Mechanism Of Negative Regulation Of Anthocyanin/Proanthocyanidin Biosynthesis By Transcription Factor FtMYB18 From Tartary Buckwheat

Tartary buckwheat（Fagopyrum tataricum（L）Gaertn.）,as a miscellaneous grain crop,is rich in flavonoid compounds such as rutin,anthocyanins and proanthocyanidins（PAs）,and its health function has been paid more and more attention in the world.Although rutin content is an important quality character of Tartary buckwheat,the anthocyanins and proanthocyanidins content are related to its stress resistance as well as the additional ornamental and edible value.Anthocyanins/proanthocyanidins biosynthesis is mainly regulated by R2R3-MYB SG4 subfamily transcription factors in plant.In this study,a SG4-Like subfamily gene FtMYB18 was obtained from the Tartary buckwheat genome data.In order to reveal the molecular regulation mechanism of FtMYB18 involved in anthocyanins and proanthocyanidins biosynthesis in plant,we studied its molecular characteristics,gene expression patterns,biological effects,participation in MYB-b HLH-WD40（MBW）ternary complex formation,and JAZ protein interaction in jasmine signal pathway.Particularly,we explored the effects of FtMYB18-ΔC,the mutant truncated C5 motif（TLLLF）,on its function.The main results are as follows:1.Sequence analysis showed that two introns（145-228 and 359-525 bp）and three exons（1-144,229-358 and 359-983 bp）constituted the 983bp FtMYB18 gene.FtMYB18encodes a MYB protein with 243 amino acids.Multiple sequence analysis showed that the N-terminal of FtMYB18 protein contained a b HLH motif while its C-terminal domain contained the SG4-Like conserved motifs C1 motif,C2/EAR motif and C5 motif.Phylogenetic analysis showed that FtMYB18 was grouped into a cluster with Arabidopsis anthocyanins synthesis related R2R3-MYB SG4 subfamily.The transcriptional activation assay showed that FtMYB18/FtMYB18-ΔC had independent transcriptional activation activity.Transient transfection of onion epidermis cells showed that FtMYB18/FtMYB18-ΔC was located in the cell nucleus.2.The 2457 bp sequence locating at the 5’upstream of FtMYB18 was cloned and named as P_FtMYB18,and then the vector p BI101-P_FtMYB18-GUS was constructed and transiently transfected into Tartary buckwheat leaves by Agrobacterium tumefaciens（GV3101）.Gus staining analysis showed that the transcriptional expression of reporter gene was significantly induced by UV-B and ABA（P<0.05）,and inhibited by Me JA treatment（P<0.05）.However,reporter gene expression was not significant different between experimental group and control group（P>0.05）under cold treatment.Furthermore,the q PCR analysis indicated the transcriptional expression of FtMYB18 gene showed the strong responses to Me JA and ABA（P<0.05）after the seedlings of Tartary buckwheat were treated with exogenous physicochemical factors.3.The recombinant expression vectors of p CHF3-FtMYB18/FtMYB18-ΔC-YFP were constructed and transfected into tobacco and Arabidopsis thaliana mediated by Agrobacterium tumefaciens GV3101,or Tartary buckwheat hairy root by Agrobacterium tumefaciens A4.Compared with the WT lines,appearance colour were lighter in the transgenic lines tobacco flowers,Arabidopsis seedlings/seeds and Tartary buckwheat hairy roots.Meanwhile,there was no significant change in the rutin content（P>0.05）but the anthocyanins and proanthocyanidins content was decreased significantly（P<0.05）.Furthermore,the expression of CHS and DFR genes in flavonoid metabolic pathway was significantly down-regulated（P<0.05）.However,the contents of anthocyanins,proanthocyanidins and rutin showed no significant difference between overexpressing FtMYB18-ΔC plants and WT plants（P>0.05）as well as the expression level of CHS.The luciferase reporter assay showed that FtMYB18 could significantly inhibit the transcriptional activity of FtCHS and FtDFR promoters,but the inhibition was eliminated after C5 motif deletion.4.Yeast two-hybrid experiments showed that FtMYB18/FtMYB18-ΔC could interact with both b HLH and WD40 proteins encoded by FtTT8 and FtTTG1 respectively.Furthermore,yeast three-hybrid experiment showed that FtMYB18 could interact with FtTT8-FtTTG1 complex to form MBW ternary complex.The transient transfection experiment showed that FtMYB18-FtTT8-FtTTG1 complex could decrease the anthocyanin synthesis in tobacco.However,FtMYB18-ΔC would weaken the activity of ternary complex and the inhibitory effect on anthocyanin synthesis.5.FtMYB18 could interact with FtJAZ1,FtJAZ3,FtJAZ4 and FtJAZ7 proteins in jasmonic acid signal pathway to reduce the anthocyanin accumulation.However,FtMYB18-ΔC losted the interaction with FtJAZ3 and FtJAZ4 as well as the inhibitory effect on anthocyanins synthesis,but it didn’t affect the interaction with FtJAZ1和FtJAZ7and the inhibitory function.In summary,FtMYB18,as a SG4-Like-MYB transcription factor with the unique function,could not only mediate the MBW complex formation and the interaction with JAZs protein,but also suppress the synthesis of anthocyanins and proanthocyanidins in plant through down-regulating the expression of CHS and DFR genes.Particluarly,FtMYB18-ΔC,the mutant truncated C5,could weaken or eliminate the above effects.