Physiological And Molecular Mechanisms Of Nitraria Billardieri In Response To Salt Stress

Salt stress is a major environmental factor affecting plant growth,development and geographical distribution.It was reported that more than 6%of the world's land is affected by salt,and there is a growing trend.Halophytes can survive in saline environment and have special salt tolerance adaptation mechanism,thus avoiding salt damage.Halophytes are ideal materials for studying salt tolerance mechanism.Nitraria billardieri is a shrub of Nitraria L.belonging to the family Nitrariaceae.It is a halophyte plant distributed in Australia.Besides its strong salt tolerance,N.billardieri is widely used in the improvement of ecological environment and vegetation in saline-alkali soil,and has potential breeding value.However,the mechanism of salt tolerance in seedlings of N.billardieri has not been reported.Therefore,elucidatingthe physiological and molecular mechanism of salt tolerance of N.billardieri has theoretical and practical significance for cultivating new varieties of salt tolerant plants and utilizing saline soil.In this study,200 mM NaCl solution was used to treat the seedlings of N.billardieri under salt stress.The changes of growth and physiological characteristics of N.billardieri seedlings with stress at the initial stage of salt stress were investigated.The differential expression profiles of protein and gene in leaves of N.billardieri under salt stress were analyzed by proteome and transcriptome techniques,and the differences of protein and gene expression profiles were compared by bioinformatics method.The mechanism of salt tolerance of N.billardieri was preliminarily discussed.The main results are as follows:1.The seedlings of N.billardieri were treated with salt stress of different concentration and time,and their phenotypes were observed.Medium concentration(200 mM)was selected as the salt stress condition for subsequent experiments.The physiological indexes of N.billardieri seedlings under medium salt stress were determined.It was found that there was slight curl at the edge of leaves of N.billardieri after 6 to 9 days of treatment.The fresh weight of the leaves decreased by 5.52%and 13.48%respectively compared with that after 3 days of salt treatment,and the water content also decreased at the later stage of stress.The results showed that the growth of N.billardieri seedlings was not seriously affected by salt stress for a short time,and the seedlings gradually showed stress injury after 6 to 9 days of treatment.2.The physiological indexes of N.billardieri seedlings were further analyzed at 0,3,6 and 9 days of salt stress treatment.The plasma membrane permeability and malondialdehyde(MDA)content in the leaves increased gradually with the prolongation of stress treatment time(within a certain time range),and the permeability of cell membrane was gradually destroyed.Stomatal conductance(Gs),intercellular CO2 concentration(Ci),transpiration rate(Tr)and net photosynthetic rate(Pn)decreased gradually.The activities of antioxidant enzymes such as superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)increased gradually.The above experiments indicate that the morphological changes of N.billardieri in response to salt stress may be related to its physiological and biochemical responses to salt stress.3.In order to study the molecular mechanism of N.billardieri responding to salt stress,the changes of protein expression profiles in leaves after 9 days of salt stress treatment were analyzed by iTRAQ technique.A total of 839 proteins were identified and 130 proteins with significant difference in expression abundance were obtained.Functional annotation of differentially expressed proteins revealed that proteins related to signal transduction,carbohydrate metabolism,protein metabolism and redox balance played an important role in response to salt stress in N.billardieri.Compared with the control,the differentially expressed proteins under salt stress showed significant enrichment in GO items such as cell nitrogen synthesis,organic nitrogen synthesis,metal ion binding and mitochondria.KEGG enrichment analysis showed that salt stress affected the expression of antibiotic biosynthesis,carbohydrate metabolism and photosynthesis-related proteins in the leaves of N.billardieri.It is concluded that salt stress affects photosynthesis of leaves by regulating photosynthesis and expression of carbon sequestration related proteins.A group of interacting proteins were detected in the protein interaction network.Five of them,SHM3,SHM4,GLT1,NTRC and ACLB-2,may play an important role in the response of N.billardieri leaves to salt stress.4.Then,transcriptome sequencing was performed on leaves of N.billardieri treated with salt stress for 1,3,5 and 7 days and corresponding control leaves.A total of 168463 Unigenes were obtained,of which 60%were annotated.The number of differentially expressed genes was 276,218,181 and 196 in the leaves of N.billardieri at 1,3,5 and 7 days of salt treatment,respectively.The number of differentially expressed genes decreased gradually with the extension of salt stress time,which may be related to the gradual adaptation of N.billardieri to salt stress.GO functional annotation and KEGG metabolic pathway enrichment analysis showed that salt stress mainly affected the biological processes of amino acid metabolism,oxidative phosphorylation,signal transduction and secondary metabolism.Differential gene interaction network analysis showed that genes related to amino acid metabolism,photosynthesis,signal transduction and sugar metabolism were mainly up-regulated in the early stage of salt stress to enhance plant energy metabolism to cope with salt stress,and down-regulated in the later stage,indicating that with the increase of stress,plant metabolic activities were inhibited,thereby reducing injury.In this study,halophyte N.billardieri was taken as the research object.By comparing the phenotype,physiological and biochemical indicators,protein and gene differential expression profiles of salt stress treatment with those of control,the mechanism of response of N.billardieri to salt stress was studied at physiological,biochemical and molecular levels for the first time,which provided a basis for exploring the mechanism of halophyte adapting to salt stress environment.In addition,the differentially expressed proteins and genes identified in this study,as well as important network-centric proteins,will be the basis for further elaborating the response of N.billardieri to salt stress and cultivating new salt-tolerant varieties.