Functional Characterization Of Diacylglycerol Acyltransferase And WRINKLED1 In Soybean

Soybean [Glycine max(L.)Merr] is one of the largest oilseed crops in the world,providing about 28% edible vegetable oils for human.Soybean oil is the best source of essential fatty acid for human because soybean oil is rich in lecithin,B vitamins,and inorganic salt.Soybean oil is a good edible vegetable oil with high nutritional value.However,compared to other oilseed crops,such as rapeseed with 40% oil content and peanut with 50% oil content,there is large potential to improve the oil content in soybean seeds.The demand of vegetable oil is increasing with the increase of the population and industrialization.In oilseed crop,such as soybean,triacylglycerols(TAGs)are the most common storage lipids in seed as the form of lipid droplets.TAGs also provide human with foods and nutrients.They are regarded as significant resources with a lot of potential to be manipulated for biofuels and bioproducts.Diacylglycerol acyltransferases(DGATs)play a key role in plant triacylglycerol(TAG)biosynthesis.There are 10 DGATs in soybean,and we cloned two different types of DGATs from soybean.Type 1 and 2 DGATs from soybean were characterized for their functions in TAG biosynthesis and physiological roles.GmDGAT1 A is highly expressed in seeds while GmDGAT2 D is mainly expressed in floral organ.Both GmDGAT1 A and GmDGAT2 D were localized to the endoplasmic reticulum and complemented the TAG deficiency of a yeast mutant H1246.Overexpression of both GmDGATs in Arabidopsis and soybean hairy root promoted seed oil accumulation,whereas,they showed differences in the preference of substrate utilization.GmDGAT1A-transgenic hairy roots synthesized more C18:3-TAG,whereas GmDGAT2 D preferred to use C18:2-acyl CoA for TAG synthesis.Overexpression GmDGAT2 D promoted C18:2-TAG in wild-type Arabidopsis but enhanced C18:1-TAG production in rod1 mutant seeds with decreased C18:3-TAG.However,GmDGAT1 A enhanced C18:3-TAG and reduced C20:1-TAG contents.The different substrate preferences of two DGATs may confer diverse fatty acid profiles in soybean oils.GmDGAT1 s play a role in usual seed TAG production and also involved in responses to environmental and hormonal cues.GmDGAT2 D was up-regulated by cold and heat stress and ABA and it was repressed by insect biting and jasmonate.GmDGAT1 A showed limited responses to stresses.Two type of GmDGATs showed different expression patterns under biological and abiotic stress condition,indicating the differences of their potential physiological functions in different tissues.The WRINKLED1(WRI1)gene encodes an APETALA2/ethylene-responsive element-binding transcription factor involved in the control of metabolism,particularly glycolysis and fatty acid biosynthesis in developing seeds.Soybean genome contains at least 15 WRI orthologues and we identified GmWRI1 a and GmWRI1 b as the WRI1 homologs in soybean.Among them,GmWRI1 a and b,with their alternative splicing(AS)products a' and b',respectively,were highly expressed in developing seeds and nodules.GmWRI1 a and b and AS forms b' differently promoted TAG accumulation in Arabidopsis wild-type and atwri1 mutant seeds.Ectopic expression of GmWRI1 s resulted in reduced soluble sugars and enhanced TAG contents.Transcriptome analysis revealed that 15 genes containing AW-boxes motif in their promoters were targeted by GmWRI1 s,including glycolysis and FA and TAG biosynthesis genes.Although GmWRI1 a,GmWRI1b and GmWRI1 b ' were all nucleus-located,only GmWRI1 a,but not GmWRI1 b or b',trans-activated targeted genes.Therefore,GmWRI1 a and GmWRI1 b are likely to regulate downstream genes in different ways.GmWRI1 s was not only involved in the seed oil accumulation,but also participated in the formation process of nodule which is unique to legumes.Overexpression of GmWRI1 s led to increased nodule numbers whereas GmWRI1 knockdown resulted in fewer nodules,coinciding with altered expression of nodulation genes.GmWRI1-regulated metabolism including starch degradation,glycolysis,and lipid metabolism were critical for nodulation.Our results indicate that GmWRI1 is required in soybean nodulation by regulating glycolysis and FA biosynthesis to provide rhizobia with carbon sources for proliferation and nodulation.