Henk Hilhorst, Wageningen University,

Genetic and molecular basis of chlorophyllretention in soybean seed

Edvaldo A Amaral da Silva Universidade Estadual Paulista (FCA)

Collaborators in Brazil:José França-Neto

Fernando HenningFrancisco Carlos Krzyzanowski

PhD student: Daiani Ajala Luccas

Post-doc: Renake N. Teixeira

Seed physiology, technology, biochemistry Molecular-genetic aspects

José França-Neto Fernando Henning

• Mature/ripe seeds remain green

• Impairment of chlorophyll degradation

• Reported for many years

The “green seed problem”

Yellow seeds Green seeds

• Big losses for growers

• Frequently reported – soybean and canola (Brassica

napus)

• Global climate changes

• Introduction of soybean to low latitudes

• Brazil – 2º largest soybean producer

• Breeding programs

Why is chlorophyll retention a problem to be

considered?

Source: targetmap.com. 2012 world soybean

production.

World Soybean Production in 2012

Normal conditions

Chlorophyll is completely degraded during maturation

nd nd

Causes of Chlorophyll Retention

• High temperatures

• Drought

Genetic Components

• Severe rust

• Chemical desiccants

nd = not detected

+

• Longevity (storability)

• Vigor

• Viability

Presence of chlorophyll in seeds = lower seed quality

www.corn.agronomy.wisc.edu/

Green Seeds:

•Reduction of up to 3 % point of total amount of oil•Higher acidity•Higher refining cost•Lower quality in storage → light - oxidation

Oxidation of the oil is influenced by the composition of fatty acids, oil processing, light, temperature,

concentration and type of oxygen, free fatty acids, mono and diacylglycerols, transition metals, peroxides,

thermally oxidized compounds, pigments and antioxidants.

Presence of chlorophyll in seeds = lower oil quality

Ch

loro

ph

yll

(mg

/kg)

Oxi

dat

ive

Stab

ility

11

0°C

(h

)

Tocopherols and Tocotrienols (mg/100g)

67.44 118.49

Green Seeds Yellow Seeds Green Seeds Yellow Seeds

Example: Cultivar W799

8.57

0.14

4.28

8.66

MINISTÉRIO DA AGRICULTURA, PECUÁRIA E ABASTECIMENTO

IN Nº 49, of DECEMBER 22, 2006 (BRASIL, 2006): define the characteristics of

identity and quality of refined vegetable oils.

IN Nº 11, of MAY 15, 2007 (BRASIL, 2007): establish the

Soybean Technical Regulation.

Sensory characteristics, color, impurities, composition of fatty acids, stability index, peroxide index, unsaponifiable matter, smoke point, etc ...

Green SeedIn-natura consumption: up to 4%

Other uses: up to 8%

Oil quality

How to address the green seed problem?

Some research questions

1. Does chlorophyll directly affect soybeanseed quality? Or is it just a marker for thestage of seed ripening?

2. Why do seeds have chlorophyll, if it ispotentially damaging? Biological role?

3. Can we dissect the environmental from thegenetic causes of green seeds?

Approaching the

Green Seed

Problem

STEP 1

Chlorophyll vs seed/oil

quality

STEP 2

Molecular characterization

of chlorophyll retention

STEP 3

Molecular control of

chlorophyll degradation

STEP 4

Molecular markers for

breeding/genes for genetic

modification

Some details of experimental approach

Environmental vs. genetic component (genotype)

of seed and oil quality

www.caloriesecrets.net

42 Lots/13 cultivars of soybean were harvested in

different regions and climatic conditions of Brazil

Percentage of green seeds was determined

(any sign of green color the seeds were classified as

‘green’)

Seed lots with the highest and the lowest percentage of green seeds from each

cultivar were selected.

CV SYN1059 RR Lot Place % Green Seeds

Lot 1 SC559102 R CRISTALINA-GO 12

Lot 2 SC559207 R CRISTALINA-GO 21

Lot 3 SC559516 A CRISTALINA-GO 3

Lot 7 SC5595024 A IPAMERI-GO 5

From 42 Lots 24 Lots

For example:

First selection

CV SYN1059 RR Lot Place % Green Seeds

Lot 2 SC559207 R CRISTALINA-GO 20.75

Lot 3 SC559516 A CRISTALINA-GO 2.75

CV 71MF00 RR Lot Place % Green Seeds

Lot 5 SC571723 R CRISTALINA-GO 15.25

Lot 6 SC5716027 A IPAMERI-GO 4.25

CV DM2366 IPRO Lote Local % Sementes Verdes

Lot 8 SC5663025 A PLANALTINA 20.25

Lot 9 SC566414 R CRISTALINA-GO 74.5

CV NA5909 R6 Lot Place % Green Seeds

Lot 12 x RIO VERDE - GO 7.75

Lot 35 9 Ubiratã-PR 22.5

Lot 37 NC 150065015 PARAÚNA - GO 55

CV NS 5959 Lot Place % Green Seeds

Lot 18 FJH23CC00018106 CAMPOS NOVOS- SC 3

Lot 34 32 Ubiratã-PR 66.5

CV DESAFIO 8473R5F Lot Place % Green Seeds

Lot 15 x RIO VERDE - GO 2.75

Lot 39 BD 400055015 PARAÚNA - GO 22

Lot 40 BD 450360015 RIO VERDE - GO 22

CV W799 RR Lot Place % Green Seeds

Lot 16 x RIO VERDE - GO 34.25

Lot 17 x RIO VERDE - GO 11

CV M6972 IPRO Lot Place % Green Seeds

Lot 20 6972465002 APROSMAT- MT 17.75

Lot 24 6972265001/58 APROSMAT- MT 0

CV 97Y07 Lot Place % Green Seeds

Lot 28 2396720 Cristalina-GO 12.25

Lot 29 2396722 Sítio D'Abadia-GO 0.75

CV AS3570 IPRO Lot Place % Green Seeds

Lot 10 x CURITIBANOS- SC 26.75

CV GNZ6905 RR Lot Place % Green Seeds

Lot 14 x RIO VERDE - GO 12.75

CV BG4272 Lot Place % Green Seeds

Lot 31 2393434 Formoso-MG 22.75

CV 6563 RSF IPRO Lot Place % Green Seeds

Lot 42 DS 200052515 RIO VERDE - GO 21.25

Seed physiological and seed oil quality were

assessed and related to % of green seeds.

24 Lots/13 Cultivars

First selection

80706050403020100

100

90

80

70

60

50

40

30

20

Green Seeds (%)

Germ

inati

on

(%

)

More than 8%

Until 8%

Correlation Germination (%) x Green Seeds (%)

r: - 0.7907

Seed lots with higher percentage of green seeds → Lower germination

>8%< 8%

PC1 and PC2 explained 93% of the variation.

First Component (73,8%)

Seco

nd

Co

mp

on

en

t (1

9,2

%)

Germination (%)Normal Seedlings (%)

Abnormal Seedlings (%)Dead Seeds (%)

Root Lenght (cm)Shoot Lenght (cm)

Green and yellow seeds → Significantdifference in seed quality

Dead Seeds (%)

Germ

inati

on

(%

)

6050403020100

100

90

80

70

60

50

40

30

>

–

–

–

–

< 0

0 15

15 30

30 45

45 60

60

Seeds (%)

Green

Percentage of Green Seeds and MortalitySeed lots with higher percentage of green seeds → Higher mortality

Influence of environment and genotype on the production of

green soybean seeds

From 24 Lots 8 Lots

Second selection

NA 5909

Desafio 8473R5F

NA 5909

Desafio 8473R5F

Environmental Influence: same cultivar from various locations

Second selection

Paraúna-GO

Ubiratã-PR

Paraúna-GO

Rio Verde-GO

Genotype Influence: same location but different cultivars

Second selection

NA 5909

NS 5959

NA 5909

Desafio 8473R5F

W799

Desafio 8473R5F

DM 2366

97Y07

Ubiratã-PR

Paraúna-GO

Rio Verde-GO

Cristalina-GO

2 Cultivars 2

Environmental

Conditions

X

Seed Quality

Chlorophyll Content

Gene Expression

Characterization of Green

Seed Production

Experimental setup molecular approach

Susceptible

X

Tolerant

Non-stressed

X

Stressed

(heat and drought)

Harvesting

3 Stages of Maturation

R6 R7 R8

Multiple gene ontologies were enriched in soybean seeds produced under stress. Among the top-ranking ontologies was the photosynthetic process.

NYC1_1, PPH2, D1 and D2 are the most affected genes instressed/green seeds of the susceptible cultivar and maybe the major cause of the chlorophyll retention insoybean seeds.

Expression analysis suggests higher levels of the photosynthetic proteins D1, D2, A1 and A2 in stressed/green seeds.

Higher expression of SGR1 (D1), SGR2 (D2), NYC1_1 and PPH2 in fully mature seeds of the tolerant cultivar allows

these seeds to cope with stressful conditions and complete chlorophyll degradation.

Main conclusion

On-going

Stay-green phenotypes

• Mutation - chlorophyll retention in leaves and seeds

(Sato et al., 2007) (Delmas et al., 2013)

• d1d1d2d2 soybean mutant (Guiamét et al, 1991)

• Various stay-green mutants in Arabidopsis

Arabidopsis thaliana

• Search for sequence differences – genes and promoters

(tolerant vs susceptible) GENOSOJA consortium

• Overexpression of SGR1 and SGR2 and other (Arabidopsis

and soybean); constructs ready

• Soybean field experiment with these transgenes

Effect on plant senescence, seed and oil quality?

Planned:

Molecular Control of Chlorophyll Retention

Dekkers et al., 2016

Follow up

1. Further characterization of upstream molecular regulation of chlorophyll degradation

2. Expand survey of green seed problem in Brazil and include meteorological data and soil properties during seed filling and ripening

3. Validation of more candidate genes (derived from upstream candidates) in Arabidopsis and soybean.

4. Derive from genome sequences of multiple soybean cultivars (tolerant and susceptible) sequences of candidate genes and potentially causal single nucleotide polymorphisms (SNPs) GENOSOJA.

5. Develop approaches for molecular breeding and genetic modification (including CRISPR-cas technology) to reduce green seed problem in the field

www.agrodaily.com

Collaborators in follow-up project

José França-NetoFernando Henning

Liliane MarciaFrancisco C. Krzyzanowski

Edvaldo A Amaralda Silva

Henk WM Hilhorst

Acknowledgements