Functional Characterization Of Populus PtrWOX13 During The Secondary Cell Wall Formation

WOX is a family of plant-specific transcription factors,and its family contains a homeobox domain(HD)of 60 amino acids "helix-loop-helix-turn-helix",which is also a binding domain to a specific DNA sequence.Arabidopsis thaliana AtWOX13 is the third branch of the three major branches of the WOX gene family.It is mainly involved in the biological processes of stem cell formation,activating cell growth,and promoting the formation of embryos during fruit development.Previous bioinformatics analysis found that At WOX13 is involved in the regulation of Arabidopsis secondary cell wall(SCW)-related genes.In order to confirm the above analysis,this study cloned the homologous gene Potri.005G101800.1 of AtWOX13 from Populus trichocarpa,named it PtrWOX13.A preliminary study on its biological function in the form of SWC of poplar was carried out and the main conclusions are as follows:1.Populus trichocarpa different tissue specific expression analysis found,PtrWOX13 can be expressed in multiple tissues,but in the transition xylem,secondary xylem and phloem tissue,the expression level of SCW is relatively high in the tissue which forms exuberant.The GUS activity of GUS transiently infected tobacco driven by PtrWOX13 promoter staining analysis showed that the roots and veins of transgenic tobacco were deeply stained in the strong part of SCW.The above results preliminarily indicate that PtrWOX13 may be involved in the formation of poplar SCW.2.A 747bp CDs sequence of PtrWOX13 gene was cloned from the xylem of Populus trichocarpa,which encodes 248 amino acids,and at the amino acid sequence 102-169,WOX family comprising a length of 67 amino acids specific " helix-loop-helix-turn-helix " conserved domain.Subcellular localization studies indicate that PtrWOX13 is localized in the nucleus.Furthermore,verification of the self-activating activity of transcription factors indicated that PtrWOXl 3 has transcriptional autoactivation activity.The above results indicate that PtrWOX13 is a transcription-activated transcription factor that is localized to the nucleus.3.Yeast single-hybrid experiments showed that PtrWOX13 could specifically bind to the high-frequency components BoxI,Wbox and TCA in the downstream regulatory target genes C4H,PAL2,4CL3 and CCoAOMT promoters predicted by bioinformatics,suggesting that PtrWOX13 may regulate these genes.4.Over-expression and dominant inhibition of PtrWOXl3 transgenic Populus euphratica growth traits showed that the over-expressed transgenic poplars increased by 4.27%and 1.15%,the number of stem segments increased by 10.97%and 7.3%,and the ground diameter was significantly 4.11%and 3.27%.Dominant inhibition of transgenic poplar plant height decreased by 7.40%,4.44%;stem number decreased significantly by 5.49%,1.82%,while the ground diameter increased significantly by 3.41%,3.89%.5.Over-expression and dominant inhibition of PtrWOX13 transgenic poplar stem lignin histochemical staining analysis showed that the depth staining of lignin staining in the over-expressed transgenic poplar stems was not significantly different from the control,indicating that there was no significant change in lignin content;dominant inhibition of transgene lignin staining of poplar was strong,the staining area was enlarged,and the core of the medulla was weakened,indicating that the lignin content and lignification degree showed an increasing trend.Cellulose staining studies showed that overexpression of transgenic poplar increased color,indicating increased cellulose content;dominant inhibition of transgenic poplar staining area and intensity decreased to a different extent,indicating a decrease in cellulose content.The results of hemicellulose staining showed that over-expression and dominant inhibition of transgenic poplar staining were deep,indicating that the hemicellulose content of both transgenic lines showed an increasing trend.The content of cell wall components in stems showed that the lignin content of over-expressed transgenic lines decreased by 18.51%,the cellulose content increased by 2.77%,the hemicellulose content increased by 3.97%,and the dominant inhibition of transgenic lines increased by 10.91%.The cellulose content was reduced by 1.92%and the hemicellulose content was increased by 2%.The fiber traits showed that the fiber length and width of the overexpressing transgenic lines decreased by 11.34%and 11.15%,and the length and width of the dominant inhibition transgenic lines decreased by 20.53%and 30.53%.Scanning electron microscopy showed that the thickness of xylem cellulose SCW increased by 18.97%and 13.1%,and the phloem cellulose SCW thickness increased significantly,forming a similar "G-lay" structure.6.Quantitative PCR analysis of overexpression and dominant inhibition of PtrWOX13 transgenic poplar showed that the expression levels of overexpressing transgenic lines CESA4,CESA7 and CEAS8 increased significantly,IRX9 increased significantly,IRX10 decreased significantly,The expression levels of PALI,PAL4,HCT,CSE2,C4H2,CAD1,CCR2 and F5H were significantly increased,while CCoAOMT was significantly decreased;the expression of CESA4,CESA7 and CEAS8 was significantly decreased in transgenic lines,IRX9 was significantly increased,IRX10 was significantly decreased,and PALI,PAL4,HCT,CSE2,C4H2,CAD1 and CCR2 were significantly decreased,CCoAOMT and F5H decreased significantly.The expression changes of the above genes were consistent with the changes in histochemical staining and cell wall component content.The above results indicate that PtrWOXI3 participates in the formation of poplar SCW by directly or indirectly regulating the expression of the above genes.