Functional Characterization Of The Poplar Transcription Factors MYB189 And MYB031 Involved In Secondary Cell Wall Biosynthesis

Wood,as important materials for building,furnitures and bioenergy,has been widely applied in industrial production and human life.Wood develops from the secondary xylem of stem and at the cellular level mainly composed of plant cell walls.The primary and secondary cell walls(SCW)make up the cell wall.Among them,the biomass accumulated in the SCW accounts for the vast majority of total plant biomass.Therefore,basic research on the mechanism of plant SCW biosynthesis not only has important scientific significance,but also lays a solid foundation for breeding new varieties that meet the needs of hunman life and industrial production.In recent decades,many studies have found that many transcription factors,key enzyme genes and microRNAs are involved in the regulation of SCW formation.Among the studies,it is clearly that SCW biosynthesis is regulated by a complex multi-level transcriptional regulation network.Numerous NAC and MYB family transcription factors participate in the network.The NAC transcription factors,as the primary master switchs,directly regulate the expression of downstream MYB transcripton factors or key enzyme genes by binding to the SNBE(Secondary wall NAC Binding Elements)sites on the promoters region of these genes,or by activating the secondary switches or downstream MYB transcripton factors,thereby regulating the expression of key enzyme genes and programmed cell death genes.MYB transcription factors promote transcription through SMREs(Secondary wall MYB Binding Elements)in the promoter regions of the target genes.As one of the most fast-growing trees distrubied in world wide,poplar is a good model plant for SCW biosynthesis following the whole genome sequence release of Populus trichocarpa,Populus euphratica and Populus alba var.pyramidalis,and genetic transformation and regeneration system establishment of many species.Although there are at least 192 R2R3-MYB transcription factors in poplar,only part of them have been shown to be involved in the process of SCW regulation.Since the SCW biosynthesis is a fine-tuning and complex process,it is essential to research the function of other members due to their functional diversity.Combined with phylogenetic analysis,expression profile analysis and previous studies,two pair of homologous transcription factors,MYB158/189 and MYB026/031,were screened out to research their roles in SCW biosynthesis.After the amino acid sequence alignment and sequence consistency analysis,two representative factors,MYB189 and MYB031 were selected for deeply study.A variety of experimental methods in genetics,cytology and biochemistry were used to study the function of MYB189 and MYB031 in SCW formation of Arabidopsis and poplar.The results are as follows:(1)MYB189 and MYB031 may regulate the biosynthesis of SCW in different ways.MYB189 and MYB031 with the higher expression levels in xylem may play a role in the regulation of SCW biosynthesis.By subcellular localization,we found that both porteins are located in nuclear.The transcription activation assays showed that MYB031 is a transcriptional activator,while MYB189 has no transcription activity.The results suggested that MYB189 and MYB031 may be involved in the regulation of secondary wall biosynthesis via different ways.(2)MYB189 is an active repressor and is mainly expressed in stem and root.Phylogeneticanalysis showed that MYB189 belonds to C49 subfamily which is relatively distant from the C4 phenylpropanoid biosynthesis repressors subfamily.Amino acid sequence alignment among MYB189 and other members of MYB C4 subfamily reveals that they share a highly conserved R2-R3 domain at the N-terminal regions,but have divergent at the C-terminal ends.Interestingly,MYB189 don’t contain the classical repression domains,such as EAR and TLLLFR.In order to determine whether MYB189 is a repressor or not,MYB189 was fused with the VP16 activation motif for transcription activation analysis,and the results confirmed that MYB189 is a transcription repressor.A series of truncation fragment of MYB189 fused with the VP16 activation motif had been transformed into yeast for repression domain screening,and the repressive domain in MYB189 was determined,which was GDDYGNHGMIKKE.To explore the expression profiles of MYB189 in poplar,Quantitative real-time RT-PCR(qRT-PCR)analysis and MYB189 promoter-driven GUS activity showed that MYB189 was highly expressed in stem and roots.(3)MYB189 represses the development of SCW in Arabidopsis.To investigate the function of MYB189,the overexpression vector of MYB189 was firstly transformed into the wild type of Arabidopsis,and the homozygous transgenic plants were obtained and studied.The transgenic plants showed thinner and pendent inflorescence stems.After staining with phloroglucinol-HCl,the cross sections of transgenic plants inflorescence stems showed retardated vascular tissue and narrow xylem,which lead to the reduction in mechanical resistance of stems.We further examine the expression of SCW biosynthetic genes in MYB189-OE lines by qRT-PCR.The results suggested that MYB189 may negatively regulate SCW biosynthesis by down-regulating synthetic genes of lignin,cellulose and xylan in Arabidopsis.These results indicated that MYB189 negatively regulates SCW biosynthesis.(4)MYB189 negatively regulates the SCW biosynthesis in poplar stems.To determine whether MYB189 carries out similar functions in poplar,the transgenic Populus tomentosa plants of over-expressing MYB189 were obtained and the growth of transgenic plants was suppressed with significantly lower biomass accumulation,and lodging was observed in these plants.Microscopy on stem sections showed that the cell wall was significantly thinner in fiber cells in the xylem of transgenic plants,and chemical analysis showed significantly lower contents of cellulose,lignin and xylan in transgenic plants.In order to study the molecular regulation mechanism of MYB189 in the deposition of SCW components,RNA-Seq and qRT-PCR analysis had been used in this study.The results showed that many key enzyme genes involved in the biosynthesis of lignin,cellulose and xylan,as well as master switch genes of secondary wall biosynthesis have been down-regulated in transgenic plants.The RNAi interference expression vector of MYB189 had been constructed and transformed to poplar,but there was no significant difference between wild type and transgenic plants.It indicated that there may be redundant genes of MYB189 in poplar.(5)MYB189 directly binds to the promoters of WNDs and SCW biosynthetic gene and suppresses their expression.To investigate the mechanism in more details,the promoter of SCW biosynthetic genes have been analyzed,and several SMRE elements were found in the upstream promoter regions of SCW biosynthetic genes,C4H2,COMT2,GT43 B and CesA2 B.Chromatin immunoprecipitation(ChIP)-qPCR assays and transient co-expression experiments showed that MYB189 could directly bind to the promoters of SCW biosynthetic genes and repress their expression.Further,qRT-PCR analysis also revealed that over-expression of MYB189 could down-regulate the expression of wood-associated NAC transcription factors(WNDs).We also found that there are several SMRE elements in the upstream promoter regions of WND2A/2B.ChIP-qPCR assays was carried out,and the result demonstrated that MYB189 was able to directly bind with the promoter regions of WND2 A and WND2 B.Transient co-expression experiments also showed that MYB189 could negatively regulate the expression levels of WND2 A and WND2 B.In summary,the results demonstrated that MYB189 could suppress the development of SCW in poplar through directly repress the expressing of WND2A/2B,C4H2,COMT2,GT43 B and CesA2 B.(6)MYB031 is mainly expressed in xylem and root.Tissue expression profile analysis indicated that among the four homologues,MYB031 had the highest level in the xylem,suggesting that MYB031 may be specially involved in the regulation of xylem development.Previous studies have found that MYB031 is a transcriptional activator,indicating that it plays a role in SCW biosynthesis in a different way compare with MYB189.So,it makes sense to select MYB031 for in depth study.qRT-PCR in different tissues revealed that it is mainly expressed in xylem.MYB031 promoter-driven GUS activity showed that MYB031 was highly expressed in the cambium of stems and leaves.(7)Overexpression of MYB031 represses the SCW biosynthesis in poplar stems.However,plants growth was inhibited and leaves showed a curled shape in transgenic poplar plants over-expressing MYB031.Cross sections and chemical staining of leaf veins showed that the development of xylem in veins and petioles was inhibited in transgenic plants.The abnormal development of veins in leaves led to the change of angle between veins and leaves,resulting in uneven leaves in transgenic poplar.Microscopy analysis of stem sections showed that overexpression of MYB031 lead to a decrease in layers of cells and thinner walls in fiber and vessel cells of xylem.Moreover,cell wall composition analysis revealed that the contents of lignin,cellulose and xylan were reduced in the transgenic plants.High-throughtput sequencing and qRT-PCR assay also showed that the expression of key enzyme genes involved in lignin,cellulose and xylan biosynthesis were down-regulated in MYB031 overexpressed plants.In addition,the expression of the upstream primary master switch genes WNDs and secondary master switch genes MYBs were significantly down-regulated.(8)Suppress the expression of MYB031 active the SCW biosynthesis in poplar stems.For further verify the function of MYB031,MYB031-RNAi transgene plants were obtained and ectopic deposition of lignin in pith was found.Moreover,Cell wall composition analysis revealed that the contents of lignin,cellulose and xylan were all slightly increased in the transgenic plants.The results of qRT-PCR also showed that the expression of many SCW associated WNDs,MYBs and key enzyme genes were upregulated in MYB031-RNAi transgene plants.(9)MYB031 negatively regulates the SCW development by interacting with JAZ1.In order to study the molecular regulation mechanism of MYB031,through yeast two-hybrid library screening,it was found that MYB031 could interact with JAZ1(JASMONATE-ZIM-DOMAIN PROTEIN 1)protein,which is involved in signal transduction pathway of JAs.And the result was further verified by two-hybridization and BiFC analysis in vitro and in vivo.To research whether MYB031 can negatively regulate the biosynthesis of secondary walls by interacting with JAZ1,transient expression experiments were introduced into the tobacco leaves.The results showed that MYB031 inhibited the expression of WNDs by interacting with JAZ1.At the same time,stem cross sections and chemical staining results of wild type and MYB031-OE plants after MeJA treatment for 30 days,showed that the inhibition of SCW deposition in MYB031-OE plants had been relived.The results suggest that MYB031,as a transcription activator,repress the development of SCW by interacting with JAZ1.Although MYB189 and MYB031 are in the same subgroup,they negatively regulate the biosynthesis of SCW in different ways.MYB189,as an inhibitor,repress the deposition of SCW by directly regulating the expression of key enzyme genes and WNDs genes,while MYB031,as an activator,repress the expression of WNDs genes by interacting with JAZ1,thereby inhibiting the deposition of SCW.The results of this dissertation provide a new mechanism for regulating the SCW biosynthesis by MYB transcription factor in poplars,and enrich the regulation network of SCW biosynthesis.