Study On Salt Stress Response Regulation Mechanisms Of Carex Rigescens Basis On Proteomics Analysis

Salt stress is a major abiotic stress limiting the growth and development of many plant species.Plant species in salinized soil areas have evolved systematic salt response and resistant mechanism to adapt to salt stress.Investigating on the function of plant salt-responsive proteins under salinity is significant to reveal the plant’s salt-tolerance regulation mechanism and improve plant breeding on salt-tolerance.Carex rigescens is an important turfgrass and widely spread in northern China,with the ability of low-maintenance and tolerance to various abiotic stress conditions.In this study,C.rigescens,was used as the plant material,for the observation of growth,the determination of physiological and biochemical parameters,the sequencing of transcription,the labeling and quantitating of proteome,and the transcriptional expression pattern analysis of the candidate salt-responsive proteins,under salt stress,with expectation to lay a theoretical foundation for plant salt-responsive regulation and salt-tolerance mechanism.The results showed as followings:In the assay of the observation of growth,and the determination of physiological and biochemical parameters of C.rigescens under salt stress,Huanghua showed higher TQ,RWC,Fv/Fm,and proline content,and lower reactive oxygens(O2-and H2O2),REL,and MDA content,illustrating Huanghua is more salt-tolerant,which shows better growth and physiologyl performance compared to Lvping No.1 under salt stress.Furthermore,higher ratio of GSH/GSSG,activity of GPX and GR,and lower ratio of AsA/DHA,activity of APX are measured in Huanghua compared to Lvping No.1,indicates the different changes of non-enzymatic oxidants and antioxidant enzymes between the two C.rigescens genotypes under salt stress.Moreover,in the comparison of the two C.rigescens genotypes,Huanghua has the lower lignin content,higher flavonoid and melatonin content,suggests the differences of metabolites under salt stress in the two C,rigescens genotypes.In addition,the ion content of Ca2+,K+ and Na+ in leaf and root,the ratios of root and shoot,and the ratios of Na+ and K+ in Huanghua under early-stage salt treatment(48 h)are significantly changed,shows that the salt-responsive regulation mechanism in the early-stage is very crucial.In the assay of sequencing of transcription in C.rigescens under salt stress,195,275 of the UniGenes were obtained utilizing transcriptome sequencing and de novo assembly,and 89,614 of them were annotated using UniProt database.Furthermore,the UniGenes were annotated and enriched using GO,COG,and KEGG database.A number of 6,291 UniGenes were screened out as the DEGs,and 1,503 and 3,991 of them were specifically expressed in the tissues of leaf and root respectively,which accounted for 87.3%of the total.DEGs were annotated and enriched to diverse functions and pathways using GO and KEGG database,which showing larger amount and more abundant function pathways in the root tissue compared to leaf.This assay indcated that the root tissue might be more active than leaf in salt-responsive regulation of C.rigescens under salt stress,the function and pathway of DEGs between leaf and root are bit different.In the assay of labeling and quantitating of proteome in C.rigescens under salt stress,total of 9,258 proteins were obtained.Among them,1,893 proteins are differently expressed,including 993 from the leaf and 900 from the root.However,94.9%of the DEPs are specifically expressed in root or leaf tissue.The DEPs are functioned to biological processes(mainly of metabolic process,cellular process,and single-organism process),cellular components(mainly of binding,and catalyic activity),and molecular function(mainly of cell,and organelle)using GO database.The KEGG pathways enrichment analysis of DEPs are mainly includes ribosome,carbon metabolism,biosynthesis of amino acids,and etc in leaf.While spliceosome,biosynthesis of amino acids,RNA transport,ribosome,andetc mainly included in root.The enriched KEGG pathways are further classified into 5 functional categories for analysis:carbohydrate and energy metabolism,photosynthesis and electron transport chain,signal sensing and transduction,protein synthesis,and secondary metabolism.Specifically,the glycolysis process,oxidative phosphorylation,and Ca2+ related signaling pathways in roots are strengthened for energy generation and signal transduction;photosynthesis and hormone signal transduction are more active in leaves for light harvesting and hormone regulation;protein synthesis and secondary metabolism-related processes are changed with different patterns in tissues.In addition,ketosphingosine reductase(KRSD)and sodium-coupled neutral amino acid transporter-2(SNAT2)and their involved sphingolipid metabolism pathway could play crucial role in salt-responsive regulation process in C.rigescens under salt stress.In the assay of the transcriptional expression pattern analysis of the candidate salt-responsive proteins in C.rigescens under salt stress,genes of antioxidant related GPX and GST,Ca2+ signaling related CBL and CML,phytohormone response signaling related PYL、SnRK2 and COI-1,phenylpropanoids synthesis and metabolism related COMT and CCoAOMT are rapider and more effective induced in Huanghua compared to Lvping No.1.However,genes of antioxidant related APX,phenylpropanoids synthesis and metabolism related CHS.4CL and C4H are higher expressed in Lvping No.1 compared to Huanghua.Combined with the determination results of non-enzymatic oxidants,antioxidant enzymes,and some metabolites,it concluded that Huanghua is more active and efficient in ROS scavenging,Ca2+binding,and phytohormone response than Lvping No.1,while Lvping No.1 showed relatively higher phenylpropanoid synthesis,using flavonoid and lignin as supplements for the inadequate ROS-scavenging capacity and the development of vascular tissues.Additionally,the rapid induction of caffeic acid O-methyltransferase(COMT)and the significantly higher content of melatonin,which directly produced the action of COMT,are probably contributing to the antioxidation and the ROS scavenging under salt stress response regulation process,indicating the important role of COMT and melatonin in salt tolerance mechanism in Huanghua.