GmMYBA2 And GmMYBR Function In A Feedback Loop To Control Pigmentation Of Seed Coat In Soybean

Soybean(Glycine max)is economically one of the most important crops providing valuable vegetable protein,oil and other ingredients for humans and livestock.It is first planted in China and then widely cultivated all over the world.The amino acid distribution of soybean protein in seeds resembles that in animal protein,making soybean a better protein resource than most other vegetable sources.It is widely accepted that modern cultivated soybean is domesticated from wild soybean(Glycine soja Sieb.& Zucc.)although the domestication origin is still mysterious.As a primary organ depositing oil,protein and other nutrients,the quality of soybean seeds might be the selecting target during soybean domestication history,and the seed coat color is one of the domesticated traits as it changes from black in wild soybean(Glycine soja)to brown,yellow,green and black color in most cultivars.Generally,flavonoids are the main substances that affect the seed coat color of soybeans.Among them,anthocyanins are mainly accumulated in black seed coat and procyanidins in brown seed coat.Their accumulation confers plant with pleiotropic benefits such as defending against a variety of stresses,attracting pollinators or seed distributors,and is also beneficial to human health.Black beans can prevent obesity and diabetes as the high seed coat flavonoids result in enhancing energy consumption and inhibiting inflammatory response.However,how the seed coat colors associate with other seed qualities such as oil or protein content is still poorly understood,and the clarification of the molecular basis of seed coat pigmentation is a prerequisite to address this question.Previous forward genetic studies have partly deciphered several loci responsible for soybean seed coat coloration such as T(Tawny),I(Inhibitor),W1,O and R,which are co-segregated with F3'H,CHS cluster,F3'5'H,ANR and R2R3 MYB activator,respectively.Although different combinations with either dominant or recessive alleles of the loci might partly account for some of the complicated soybean seed coat coloration patterns,the transcription regulation network of these major loci are not well deciphered.Therefore,to find transcription factors that can regulate the color of soybean seed coat and to understand how they work is the key factor to study the transcriptional regulation mechanism of soybean seed coat color formation.In this study,two transcription factors regulating seed coat color are select from the soybean MYB transcription factor database as transcriptional activators and transcriptional suppressors,respectively.Then the anthocyanin transcriptional regulation network of soybean seed coat is analyzed by transient protoplast transfection of Arabidopsis and soybean,genetic transformation,HPLC-ESI-MS,Ch IP-q PCR,Yeast two-hybrid assay,EMSA,etc.It laid a foundation for analyzing the color formation mechanism of soybean seed coat.The main results were as follows:Here,two major regulators,Gm MYBA2 and Gm MYBR,were functionally characterized as anthocyanin activator and repressor,respectively.Ectopic expression of Gm MYBA2 in soybean hairy roots conferred the enhanced accumulation of delphinidin-and cyanidin-types of anthocyanins in W1 t and w1 T backgrounds,respectively.The root of soybean hair appeared purple and brownish color,respectively.Gm MYBA2-OE activated F3 H,F3'5'H,UGT and other anthocyanin genes.The seed coat pigmentation of Gm MYBA2-OE transgenic plants in W1 background mimicked the imperfect black phenotype(W1/w1,i,R,t),suggesting that Gm MYBA2 was responsible for R locus.A series of Molecular and biochemical analysis showed that Gm MYBA2 could directly bind to the promoter of the structural genes Gm F3'5'H,Gm UGT78K2 and the regulatory gene Gm MYBR to activate their expression.In addition,experiment of transient protoplast transfection,Bi FC,Y2 H verified that Gm MYBA2 and Gm MYBR could interact with Gm TT8 a,respectively.However,Gm MYBR was a transcriptional inhibitor,in the Gm MYBR-OE transgenic plants expression levels of anthocyanin synthesis related genes showed a significant decrease.Gm MYBA2 and Gm MYBR might form a feedback loop to fine-tune seed coat coloration,which was confirmed in transgenic soybeans.Both Gm TT8 a and Gm MYBR that were activated by Gm MYBA2 in turn enhanced and obstructed the formation of Gm MYBA2-Gm TT8 a module,respectively.The results revealed the sophisticated regulatory network underlying the soybean seed coat pigmentation loci and shed light on the understanding of the seed coat coloration and other seed inclusions.