Function Analysis Of Autophagy-related Genes And Aquaporin In Response To Salt And Drought Stress In Populus

Plants always encounter varieties of environment stresses such as salt stress and drought stress during their growth and development stages.These abiotic stresses seriously stunted plant growth and bring serious losses to agricultural and forestry production.Therefore,it is of great significance for agricultural and forestry production to reveal the mechanism of plant stress resistance.In order to avoid the damage resulted from stress conditions,a series of adaptive pathway have been generated in plants,such as degrading damaged proteins and organelles and decreasing transpiration rate.Autophagy and aquaporins play key roles in plant response to abiotic stress.Poplar,as a woody model plant,has been widely used in stress response studies.However,its response mechanism in the process of autophagy and the H₂O₂transport mechanism involved by aquaporins in salt and drought resistance is still unclear.In this study,poplar autophagy related genes ATG(autophagy-associated genes),Pag NBR1(neighbor of BRCA1)and Pag PIP2;10(plasma membrane intrinsic proteins)were studied.Autophagy related genes were identified on the whole genome,Pag ATG18a,Pag NBR1 and Pag PIP2;10 transgenic lines were generated and the response mechanism of poplar under salt and drought stress was analyzed.The main conclusions were as follows:1.In this study,a total of 49 ATG were isolated from 19 chromosomes in the poplar genome.In terms of structure of these genes,there are similar quantity of introns or exons among all subfamily members.Tissue expression patterns showed that there were significant differences in root,xylem,mature leaves,extended leaves,seeds and blotches among the subfamilies.After ABA and BR treatment,the expression levels of ATG18 subfamily members was higher than that of other subfamilies.The expression of ATG18a in all members of the ATG18 subfamily was the highest.2.The expression of Pag ATG18a was mainly enriched in leaves.Transient transformation of Pag ATG18a in poplar leaves showed that it was mainly targeted to cytoplasm and nucleus.Overexpression of Pag ATG18a signally improve the salt tolerance of poplars.The transgenic poplar maintained higher leaf water content and photosynthesis rate under salt stress.Except that,the activities of SOD,POD and CAT of Pag ATG18a transgenic lines increased,and the oxidative damage of cell membrane in Pag ATG18a was reduced.3.The function of autophagy receptor gene Pag NBR1 in response to salt stress in poplars was analyzed and results showed that overexpression of Pag NBR1 in poplar signally enhanced salt stress tolerance.Compared with wild type(WT)plants,the overexpression lines exhibited higher enzyme activity in scavenging ROS,and higher net photosynthesis rates under salt stress.Furthermore,co-localization and yeast two-hybrid analysis revealed that Pag NBR1 was localized in the autophagosomes and could interact with ATG8.Pag NBR1 transgenic poplars accumulated more autophagosomes and exhibited higher expression of ATG8s,resulting in less accumulation of insoluble protein and insoluble ubiquitinated protein compared to WT under salt stress.The accumulation of insoluble protein and insoluble ubiquitinated protein was similar after treatment of autophagy inhibitor Con A in WT and transgenic lines.In summary,our results implied that Pag NBR1 is an important selective autophagy receptor in poplar and confered salt tolerance by accelerating antioxidant system activity and autophagy activity.4.The Pag PIP2;10 can be induced by dehydration stress,and the proteins encoded by them are mainly located on the cell membrane.Overexpression of Pag PIP2;10 in poplar enhanced drought stress tolerance.Actually,overexpression of Pag PIP2;10 promoted the stomatal closure in transgenic lines and reduced transpiration,which result to the higher water content in transgenic line than WT.Further analysis found that ROS accumulated more in guard cells of transgenic lines that WT,which helps stomatal closure after ABA treatment.Except that,the root hydraulic conductance of transgenic lines were higher than that of WT plants.Taken together,these results implied that Pag PIP2;10 played a positive role in enhancing drought stress via enhancing water retaining capacity under drought stress.In summary,the autophagy-related genes Pag ATG,Pag NBR1 and aquaporin-coding gene PIP2;10in poplar were studied in this study.The results showed that Pag ATG,Pag NBR1 and Pag PIP2;10 can be induced under stress condition.Overexpression of Pag ATG18a and Pag NBR1 increased the tolerance of poplar to salt stress by enhancing the antioxidant activity and autophagy process.Overexpression of Pag PIP2;10 promoted the accumulation of ROS in stomata under drought stress,which in turn promoted the closure of stomata and enhanced poplar tolerance to drought stress.This study provides genetic resources and theoretical basis for poplar stress resistance breeding.