factors affecting isoflavone concentration in soybean glycine max l

8/19/2019 Factors Affecting Isoflavone Concentration in Soybean Glycine Max L.

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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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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.

V111


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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

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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

XVll


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List

o

Appendices

Appendix 1

Description of development stages

of

soybean 3

XV


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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

1


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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.

2


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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.

3


23/154

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

4


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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

5


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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

6


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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

8


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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

9


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30/154

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.


31/154

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.

12


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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

3


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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

14


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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.

5


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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.

6


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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,

17


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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

8


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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.

9


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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.

2


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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.

2


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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

22


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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

23


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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.

30


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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


53/154

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

factors affecting isoflavone concentration in soybean glycine max l

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