Molecular Regulation Mechanism Of Salt Resistance And Drought Tolerance In Autotetraploid Sour Jujube

Chinese jujube is an important fruit tree for large-scale industrialization in China.The rapid changes in the global climate have led to aggravation of land drought and soil salinization,which seriously threatens the growth of jujube trees and the yield of jujube fruit.Breeding jujube rootstocks with enhanced stress resistance for grafting jujube trees is a major strategic demand for improving the stress resistance of jujube trees and promoting the healthy development of the jujube industry.In the previous research,a new germplasm of autotetraploid sour jujube was obtained and named W219.Preliminary research found that the leaves of this germplasm became thicker and larger,the leaf color deepened,the stem became thicker and the internodes became shorter.In this study,W219 was used as research object and the diploid was control to analyze its phenotype,salt resistance and drought tolerance.On this basis,the molecular basis of salt resistance and drought tolerance variation in W219 was systematically analyzed at the physiological and molecular levels,and the key genes were screened for functional analysis in sour jujube.This study will reveal the regulation mechanism of salt resistance and drought tolerance variation in autotetraploid sour jujube to a certain extent,could provide an important basis for clarifying the character variation mechanism after tree polyploidization,and lay a theoretical foundation for the further utilization of autotetraploid sour jujube germplasm W219 as salt resistance and drought tolerance rootstock.The main results are as follows:(1)Chromosome doubling significantly changed the leaf characters in autotetraploid compared with its diploid.The results showed that the epidermal cells of W219 were significantly larger than diploids,and the thickness rate of palisade and sponge tissue in W219 showed significantly higher than that in diploids;at the same time,the number of veins xylem vessels and parenchyma cells were significantly higher in W219 than in diploid.Furthermore,compared with the diploid,the cuticle thickness and cuticular wax content were significantly increased in the W219 leaves.Meanwhile,13 differential genes related to wax synthesis and transport were differentially expressed between diploid and W219 leaves.Further analysis showed that the adaxial side of W219 young leaves was densely distributed and regularly arranged in radiating platelet-shaped cuticle wax crystals,while the diploid wax crystal arrangement was sparse and irregular;lamellar wax crystals were attached to the abaxial side of mature leaves in W219 but almost no wax crystals in diploids.Moreover,more chlorophyll,soluble sugar and soluble protein content were detected in W219 than in diploid.Many key genes involved in starch metabolism,amino acid metabolism,hormone synthesis and signaling were differentially expressed in diploids and W219,among which osmotic regulation related genes were highly expressed in W219.Therefore,phenotypic characteristics variation may be potential factors affecting the stress resistance of autotetraploid,and the differences of physiological characteristics and gene expression are the important basis for explaining the variation of autotetraploid traits.(2)The autotetraploid showed strong adaptability to long-term salt stress than diploid.Almost all the physiological inicators in W219 were higher than that in diploid after different time of stress treatment,especially the proline content and CAT activity were higher than those of diploids after 14 d of salt stress.Based on RNA-seq,the response of W219 was significantly improved by long-term salt stress treatment.Under 2 d of salt stress,more differential genes in W219 were annotated in stress response pathways,such as antioxidant activity,signal transduction and transcription factors such as AP2/ERF-ERF,MYB and NAC.Meanwhile,the differential expression folds of genes involved in the osmotic regulators synthesis and transport in W219 were significantly higher than those in diploids after salt stress.In addition,the interaction network involved in stress response in W219 under salt stress was much more complex than that in diploid.In general,autotetraploid sour jujube showed a more complex,diverse and sustained response mechanism under salt stress.(3)Under drought conditions,autotetraploid showed stronger drought tolerance and ability to restore growth than diploid.The lower cuticle permeability of W219 leaves may lead to the reduction of non stomatal water loss.After drought treatment,W219 accumulated more osmotic regulators and had a lower degree of photosynthetic damage.Meanwhile,W219 accumulated lower levels of peroxides,and the differential genes related to ROS scavenging,such as SOD and LACS7 were only differentially expressed in W219 after drought treatment.After long-term drought treatment,the number of differential genes in W219 was significantly higher than that in diploid,especially the genes encoding transcription factors such as WRKY,NAC and MYB.After 48 h of drought stress,the types of differential transcription factor detected in W219 were more than twice that in diploid.Finally,7 hub genes co-expressed with 18 NAC genes highly expressed in W219 were excavated.(4)Based on bioinformatics analysis,55 members of jujube NAC transcription factor family were identified in jujube genome.According to the the analysis of phylogeny,cis-acting elements in ZjNAC promoter and gene differential expression,ZjNAC6,ZjNAC32,ZjNAC47,ZjNAC52 and ZjNAC55 in response to abiotic stress and ZjNAC32,ZjNAC36 and ZjNAC52 related to tissue maturation or senescence were selected as candidate genes.(5)The function of ZjNAC52 in sour jujube was studied.Transgenic lines over-expressing ZjNAC52 were obtained by leaf discs transformation of sour jujube.Overexpressing ZjNAC52 significantly increased the expression level of lignin synthesis related genes PRX52,4CL1 and CAD1 in sour jujube,and the width of stem secondary xylem of transgenic plants increased significantly.After drought and salinity treatment,the activities of SOD,POD and CAT in transgenic plants were significantly higher than those in wild-type plants,while the degree of membrane lipid peroxidation was always significantly lower than that in wild-type plants,indicating that ZjNAC52 can improve salt resistance and drought tolerance through antioxidant function.(6)DAP-seq showed that the number of target genes of ZjNAC52 in diploid and W219 after drought stress was 236 and 321 respectively,and the identified target gene enrichment motif characteristics were different.Five motifs were detected in the two ploidy target genes,the most reliable motif were all characterized by CTTNAAG,and three motifs were different.Functional analysis shows that more targets annotated to xylem development,secondary wall synthesis,cell death and DNA replication pathways were detected in W219.SMR1 plays distinct roles in cell proliferation and differentiation.Under drought stress,the binding peak score and signal value of ZjNAC52 to ZjSMR1 in W219 was higher than that in diploid,binding motif ABRE was detected in W219,and the fold change of ZjNAC52 and ZjSMR1 after drought stress was greater in W219 than that in diploid.Through yeast one hybrid test and the expression level analysis,it is preliminarily proved that ZjNAC52 may positive targeted regulation of ZjSMR1 expression.Therefore,the enhanced hydraulic conductivity mediated by ZjNAC52 and its targeted regulation of xylem vessel development-related genes may be one of the important reasons for the autotetraploid sour jujube stress resistance.(7)ZjGR-RBP1 was selected from NAC transcription factor gene regulatory network to study function.ZjGR-RBP1 was localized in the nucleus,stably expressed in different tissues,and up-regulated in response to short-term salt stress and drought stress.The overexpressing-ZjGR-RBP1 lines showed higher SOD,POD and CAT activities and lower degree of membrane lipid peroxidation than wild-type plants under drought and salinity conditions,indicating that ZjGR-RBP1 can improve jujube salt resistance and drought tolerance by improving the antioxidant capacity.In addition,the expression of ZjNAC52 was increased in lines over-expressing ZjGR-RBP1,which may indirectly affect plant stress resistance as an putative upstream regulator of ZjNAC52.