Isolation And Functional Characterization Of Upstream Transcription Factors In Regulating Monolignol Biosynthetic Pathway Gene CAld5H2 In Poplar

As the fourth energy after coal,petroleum and natural gas,biomass energy is derived from plant photosynthesis directly or indirectly and is a potential treasure.As the carrier of biomass energy,the main groups of biomass are lignin and cellulose.Populus,as the most widely planted fast-growing and productive tree in the world,is regarded as the direction of the development of second-generation biomass energy,along with lignocellulosic plants such as Miscanthus,Panicum virgatum and Eucalyptus.Pretreatment process during lignocellulos biomass production is time consuming,and causes high energy consumption and serious environmental pollution.Hign lignin content is a limiting factor in the pretreatment process and must be removed.Using molecular techniques to reduce lignin content and change lignin structure makes it easy to separate lignin from cellulose,and improved pretreatment methods can promote efficient conversion and utilization of biomass energy.Lignin is a complex phenolic polymer formed by the polymerization of three monolignols: syringyl(S),guaiacyl(G)and p-coumaryl(H)alcohol.Syingyl lignins have advantages over guaiacyl lignins in improving biofuel production.CAld5 H is the key enzyme in the process of S monolignol synthesis.Yeast one hybridization was used to decipher transcription factors which take part in specific regulation of CAld5 H expression in the process of S monolignol synthesis and genetic engineering was used to change the composition and structure of lignin.The research aims to understand the lignin synthesis transcriptional regulatory mechanism and provide theoretical guidance for the improvement of biomass energy traits.Using RNA sequencing,227 xylem specifically expressed transcription factors were identified by camparing xylem,phloem,leaf and stem tip tissue transcriptome.12 upstream transcription factors regulating CAld5H2,which encodes the key enzyme for S monolignol biosynthesis in poplar,were identified through yeast one hybridization and co-expression analysis.Furthermore,12 TFs were discovered to bind to the promoter around 600 bp before the transcriptional initiation site.Yeast one hybridization was used to identify which TF among 12 could bind to the promoter of genes encoding monolignol biosyntheticpathway enzymes in poplar,including CAld5H2,CAld5H1,CAD1,C3H3,COMT2,CCR2,PAL4,HCT1,4CL3,4CL5 and C4H2.It was found that three BEL1-like family members BLH6 a,BLH6b and BLH2 could bind to the promoters of CAld5 H family members specifically..BLH6 a and BLH6 b bound to the CAld5H2 promoter,while BLH2 bound to both CAld5H1 and CAld5H2 promoters.These results suggest that three BELL transcription factors are specific regulators of CAld5 H genes for S monolignol biosynthesis.Transient transformation of tobacco by dual-luciferase reporter assay system showed that BEL1-like homeodomain protein BLH6 a significantly inhibited the expression of reporter LUC gene.Dominant repression of BLH6 a function didn't affect plant height and leaf shape.However,it blocked the secondary wall formation in young stems.TBO staining of stem cross sections showed that the secondary wall thickness of the transgenic plants was reduced in soft stem,which was especially obvious during the initial xylem formation and consistent with the results of RNA in-situ hybridization of BLH6 a.Further,luciferase complementation imaging analysis showed extensive protein-protein interactions among 12 TFs.Our results demonstrate that BLH6 a is a negative regulator of CAld5H2 and implicate a combinatorial regulation of multiple TFs on S monolignol biosynthesis in poplar.The expression levels of CAld5H2,CAld5H1,CAD1,C3H3,COMT2,CCR2,PAL4,HCT1,4CL3,4CL5,C4H2 and BLH6 a in differentiating xylem of transgenic lines and wild type were determined by q RT-PCR.Compared with wild-type,the transcript abundance of CAld5H2 in transgenic lines #29 and #2 was significantly reduced,and the transcript abundance of CAld5H2 in transgenic lines #29 and #2 was reduced by 62.9% and 37.7% respectively,which was consistent with the higher expression level of BLH6a:SRDX in transgenic line #29.No significant changes were observed for CAld5H1 expression level in the transgenics.Total lignin content in annual transgenic wood showed a similar level as wild-type.Maule and Wiesner staining of 2 months old stem cross sections showed that S lignins were more concentrated in fiber cells.Maule staining showed that the color of xylem cells in wild type was obviously more red than transgenic plants which means that S lignin content in the transgenic wood may decrease and lead to the reduction of S/G ratio.Therefore,as an inhibitory factor,BLH6 a directly regulates secondary wall formation by regulating CAld5H2 gene expression and may change the lignin structure.The purpose of this study is to identify the transcription factors involved in S monolignol synthesis and to understand the molecular mechanism of trancriptional control on lignin biosynthesis.We aim to provide theoretical basis for the creation of new varieties with low energy consumption and high conversion efficiency by changing S monolignol content(S/G ratio)in wood using genetic engineering.Thus,new tree varieties with low energy consumption and high conversion efficiency are cultivated and applied to the industrial production practices such as biofuel extraction,pulping and papermaking.