Functional Analysis Of WOX And HSP17.8 In Poplar Development And Stress Resistance

WUSCHEL-related homeobox(WOX)transcription factor family is unique to plant,which are involved in the stem cells maintaining,the growth of lateral organs,the de novo root regeneration of the explant,and other development process.And Heat shock protein(HSP)is a kind of protective protein andplay importantroles in stress tolerance and environment adaptation.In this study,PtoWUSa,PtoWOX4 a,PtoWOX5aand PtrHSP17.8were chosen as research object.Their amino acid sequence identity and evolutionary relationships were analyzed,and the expression pattern were studied though the qRT-PCR and GUS staining methods.The genetic effects of changing the expression of these genes on the development of leaf,adventitious root and histological anatomy structure were explored in the transgenic poplar and Arabidopsis.Co-expression network were conducted,and the expression of the genes from co-expression network and candidate genes were detected in the transgenic plants,which reveal the molecular mechanism of PtoWUSa,PtoWOX4 a,PtoWOX5aand PtrHSP17.8in secondary vascular,AR development and plant stress tolerance.The main results were listed as follows:1.PtoWUS,PtoWOX4 a and PtoWOX5 a are belong to the WUS/modern clade.All of them have Homedomain and WUS domain.Besides,PtoWUSa and PtoWOX5 a have the ERF-like domain in the C-terminal end.They share 29%-99% amino acid sequence identity with other WOX genns from different species.2.The expression patterns of PtoWUS,PtoWOX4 a and PtoWOX5 a were analyzed by qRT-PCR and GUS staining.The results showed that these three genes are all expressed in the vascular tissues of leaf.PtoWUSa and PtoWOX4 a are highest accumulation in stem,especially in cambium and phloem.The GUS signaling in shoot apical meristem was observed in PtoWUSa,but not in PtoWOX4 a.All these three genes were expressed in adventitious root.However,PtoWUSa and PtoWOX5 a were expressed in the adventitious and lateral root(LR)tip,while PtoWOX4 a was in column of root.During AR and LR regeneration from the stem segment,PtoWOX5 a was specifically expressed in the AR tip(ART)and LR tip(LRT).3.Overexpression of PtoWUS,PtoWOX4 a and PtoWOX5 a led to deformed leaves in poplar.Compared with Ctrl plants,leaf margins curled to the inside,the length and area were reduced,and the width disorderly changed in the transgenic lines.qRT-PCR analysis indicated the expression of the YABBY1/3d,KAN1 a,KAN1c and AS2 which are involved in the leaf development were reduced.These results indicated that PtoWUS,PtoWOX4 a and PtoWOX5 a have a conserved function in regulating leaf developments.4.Ectopic expression of PtoWUS,PtoWOX4 a and PtoWOX5 a cause great phenotypic changes in poplar root,which mainly reflected in increased AR number,decreased AR length.The ARTs and LRTs of Ctrl plants were sharp and small,but the swollen and enlarged ARTs and LRTs were observed and the distribution of LRs was disordered in PtoWUSa and PtoWOX5a-overexpression transgenic seedlings.Ectopic expression of PtoWUS in Arabidopsis,the length of the main root were reduced and the main root lost gravitropic,but the length of LR were inceased.The abnormal QC cell shape in the atwox5 mutant was recovered by complementary expression of PtoWOX5 a.These results showed that PtoWOX5 a could substitute for AtWOX5 to rescue the abnormal QC cells in the atwox5 mutant.In PtoWOX4a-overexpression,enlarged AR tip,increased root diameter and thicker xylem layer were observed.5.The growth index of PtoWUSa and PtoWOX4 a overexpression plants were measured.The transgenic plant showed decreased internode number and dwarf phenotype.Moreover,overexpression of PtoWUSa and PtoWOX4 a led to a significant increase the number of xylem cells and decrease the number of pith parenchyma cells.The number and area of the phloem fiber decreased in the PtoWOX4a-overexpression plants,while these phenotypes did not observed in PtoWUSa-overexpression plants.Interestingly,overexpression of PtoWUSa and PtoWOX4 a enhanced the plant response to the gravity.Overexpression of PtoWUSa and PtoWOX4 a with different specific promoters showed dwarf and increased the number of xylem cell.These results indicated that PtoWUSa and PtoWOX4 a have conserved function in regulating the secondary vascular development.6.The co-expression network of PtrWUSa and PtrWOX4 a were conducted.The genes that related to sugar transport and metabolism,plant hormal and other function genes were detected in the network.The expressions of these genes and some candidate genes were regulated in the stem of PtoWUSa and PtoWOX4 a transgenetic plants,respectively.The expression of PIN1,PIN3 and WOX11/12 were effected in the AR of PtoWUSa and PtoWOX4 a transgenetic plants.Furthermore,PtoWOX4 a also regulate the expression of the xylem development related genes to control the AR secondary growth.7.The expressions of the CYCD and CDF4 were regulated in the AR of PtoWOX5a-ovexpression plants.In the co-expression network,56 genes have been shown to be involved with root development.The expression levels of 20 genes were up-regulated,and 32 genes were down-regulated in the 52 unambiguously detected genes.These data suggested that PtoWOX5 acooperates with a series of functional genes to regulate poplar root development.8.The gene expression profile of PtrHSP17.8 in various tissues showed that it was significantly accumulated in stem and root,the expression of PtrHSP17.8 could be induced by various abiotic stresses and significantly activated by heat stress.Overexpression of PtrHSP17.8 enhanced the tolerance to heat and salt stresses in Arabidopsis.The seedling survival rate,root length,relative water content,antioxidative enzyme activities,proline,and soluble sugar content were increased in transgenic Arabidopsis under heat and salt stresses,but not in normal condition.The co-expression network of PtrHSP17.8 were constructed and demonstrated many stress responsive genes included.The stress-related genes in the co-expression network were up-regulated in the PtrHSP17.8 overexpression seedlings.These results suggest that PtrHSP17.8 confers heat and salt tolerances in plants.Here,the role of WOXand HSP genes in wood formation,adventitious root(AR)development and stress tolerance were studied.These resultswill provide basis for comprehensive research on the mechanism of WOX genes and HSPin different species and also provide theoretical significance and potential application value for improving poplar wood property,wood production rate,the success rate of cuttings rooting and plant resistance breeding.