Genetic Mechanism And Gene Regulation Network Of Flavonoid Pigmentation In Rice(Oryza Sativa L.)

The floral organs can be painted as purple,brown or red color due to the accumulation of flavonoids in rice plants.The flavonoids are also widely distributed in vegetative organs such as leaf,sheath,stem and so on.However,the molecular mechanism for specific organ pigmentation is far from clear in rice.Here,we systematically clarify the C-S-A gene module in pigmentation of rice hull from the perspective of genetics,molecular biology,metabolism and evolution.Under the theoretical basis of this model,we further investigate the genetic mechanism of stem pigmentation and clone the related gene.In addition,the preliminary work is carried on to extend applications based on C-S-A gene model.In C-S-A gene system,three kinds of genes that participated in the regulation of flavonoid pigmentation are C1,S1 and A1.C1 encodes a R2R3-MYB transcription factor and acts as a switch on the color-producing process.S1 encodes a bHLH protein and functions in a tissue-specific pattern.A1 encodes a dihydro-flavonol reductase(DFR)and is responsible for anthocyanin biosynthesis.C1 and A1 are key factors for color formation.They together determine whether color could be produced and which kind of color to be shown.The brown color could appear in apiculus under the independent function of C1,while purple color formation needs co-function of C1 and A1.If S1 additionally participates in the coloration process,the brown or purple color could occur in hull.The molecular basis of C-S-A gene regulation system is revealled by series of molecular biology experiments.The R3 repeat of C1 and the N terminal conserved region of S1 can interact with each other to regulate the expression of downstream structural genes.If mutation occurs in the R3 repeat of C1,the interaction between C1 and S1 will be weaken significantly.It causes the dramatically reduced expression of structural genes and at last color disappears.Trans-activation assays testify that co-regulation of C1 and S1 could strikingly enhance the transcription activity of structural genes(CHS,CHI,F3H,F3 ’H,A1,ANS).It is also clarified that C1 and S1 can directly bind to the DNA motif in the promoter region of A1.This indicates the direct hierarchy regulation between these two kinds of genes.The purple color is formed by the accumulation of cyanidin 3-O-glucoside,one kind of anthocyanin,which is synthesized under the catalyzation of A1 encoded reductase.If A1 losses or changes its function in the pathway,the synthesis of C3G is blocked.The main flavonoids are flavonols and flavanones that located on the branches upstream of the A1,and also the other kinds of anthocyanin,delphinidin 3-O-glucoside,that different with C3G.The overlay of these metabolites causes the brown color formation.Via the investigation of Cl and Al combination haplotypes in natural rice germplasm,we find that the gene model is universal in regulation of color formation in rice varieties.In addition,we find that varieties with brown colors almost all belong to the sub-group of the temperate japonica,this specificity is highly correlated with the functional mutations on Al gene which only occurr in temperate japonica.By phylogenetic analysis using the genotype of functional mutation sites of C1 and A1,we clarify that rice color diversification patterns are different between indica and japonica.In indica,purple color changes to colorless type directly.But in japonica,it has three ways to turn its color.One way is pruple color changes to brown and then changes to colorless,the second way is purple color directly changes to colorless phenotype and the third way is purple color changes to brown without further variations.Neutral tests reveal that C1 and A1 did not undergo selection during domestication from wild rice to cultivar,but experienced artificial selection after the diversification process of cultivars.This directional selection is resulted from the disappearance or changement of colors that caused by the functional mutations on C1 and A1.The phylogenetic tree clearly shows that indica and japonica are divided into different clades with their recent wild ancestors located in different areas.The independent origin and evolution of rice color is clearly uncovered by the minimum spanning tree.It also indicates the independent origin of indica and japonica,with their recent wild ancestors geographically originated from different districts.According to the C-S-A gene model,we construct the segregated population for stem pigmentation on purpose.By map-based cloning,we find the specific gene for stem pigmentation is still on S1 locus and consider as an allele of S1,name it S1-j.Transformation test by knock-out of this gene in parent with purple-colored stem using crispr-cas9 system and over-expression of S1 in PH NIL clarify the function of S1-j in regulation of the stem coloration.From the above,we put forward the C-S-A model in rice color formation by the amount of experimental evidence and establish the theory basis for the deep studying in rice flavonoid biosynthesis.Meantime,it provides theoretical guidance for applying this gene model in breeding process to cultivate new rice varieties with healthcare function.