Regulation Of Flavonoids In Strawberry Fruits By R2R3-FaMYB5/10 Dominated MYB-bHLH-WD40 Ternary Complexes

Strawberry(Fragaria × ananassa Duch.)is a perennial herb in the Rosaceae family,the edible part of a strawberry is developed from the receptacle with a large number of flavonoids.In unripe fruit proanthocyanidins(PAs,also named condensed tannins)are the mainstay,while in ripe strawberry fruit mainly contains anthocyanins.In the process of plant growth and development,flavonoids help plants resist stresses from the external environment.At the same time,flavonoids play an important role in human health as medicinal,health and nutrition components.Therefore,it has always been the goal of strawberry breeders to increase the content of flavonoids.The biosynthesis and accumulation of plant flavonoids involves a complex regulatory network,in which MYB transcription factor plays a central role.At present,studies on the Rosaceae mainly focus on MYB10 and its alleles,little is known about other MYBs and the MYB-b HLH-WD40(MBW)ternary complex involved in this process.In this study,cultivated strawberry’Benihoppe’,’Xiaobai’,’Toyonoka’,’Snowwhite’ and tobacco(Nicotiana benthamiana)were used as plant materials.Bioinformatics analysis,transient overexpression,Y2 H,BiFC,Dual-luciferase assay,EMSA,Western blot,RNA-seq and metabolome sequencing were used to explore the regulation mechanism of MYBs,b HLHs and WD40 s transcription factors and MBW complexes on flavonoid metabolism in strawberry fruits.The main research results are as follows:1.A total of 15 candidate MBW members were selected and amplified from cultivated strawberries,including six MYB TFs(FaMYB1/9/10/11,R3-FaMYB5,and R2R3-FaMYB5),six b HLH TFs(FaEGL3,Fab HLH33/33B/3,FaMYC1,and FaGL3)and three WD40 TFs(FaTTG1,FaLWD1,and FaLWD1-like).A novel FaMYB5 TF with an intact R2R3 domain was amplified.Further analysis of the expression patterns at different developmental stages in ’Benihoppe’ and ’Xiaobai’ revealed that they were expressed at all developmental stages.FaMYB1 was gradually elevated in ‘Xiaobai’,R2R3-FaMYB5 was constitutively expressed in different developmental stages,which was strikingly contrasting to the fruit specific expression patterns of FaMYB10,and FaMYB9/11 were mainly expressed in green fruit.2.Transient overexpression of these 15 TFs was performed in strawberry fruits,and it was found that R2R3-FaMYB5,FaMYB9/10/11 promoted the accumulation of Cy3 G and Pg3 G in ’Xiaobai’ flesh,and restored the accumulation of Cy3 G in ’Benihoppe’ flesh,thus significantly increasing anthocyanin content in strawberry fruit.The content of PAs in the above overexpressed samples was also increased.3.Y2 H and BiFC assays confirmed that R2R3-FaMYB5/10,FaEGL3 and FaLWD1/FaLWD1-like proteins interacted with each other and formed MBW complex to regulate flavonoid metabolism in strawberry.R2R3-FaMYB5/10+FaEGL3,R2R3-FaMYB5/10+FaEGL3+FaLWD1 and R2R3-FaMYB5/10+FaEGL3+FaLWD1-like complexes greatly promoted the accumulation of anthocyanins and PAs in strawberry fruits.The regulatory intensity on structural genes in the metabolic pathway was stronger than that of individual complex member.4.The positive effect of FaMYB10 on flavonoids was achieved by up-regulating the expression of almost all structural genes(except LAR)in the metabolic pathway.In contrast,R2R3-FaMYB5 was more specific,only PAL,LAR,TT12,AHA10,C4 H,F3’H and LAR can be affected.The promotion of anthocyanin by these two MYBs is mainly through significantly upregulate the expression level of C4 H and F3’H.R2R3-FaMYB5 increased PAs content via LAR branch,while FaMYB10 through ANR branch.It is noteworthy that both FaMYB9 and FaMYB11 tremendously elicited the accumulation of PAs by up-regulating the expression levels of both LAR and ANR.5.RNA-seq and metabolome analyses were carry out on R2R3-FaMYB5 overexpressed samples.Based on RNA-seq data,6896 differentially expressed m RNAs,489 differentially expressed lnc RNAs,37 differentially expressed circ RNAs and 12 differentially expressed micro RNAs were screened.Consistent with the q PCR results,the expression levels of most flavonoid biosynthesis genes,including early and late structural genes and transporters,were increased,with C4 H,F3’H and LAR increasing the most.Dual-luciferase analysis showed that R2R3-FaMYB5 enhanced the activity of F3’H and LAR promoter.Further EMSA was used to confirm that R2R3-FaMYB5 could directly bind to the MYB binding site(AACCTAA)on the F3’H promoter and the MYB binding site(ACCAACAACCAAA)on the LAR promoter.In addition,differentially expressed lnc RNA TCONS＿00102355 and TCONS＿00090344 were predicted to regulate transcription level of R2R3-FaMYB5.Metabolome results showed that overexpression of R2R3-FaMYB5 not only increased the content of common anthocyanins in strawberry,but also up-regulated many flavonoid compounds such as quercetin glycoside and genistin.In addition,the intermediate metabolites of lignin biosynthesis,such as caffeic acid and coumarin,were also significantly increased.6.Overexpression of R2R3-FaMYB5 in the myb10 mutant ’Snowwhite’ was found to fail to restore anthocyanin accumulation in the flesh.q PCR and RNA-seq results indicated that R2R3-FaMYB5 significantly promoted the expression of most structural genes involved in anthocyanin metabolism,including PAL,C4 H,F3’H and LAR,However,compared with FaMYB10,R2R3-FaMYB5 could not affect the expression of the anthocyanin transporter TT19.Co-overexpression of R2R3-FaMYB5 and FaTT19 in’Snowwhite’ restored partial pigment accumulation.7.Lnc RNA-myb5 affected anthocyanin and PAs through positive feedback regulation of R2R3-FaMYB5 on transcription level.FaMYB1 inhibited the facilitative effect of R2R3-FaMYB5 to a certain degree by weakening the activation intensity of R2R3-FaMYB5 on promoter F3’H and LAR.FaMYB44-like directed the metabolic flow from the phenylpropane pathway to the lignin branch,which offset the positive effect of R2R3-FaMYB5 on anthocyanin.FaBT2 interacted with R2R3-FaMYB5 and mediated the degradation of R2R3-FaMYB5 protein through the ubiquitin/26 S proteasome pathway.