Genome-wide Characterization Of AQP Gene Family And Functional Analysis On Stress Resistance Of EsPIP1;2 And EsPIP2;1 In Eutrema Salsugineum

Eutrema salsugineum is a halophytes plant and belong to Brassicaceae.which is relatively close to Arabidopsis thaliana.It is quite tolerant to salt,drought and cold stresses,with a small individual,short life cycle,self-pollination,large number of seeds,and small genome.Recently,it has been considered to be a halophyte model plant for investigating the mechanism of plant resistance to stress.Therefore,excavation of stress-resistant genes from E.salsugineum has great significance to improve plant adaptability of s multiple stresses.Previous studies have shown that plant aquaporins(AQPs)play an important role in response to abiotic stress.AQPs serve as water channel proteins and belong to major intrinsic proteins(MIPs)family,functioning in rapidly and selectively transporting water and other small solutes across biological membranes.The AQPs gene family of many plants has been excavated and sorted.However,the research on E.salsugineum AQPs gene family is still in its infancy.This study used the E.salsugienum genomic database in combination with bioinformatics to analyze the genome-wide characteristics of E.salsugienum AQPs.Based on the gene expression results,EsPIP1:2 and EsPIP2:1 genes were selected,and their sequence characteristics,subcellular localizations,water transfer activities,and stress resistance functions were analyzed.Our work provided theoretical basis for studying on E.salsugineum AQPs.The main research results as follow:(1)To extensively identify AQPs in E.salsguineum,HMM profile of MIP domain(PF00230)and NCBI BLAST tool were used.As a result,a total of 35 putative EsAQPs were identified for further analysis.Based on the phylogenetic analysis,we found that the identified EsAQPs had very high similarity with AtAQPs which could be grouped into four subfamilies,including twelve PIPs,eleven TIPs,nine NIPs and three SIPs.In addition,the EsPIP subfamily was further divided into two classes(five EsPIP1s and seven EsPIP2s),the EsTIP subfamily into five classes(three EsTIP1s,four EsTIP2s,two EsTIP3s,one EsTIP4s and one EsTIP5s),the EsNIP subfamily into seven classes(1 EsNIP1s,1 EsNIP2s,1 EsNIP3s,three EsNIP4s,one EsNIP5s,one EsNIP6s and one EsNIP7s),and the EsSIP subfamily into two classes(two EsSIP1s and one EsSIP2s).The chromosomal locations of 34 EsAQP genes were randomly assigned to the seven chromosomes.All EsAQPs had two complete NPA conserved motifs and six transmembrane domains.Analysis of the gene structure,conserved domains,and characteristic sites showed that EsAQPs had structural diversity and subfamily specificity.(2)Analysis of the expression levels of EsAQP genes in different tissues including root,stem,leave,flower and siliquer showed that the expression of all the 35 EsAQPs could be detected in all tissues.Among them,the most abundant transcripts were EsPIPs and a few EsTIPs(EsTIP1;1 and EsTIP1;2).indicated that these genes are important in the growth and development of E.salsugineum.In addition,we found that EsTIP3;1 and EsTIP3;2 were highly expressed in silique specifically,which indicated that EsTIPs may also play an important role in seed development and maturation.(3)Quantitative results of EsAQP genes under abiotic stresses(salt,drought and cold stress)showed that most EsAOPs related to abiotic stresses.The expression pattern of most EsAQP genes in different abiotic stresses shown as:the expression was up-regulated at the beginning of the stress,and then gradually decreased with the increase of the stress time,but the expression level returned to the control level or higher than the control in the later period.EsPIP1;2,EsPIP1;3,EsPIP2;1,EsPIP2;7,EsTIP1;2 and EsTIP2;1 were all significantly differential expressed under different abiotic stresses and high abundant in different tissues.These results suggest that the EsAQP genes may play an important role in stress response.(4)The E.salsugineum EsPIP1;2 and EsPIP2;1 were typical PIPs.which were most closely related to AtPIP1;2 and AtPIP2;1,respectively.The onion epidermis and Arabidopsis protoplast transient expression systems were used to verify the subcellular localization of EsPIP1;2 and EsPIP2;1,and the results showed that they were both localized on the plasma membrane.The water transfer activities of EsPIP1;2 and EsPIP2;1 were observed by Xenopus oocyte expression system,and we found that both of them were functional AQP with water channel activity.The water transfer activity of EsPIP2;1 was greater than EsPIP1;2.In addition,the co-expression of EsPIP;2 and EsPIP2;1 could improve water transfer activity.(5)The growth of transgenic EsPIP1;2 and wild-type Arabidopsis under salt and drought stress were compared,and the results showed that heterologous expression of EsPIP1;2 gene could significantly enhance the tolerance of transgenic Arabidopsis to salt and drought stress.Under stress conditions,seed germination and root growth of transgenic Arabidopsis were significantly better than those of wild type.Under salt stress,the transgenic lines had lower H2O2,malondialdehyde(MDA)content,ion leakage(IL),and a higher K+/Na+ ratio;under drought stress,the transgenic lines had significantly higher content of proline and antioxidant enzymes(CAT,POD and SOD)than those of wild type,and the content of H2O2,MDA and IL were significantly lower than those of wild type.These results indicated that overexpression of EsPIP1;2 could reduce lipid peroxidation and membrane damage in transgenic Arabidopsis,thereby increased the tolerance of transgenic Arabidopsis to salt and drought stress.In addition,overexpression of EsPIP1;2 could increase the antioxidant enzyme activity of transgenic Arabidopsis and promote osmotic regulation to enhance resistance to drought stress,and balance the K+/Na+ ratio to enhance resistance of transgenic Arabidopsis to salt stress.(6)Phenotypic analysis showed that overexpression of EsPIP2;1 could enhance the tolerance of transgenic Arabidopsis to salt and drought stress.Under salt stress,the transgenic lines had lower H2O2,MDA,IL,higher chlorophyll content,Fv/Fm,NPQ,K+/Na+ratio and the expression of genes related to salt stress.These results suggested that EsPIP2;1 transgenic Arabidopsis could promote the expression of stress-related genes,reduce the production of ROS and the extent of membrane damage,maintain a higher photosynthetic rate and ion equilibrium,to improve the tolerance of transgenic Arabidopsis to salt stress.Under drought stress,transgenic lines had higher antioxidant enzyme activity,proline content,relative water content and the expression of genes related to drought stress,lower H2O2,MDA and IL,and water loss rates.It suggested that overexpression of EsPIP2;1 could improve transgenic Arabidopsis antioxidant enzyme activity,promote osmotic adjustment,and maintain water content,reduce lipid peroxidation and membrane damage,and improve the tolerance of transgenic Arabidopsis to drought stress.In addition,the germination rate and root length of transgenic Arabidopsis thaliana were significantly higher than those of wild type under ABA treatment,and EsPIP2;1 may be involved in maintaining the stomatal aperture under ABA stress.This study not only provided a full-scale bioinformation on AQPs in E.salsugineum,but also preliminary revealed the role of EsPIP1;2 and EsPIP2;1 in plant stress tolerance,which would be helpful for further studies on the molecular regulatory mechanism of stress tolerance mediated by AQP genes.