Screening for salt-responsive proteins in two contrasting alfalfa cultivars using a comparative proteome approach

• Two alfalfa cultivars differing in salt tolerance were exposed to NaCl treatment.
• Distinct physiological and metabolic changes were observed.
• Na+, K+, Ca2+ and osmolites accumulation pattern were distinct in two cultivars.
• Salt-induced proteome profiles changes and their roles were discussed.

A comparative proteomic approach was carried out between two contrasting alfalfa cultivars, nonomu (NM-801; salt tolerant) and vernal (VN; salt intolerant) in terms of salt tolerance. Seedlings were subjected to salt stress (50 and 100 mM NaCl) for three days. Several physiological parameters (leaf water, chlorophyll, root Na+, K+, and Ca2+) and root proteome profile were analyzed. Comparison of physiological status revealed that NM-801 is more tolerant to salt than VN. Eighty three differentially expressed proteins were found on 2-DE maps, of which 50 were identified by MALDI-TOF or MALDI-TOF/TOF mass spectrometry. These proteins were involved in ion homeostasis, protein turnover and signaling, protein folding, cell wall components, carbohydrate and energy metabolism, reactive oxygen species regulation and detoxification, and purine and fatty acid metabolism. The comparative proteome analysis showed that 33 salt-responsive proteins were significantly changed in both cultivars, while 17 (14 in VN and 3 in NM-801) were cultivar-specific. Peroxidase, protein disulfide-isomerase, NAD synthetase, and isoflavone reductase were up-regulated significantly only in NM-801 in all salt concentrations. In addition, we identified novel proteins including NAD synthetase and biotin carboxylase-3 that were not reported previously as salt-responsive. Taken together, these results provide new insights of salt stress tolerance in alfalfa.