| The vast area of forest land planted in the northern region of China has an important position for the development of national forestry.Heilongjiang province,as a traditional forestry province,has also been facing the problems of shrinking forest land area,serious degradation of good germplasm resources of forest trees and soil fertility of forest land,and increasing salinization and desertification of soil in recent years.The discovery of genes related to forest tree resistance and promotion of growth and development regulation,combined with the current molecular biology technology for genetic breeding improvement of forest trees,is considered as one of the most rapid and effective ways to improve the resistance of woody plants.The phospholipase C are key enzymes in phospholipid signaling in plant growth,development,biotic,and abiotic stress responses.So far,many PI-PLC（Phosphoinositide-specific PLC）genes have been cloned from various plant species and their functions have been analyzed.However,the current understanding of the PI-PLC signal transduction pathway in woody plants is limited,and the function of PI-PLC in plant abiotic stress responses still needs to be experimentally verified.In our previous study,PsnPI-PLC6 was screened by yeast two-hybrid assay of ERF transcription factors.And stress tolerance analysis was further carried out based on the cloning of PsnPI-PLC6 gene.This study lays a foundation for further revealing the physiological and biochemical mechanisms of PI-PLC gene in regulating plant response to stress,and it also provides a theoretical and practical basis for the study on molecular breeding of poplar for resistance.The study found that:1.Seven PtPI-PLCs were identified according to the unique conserved domain（EF/X/Y/C2 domain）.The PI-PLC gene structure in poplar is conserved.The PI-PLC gene family of populus has the same motif distribution,a number of cis-acting elements with stress-related effects are found in the 2000 bp region upstream of the start codon.2.The q RT-PCR expression pattern analysis showed that PsnPI-PLC6 was responsive to abiotic stress,which implied that the expression of PsnPI-PLC6 showed spatiotemporal specificity.3.Subcellular localization analysis showed that PsnPI-PLC6 was localized in the plasma membrane and cytoplasm.PsnPI-PLC6 showed a Ca2+dependent hydrolytic activity.Its maximum activity toward PI was at 10μmol/L Ca2+concentration with the optimum pH at 6.8.4.Physiological and biochemical results showed overexpression of PsnPI-PLC6 in tobacco increase abiotic stress tolerance.To gain insight into the salt response mechanism of PsnPI-PLC6,we further analyzed the RNA-seq data using transgenic tobacco against WT.The DEGs were mapped to the corresponding GO terms and KEGG pathways.Gene ontology analysis showed that the principle enriched GO terms include“integral component of membrane”（CC）,“metal ion binding”（MF）,“carbohydrate metabolic process”（BP）,and“metabolic process”（BP）,which are all closely associated with the functions of the membrane.In addition,overexpression of PsnPI-PLC6 also significantly affected the expression of the genes encoding ABA 8’hydroxylase ABH and dehydratase TAS14,which suggests that phospholipase C may be directly related to the regulation of endogenous ABA content in plants.This study provides new insights into the functional analysis of poplar PI-PLC genes in response to abiotic stress. |
