Characteration And Analysis Of Salt-Stress Related Proteins And MiRNA In Medicago Sativa And Medicago Truncatula

Salt stress is one of the most significant abiotic stresses to cause crop failureworldwide. So far, many salt stress induced genes and improving salt tolerance geneshave been characterized in many plants. In saline lands, plant root growth and functionare determined by the action of environmental salt stress through specific genes thatadapt root development to the restrictive condition. Medicago sativa and Medicagotruncatula are two important legumes for one is a primary forage, the other is a modellegume. To understand better NaCl stress responses in Medicago. sativa (Zhongmu-1)and Medicago. truncatula (Jamalong A-17) roots, a comparative proteomic analysis ofroots that had been exposed to300mM NaCl for0h and10h was conducted. Changesin the abundance of protein species within roots were examined using two-dimensionalelectrophoresis. Among the>1000protein spots reproducibly detected on M. sativaroots gel, the abundance of40protein spots decreased and53increased, at10h timepoints, in response to NaCl treatment. Among the>1000protein spots reproduciblydetected on M. truncatula roots gel, the abundance of8protein spots decreased and22increased, at10h time points, in response to NaCl treatment. Through tandem massspectrometry, identity was assigned to60and26of the differentially abundant spots inM. sativa (Zhongmu-1) and M. truncatula (Jamalong A-17) roots. The proteinsidentified included many previously characterized stress-responsive proteins and someprocesses including iron transport, catalysis, DNA or RNA binding and so on.In plant, many functions are regulated in transcriptional and posttranscriptionallevels. Small21-to24-nucleotide RNAs, notably the microRNA (miRNA), areemerging as a posttranscriptional regulation mechanism. Four small RNA librariesconstructed from roots of Zhongmu-1(M. sativa, salt tolerance) and Jamalong A-17(M.truncatula, salt sensitive). High-throughput sequencing was used to sequence the smallRNAs in these libraries.659and677conserved miRNA belong to250miRNA familieswere identified in Zhongmu-1and Jamalong A-17, as well as189and218novelcandidate miRNAs in were identified in Zhongmu-1and Jamalong A-17. Statisticalanalysis on sequencing reads abundance revealed specific miRNA showing contrastingexpression patterns in Zhongmu-1and Jamalong A-17roots.The differentiallyexpressed conserved and novel miRNAs may target a large variety of mRNAs, some of which may play key roles in salt stress regulation. The spatial salt stress regulation ofmiRNAs may determine specialization of regulatory RNA networks in plant.In this study one new salt induced M. sativa (alfalfa) gene (MsRCI2A) was clonedand characterized based on its EST, which shows high homology to yeast PMP3gene(plasma membrane protein3) and Arabidopsis AtRCI2A. Sequence comparisons revealthat five genes (MtRCI2A-E) show high homology to MsRCI2A in M. truncatulagenome. MsRCI2A and MtRCI2A-E all encode small, highly hydrophobic proteinscontaining two putative transmembrane domains, and mostly localize in the plasmamembrane. Transcription analysis result suggests that MsRCI2A and MtRCI2A-D genesare highly induced by salt stress. Expression of MsRCI2A and MtRCI2A-C in yeastmutants lacking the PMP3gene can functionally complement the membranehyperpolarization and salt sensitivity phenotypes resulting from PMP3deletion. Toinvestigate whether the overexpression of RCI2-ralated gene causes an enhancedsalt-tolerant phenotype, MsRCI2A is overexpressed in Arabidopsis thaliana. The resultreveals that the salt tolerance of MsRCI2A-overexpressing transgenic plants is improvedcomparing to the wild type under high salinity treatment.Many glycine-rich proteins have been implicated in plant responses toenvironmental stresses, but the function and importance of some GRPs in stressresponses are largely unknown. In this study a novel salt induced glycine-rich proteingene (MsGRP) was isolated from alfalfa. Compared with some glycine-rich RNAbinding proteins, MsGRP contains no RRM motif and localizes in the cell membrane orcell wall according to the subcellular localization result. MsGRP mRNA is induced bysalt, ABA and drought stresses in alfalfa seedlings, and its overexpression driven by aconstitutive cauliflower mosaic virus-35S promoter in Arabidopsis plants conferssalinity and ABA sensitivity, as compared with WT plants. MsGRP retards seedgermination and seedling growth of transgenic Arabidopsis plants under salt and ABAtreatments, implying that MsGRP may affect germination and growth through an ABAdependent regulation pathway. These results provide indirect evidence indicating thatMsGRP plays important roles in seed germination and seedling growth of alfalfa undersome abiotic stress conditions.