Molecular Mechanism Of PuHox52-Regulated Adventitious Root Formation In Populus Ussuriensis

ARs are formed on non-root tissues(such as stems and leaves)of plants.The intact plants obtained by adventitious roots(ARs)can maintain the excellent traits of the mother plants and greatly shorten the breeding cycle.Therefore,cutting rooting is a method of vegetative propagation extensively used in many important agricultural,horticultural and forest plants.Cutting rooting play an important role in the genetic improvement of forest trees.Therefore,it is very urgent and necessary to study the molecular mechanism of the formation of ARs in woody plants.1.The effects of methyl jasmonate(MeJA)and a JA biosynthesis inhibitor,salicylhydroxamic acid(SHAM),were tested on AR formation in Populus ussuriensis cuttings.The results showed when cuttings were treated with the MeJA concentrations of 0.5-10 ?M,the number of ARs were significantly increased.On the contrary,when cuttings were treated with 30-100?M SHAM,the number of ARs reduced significantly.2.RNA-seq analysis of non-treated and MeJA treatment in P.ussuriensis showed that PuHox52 belong to the HD-Zip gene family was significantly up-regulated in the early stage of AR formation.3.Subcellular localization results showed that PuHox52-eGFP was preferentially localized to the nucleus.Transcriptional activation assay demonstrated that the activation region of PuHox52 was located at the N-terminal region(NTR,1-28 aa).Analysis of cis-elements revealed that the promoter of PuHox52 contains eight MeJA response elements.We generated PuHox52pro::GUS transgenic lines to monitor PzHox52 expression in.ussuriensis.The promoter activity of PuHox52 was overwhelmingly expressed at the stem bases of cuttings.QRT-PCR analysis of whole AR formation process(0-120 h)revealed an overall up-regulated process of PuHox52 with a climbing phase ranging from 0 to 6 h and then a declination phase ranging from 6 to 120 h.After treatment with NAA,ACC,MeJA and SHAM,the expression of PuHox52 was significantly down-regulated only on SHAM treatment,while other hormones did not cause changes.This further demonstrates that PuHox52 may be involved in the AR formation and is only induced by endogenous JAs signal.4.We found the transgenic lines that overexpressed PuHox52(35Spro::PuHox52)showed 1-2 days earlier root emergence,the number of ARs and root biomass increased significantly,and the length of ARs became longer in comparison with WT.In PuHox52-suppresion lines,PuHox52-RNAi and PuHox52-SRDX,showed a delay of 1-2 days in AR formation,the number of ARs and roots decreased significantly,and the root length became shorter as comparison with WT.No matter PuHox52 overexpression or suppression lines,there were no significant changes in the number of ARs when transgenics lines were subjected to 50 ?M SHAM treatment,suggesting PuHox52 is a positive regulator of adventitious rooting in poplar downstream of JAs signaling.5.Transcriptome sequencing was performed for the cuttting of PuHox52 overexpressed lines and WT plants at different stages of AR formation.It was found that a large number of genes were involved in wound,hormone response,root development and other processes when GO annotation was performed by DGEs.In this study,a analysis of DEGs was used to construct a multi-layered hierarchical regulatory network(ML-hGRN),PuHox52,which acted as regulator in orchestrating AR formation.Meanwhile,we found that 15 important PuHox52 target genes associated with plant hormones,growth and development.6.The dual-luciferase reporter assay,EMSA,yeast one-hybrid and ChIP-qPCR confirmed that among the 15 PuHox52 target genes,PuHox52 can binding 9 gene promoters(PuAGL12,PubHLH137,PuHAT2,PuIAA7,PuLBD21,PuMYC2,PuWRKY51,PuWRKY70 and PuIAA14),except for PuZFP14,PuHB13,PuLBD22,PuNAC038,PuHD-like and PuMYB3.These results indicated that ML-hGRN has high credibility.7.QRT-PCR showed that the transcription level of PuAGL12,the target gene of PuHox52 of P.ussuriensis,was significantly increased in PuHox52-overexpressing lines(35Spro::PuHox52),while significantly decreased in PuHox52-suppressing lines(PuHox52-RNAi and PuHox52-SRDX)as compared with WT.PuAGL12 was significantly induced with the prolongation of rooting time.Further functional verification test results showed that PuAGL12 overexpression promoted AR occurrence.This study can provide important theoretical basis and genetic resources for the analysis of molecular regulatory mechanism of AR development as well as the genetic improvement of rooting ability of poplar.