The Transcriptional Regulatory Mechanism Of The Poplar Transcription Factor MYB6 Involved In Flavonoid And Lignin Biosynthesis

Secondary metabolites play vital roles in plant growth,development and defense against the environmental stress.As a class of secondary metabolites ubiquitously found in plants,phenylpropanoids(including anthocyanins,tannins and lignins)also play important roles in plant growth and development.In study of genetic breeding of woody plants,increasing the content of anthocyanins and tannins in plants can enhance their ability to defense against both biotic and abiotic stresses.On the other hand,adjusting the lignin content or components in plants is conducive to make better use of timber resources.Previous studies have revealed that MYB transcription factor is a large family in plants,and multiple members were able to effectively regulate the biosynthesis of flavonoid and lignin.However,in woody plants,just a few MYB transcription factors were demonstrated to play roles in flavonoid and lignin biosynthesis.As the most important economic tree in human production and life,poplar has become one of the major trees for plantation in China.With the release of its whole genome sequence and the establishment of genetic transformation and regeneration system,poplar has become a model organism for tree genomics studies,which greatly promotes studies in molecular biology and genetic breeding of woody plants.It has been shown that there were at least 192 R2R3-MYB transcription factors in the poplar genome,just a few were demonstrated to play roles in phenylpropanoids biosynthesis,however,no one has been reported to regulate both flavonoid biosynthesis and secondary wall formation.Based on the above information,the present study identified one R2R3-MYB transcription factor named MYB6 may simultaneously influence both flavonoid biosynthesis and secondary wall formation out of the 20 poplar R2R3-MYB candidates that may possibly be involved in phenylpropanoids biosynthesis regulation through bioinformatics analysis.Then MYB6 was studied in poplar and Arabidopsis toelucidate its role and mechanism in regulating both flavonoid and lignin biosynthesis by genetic and biochemical methods.The major results are as follows:(1)The poplar MYB6 may act as a R2R3-MYB transcriptional activator and be involved in the regulation of anthocyanin,tannin and lignin synthesis.Phylogenetic analysis of a plethora of R2R3-MYB transcription factors from various species that have been previously reported to play roles in regulating phenylpropanoid biosynthesis together with R2R3-MYBs from poplar.It revealed that20 candidate R2R3-MYBs were involved in phenylpropanoid biosynthesis,and poplar MYB6 and MYB126 were closely related to VvMYB5 a and VvMYB5 b.Further sequence alignment unraveled a higher homology of poplar MYB6 with VvMYB5 a and VvMYB5 b,making MYB6 a more promising candidate.The full-length MYB6 gene was cloned from Populus tomentosa,which encodes a 316-AA peptide with a typical R2R3 domain as well as a conserved motif for interaction with bHLH proteins.In addition,a promoter fragment of 1323 bp upstream of the gene was also cloned,where a SMRE(Secondary wall MYB-Responsive Element)element was discovered.Expression profiling via quantitative real-time RT-PCR analysis showed that MYB6 was expressed in young leaf at the highest level and also highly expressed in root and stem.Moreover,the promoter fragment of MYB6 was fused with a GUS reporter gene and transformed into P.tomentosa,and GUS staining indicated that MYB6 was highly expressed in young leaf,root and stem,while in mature leaf,it was expressed at the lowest level and mainly found in the veins.Further,MYB6 was fused with the GFP gene and transiently transformed into onion epidermal cells.Confocal laser scanning microscopy revealed that the fusion protein was specifically localized to the nucleus.Additionally,yeast one hybrid analysis suggested that MYB6 possessed a transcription activation activity.All these results indicate that MYB6 may be involved in both flavonoid and lignin biosynthesis as a nuclear localized transcription activator.(2)The transgenic method confirmed that MYB6 can positively regulate the synthesis of tannin and anthocyanin in poplar but inhibit the synthesis of lignin.To verify the biological function of MYB6,over-expression vectors for MYB6 and MYB6 fused with an repressor named MYB6-SRDX were constructed and transformed into wild type P.tomentosa.In transgenic plants overexpressing MYB6,red leaves were found,with increased anthocyanin and tannin contents,while fewer layers of xylemcells,thinner cell wall and lower lignin content were found.In transformants overexpressing MYB6-SRDX,the contents of anthocyanin,tannin and lignin were all decreased.Quantitative real-time RT-PCR revealed that the trends in anthocyanin,tannin and lignin content were consistent with the changes in expression levels of key enzyme genes.These results suggest that MYB6 may promote the accumulation of anthocyanin and tannin,but repress the lignin content and affect the development of secondary wall in poplar via influencing the expression of key enzyme genes.(3)Yeast two-hybrid experiments demonstrated that MYB6 interacts with bHLH and KNAT7,and the key domain involved in the interaction of MYB6 is the R3 domain.To explore the transcriptional regulatory mechanism of MYB6 acting as a transcriptional activator to negatively regulate the lignin biosynthesis.A yeast two hybrid screening was carried out with a MYB6 protein with its transcriptional activation activity removed fused to GAL4-AD as the bait.The yeast two hybrid analysis revealed that MYB6 could interact with GL3,a bHLH(Basichelix–loop–helix)transcription factor that is involved in anthocyanin biosynthesis and trichome development and KNAT7,a transcriptional repressor for plant lignin biosynthesis.The interactions were further confirmed by bimolecular fluorescence complementation analysis in plant cells.Previous studies have shown that the key region of the interaction between MYB and bHLH transcription factor is located within the R3 domain of MYB transcription factor.Moreover,a series of truncations for MYB6 and KNAT7 were done for further yeast two hybrid analysis,and the results indicated that the key interaction zone located within the C-terminus of the R3 domain in MYB6.Furthermore,mutation of C-terminus of the R3 domain in MYB6 was confirmed by yeast two-hybrid experiments.The mutant MYB6R3 m could not interact with KNAT7,but did not affect the interaction with GL3.These results indicate that MYB6 may regulate the biosynthesis of flavonoid by interacting with GL3 to form MBW(MYB-bHLH-WD40)complex,while it may interact with KNAT7 to regulate the biosynthesis of lignin thereby influence the formation of secondary wall.(4)Biochemical methods demonstrated that MYB6 forms a MBW complex with GL3 and TTG1 to regulate the flavonoid synthesis pathway,while interacts with KNAT7 to negatively regulate the lignin biometabolism pathway.To validate the influence on the expression of key enzyme genes in flavonoid and lignin biosynthesis of MYB6,MYB6 was transiently co-expressed with the promoters of the key enzyme genes in tobacco leaves,and the results indicated that MYB6 could activate the expression of PAL1,an upstream structural gene in the phenylpropanoid biosynthesis pathway,DFR2 and LAR1,two key enzyme genes in anthocyanin and tannin biosynthesis and CCoAOMT1,a key enzyme gene in lignin biosynthesis.Further electrophoretic mobility shift assay(EMSA)revealed that MYB6 could bind to AC elements within these promoter sequences and promote transcription.When co-expressed in tobacco leaves,GL3 and TTG1 that encodes a WD40 protein could significantly enhance the regulatory effect of MYB6 on DFR2 and a weak enhance was observed for CCoAOMT1 expression,while in the presence of KNAT7,MYB6 could not activate the transcription of DRF2,and an increased repression was observed for CCoAOMT1 expression.These results indicate that MYB6 could be transformed into a transcription repressor by the interaction between MYB6 and KNAT7.Taken together,MYB6 interacts with GL3 and TTG1 to form an MBW complex for the positive regulation of flavonoid biosynthesis,while it interacts with KNAT7 to negatively regulate the biosynthesis of lignin,thereby influence the formation of plant secondary cell wall.(5)Genetic methods validate that MYB6 negatively regulates the biosynthesis of lignin in Arabidopsis dependent on its interaction with KNAT7.Previous studies have shown that Arabidopsis MYB75(PAP1)positively regulated flavonoid biosynthesis,while the phenotypes of thickened secondary wall and increased lignin contents were shown in myb75 mutant,and the interaction between MYB75 and KNAT7 was confirmed.The secondary wall was also significantly thickened in the knat7 mutant,suggesting that the MYB transcription factor may act synergistically with the KNAT7 transcription factor to inhibit lignin synthesis.We utilized an Arabidopsis knat7 mutant to confirm the influence of MYB6 on plant secondary cell wall formation.Vectors for over-expression of poplar MYB6 and MYB6R3 m were separately transformed into wild type and knat7 Arabidopsis plants.In comparison with wild type Arabidopsis plants,MYB6-OE lines showed fewer layers of cells and thinner cell walls in the interfascicular fibers and vascular bundles of the inflorescence,while more layers of cells and thicker cell walls were observed in MYB6R3m-OE transgenic lines.As compared with knat7 mutants,more layers of cells and thicker cell walls were observedin the interfascicular fibers and vascular bundles in the inflorescence of MYB6-OE/knat7 transgenic plants.These results indicate that the repressive effect of MYB6 on lignin biosynthesis may rely on its interaction with KNAT7.In summary,on one hand,the poplar transcription factor MYB6 could form an MBW complex with GL3 and TTG1 to activate the expression of key enzyme genes in anthocyanin and tannin biosynthesis pathways,thereby increase the contents of anthocyanin and tannin.On the other hand,MYB6 could interact with KNAT7 so as to repress the expression of key enzyme genes downstream of the lignin biosynthesis pathway and lower lignin content and influence the formation of secondary cell wall.Finally,MYB6 was able to interact with specific proteins to regulate different branches of the phenylpropanoid pathway in poplars,and maintain a dynamic balance between pathways downstream of the phenylpropanoid biosynthesis.The results of this dissertation provide new molecular evidence for clarifying the genetic mechanism of the MYB transcription factor regulating the phenylpropanoid pathway in poplars.