993;
Copeland et al., 1993). Various agronomie practices could improve production of
soybean in North America. Previous studies with soybeans have shown that the time
of sowing (Boerma and Ashley, 1982; Ashlock et al., 2000), plant density (Nelson
and Weaver, 1980; Chen et al., 1992), row spacing (Board et al., 1992; Frederick et
al., 1998), and rates and methods
of
applying fertilizer (Yin and Vyn, 2002; Yin and
Vyn, 2003) can significantly affect grain yields.
2 2 Nutraceuticals: Definition
Nutraceuticals can be defined as any non-toxic food component that has health
promoting, disease preventing
or
medicinal properties (Camire et al., 2003). Such
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products may include any natural bioactive chemical compounds including
isoflavones (Messina and Messina, 1994). There has been n exponential increase in
interest in nutraceuticals during the last decade. Currently, most plant extracts and
isoflavones used by the nutraceutical sector in Canada are imported from the USA,
Europe, Australia, and Asia. The market for nutraceuticals currently has a growth rate
of
15
yea{l, which is well above that
of
other related industries such as the
pharmaceutical and conventional food industries. t is currently believed that the
demand for nutraceuticals and functional foods in Canada is in the range of 1-2 billion
Canadian
,
though estimates depend
on
the definition
of
the industry (Wolfe, 2002).
An
example ofthis growing interest in nutraceuticals is the recent creation of the
Institut des Nutraceutiques et des Aliments Fonctionnels in Quebec.
2 3 Benefits and importance
o
soybean isoflavone
Soybean is a key species used by the nutraceutical industry.
t
contains isoflavones,
which have important beneficial effects
on
human health. Isoflavones is one of
subgroups
of
flavonoids, which are found mainly in legumes. Flavonoids are natural
products that are widely distributed in the plant kingdom, it consist of 6 major
subgroups: chalcone, flavone, flavonol, flavanone, anthocyanins and isoflavone.
Twelve key isoflavones are found in soybean, including three aglycones (daidzein,
genistein, and glycitein), their glycosides, and their corresponding acetyl and malonyl
derivatives.
Soybean isoflavones are thought to have several beneficial effects including
antiatherosclerotic, antioxidative, antitumorial, and antiestrogenic activities (Messina
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and Messina, 1994). Epidemiological data have demonstrated that consumption
of
soybean might contribute to the low incidence
of
breast cancer in apanese women
(Adlercreutz et al., 1993). Similar results were found in Singapore where a
correlation between high dietary soy consumption and lower breast cancer risk was
established (Lee et al., 1991). Soybean extracts have been successfully used to
decrease discomfort associated with menopause (Setchell and Cole, 2003). Recent
studies have also demonstrated that soybean isoflavones have beneficial effects on
diabetes and renal diseases (Ranich et al., 2001).
These properties and effects led to the incorporation
of
soybean isoflavone
extracts in a range of commercial functional foods and to the development of
numerous non-prescription food supplements (Setchel and Cole, 2003). There has.
thus been an explosion in the use
of
soybean products and soybean extracts, and a
concurrent increased demand for soybean with high isoflavone concentrations.
Nutraceutical manufacturers and processors require soybean with a certain isoflavone
concentration, and thus
p y
premium for high-isoflavone soybeans (i.e.,
8
to
36 CAN per metric ton, representing a 6 to 13% premium over conventional soybean
prices) (http://web.aces.uiuc.edu/value/factsheets/soy/fact-isoflavone-soy.htm).
The production of soybean for the nutraceutical sector is thus an interesting
value-added niche market for soybean producers
of
eastem Canada. Although there
have been sorne attempts to genetically modify soybean for increased isoflavone
synthesis (e.g., Yu et al., 2003), currently, only non-GMO soybeans are accepted for
high isoflavone soybean production. t is therefore essential to identify management
strategies and non-GMO technologies that will maximize isoflavone concentration in
soybean.
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2.4. Funetions of isoflavones in plants
Isoflavones have several important functions in plants, including key roles in
pathogenic and symbiotic plant-microbe interactions Stafford, 1977; Long, 1989;
Ols son et al., 1998) and plant fertility Yistra, 1992). The first evidence for the
connection between isoflavones and fertility followed studies
of
a naturally occurring
mutant in corn that was deficient in pollen chalcone synthase activity and was self
sterile Coe et al., 1981). Mo
t
al. 1992) found in both corn and petunia plants
deficient in flavonols synthesis which produced pollen that either failed to germinate
during pollen tube formation. Isoflavones are also involved in the communication
process between legumes and rhizobia that lead to nodulation and N
fixation. In this
two-way interaction, isoflavones act as chemoattractants, and regulate gene
expression in the rhizobia resulting in the production of lipo-chitooligosacharides
LCOs). These LCOs act as bacterium-to-plant signaIs triggering the expression in
plants of many genes responsible for nodule formation Long, 1989).
Phenolic compounds, including isoflavones, contribute to disease resistance
mechanisms in plants. There are numerous reports of an increase of total phenolic
compounds in response to pathogen attack; for example Mohr and Cahill 2001)
reported an increase of isoflavone in soybean seedling root tissues in response to
Phytophthora sojae Phenolic compounds may accumulate as inducible defence
agents phytoalexins) as a result of microbial attack. Phytoalexins are usually
produced and accumulate post-infection, although they might be constitutively
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expressed and present at low concentrations in the plant at any given time (Strack,
1997).
The levels
of
flavonoids in plants are influenced by numerous factors including
stress (biotic and abiotic), phenology (maturity), disturbance or defoliation, and
genotype (Tiller et al., 1994; Saloniemi et al., 1995; Tsukamoto et al., 1995; Vetter,
1995; Hoeck et al., 2000). Stress conditions, such as, excessive UV, microbial
infections, mechanical wounding of the plant, chemicals such as heavy metals and
pesticides n induce the biosynthesis of phenolic compounds including isoflavone
(Balakumar et al., 1993; Tiller et al., 1994; Tsukamoto et al., 1995; Parr and Rhodes,
1996; Mandavia et al., 1997). Thus, it appears that numerous factors impact the
content phenolic compounds in plants including isoflavones.
2 5 Factors affecting isoflavones concentration in plants
2 5 1 Genetic factors
Isoflavone content and profile vary considerably from one species to another. Studies
with soybean, red clover, and alfalfa have reported that isoflavone concentration may
also vary considerably among cultivars
of
a given species (Saloniemi et al., 1995;
Vetter, 1995; Hoeck et al., 2000). Eldridge and Kwolek (1983) found that total
isoflavone content
of
soybean seed varied from 1160 to 3090 .tg g
1
among four
cultivars grown in the same environment in Iowa. Seguin et al. (2004a) observed
similar results in Quebec and found that seed total and individual isoflavone
concentrations were affected by cultivars, which interacted with site and year. Despite
cultivar and cultivar by environment effects, they found that S08-80 and Proteina
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2.5.2. Correlation between isoflavones concentrations and other important
agronomie factors.
The reationship between isoflavones concentrations and other agronomic characteristics
is
of
imortance in breeding and selection programs. The presence of positive
correlations indicates that selection
of
several desirable traits can be done concurrently.
Results from the few studies having investigted correlations between isoflavones and
other seed characteristics are sometimes conflicting. Positive correlation between
isoflavones concentrations and seed yield, 100-seed weight, and protein were reported
by Seguin et al. 2004a), Primomo et al. 2005) and Yin and Vyn 2005); while,
negative correlations between specific isoflavones and seed yield, days to maturity, and
plant height were reported by Wang et al. 2000), and between total isoflavones and
prote n by Chiari et al. 2004).
According to Charron t al. 2005), correlations between isoflavones and protein
and oil might vary depending on the cultivar. In a trial conducted with
7
cultivars at
three locations in Tennessee, they observed only week negative correlations between
isoflavones and oil concentrations across sites and cultivars. However, strong negative
correlations between oil and isoflavones were observed for 6 cultivars, while 5 cultivars
had strong positive correlations between isoflavones and prote in content. Authors
suggested that these specific cultivars should be used as a germplasm source in future
breeding efforts.
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2.5.3. Environmental and management factors
Given that environmental and genotype by environment effects have a large influence
on isoflavone content of soybean it is essential to understand how t is affected by
specifie biotic and abiotic factors so that growing conditions that maximize the
concentration and yield
of
isoflavones can be identified. There is evidence that a
range of factors including: soil moisture levels Chaves et al., 1997), pest pressure
Parr and Rhodes, 1996), temperature Tsukamoto et al., 1995), mineraI nutrition
Tiller et al., 1994), and light quality Kubasek et al., 1992; Stapleton, 1992) may
affect isoflavone concentrations in a range
of
species including soybean.
2.5.3.1. Temperature
Temperature is one of the most important factors affecting the synthesis of
isoflavones. In Japan, Tsukamoto et al. 1995) reported that the cultivar Lee had
seed isoflavone concentrations 5.8 times lower when sown in May than when sown in
July. Differences between seeding dates were attributed to resulting differences in
temperatures at the time ofpod filling and which were higher with the May seeding.
They also reported that, in greenhouse trials, seeds that matured at low temperature
daytime
25 oC
and night time 10°C) had greater isoflavones content than seeds that
matured at high temperatures daytime 38 Oc and night time 28 OC . Isoflavone
contents of cotyledons exhibited a large response to temperature during seed fill, but
the isoflavone content
of
hypocotyls remained relatively constant across a range of
temperatures.
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In a study conducted by Carrao-Panizzi et al. (1998) in different regions of
Brazil, it was reported that the highest isoflavone concentrations were observed in
seeds of soybean plants grown in locations with cooler temperatures (high latitudes) .
when compared to locations with warmer temperatures (low latitudes). They also
reported that temperature during seed development was one of the major factors
affecting seed total isoflavone concentrations. Consequently, eastem Canada, where
temperatures are cooler during seed filling than in South America or most regions of
the USA could have an advantage for isoflavone production over these other soybean
producing regions.
2.5.3.2.
ater
stress
Studies on the effects of water stress on the production of flavonoids are contradictory
and appear to be related to the intensity of the stress (Homer, 1990). From greenhouse
trials, Karen and Carol (2001) reported that during periods
ofwater
stress the
isoflavone content
of
soybean plants can be increased compared to non-stressed
conditions. However, other trials suggest that well-watered plants produce more
isoflavones than plants watered with an irrigation regime that mets 30 of the
evapotranspiration demand of plants. Drought stress lowered isoflavone levels by 5 to
50 percent depending on the cultivar (Nelson et al., 2002). Similarly, in field
experiments, Estiarte et al. (1999) reported that well-irrigated wheat plants (100
replacement of potential evapotranspiration) had higher flavonoid concentrations than
those half watered throughout the growth cycle. In a study conducted by Bennett et al.
(2004) in Missouri, it was reported that the levels of isoflavones in soybean seeds are
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increased
by
irrigation. However, studies conducted in another region
of
the United
States Kansas) found that isoflavone content
of
soybean was higher under rainfed
conditions than under irrigation Swanson
et
al., 2004).
No
studies have however yet evaluated the effect
of
different irrigation regimes
on isoflavone concentrations in mature soybean seeds, nor has the exact relation
between soil water potential and isoflavone levels been established.
2 5 3 3 Soil fertility and fertilization
t
is generally said that plants respond to sub-optimal soil fertility levels by increasing
the synthesis and accumulation of flavonoids in their tissues. However, results of
studies investigating the effects
of
fertilization on isoflavone concentration are
contradictory; response may vary wiih species, elements and level of the stress.
Seguin et al. 2003) found that K, P, Sand B fertilization had limited impact on
soybean seed isoflavone content in fields with medium to high soil fertility.
On
the
other hand,
Vyn
et al. 2002) observed positive effects
of
fertilization
on
isoflavone
concentration of soybean seed on low- to medium-testing K soils. Carpena
et
al.
1982) reported that tomato response may vary depending on the element. They
reported that while B deficiencies result in increased flavonoid accumulation in
leaves, P and
Mn
deficiencÎes do not affect the total flavonoid content but rather
affect the types offlavonoid present. Stout et al. 1998) reported that, in greenhouse
trials, tomato
leaf
grown at low N availability had greater flavonoid content. Most
studies on the effects
of
mineraI nutrition have been conducted under controlled
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conditions and there is st illiimited information on the impact of fertilization strategies
on concentrations and yields
of
isoflavones in field-grown soybean.
2 5 3 4 Light
In general, light stimulates the synthesis of flavonoids. Various researchers have
reported an increased production
of
flavonoids with UV radiation stress Caldwell et
al., 2005; Schmelzer et al., 1988; Tevini t al., 1991; Kubasek et al., 1992). It has
been reported that plant flavonoid biosynthesis genes are transcriptionally activated
by light, where isoflavones may provide defence against ultraviolet light Stapleton,
1992). Hughes et al. 1999) found that exposure of root systems of aIder plants to
light can promote the synthesis of flavonoids. They also observed increased levels of
isoflavones in plants supplemented with UV light. While investigating the role of
ecological variables in the seasonal variation of isoflavone content of istus ladanifer
exudate, Chaves t al. 1997) found a two- to four-fold increase in summer, as
compared to spring; authors attributing such increases to differences in light intensity
and quality.
Research on the effect of light on isoflavone concentrations in soybean remains
extremely limited and often anecdotal. Studies conducted with soybean seedlings
demonstrated that concentrations were positively correlated with light duration Sun
et al., 1998); similar results were aiso reported for seed concentrations
of
field-grown
plants
Li t
al., 2004). According to Kirakosyan
t
al. 2006) response could however
depend on the cultivar.
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2 5 3 5 Pests
Many studies have demonstrated the importance of phenolic compounds, including
isoflavones, in plant defence response Harbome, 1991; Appel, 1993). The synthesis
of phenolic compounds is increased in plant tissues following infection by pathogenic
organisms or feeding by herbivores parr and Rhodes, 1996). t has been shown that
isoflavones, including those found in soybean, have antifungal and antioxidant
activities Pratt and Birac, 1997). The accumulation
of isoflavones including
genistein is induced by wounding ofsoybean Karen and Carol, 2001). Recently,
Lozovaya et al. 2004) studied the biochemical response
of
soybean roots to
usarium
sol ni infection, and concluded that usarium sol ni inoculation of soybean roots in
soil induces the synthesis
of
isoflavones in seedlings. The impact of pests on
isoflavone content of mature soybean seeds however has not yet been studied.
2 5 3 6 Natural Inducers
Being involved in plant defence response and plant-microbe interactions, flavonoid
production by plants including isoflavones) may be increased when plants recognize
certain molecules or structures that characterize a pathogen or a symbiont; such
compounds are known
as
inducers or elicitors. Consequently the use ofbiotic or
abiotic elicitors of plant defence response has been evaluated for a number
of
years as
a pest biocontrol strategy Tahvonen, 1988; Lafontaine and Benhamou, 1996;
Benhamou et al., 1998; Duzan, 2004), and more recently as a means ofincreasing the
production of compounds
ofv lue
to the nutraceutical industry.
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Kneer
et
al. (1999) demonstrated that the exogenous application
of
natural
elicitors to roots
of
upinus luteus L, inc1uding lipochito-oligosaccharide (LCOs)
produced by rhizobia, chitosans (i.e., deacylated chitin), and salicyclic acid (i.e., a
compound involved
in
plants' systemic response to pathogens), resulted in an increase
in the synthe sis and root concentration of the isoflavone genistein. The response was
dose dependant but was observed
in
aIl cases at very low concentrations varying with
the elicitor but being as low as 100 lM. Injections ofpurified yeast cell wall (which
contain chitin polymers) increased flavonoid content in the foliage of upinus albus
1
(Bednarek et al., 2001). Gagnon and Ibrahin (1997) also reported a marked
increase in isoflavone content of upinus albus 1 seedlings upon treatment
of
seeds
with yeast extract and chitosan. Actinomycetes are organisms that have been reported
to have antagonist effects
on
sorne pathogens and have been successfully used as
biocontrol agents; it has been suggested that sorne
of
their properties may be
associated with an induction
of
plant defence responses, although such properties
remain to be demonstrated (Beausejour et al., 2003)
It
thus seems possible to envision the utilisation of natural elicitors as a means
of increasing isoflavone synthesis and content in mature soybean seeds, however, it
has not
yet een
evaluated. Consequently, there is currently no information available
on particular elicitors that could be effective; neither the optimal time nor the
application doses are known.
2 5 3 7 Role
o
abiotic inducers and flavonoids in plant resistance
As for biotic inducers, several studies demonstrated that abiotic inducers can also
induce biochemical changes that allow the plant to decrease disease occurrence (Kuc,
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1995; Karban et al., 1999; lnbar et al., 1999). Field studies demonstrated that abiotic
elicitors including Probenazole and KeyPlex 350 significantly reduced bacterial spot
and early blight occurrence lnbar et al., 1998). lnbar et al. 1998) also, demonstrated
that Benzothiadiazole BTH) supplied cross-resistance and significantly decreased the
occurrence ofbacterial spot Xanthomonas campestris pv), early blight Alternaria
solani), leafmold Fulviafulva), and leafminer larval densities Liriomyza spp.).
Guleria and Kumar 2006) recently reported that BTH induced high levels
of
pathogenesis-related proteins in mustard plant.
Salicylic acid plays a key role in both systemic acquired resistance and as an
inducer of the oxidase protein in tobacco cell suspensions Ryals et al., 1996; Shirasu et
al., 1997). Du and Klessig 1997) reported that exogenous application of salicylic acid
also induces PR pathogenesis-related proteins) gene expression and increased disease
resistance in tobacco. On the other hand, Ervin et al., 2004) reported that exogenous
application of SA to Kentucky bluegrass could be used as mean of increasing
antioxidant activity and pigment content which were correlated with less le f injury
against UV light
The role of flavonoids in plant resistance has been extensively studied. Previous
research showed that several insects are sensitive to flavonoids Brignolas et al., 1998;
Berhow and Vaughn, 1999; Hoffmann-Campo et al., 2001; Widstrom and Snook, 2001;
Haribal and Feeny, 2003; Thoison et al., 2004; Chen et al., 2004). Nevertheless,
Nykanen and Koricheva 2004) showed that flavonoids do not perform as broad
spectrum defensive compounds.
Abiotic and biotic signaIs are mostly perceived by membrane-Iocalized
receptors that transduce those signaIs inside plant cells to initiate defense responses Yin
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et al., 2002). Knight 2000) reported that calcium is a key point
o
signalling cross-talk
as it can be elicited by numerous abiotic and biotic factors. On the other hand, Xiong et
al. 2002) reported that plant hormones including abscisic acid AB A), ethylene, and
salicylic acid SA) can consecutively; start a second round o signalling that can control
exact sets o stress.
2 5 3 8 Plant maturity
The concentration and yield
o
isoflavones and other phenolic compounds in plants
varies among tissues. Alfalfa stores the largest amounts
o
isoflavones in the seed coat
Hartwig et al., 1990), whereas in soybean the amounts
o
isoflavones are much larger
in the cotyledons than in the hypocotyls Tsukamoto et al., 1995). In a study
conducted by Bordignon et al. 2004) to evaluate the effects o pod position
on
soybean seed isoflavone concentration, it was found that isoflavone concentration was
lower in seeds collected from the top part o the plants and higher in seeds from the
bottom parts. In study conducted by Nakamura et al. 2001) t was determined that the
content and composition
o
isoflavone in mature
or
immature soybean seeds, it was
found that isoflavone concentrations on a dry matter basis were highest in mature
seeds.
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Preface
t
bapter 3
A manuscript based
on
the following chapter was submitted for publication in the
Canadian Journal
o
Plant Science. Although aIl the work presented herein is the
responsibility o the candidate, the project was supervised by Dr. Philippe Seguin,
Department
o
Plant Science, Macdonald Campus o McGill University. The
manuscript is co-authored by the candidate and Dr. Philippe Seguin. Dr. Seguin
provided funds and assistance for this research, including supervisory guidance and
the reviewing
o
the manuscript. Authors contributions are described in detail earlier
in the section describing the contributions o authors.
This chapter determines the main effects and interactions o several agronomie
factors on soybean isoflavone concentrations. Factors studied include date o seeding,
row spacing and weed pressure.
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hapter
3
Effects of seeding date, row spacing,
and
weeds on soybean isoflavones
concentrations
3.1. Snmmary
Soybean
[ lycine
m x L.) Merr.] seeds contain isoflavones that may have positive
impacts on human health. Field experiments were conducted in 2003/4 in Québec,
Canada to determine the effects of seeding date (late May and mid June), row
spacing (20-, 40-
and
60-cm) and weeds (presence or absence) on soybean
isoflavone concentrations and yield. Total and individual isoflavone concentrations
were determined by HPLC. Seed yield, and oil and crude prote in (CP)
concentrations were concurrently determined. Year, seeding date, and weeds
affected total and individual isoflavone concentrations, while row spacing had no
effect. Total isoflavone concentration was 84 greater in 2003 than 2004. Seeding
in mid June increased isoflavone concentration by 38 , compared to seeding in
May. The presence ofweeds increased total isoflavone concentrations
by
9 . Year,
row spacing, and weeds signif icantly affected seed yields. Seed yields were greatest
in 2004, at 20- or 40-cm row spacing, and in the absence
of
weeds. Seeding date
affected CP and oil concentrations. Greater CP concentration was observed with
earlier seeding, the reverse was observed for oil. Weeds also affected CP and oil
concentrations: higher P and oil concentrations were observed in weedy and weed
free plots, respectively. Total isoflavone yield was affected by aIl factors evaluated.
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Yield was greater in 2003 than 2004, with mid June rather than late May seeding,
when seeded at row spacing
of20
or 40- than 60-cm, and without weeds. Finally,
negative correlations were observed between isoflavone concentrations and CP
concentration and seed yield.
t
thus seems that certain agronomic practices may
need to be tailored specifically for isoflavone production if concentrations in
soybean are to be maximized.
3.2. ntroduction
Soybean is a key species used by the nutraceutical industry. t contains isoflavones,
which may have important beneficial effects on human health. Three major groups
of isoflavones are found in soybeap inc1uding daidzein, genistein, and glycitein.
Soybean isoflavones are thought to have several beneficial effects that include
reducing menopausal symptoms, certain cancers, and cardiovascular diseases
Messina and Messina, 1994). These properties led to the incorporation
of
soybean
and soybean extracts into a range of commercial functional foods and to the
development of a range of non-prescription food supplements Setchell and Cole,
2003).
The isoflavone concentration in soybean seeds s in part genetically
determined, environmental and genotype x environment effects also greatly affecting
isoflavone concentrations Meksem et al., 2001; Lee et al., 2003). Environmental
factors reported to affect isoflavone concentrations in soybean inc1ude: temperature,
soil moisture levels, soil fertility, CO
2
levels, light quality, and pest occurrence
Tsukamoto et al., 1995; Vyn et al., 2002; Bennett et al., 2004; Li et al., 2004;
Lozovaya et al., 2004; Kim et al., 2005b; Lozovaya et al., 2005). Several studies
have reported tempe rature as being one key environmental factor affecting soybean
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isoflavone concentrations. Tsukamoto et al. (1995) reported from greenhouse trials
that seeds maturing at low temperature (daytime 25 oC and night time 10°C) had
greater isoflavone content than seeds maturing at high temperatures (daytime 38
c
and night time 28
oC .
Similarly Lozovaya et al. (2005) reported two- to three-fold
differences between soybean plants grown under different temperature regimen, with
greater concentrations observed in seeds
of
plants grown at lower temperatures.
They also reported higher isoflavone concentrations in seeds
of
plants grown from
R6 (full seed - green bean) in soil at 70
of
its water holding capacity compared to
plants grown at a 30 lower holding capacity. Kim et al. (2005) reported greater
isoflavone concentration in seeds
of
soybean grown at higher O
2
levels (i.e., 650
vs. 360 Ilmol morl ofC02). FinaIly, stresses such as wounding or pests may also
increase soybean isoflavone concentrations (Lozovaya et al., 2005; Wegulo et al.,
2005).
If
environmental factors effects on soybean isoflavone concentrations have
been weIl documented and researched, information on the effects
of
specific
agronomic practices remains limited. Agronomic practices may indirectly affect
isoflavone concentrations of soybean
by
altering micro-environmental conditions
and abiotic and biotic factors to which plants are exposed. Fertilization in low
fertility soils could potentially increase isoflavone concentrations. Vyn et al. (2002)
indeed reported that K fertilization in low K -test soils increased isoflavone
concentrations. However, Seguin and Zheng (2006) failed to observe K, P,
S,
or B
fertilization effects in highly fertile soils, while Kim et al. (2005b) reported that N
fertilization
(40
kg N ha-
I
could reduce soybean isoflavone concentrations.
Tsukamoto et al. (1995) in Japan reported soybean isoflavone content that was up to
5.8 times higher when sown in July than in May. Vyn et al. (2002) reported in one
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study that soybean isoflavone concentrations were not affected by row spacing.
Finally, irrigation has been reported to increase isoflavone concentrations in sorne
cases (Bennett et al., 2004).
The objective
of
this study was to determine the main effects and interactions
of
several agronomic factors on soybean isoflavone concentrations. Factors studied
include date of seeding, row spacing and weed pressure.
3.3. Material and methods
3.3.1. Site description and management
Field experiments were established in 2003 and 2004 in Sainte-Anne-de-Bellevue,
QC, Canada (45°25'45 N lat., 73°56'00 W long.). The soil type in both years was a
Macdonald clay loam (Dark Gray Gleysolic). Treatments were assigned to a
randomised complete block design in a split-split-plot arrangement with four
replications. Seeding dates [late May (22 May 2003 and 31 May 2004) and mid June
(18 June 2003 and 22 June 2004)] were randomly assigned to main plots in each
replicate; row spacing (20-, 40- and 60-cm) to sub plots, and weed treatments (weedy
and weed-free) to sub-sub plots.
Plots were fertilized with 20 kg ha
t
ofN, and sufficient P and (i.e., 30 kg
ha
t
ofboth P205 and
2
0)
during field preparation prior to seeding as recommended
locally based on soil tests (CRAAQ 2003). Seeding
of
the cultivar AC Proteina' was
done in aIl plots by hand at a rate of 50 plants m
2
to an average depth of 3 cm, with
appropriate rhizobial inoculant added at time of seeding (Nitragin, Milwaukee, WI).
Sub-sub-plots were 2.2 x 4.5 m. In weed-free plots weeding was done manually every
other week during the entire growing season. Dominant weed species in both years
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were ragweed
Ambrosia artemisiifolia),
crabgrass
Digitaria sanguinalis),
and foxtail
Setaria spp). Weed density or biomass in the weedy plots was not determined,
however, weed coyer was estimated visually in one replicate at the onset of podding in
both years. Weed coyer varied between 20 and 40 and, as expected, was positively
associated with row width.
Soybean was harvested with a self-propelled combine when plants from all
plots had reached physiological maturity (i.e., 19 September 2003 and 17 September
2004) to determine seed yield, and isoflavone, crude prote in, and oil concentrations
and yields per hectare. Whole sub-sub-plots were harvested. Weather data for the
growing season in both years were retrieved from a nearby weather station (Fig. 3.1).
3.3.2. Isoflavone extraction
and PLC
analyses
Following harvest, seeds were stored at room temperature and, within one month,
were extracted for determination of isoflavone concentrations. Extraction was do ne
using a modified version
ofthe
protocol
ofVyn
et al. (2002), which relies on acid
hydrolysis of the 12 major isoflavones found in soybean seeds to their aglycone forms
(Le., daidzein, genistein, and glycitein). In summary, a 0.25 g sub-sample from a 60 g
finely ground seed sample was hydrolysed in a mixture of pure HCI (2 ml) and
ethanol (10 ml) by boiling for 2 h (Pettersson and Kiessling 1984; Choi et al. 2000).
Samples were then cooled and centrifuged at 10,000 rpm for
10
min.
Daidzein, genistein, and glycitein were separatedby HPLC using a Waters
chromatograph system (Waters, Milford, MA), equipped with two mode1510 pumps,
a WISP 712 autosampler and a
UV
mode1441 absorbance detector. Fifty J lL
of
each
extract were used for the analysis. The separation was carried out on a C 18 reversed
phase column (Bondapak, 3.9 300 mm, Millipore, Milford, MA, USA). Elution of
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isoflavones was performed using a linear gradient system from 20 methanol and
80 water, to 80 methanol and 20 water over the course of 30 min, following an
initial 5 minutes of steady elution with 20:80 methanol:water.
AU
isoflavones were
detected at 254 nm (Wang et al. 2000). Purified isoflavones [daidzein, genistein,
glycitein; (Sigma-Aldrich, Mississauga, ON, Canada)] were used as standards to
identify isoflavones on chromatograms and calculate their concentrations. The
recovery rate was > 90 .
AU concentrations were expressed
on
a dry matter (DM) basis. Concentrations
of aglycones were summed to obtain total isoflavone concentration. Isoflavone yield
per hectare was also determined by multiplying isoflavone concentrations and seed
yields.
3.3.3. Other variables measured
Seed samples from the harvested plots were also used to determine seed crude prote in
(CP), oil, and DM concentrations. Oil and CP concentrations were determined on 10-g
sub-samples of finely ground seeds from each plot using a FOSS N R Systems Model
6500 (Silver Springs, MD, USA). Yield per hectare of CP was determined by
multiplying CP concentration by the seed yield per hectare. AlI values were expressed
on a DM basis.
3.3.4. Statistical analyses
AU data were subjected to an analysis of variance (ANOVA) using the generallinear
model (GLM) procedure in SAS (Statistical Analysis Software 1989) to identify
significant treatment effects and interactions. Homoscedasticity among experiments
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was verified using the chi-square test Gomez and Gomez 1984). Data were then
analyzed in a combined analysis that regrouped years, date
of
seeding, row spacing,
and weeds McIntosh 1983). Comparisons between means were made using least
significant differences LSD) at a 0.05 probability level when ANOVA indicated
model and treatment significant effects. Pearson product-moment correlation
coefficients were calculated based on the data from all plots across the two years,
using the ORR procedure in SAS to de scribe the relationship between
aIl
variables
measured.
3.4. Results and discussion
3.4.1. Climate data
Climatic conditions differed considerably in both years Fig 3.1). In 2003,
precipitation in May was substantially greater than the 30-yr average Le., 116 vs. 7
mm); however during the rest
of
the season it was lower i.e.,
7
mm less). In 2004,
precipitation between May and September was overall comparable to the 30-yr
average, however it was
23
and 100 mm lower in June and August, and 49
mm
greater
in July than the 30-yr average. Mean monthly temperatures were on average within 1
oC of the 30-yr average in both years except in August and September 2003 Le., 2
oC
higher on average) and in September 2004 i.e., 1.5
oC
higher). Average air
temperature consistently ranged between 20 and 25
oC
between late June and late
August 2003, temperature ranging between
5
and 25 oC for the same period in 2004.
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3 4 2 Isoflavone concentrations
Year (P 0.001), date
of
seeding (P 0.001), and weed (P 0.05) main effects were
observed for total isoflavone concentrations (Table 3.1). Total isoflavone
concentration was 84% greater in 2003 than 2004, and was also 38% greater when
seeded in mid June than late May (Table 3.2). A year x seeding date interaction (P
0.01) indicated that the difference between seeding dates was greater in 2003 than in
2004. FinaIly, the presence ofwee s increased total isoflavone concentration by 9%,
when compared to weed-free plots.
Individual isoflavones responded differently to treatments. Daidzein was
only affected by the seeding date (P < 0.01), concentrations being 29% greater when
seeding occurred in mid June compared to late May. Year and seeding date main
effects (P 0.001) were observed for genistein; concentration was 2.45 times greater
in 2003 than in 2004. A year x seeding date interaction (P 0.001) indicated that
seeding date differences were only significant in 2003, the mid June seeding resulting
in 55% greater genistein than a late May seeding. Response of glycitein was more
complex, year (P 0.001), seeding date (P 0.001), and weeds (P 0.05) main
effects being observed along with seeding date x row spacing and year x weeds (P
0.05) interactions. Glycitein concentration was consistently greater in 2003 than 2004
(i.e., 2.58 times) for aIl treatment combinations. However, the seeding date
x
row
spacing interaction reflected greater concentrations with seeding in mid June than late
May for aIl row spacing except 20-cm; the year
x
weeds interaction reflected greater
concentrations in weedy plots than hand weeded ones in 2003, no differences being
observed between weed treatments in 2004.
Previous greenhouse and growth chamber experiments demonstrated that
temperature and soi moisture levels greatly affect isoflavone concentrations. Indeed,
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it has been reported from a number
o
sources that isoflavone concentrations are
greater when plants are exposed to mild temperatures and optimal soil moisture levels
Tsukamoto et al. 1995; Lozovaya et al. 2005). In contrast, in our study, we observed
greater isoflavone concentrations in the year i.e., 2003) where temperature was on
average greater and precipitations lower Fig. 3.1). t is possible that differences
between studies could be due to the confounding effects o other abiotic or biotic
factors, which may not be an issue in more controlled greenhouse or growth chamber
trials. Aiso timing
o
certain tempe rature or precipitation events could prove to be
determinant. t is not know at this point
i
certain growth stages are more susceptible
to specifie temperature
or
soil moisture conditions.
Differences we observed between seeding dates however are in accordance
with those o Tsukamoto et al. 1995) who reported, at one o two sites in Japan,
higher soybean isoflavone concentrations when sown in July when compared to
seeding in May. t was hypothesized that the higher isoflavone concentrations o later
seeding dates could result from lower temperatures and greater precipitations during
pod development and seed filling, when compared to the earlier, more typical, May
seeding.
Row spacing proved to have little to no effect on isoflavone concentrations.
These results are in accordance with those o Vyn et al. 2002) who also reported a
lack o response to row spacing. Finally, a sm aIl but consistent response to weeds was
observed for the first time. The greater concentrations we observed in weedy plots
could possibly result from a stress response or from modified micro-environmental
conditions, such as reduced light intensity or altered light quality, increased moisture
in the canopy, or ev en the possible presence o allelopathic compounds. Information
on the effects
o
these factors on soybean isoflavone concentrations although remain
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scarce. t has been suggested that sunshine hours and light duration could be positively
correlated with isoflavone concentrations in mature seeds and seedlings (Sun et al.
1998; Li et al. 2004). Light quality, which is modified by competing plants and
shading, has also been reported to affect isoflavone synthesis and accumulation in
soybean seedlings, response depending on soybean cultivars (Kirakosyan et al. 2006).
3.4.3. Seed yield
Year (P 0.001), row spacing (P 0.001), and weeds (P 0.001) main effects were
observed for seed yield; seeding date did not affect seed yield (Table 3.1). Row
spacing x weeds
(P
0.01) and year x weeds
(P
0.001) interactions illustrate
magnitude differences in the yield reduction caused by the presence o weeds
depending on years and row spacing. Yield reduction caused by weeds was greater in
2004 than 2003 41 vs. 33 ), and was greater at a row spacing o 60- than either 20-
or 40-cm 51 vs. 33 ). Overall, weeds caused a 38 reduction in yield. The greatest
yield reduction observed with the 60-cm row spacing was most likely due to a
potentially greater weed competition, as soybean plants in wider rows usually have a
slower canopy closure and hence a lower competitive ability early in the season
(Willcott et al. 1984; Lee 2006).
Seed yield was 33 greater in 2004 than 2003. The 2003 season was
substantially warmer and drier; comparable differences in soybean yield between
years were also observed in the region (Institut de la Statistique du Québec 2005a,b).
Across years, seeding dates, and weed treatments, seed yields were 25 lower when
plants were seeded at a 60-cm row spacing compared to 20- or 40-cm. Greater seed
yields in narrow row soybean were also reported by others in a range o environments
(Herbert and Litchfield 1984; Oriade et al. 1997; Bowers et al. 2000; Heatherly et al.
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2002; Hoishouser and Whittaker 2002; Lee 2006). For example, Herbert and
Litchfield (1984) reported 23 lower seed yield in soybean grown at 75- than 25-cm
row spacing. Again, the lower seed yields observed with the wider row spacing are
attributable to a slower canopy closure, lower leaf area index, and greater in-row
competition, resulting in reduced light interception and fewer pods per plants (Herbert
and Litchfield 1984; Wilcott
et
al. 1984; Lee 2006; Thelen 2006).
3 4 4 Crude protein and
o
concentrations
Crude prote in concentration was significantly affected
by
year (P 0.01), seeding date
(P 0.01), and weeds (P 0.05) main effects; however year x seeding date, year x
seeding date x row spacing, and year x weeds cross-over interactions (P 0.05) were
also observed. The year x seeding date x row spacing and year x seeding date
interactions reflected higher CP concentrations with seeding in late
ay
than mid
June, except in 2004 for 20- and 40-cm row spacing. Overall, CP was slightly greater
with seeding in late May than mid June, with concentrations of 509 and 504 g kg-l,
respectively; a similar difference was observed between years with higher CP
concentration in 2004 than 2003. These overall high CP values are explained by the
fact that the cultivar used in our experiment (i.e., AC Proteina) is a cultivar that was
selected for high CP. t has consistently ranked among cultivars with highest CP
concentration in trials conducted in eastern Canada (Seguin
et
al. 2004). Finally, the
year x weeds interaction reflected that concentration was greater in weedy than hand
weeded plots in 2003, but not in 2004. Differences were although small and were
bio logically insignificant.
Oil concentration was affected by year (P 0.001), seeding date (P 0.01),
weeds (P 0.001), and a year x seeding date interaction (P 0.05). The year x
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seeding date interaction indicates that differences between seeding dates was
significant only in 2004, during which the mid June seeding resulted in greater oil
concentration than a late May seeding. Overall, differences between treatments were
again small, oil concentration being 5 greater in 2003 than 2004, and 3 greater in
weed-free than weedy plots.
Crude protein and oil concentrations, which are negatively correlated, were
previously reported to be affected by a range of environmental conditions and
agronomic practices, inc1uding row spacing, weed control, and planting dates, reports
have however been conflicting and differences were often minor (Donovan et al.
1963; Rose 1988; Kane et al. 1997; GalaI2004).
3 4 5 Isoflavones and crude protein yields
Total isoflavone yield was only affected by main effects including year, seeding date
(P 0.05), row spacing (P 0.01), and weeds (P 0.001) (Table 3.1). Total
isoflavones yield averaged 2.16 kg h
1
across treatments and was 37 greater in 2003
than 2004,30 greater with a mid June than a late May seeding, 40 greater at 20-
and 40-cm than 60-cm row spacing, and 47 greater in weed-free than weedy plots
(Table 3.4). Although, total isoflavones concentration was greater in weedy than
weed-free plots, this did not translate into greater isoflavones yield per hectare due to
the much lower seed yield ofweedy plots. Isoflavone yield responses to years and
seeding dates paralleled responses observed for isoflavone concentrations, while that
to row spacing reflected seed yield response.
Yield responses
of
individual isoflavones varied greatly depending on the
isoflavone (Table 3.2). Year and seeding date main effects were observed for both
genistein and glycitein yields (P 0.05), reflecting in both cases greater yields in 2003
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than 2004 (average o 163 ), and with a mid June than a late May seeding (average
o
47 ) (Table 3.4). Row spacing main effects were observed for both daidzein and
genistein yields, however a year x row spacing crossover interaction was also
observed for genistein yield. Daidzein yield was 45 greater when soybean was
planted at 20- and 40-cm than at 60-cm. In the case o genistein similar differences
between row spacing were observed but only in 2003. Yield o aU isoflavones was
strongly affected by weeds (P < 0.001), greater yields o aU isoflavone being observed
in weed-free plots. Response to weeds was complicated by the presence
o
several
interactions, although only one was a crossover interaction, reflecting that genistein
response to weeds varied depending on the seeding date and row spacing.
FinaUy, CP yield was affected by year, row spacing, and weeds main effects (P
0.001), and row spacing x weeds (P 0.01) and year x weeds (P 0.001)
interactions. Crude protein yield was 36 greater in 2004 than 2003, 33 greater
with 20- and 40-cm row spacing than 60-cm, and 60 greater in weed-free than
weedy plots. The interactions implicating weeds reflected greater differences between
weed treatments in different years or at different row spacing.
3.4.6. Correlations between isoflavone concentrations and other seed
characteristics
Negative correlations (P < 0.05) were observed between seed yield and genistein,
glycitein, and total isoflavone concentrations r ranging between -0.34 and -0.40)
(Table 3.5). Similarly, negative correlations were significant (P 0.05) between CP
and individual as
weU
as total isoflavones r ranging between -0.30 and -0.44).
Positive correlations (P 0.05) were however observed between oil and genistein,
glycitein, and total isoflavone concentrations r ranging between 0.44 and 0.52). As
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expected there were positive correlations between most individual isoflavones and
total isoflavone concentrations and yield r ranging between 0.28 and 0.91). These
correlations are not surprising as individual isoflavones are aIl synthesized via the
phenylpropanoid pathway (Yu and McGonigle 2005).
The negative correlations we observed between isoflavone concentrations and
both CP concentration and seed yield are in agreement with Chiari et al. (2004) and
Wang et al. (2000) who also reported negative correlations between isoflavone
concentrations and CP concentration and/or seed yield. Other studies, however,
reported positive correlations between isoflavone concentrations and CP concentration
and/or seed yield (Seguin et al. 2004; Primomo et al. 2005; Yin and Vyn 2005).
According to Charron et al. (2005), correlations between isoflavones and CP and oil
concentrations might vary depending on the cultivar. In a trial conducted with
7