Functional Characterization Of Transcription Factors Regulating Oil Biosynthesis In Soybean (Glycine Max Merr.)

Glycine max commonly known as soybean,is a legume crop that is cultivated globally for beans production owing to their broad range commercial applications.For example,soy vegetable oil obtained from these beans serves as an important ingredient of human food besides serving as a major raw material for various industrial applications.Lipids synthesis and metabolism are the major activities of living systems,such as,triacylglycerol(TAG)serves as energy reservoir while phospholipids acts as building blocks for the formation of biological membranes which are essential for the early development and growth of plants and serve as food and nutrients for humans.Conventional approaches for enhancing oil production mainly focused on manipulation of oil production pathway genes.However,such approaches were limited by the fact that these approaches could only target some specific genes directing the regulation of particular fatty acid synthesis,degradation,and composition,etc.Such discrepancies could be overcome by manipulating the potential of transcription factors to simultaneously regulate multiple genes controlling a complex pathway,for lipid biosynthesis.Therefore,the current study was aimed to enhance oil production in soybean by manipulating ABSCISIC ACID-INSENSITIVE3(ABI3),FUSCA3(FUS3),and LEAFY COTYLEDON2(LEC2)B3 domain binding factors along with LEC1 that is a HAP3-type transcription factor(together known as LAFL-clade)essential for seed development and regulation of seed storage substances such as protein,carbohydrates,and lipids.These transcription factors were cloned in p B2GW7 destination vector through GATEWAY cloning method.The constructs were used to transform Arabidopsis and soybean cotyledons through floral-dip and hairy-root induction methods,respectively.The fatty acid analysis revealed that ectopic expression of Gm LAFL-clade modified the fatty acid composition of wild-type and respective mutant seeds.Arabidopsis seed TAG analysis revealed that lec1,lec2,fus3 and abi3 Arabidopsis mutants contained low oil content compared to their wild-type and Gm LAFL complemented seeds.Gm LEC1/atlec1 and Gm LEC2/atlec2 seeds contain 4.5% and 10% more total TAG content relative to atlec1 and atlec2 mature mutant seeds.However,these mutants demonstrated high concentration of starch and other phenolic compounds compared to wild-type.In Gm FUS3/atfus3 and Gm ABI3/atabi3 seeds an increase of 16% and 7.7% in oil was recorded compared to atfus3 and atabi3 seeds,respectively.Furthermore,mutant complementation of each single lafl mutant with respective Glycine max LAFL gene rescued seed phenotype and embryonic traits such as trichome formation and cotyledon identity.We further verified that over-expression of each Gm LAFL-clade member enhanced the total oil content of Arabidopsis seed.Specifically,Gm LEC1,Gm LEC2,Gm FUS3,and Gm ABI3 averagely increased oil production in seeds up to 13%,34%,25%,and 34% respectively,compared to wild-type.Moreover,the total seed protein was also increased in ectopically expressed Gm LEC1,Gm LEC2,and Gm FUS3 transgenic Arabidopsis seedsEctopic expression of these transcription factors in transgenic soybean hairy roots enhanced TAG production and modified the fatty acid composition compared to control(non-transformed hair-roots).Further,transcriptome data of overexpressed Gm LEC2 and Gm FUS3 hairy roots demonstrated that these transcription factors direct several key genes involved in fatty acid biosynthesis,elongation,transport,biosynthesis/catabolism of TAG and other polar lipids.Specifically,Gm FUS3 participate in oil production through repressing of transcription factors that negatively regulate fatty acid biosynthesis or LAFL genes during seed development.Gm LEC2 transcriptome data revealed that Gm LEC2 is crucial for starch metabolism by down-regulating sucrose synthases(SUS).Moreover,expression pattern of Gm LAFL in soybean seed at different developmental stages indicated their role in soybean seed development.These findings indicate that Gm LAFLclade has potential to regulate the seed storage substances filling during soybean seed development.These results emphasize the importance of LAFL genes to customize the crops for high oil production.