Molecular Mechanism Of BpVND1 Gene Regulating Growth,Stress Resistance And Wood Properties Of Betula Platyphylla

Timber is a renewable material that is widely used in the industrial production and energy production.Secondary cell wall(SCW)is the main component of wood.VND(Vascular-related NAC domain)is a key transcription factor in secondary wall biosynthesis,which plays an important role in regulating plant secondary growth and wood formation.However,only a few VND1 genes have been characterized.In this study,the function and molecular mechanism of BpVND1 gene involved in growth,stress resistance and wood properties of Betula platyphylla were revealed.1.BpVND1 protein is located in the nucleus.The expression of Bp VND1 is tissue-specific,is the highest in the mature stem,followed by in the mature leaf,similar level in the young leaf and the young stem,and the lowest in the roots.These results suggested that it may be related with plant growth and development.In addition,NaCl and mannitol stress can induce the expression of Bp VND1,suggesting that it is involved in salt alkali and osmotic stress.2.The Bp VND1 gene was transferred into the genome of B.platyphylla.The transgenic B.platyphylla plants of BpVND1 overexpression(OE)and silence expression(SE)were generated.Under NaCl and mannitol stress conditions,BpVND1 gene overexpression lines showed improved tolerance.BpVND1 improve salt and drought stress tolerance mainly by enhancing the capability of scavenging reactive oxygen species(ROS),reducing cell death and activating proline biosynthesis.3.The stem segments of transgenic and wild-type B.platyphylla were transected.The results of scanning electron microscope,safranine and toluidine blue staining showed that the xylem cell wall of OE plants was thicker than that of wild-type plants.The thickness of cell wall and the cell wall ratio of wood fiber were measured,and OE plants had the thickest secondary wall and the highest rate of wood cell walls,followed by WT,SE plants had the thinnest secondary wall and the lowest rate of wood cell walls.It is suggested that BpVND1 can promote the formation of secondary cell wall of B.platyphylla.4.The growth of 1-year-old transgenic birch were analyzed.The growth of SE transgenic plants were significantly improved,and the plant height,ground diameter and fresh weight were significantly higher than those of WT plants,while the plant height and fresh weight of OE plants were similar to those of WT plants,and ground diameter was higher than that of WT plants.The content of lignin and holocellulose in OE birch is higher than that of WT plants,and the content of holocellulose is lower than that of WT plants;the content of holocellulose in SE birch is higher than that of WT plants,and the content of lignin is lower than that of WT plants.Compared with the WT plants,the fiber length of SE transgenic birch increased significantly,the fiber width difference was not obvious,and the length width ratio increased significantly;the fiber length of OE transgenic birch became shorter,the fiber width and the length width ratio were not obvious different.5.BpVND1 can not only activate the expression of a series of genes related to secondary wall synthesis,but also positively regulate many MYB transcription factors,which are involved in the regulation of secondary wall synthesis.ChIP PCR showed that BpVND1 could directly regulate the expression of MYB54,MYB83,CCR1 CCoAOMT and 4CL1 genes to regulate secondary wall synthesis.6.In order to study the gene regulation of secondary wall synthesis and growth of B.platyphylla,the RNA-seq of lines of OE4,SE8 and WT were sequenced.There are 516 differentially expressed genes(DEGs)between OE4 and WT,including 251 genes were up-regulated and 265 were down-regulated.Among the up-regulated genes,25 are related to cell wall thickening,resistance enhancement and lignin increase.Among the down regulated genes,20 were related to cellulose and growth decline.Pathway enrichment analysis showed that the number of DEGs in the pathway of phenylpropane biosynthesis was the largest.Compared with WT,SE8 has 156 DEGs,of which,55 are up-regulated and 101 are down-regulated.Pathway enrichment analysis showed that the number of differentially expressed genes in plant hormone signal transduction pathway was the largest.Three genes of Gretchenhagen 3(GH3s)were down-regulated,which may be related to the rapid growth and biomass increase of plants.This experiment systematically characterized the function and regulation mechanism of BpVND1 gene in the process of growth,response to abiotic stress and secondary cell wall development,which provideds a theoretical basis for the genetic improvement of B.platyphylla and the regulation mechanism of wood formation.