The Negative Regulational Molecular Mechanism Of The Poplar Transcription Factor MYB93 Involved In Flavonoids And Lignin Biosynthesis

Phenylpropanoids(including anthocyanins,tannins,flavonols and lignin)are important secondary metabolites in poplar.Not only the broad participation of poplar insect resistance,disease resistance,resist ultraviolet radiation and anti-aging and other physiological processes,it is an important component of poplar wood.Therefore,to elucidate the regulatory mechanism of flavonoids and lignin biosynthesis have great scientific significance and bright industry prospects.Based on the previous studies,a R2R3-MYB transcription factor MYB93,which may play roles in flavonoids and lignin biosynthesis,was screened and identified.In order to analyze the function of MYB93 in poplar,various experimental methods including cell biology,molecular genetics and biochemistry had been performed.The main findings are as follows:(1)MYB93 is a transcriptional repressor which localizes in nucleus,and has high expression levels in leaves and stemsqRT-PCR analysis showed that MYB93 has very high expression levels in anthocyanins-,tannins-rich tissues(leaves),and lignin-rich tissue(stems);the MYB93 promoter was linked to GUS reporter gene,and the vector was transformed into wild-type poplar.GUS staining analysis showed that MYB93 had the deepest staining in the old leaves and stems,which was consistent with qRT-PCR analysis.And the results indicated that the positions of MYB93 expression were overlap with the positions of anthocyanin,tannin and lignin synthesis.Subcellular localization demonstrated that MYB93 localizes in the nucleus;yeast self-activation analysis showed that MYB93 possessed transcription inhibitory activity.These results suggest that MYB93 may be involved in the regulation of anthocyanin,tannin and lignin biosynthesis and act as a transcriptional repressor.(2)MYB93 inhibits the biosynthesis of anthocyanins,tannins and ligninTo elucidate the biological function of MYB93,the overexpression and CRISPR/Cas9 knock-out vectors were constructed and transformed into wild-type popular.Phenotypic observation and histochemical staining analysis showed that the content of anthocyanins and tannins in the over-expressed transgenic poplars was significantly reduced.In the knockout poplar plants,the content of anthocyanins and tannins was increased;Meanwhile,MYB93 over-expressed transgenic plants became shorter,the diameter of stems became thinner,the cell layers of xylem cells in stems decreased,the lignin autofluorescence weakened,and the lignin content decreased;Knockout lines showed an opposite trend,but there was no statistically significant difference with wild-type popular.qRT-PCR analysis showed that the expression levels of key enzymes in multiple anthocyanin,tannin and lignin synthesis pathways were significantly down-regulated in transgenic poplars overexpressing MYB93,and their expression levels showed an opposite trend in MYB93 knockout lines.It indicated that the effects of MYB93 on anthocyanins,tannins and lignin biosynthesis is mainly achieved by regulating the expression of key enzyme genes.(3)MYB93 relys on the EAR domain to supress the expression of key enzyme genes via regulating promoters of the downstream enzyme genesIn effector-reporter experiment,the MYB93 overexpression vector was co-transformed into the tobacco with the promoter vectors of key enzyme genes,which proved that MYB93 could regulate the promoters of anthocyanins,tannins and lignin biosynthesis key enzyme genes,and inhibit the expression levels of these enzyme genes,thereby inhibiting the biosynthesis of anthocyanins,tannins and lignin.Our previous studies have shown that the EAR domain plays an important role in the inhibition of anthocyanin and tannin synthesis by MYB transcription factors.The mutant ?MYB93 mutated from the EAR(LxLxL)domain and the enzyme gene promoter were subjected to the effector-reporter experiment.The results showed that ?MYB93 lost its inhibitory function after the EAR domain was mutated.It has been demonstrated that the inhibition of anthocyanin,tannin and lignin synthesis by MYB93 were accomplished by the EAR domain.(4)MYB93 forms an MBW complex with TT8 and TTG1,which can enhance the inhibition of MYB93 on anthocyanin and tannin biosynthesisStudies have shown that in flavonoid biosynthesis,MYB transcription factors,bHLH transcription factors and WD40 proteins can form MBW complex to enhance the activity of activation or inhibition of the downstream genes of MYB transcription factors.The effector-reporter experiment showed that MYB93 enhanced the inhibitory effects on key enzyme genes in the flavonoid pathway when TT8 and TTG1 were added.Yeast two-hybrid experiments showed that MYB93 could interact directly with TT8,and TT8 acted as a bridge to form MBW complex with MYB93 and TTG1 to negatively regulate the biosynthesis of flavonoid pathway.(5)Function differentiation analysis of MYB93 and MYB57Both MYB93 and MYB57 are homologous genes of Arabidopsis AtMYB4,but MYB93 can inhibit the biosynthesis of anthocyanins,tannins and lignin,while MYB57 can only inhibit the synthesis of anthocyanins and tannins.There are significant differences in functionality.Tissue expression specificity analysis showed that MYB93 had higher expression levels in leaves,xylem and phloem,while MYB57 had higher expression levels only in leaves and very low levels in xylem and phloem,suggesting that it does not participate in lignin synthetic regulation.qRT-PCR and transient expression analysis showed that MYB57 could not bind to the promoter of the key enzyme gene of lignin synthesis and regulate its expression.In conclusion,this study preliminarily demonstrated that MYB93 acts as a transcriptional repressor,which forms an MBW complex with TT8 and TTG1,inhibits the expression activity of key enzyme genes in the flavonoid synthesis pathway,and thus negatively regulates anthocyanins and tannins biosynthesis.At the same time,MYB93 inhibits the biosynthesis of lignin by down-regulating the expression of key enzyme genes in the lignin synthesis pathway.These results provide new evidence for the molecular genetic mechanism of phenylpropanoid metabolism in poplar,and lays a foundation for forest breeding and wood optimization.