Functional Characterization Of PtoDWF4 In Secondary Cell Wall Biosysnthesis In Populus

Populus spp.is one of our main forest species,shelterbelts and green trees.With the completion of sequencing of poplar genomes,poplar has been widely used as a woody model plant for molecular biology research.However,the mechanism for the regulation of secondary wall in poplar is not well understood at present.Brsinosteroids(BRs),a group of steroids found in plants in the 1970 s.The study found that BRs play an important role in the whole growth and development of plants,including cell elongation,division and differentiation,cell wall synthesis,vascular differentiation,light morphogenesis,seed dormancy and germination,root development,senescence and biological and abiotic stress.The current research on the effects of BR on plant growth and development is mainly concentrated in herbaceous plants such as rice,pea,tobacco and Arabidopsis thaliana,and the effect in woody plants is rarely reported.It has been confirmed in the herbaceous plants that BRs affect the formation of secondary walls.However,the role of BRs in the secondary wall formation of woody plants is unclear.DWF4 is a key enzyme gene in the BR biosynthetic pathway,and its mutants will lead to plants showing dwarfing and other characteristics.While the exogenous application of BLor BRs can restore DWF4 gene mutation caused by the phenotype.This indicated that DWF4 gene plays an important role in the BRs biosynthesis and the normal growth and development of plants.In this paper,In order to investigate the function and mechanism of the secondary wall synthesis of BRs in poplars,Pto DWF4 was cloned from Populus tomentosa,and its biological function was studied in the regulation of secondary cell wall biosynthesis of poplar.The results are as follows:(1)Pto DWF4 encodes a protein consisting of 488 amino acids.The phylogenetic tree analysis showed that the similarity of the protein was 74.08% and 98.57%respectively with that of A.thaliana and P.trichocarpa,and Pto DWF4 had a conservedfunctional domain of cytochrome P450 family.(2)Quantitative RT-PCR analysis of Pto DWF4 gene expression profile showed that Pto DWF4 was widely expressed all tissues examined,including root,stem,xylem,phloem,young leaves and old leaves of P.tomentosa,and especially highly in xylem of stem,suggesting that Pto DWF4 might participated in the control of the xylem in the secondary wall of the plant.(3)Overexpression of Pto DWF4 in poplar promoted the increase of plant height,stem diameter and increased the biomass of transgenic poplar plants.However,The Pto DWF4 mutants obtained by CRISPR / Cas9 targeting gene editing showed the short stature and narrow stems,indicating that Pto DWF4 may be involved in the biosynthesis of secondary wall of poplar.(4)The histological sections of the plants were stained with toluidine blue,and the number of lignin layers in the stems of the overexpression plants was significantly higher than that of the control while in knock-out transgenic poplars showed the opposite phenotype,indicating that Pto DWF4 was involved in the synthesis and regulation of the secondary xylem of stem.(5)Secondary cell wall component analysis showed that the contents of lignin,xylan and cellulose in overexpression of Pto DWF4 in poplar were notably up-regulate,and the knock-out of Pto DWF4 was significantly down-regulated.The above results demonstrate that Pto DWF4 plays a positive role in the secondary cell wall synthesis of poplar.(6)Quantitative RT-PCR analysis showed that the relative expression level of DWF4 downstream gene was up-regulated in the overexpression transgenic plants,and there was a certain decrease in knock-out plants.The structural genes related to lignin,xylan and cellulose synthesis in overexpression of Pto DWF4 plants were all up-regulated.On the contrary,these genes were significantly down-regulated in knockout plants.Similar results were found in the detection of secondary cell wall synthesis related NAC / MYB transcription factors.The above experimental results show that Pto DWF4 gene is involved in the formation of secondary cell wall of poplar.In conclusion,the key enzyme gene Pto DWF4 in BR synthesis pathway was cloned and analyzed by transgenic technique,gene knockout technique,histochemistry and molecular experiment.It is speculated that Pto DWF4 may regulate the formation of secondary wall of poplar by activating multiple key enzyme genes and related transcription factors in the secondary wall synthesis pathway.This study provides atheoretical basis for the genetic improvement of poplar.