Molecular Mechanism Of PdeWRKY65-UGT75L28 Module Regulating Lignin And Callus Formation In Poplar

The lignification of plant cells is the process of lignin accumulation,and lignification will provide rigidity and mechanical support for plants.The formation of plant callus is the process of new cell generation and accumulation,but the relationship between the two biological processes is not clear.In previous study,UGT75L28 that encodes a glycosyltransferase was identified.Its biochemical function,was confirmed by in vitro enzymatic reaction.By screening of the upstream transcription factors(TF)of UGT75L28 and generating of their overexpressed(OE)and reduced expressed(RE)transgenic lines of poplar,their regulatory functions on lignification and callus formation were verified.The main findings of this study are as follows.1.The protein of UGT75L28 was expressed and purified in E.coli by in vitro protein expression system.Enzymatic reactions using various compounds as substrates was performed to confirm that UGT75L28 can only glycosylate the lignin monomer precursor coniferaldehyde that is one of substrates in the lignin synthesis pathway.This result suggests that UGT75L28 may be involved in the regulation of poplar lignin biosynthesis.2.By generating of p UGT75L28::GUS poplar transgenic lines,the positive lines were stained with GUS.It was found that UGT75L28 was mainly expressed in poplar leaf petioles,leaf veins and young shoots.Quantitative PCR(RT-qPCR)analysis in different tissues of poplar wild-type(WT)confirmed that UGT75L28 has a higher relative expression level in poplar petioles.We then created OE-UGT75L28 and RE-UGT75L28 poplar transgenic lines.The phenotypic assays of these transgenic lines showed that the degree of petiole lignification was reduced in the OE-UGT75L28 poplar transgenic line,while the RE-UGT75L28 line showed an opposite phenotype.These results suggest that UGT75L28 is involved in the biological process of lignin biosynthesis in poplar cells via glycosylating the monomeric lignin precursors.3.Explants of OE-UGT75L28 showed enhanced callus production,while its REUGT75L28 lines showed reduced callus production.GUS staining of p UGT75L28::GUS poplar transgenic lines showed that this gene can respond to both mechanical wound and callus induction medium(CIM)at the expression level.Based on the above results,we speculated that UGT75L28 might be involved in callus formation in poplar through delignification.4.A WRKY TF PdeWRKY65 was screened by yeast one-hybrid(Y1H)assay.Through point-to-point Y1 H,gel mobility arrest assay(EMSA),tobacco co-expression of dual luciferase and GUS,and co-immunoprecipitation PCR(Ch IP-qPCR)analysis,it was confirmed that PdeWRKY65 can directly bind to the promoter of UGT75L28 and positively regulate the expression of UGT75L28.5.Created OE-PdeWRKY65 and RE-PdeWRKY65 poplar transgenic lines.In the OE-PdeWRKY65 lines,it was found that petiole lignification was repressed and the callus formation was promoted;in the RE-PdeWRKY65 lines,it was found that petiole lignification was promoted and the callus formation was repressed.Addtionally,it was also found that the lignin content in the callus produced by the OE-UGT75L28 and OEPdeWRKY65 explants was significantly reduced;while the lignin content in the callus produced by the RE-UGT75L28 and RE-PdeWRKY65 was significantly increased.These results indicate that the PdeWRKY65 TF positively regulates the expression of UGT75L28,and then participates in the lignification and callus formation of poplar cells.6.In order to study whether the molecular PdeWRKY65-UGT75L28 gene module dependently or independently regulates the two phenotypes of poplar cell lignification and callus formation,we measured the lignin content in poplar WT callus formation.We detected a decreased lignification in poplar WT explants,as well as a decrease of the lignin monomer precursor coniferaldehyde.Based on the above results,we speculated that the expression of PdeWRKY65 was activated upon mechanical wounds and CIM.The increased expression of PdeWRKY65 resulted in an increased expression of UGT75L28.Therefore,the glycosylation of coniferaldehyde in explants were also enhanced and these biological processes finally increased the formatiom of callus.Based on the above findings,we uncovered a new molecular module regulating lignin biosynthesis and callus formation in poplar,and revealed that reducing lignin biosynthesis could promote callus formation in explants.According to the performances of this study,we expanded the regulatory network of plant lignin and callus formation,and provided some novel knowledge for improving these two traits in poplar in the future.