Molecular Mechanisms Underlying The Difference Of Pigmentation In Mulberry Fruits

Mulberry is the traditional food source of silkworm.The fruit of mulberry is also favored by consumers because of its gorgeous color,good taste and rich nutritional value.Mulberry fruits have a variety of colors,such as white,yellow,red,and purple.Anthocyanins are abundant in purple mulberry fruits,but not in pale(yellow or white)ones.The genes involved in anthocyanin biosynthesis in mulberry plants have been identified and cloned,and the transcriptional levels of CHS1,CHI,F3H1,F3?H1,and ANS have been shown to be correlated with anthocyanin concentrations during fruit ripening.However,neither the regulation of flavonoid biosynthesis in mulberry nor the molecular basis for the different fruit colors of mulberry is fully understood.Therefore,it is of great significance to reveal the regulation mechanism of the flavonoids pathway and the molecular mechanism of mulberry fruit color difference for improving the quality of mulberry fruit.Morus notabilis C.K.Schneid(yellow fruit),Morus alba L.cv.Hongguo2(purple fruit),and Morus alba L.cv.Baiyuwang(white fruit)were selected for this study.Metabonomics and transcriptomics were used to analyze the fruits of these cultivars,and the material basis for the color difference of the fruits was identified.Based on the M.notabilis genome and the fruit transcriptome,we rearranged the flavonoid biosynthesis pathway of mulberry fruit,and the transcription factors involved in the flavonoid metabolic pathways were identified.In vivo and in vitro assays uncovered the regulation mechanism of the flavonoids pathway and the molecular basis for the different fruit colors of mulberry.The results of this study are as follows: 1.The Material Basis of Color Differences in MulberryThe metabonomics and transcriptomics of yellow fruit(CS),white fruit(BYW)and purple fruit(HG2)were determined.Flavonoids are the main differentially accumulated metabolites among the cultivars.Purple fruit(HG2)contains the most anthocyanins,while yellow(CS)and white fruits(BYW)tend to accumulate flavones and flavonols,respectively.The expression of genes related to flavonoid biosynthesis pathway was significantly different among the fruits of cultivars.The yellow fruit(CS)is rich in anthocyanins of delphinidin type,but we did not find any F3'5'H gene belonging to CYP75 A subfamily,and two CYP75 B genes which are close to CYP75 A subfamily do not have the catalytic activity of F3'5'H.Based on these findings,we rearranged the flavonoid biosynthesis pathway of mulberry fruits.The transcript levels of the pathway genes in fruits were much lower in CS and BYW than in HG2.Correspondingly,the flavonoid contents were much lower in CS and BYW than in HG2.The key genes of anthocyanin pathway changed in CS and BYW,the carbon flow could not flow to the anthocyanin pathway normally in CS and BYW due to the lower expression of DFR,and turned to the upstream flavonol pathway for diversion,resulting in a significant decrease in the anthocyanin content and a significant increase in the proportion of flavonol in CS and BYW.Compared with BYW,the carbon flux flow to flavonol pathway in CS is also limited by lower expression of FLS gene,which cause a proportion of the carbon flux to be diverted to upstream branches,resulting in the highest proportion of flavone in CS.Therefore,the differences in the expression of the structural genes change the trend of carbon flux in the flavonoid pathway,resulting in various colors of mulberry fruit.2.Core factors of the mulberry flavonoid biosynthesis regulatory networkBased on the M.notabilis genome and the fruit transcriptome,we identified seven transcription factors involved in the regulation of flavonoid biosynthesis.MYBA-bHLH3-TTG1 regulates the biosynthesis of anthocyanins,while TT2L1 and TT2L2 work with bHLH3 or GL3 and form a MYB-bHLH-WD40(MBW)complex with TTG1 to regulate proanthocyanidin synthesis.By contrast,MYBF regulates the flavonol biosynthesis without any partner.Notably,the analysis of transcriptional regulation of the flavonoid pathway genes showed that the participation of bHLH3 was required for the activation of structural genes,including those of CHS2,CHI,F3 H,CYP75B1,DFR,and UFGT.It is suggested that bHLH3 plays a vital role in determining the biosynthesis of flavonoids in mulberry fruits.The gene products of MYBA and bHLH3 in purple fruit were no differ from that in pale-color fruit based on our transcriptome data.Although MYBA and bHLH3 participate in the regulation of anthocyanin biosynthesis,only bHLH3 transcript level are correlated with those of flavonoid pathway genes between purple and pale-color fruits,and there was no significant difference in the transcript levels of MYBA between pale and pigment-rich fruits.Taken together,all these findings imply that the active function of MYBA is dependent on bHLH3,and that bHLH3 plays a vital role in determining the biosynthesis of flavonoids in mulberry fruits.3.Interaction between regulatory mechanisms of the mulberry flavonoid pathwayIn correlation network analysis of anthocyanins and gene expression,the transcript level of MYB4 is highly correlated with that of ANS and bHLH3 and the degree of fruit coloration(i.e.,abundant transcripts in purple fruit but low levels in pale fruit).MYB4,a typical R2R3-MYB repressor belongs to the R2R3-MYB subgroup 4 family,was found to interact with bHLH3 or GL3 and then form a ternary complex with TTG1.The results of dual-luciferase reporter assays showed that addition of MYB4 to MYBA/bHLH3 and TT2L2/GL3 complexes reduced the activities of ANS and LAR promoters,respectively.Furthermore,ectopic expression of MYB4 in tobacco and A.thaliana demonstrated that it functions as a negative regulator of both anthocyanin and proanthocyanidin biosynthesis.These findings suggest that MYB4 acts as a repressor of anthocyanin and proanthocyanidin biosynthesis by disrupting the interaction between the MYB activator and bHLH.At the same time,in addition to being activated by MYBA /bHLH3,MYB4 can also be activated by proanthocyanidin activation complexes,including TT2L1/bHLH3,TT2L1/GL3,TT2L2/bHLH3,and TT2L2/GL3.These results suggest that MYB4 participates in a negative feedback mechanism to balance anthocyanin and proanthocyanidin accumulation and constructs a steady-state regulatory network for flavonoid biosynthesis together with activating factors of the anthocyanin and proanthocyanidin pathways.This both ensures adequate synthesis of anthocyanins and proanthocyanidins and avoids the cellular damage caused by excessive accumulation of flavonoids.The flavonoids homeostasis network is stable in pigment-rich fruits of mulberry and ensures the fruit accumulates desirable flavonoids.However,the abnormal expression of bHLH3 disrupts this dynamic equilibrium mechanism: MYB4 cannot be effectively activated by bHLH3,causing the feedback regulation mechanism to fail during the fruit pigmentation,and the MBW complexes dependent on bHLH3 cannot effectively activate the target genes,thereby reducing the accumulation of flavonoids.A decrease in DFR expression causes carbon flux to flow to the upstream branch,resulting in a higher proportion of flavonols in CS and BYW than in HG2.Because of the low expression of MYBF and the high expression of FNS in CS,the blockage of the anthocyanin and flavonol pathways diverts carbon flux to the upstream flavone pathway,resulting in the highest flavone content in CS.The results of our research systematically revealed the reason why mulberry fruits have various colors,which provides a unique perspective for explaining the polymorphism of plant tissue color.