Study On The Function Of PeERF1 And PeCLH2 Genes Of Populus Euphratica In Response To Salt Stress

Soil salinity is considered to be a major problem for global agricultural production and sustainability.Salt stress has now severely limited the growth,development,yield and quality of plants worldwide.Therefore,accelerating the dissection of salt tolerance mechanisms in forest trees and improving their ability to adapt to salt tolerance is an urgent need for land use,environmental sustainability and improved economic efficiency.Poplar(Populus euphratica)has long been grown in saline and arid soil environments,and is extremely adaptable to saline and arid environments,making it a typical salt-tolerant and drought-resistant model species.In this study,a series of key regulatory factors related to salt tolerance were identified based on the results of the previous genome-wide association analysis(GWAS)of poplar natural population seeds for salt tolerance,combined with the joint analysis of gene co-expression network analysis(WGCNA).Two genes,PeERF1 and PeCLH2,were selected for transgenic functional validation to investigate the mechanism of their response to salt stress.The main findings are as follows:(1)Salt stress treatment experiments were conducted on Populus euphratica seedings.The results showed that POD and SOD contents increased significantly from 0 h to 12 h under Na Cl treatment,with the highest content at 12 h in 150 m M treatment.Starting from 24 h,the contents of POD and SOD decreased with the longer treatment time and higher treatment concentration,and reached the lowest at 300 m M for 48 h.MDA analysis showed the opposite trend.It shows that the higher the treatment time and treatment concentration of plants,the more active oxygen content in plants,and the greater the damage to plants.Populus euphratica may protect cells from damage by defending against salt stress through osmotic regulation and ROS homeostasis.Transcriptome analysis identified 18,002 differentially expressed genes under salt stress,of which 8,624 genes were up-regulated and 9,378 genes were downregulated.Differential genes were significantly enriched in the GO terms extracellular region,outer encapsulation structure,cell wall,cell periphery and apoptotic layer in all groups.KEGG enrichment analysis found that phytohormone signaling,galactose metabolism,MAPK signaling pathway-plant and keratin,folinic acid and wax biosynthesis were significantly enriched in most of the comparisons.The constructed WGCNA co-expression network found that the turquoise module of 9,438 differential genes,the green module of 763 differential genes and the blue module of 4,624 differential genes were related to phenotypic traits including POD,SOD and MDA.Significant and stable correlation,two salt tolerance genes,PeERF1 and PeCLH2,were found to be key regulators related to salt tolerance.(2)To further investigate the function of the PeERF1,the PeERF1 was cloned from Populus euphratica and the sequence of the PeERF1 protein was found to have a conserved AP2 structural domain.The PeERF1 is a nuclear-localized protein and has transcriptional activation activity.Yeast one-hybrid experiments revealed that the PeERF1 specifically binds to GCC-BOX,DRE,TGG1 and TGG2 elements.The 1524 bp DNA sequence upstream of the PeERF1 in Populus euphratica and element prediction by the promoter identified many elements related to plant growth and development,light-responsive elements,response elements involved in defense against stress and growth hormone response elements.GUS staining results indicated that the PeERF1 promoter was active and could drive GUS gene expression in the top young tissues of plants,but not in stems and roots.It is not expressed in stems and roots.(3)PeERF1 overexpression and dominant inhibitor vectors were successfully constructed by Gateway method,and overexpression and dominant inhibitor transgenic plants were successfully obtained using 84 K poplar(Populus alba × P.glandulosa)callus transformation method.Under salt stress conditions,the plant height,root length,fresh weight,POD,SOD,soluble sugar content,chlorophyll content and relative expression of related resistance genes of PeERF1 overexpressing transgenic were significantly higher than those of wild type,and the MDA content and relative conductivity were significantly lower than those of wild type.The suppressed expression transgenic plants showed the opposite situation.These results suggest that the PeERF1 can enhance salt stress tolerance by improving the ability of plants to scavenge reactive oxygen species and proline accumulation under salt stress conditions.(4)Transcriptome sequencing analysis of transgenic PeERF1 overexpression(PeERF1-OE),PeERF1 inhibitory expression(PeERF1-SE)and WT under normal conditions and salt stress.1237 differentially expressed genes were identified in the results,and 126 differentially expressed genes were identified in PeERF1-OE in the control group.132 differentially expressed genes were identified in PeERF1-SE.In the treated group,PeERF1-OE identified935 differentially expressed genes,of which 207 genes were up-regulated and 728 genes were down-regulated.44 differentially expressed genes were identified by PeERF1-SE.It showed that PeERF1-OE and WT differential genes were the most abundant after salt treatment,indicating that PeERF1-OE mainly functions after salt stress.The annotation of differential genes identified GO terms related to stress and metabolism,such as hydrolase activity,response to organic nitrogen compounds,jasmonic acid metabolic process,and lignin metabolic process,etc.The results of KEGG enrichment analysis revealed that these genes were mainly involved in metabolic pathways,secondary metabolite synthesis pathways,and flavonoid biosynthesis pathways.(5)The PeCLH2 gene was cloned from Populus euphratica.It is predicted that CLH2 belongs to the superfamily of hydrolase and has a chlorophyll domain at 49-996 amino acid residues.The results of multiple sequence alignment and evolutionary analysis showed that the CLH2 protein had high homology with CLH2 proteins from P.trichocarpa,Hevea brasiliensis,Jatropha curcas,and Cherry.The subcellular localization results revealed that PeCLH2 is a nuclear localized protein.GUS staining of PPCLH2::GUS promoter transgenic showed that the PPCLH2::GUS promoter was active and could drive GUS gene expression in the young tissues of the plants.The plant height,root length and fresh weight of PeCLH2-overexpressing transgenic lines had obvious advantages over wild-type controls under salt stress conditions.The POD,SOD,soluble sugar content and chlorophyll content of PeCLH2 overexpression transgenic poplar were significantly higher than those of WT under salt stress.The results of MDA content and relative conductivity tests showed that poplar overexpressing PeCLH2 suffered significantly lower damage under salt stress than WT.The RNAi-PeCLH2 expression showed the opposite trend.The PeCLH2 overexpression transgenic lines were found to be significantly more damaged under salt stress than WT,and the RNAi-PeCLH2 transgenic were significantly less damaged under salt stress than WT.This indicates that the PeCLH2 gene has some salt tolerance.(6)Transcriptome sequencing analysis of transgenic PeCLH2-OC,PeCLH2-RC and WT under normal conditions and salt stress identified 2,190 differentially expressed genes.Under normal conditions,11 differentially expressed genes were identified in OC and 27 differentially expressed genes were identified in RC.In the treatment group,2,146 differentially expressed genes were identified in OC and 6 differentially expressed genes were identified in RC.Among them,PeCLH2-OC and WT had the most differential genes after salt treatment,indicating that PeCLH2-OC functioned mainly after salt stress.It was found that the GO terms that were significantly enriched in the differential genes were all stress-related,including responses to osmotic stress,responses to stimuli,and responses to stress.To sum up,this study screened a series of key regulatory factors of salt tolerance through transcriptome analysis of Populus euphratica in salt response and combined analysis results of WGCNA and GWAS.The candidate genes PeERF1 and PeCLH2 were identified by transformation validation of 84 K poplar for gene function.The results revealed that PeERF1 and PeCLH2 genes could improve ROS scavenging ability and reduce cell damage under salt stress.This study contributes to a better understanding of poplar response to salt and shows that co-expression networks have important potential in facilitating the identification of salt-related candidate genes.It is of great significance to further improve the salt tolerance mechanism of Populus euphratica,screen out high-quality salt tolerance genes and cultivate woody plants with excellent salt tolerance.The results are helpful to analyze the salt tolerance mechanism of poplar.This study provided important genes for salt tolerance breeding of poplar.