Genetic and molecular basis of chlorophyll retention in soybean seed

Genetic and molecular basis of chlorophyll retention in soybean seed Henk Hilhorst, Wageningen University, Edvaldo A Amaral da Silva Universidade Estadual Paulista (FCA) Collaborators in Brazil: José França-Neto Fernando Henning Francisco Carlos Krzyzanowski

PhD student: Daiani Ajala Luccas Seed physiology, technology, biochemistry Post-doc: Renake N. Teixeira Molecular-genetic aspects José França-Neto Fernando Henning

The green seed problem Yellow seeds Green seeds Reported for many years Impairment of chlorophyll degradation Mature/ripe seeds remain green

Why is chlorophyll retention a problem to be World Soybean Production in 2012 considered? 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 Source: targetmap.com. 2012 world soybean production.

Normal conditions Chlorophyll is completely degraded during maturation

Causes of Chlorophyll Retention High temperatures Drought Severe rust Chemical desiccants + Genetic Components nd nd nd = not detected

Presence of chlorophyll in seeds = lower seed quality Viability Vigor Longevity (storability) www.corn.agronomy.wisc.edu/

Presence of chlorophyll in seeds = lower oil quality 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.

Chlorophyll (mg/kg) Oxidative Stability 110 C (h) Example: Cultivar W799 Tocopherols and Tocotrienols (mg/100g) 67.44 118.49 8.57 8.66 4.28 0.14 Green Seeds Yellow Seeds Green Seeds Yellow Seeds

Oil quality 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. Sensory characteristics, color, impurities, composition of fatty acids, stability index, peroxide index, unsaponifiable matter, smoke point, etc... IN Nº 11, of MAY 15, 2007 (BRASIL, 2007): establish the Soybean Technical Regulation. Green Seed In-natura consumption: up to 4% Other uses: up to 8%

How to address the green seed problem?

Some research questions 1. Does chlorophyll directly affect soybean seed quality? Or is it just a marker for the stage of seed ripening? 2. Why do seeds have chlorophyll, if it is potentially damaging? Biological role? 3. Can we dissect the environmental from the genetic causes of green seeds?

Approaching the Green Seed Problem STEP 4 Molecular markers for breeding/genes for genetic modification STEP 1 Chlorophyll vs seed/oil quality STEP 3 Molecular control of chlorophyll degradation STEP 2 Molecular characterization of chlorophyll retention

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 )

First selection From 42 Lots 24 Lots For example: 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

First selection 24 Lots/13 Cultivars Seed physiological and seed oil quality were assessed and related to % of green seeds. 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 lots with higher percentage of green seeds Lower germination 100 90 Correlation Germination (%) x Green Seeds (%) r: - 0.7907 >8% < 8% More than 8% Until 8% 80 Germination (%) 70 60 50 40 30 20 0 10 20 30 40 50 Green Seeds (%) 60 70 80

Second Component (19,2%) Green and yellow seeds Significant difference in seed quality PC1 and PC2 explained 93% of the variation. Germination (%) Normal Seedlings (%) Abnormal Seedlings (%) Dead Seeds (%) Root Lenght (cm) Shoot Lenght (cm) First Component (73,8%)

Seed lots with higher percentage of green seeds Higher mortality Percentage of Green Seeds and Mortality Germination (%) 100 90 80 70 60 50 Green Seeds (%) < 0 0 15 15 30 30 45 45 60 > 60 40 30 0 10 20 30 40 Dead Seeds (%) 50 60

Second selection From 24 Lots 8 Lots Influence of environment and genotype on the production of green soybean seeds

Second selection Environmental Influence: same cultivar from various locations NA 5909 Paraúna-GO NA 5909 Ubiratã-PR Desafio 8473R5F Paraúna-GO Desafio 8473R5F Rio Verde-GO

Second selection Genotype Influence: same location but different cultivars Ubiratã-PR NA 5909 NS 5959 NA 5909 Paraúna-GO Desafio 8473R5F W799 Rio Verde-GO Desafio 8473R5F Cristalina-GO DM 2366 97Y07

Experimental setup molecular approach 2 Cultivars Susceptible X Tolerant X Harvesting 3 Stages of Maturation 2 Environmental Conditions Non-stressed X Stressed (heat and drought) Characterization of Green Seed Production Chlorophyll Content Seed Quality R6 R7 R8 Gene Expression

Multiple gene ontologies were enriched in soybean seeds produced under stress. Among the top-ranking ontologies was the photosynthetic process. Expression analysis suggests higher levels of the photosynthetic proteins D1, D2, A1 and A2 in stressed/green seeds. NYC1_1, PPH2, D1 and D2 are the most affected genes in stressed/green seeds of the susceptible cultivar and may be the major cause of the chlorophyll retention in soybean seeds.

Main conclusion 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.

On-going

Stay-green phenotypes Mutation - chlorophyll retention in leaves and seeds Arabidopsis thaliana (Sato et al., 2007) (Delmas et al., 2013) d1d1d2d2 soybean mutant (Guiamét et al, 1991) Various stay-green mutants in Arabidopsis

Planned: 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?

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 CRISPRcas technology) to reduce green seed problem in the field www.agrodaily.com

Collaborators in follow-up project José França-Neto Edvaldo A Amaral Fernando Henning da Silva Liliane Marcia Francisco C. Krzyzanowski Henk WM Hilhorst

Acknowledgements