Root Nodules Confer Salt Tolerance Upon Alfalfa (Medicago Sativa L.) Plants, Proteome Analysis

Salt stress is one of the main abiotic stresses than negatively affects crop growth,development and production. Alfalfa is a key legume that can symbiotic nitrogen fixationthrough nodules. To investigate the mechanism that root nodules confer alfalfa salt tolerance,we assayed the changes of physiological parameters, proteomics and relative expression ofnodules-specific proteins in aboveground and underground tissues of alfalfa seedlings withactive nodules, inactive nodules or without nodules by physiological parameters assay,iTRAQ (Isobaric Tags for Relative and Absolute Quantization) and qRT-PCR (Real-timeQuantitative Polymerase Chain Reaction) methods. The results will describe a novel approachto study alfalfa abiotic stresses tolerance.The main findings are as follow:1. Compared to alfalfa seedlings with inactive nodules or without nodules, alfalfaseedlings with active nodules had higher relative water content, dry matter, reducedglutathione, proline and soluble sugar content; higher enzyme acitivities of superoxidedismutase and catalase; lower super anion free radicals and malondialdehyde content. Theactived nodules can reduce the cell membrane damage, promote cell stability, and maintaincell homeostasis under salt stress. The alfalfa seedlings with active noduels had a strongerability of osmosis regulate and reactive oxygen scavenging. And the alfalfa seedlings withactive nodules had a stronger ability on salt tolerance.2. iTRAQ is a powerful method to detect and quantify a large number of nodule-treatedproteins in aboveground and underground tissues of alfalfa seedlings under salt stress. Weidentified a total of2,593and2,680proteins in aboveground and underground tissues ofrhizobia-treated alfalfa seedlings under salt stress respectively. And the differentiallyexpressed proteins in the aboveground and underground tissues were1,012and906respectively.3. The differentially expressed proteins in the aboveground tissues are mainly involvedin electron transfer, signal conduction, photosynthesis and energy supply. Theses proteins canreduce the ductility of cell wall and maintain the cell integrity and homeostasis. Thedifferentially expressed proteins in the underground tissues are mainly involved in proteinsynthesis, reactive oxygen scavenging, metabolism and redox. These proteins can enhance nitrogen fixation and energy supply, and maintain cell osmotic balance.4. The differentially expressed proteins are mainly involved in photosynthetic carbonfixation, phosphorylation, glutathione, phenylpropanoid biosynthesis, phenylalaninemetabolism and ribosome pathway. These proteins are mainly involved in catalytic activity,protein structural molecular activity, energy transfer and protein bindig process. These resultswill indicate that nodules can strength the ability of metabolism, energy supply, reactiveoxygen scavenging, cell integrity maintain and homeostasis. And nodules can improve alfalfasalt tolerance.5. These ten genes displayed expression patterns similar to their protein levels inqRT-PCR analysis. These results indicated the validity of iTRAQ analysis.6. The differentially expressed proteins in qRT-PCR analysis are mainly involved inphosphorylation, proteins biosynthesis, redox, energy and reactive oxygen scavengingprocesses. And these proteins play a key role on alfalfa salt tolerance.7. The process of rhizobia inoculated may induce plant stress defence and immunemechanism, which can improve alfalfa salt tolerance.