Proteomics Analysis of Salt Responsive Proteins in Alfalfa (Medicago sativa L.) Leaves by Two Dimensional Electrophoresis and MALDI-TOF MS.

Background: Salinity is a major abiotic stress that limits plant growth and development. Salinity affects several physiological and biochemical characteristics adversely, which results in oxidative stress in plant species. Plants change the protein pattern to cope with salinity stress. The identified salt-responsive proteins in alfalfa are involved in energy and metabolism, photosynthesis, regulation of carbohydrates, transcription/translation, signal transduction, stress/redox homeostasis, ion binding, and stress and defense with ROS scavenging and detoxification. Objective: The present research aimed to study the response of two contrasting alfalfa varieties to salt stress, and to identify the altered leaf proteins by two-dimensional gel electrophoresis and MALDI-TOF/TOF/MS spectrometry. Methods: Salinity stress significantly decreased shoot fresh and dry weights in both Synthetic II (salt-tolerant) and Khajeh (salt-sensitive) varieties, and K+/Na+ ratio in Khajeh, while it significantly increased K+/Na+ ratio, soluble sugars, chlorophyll a and catalase activity in Synthetic II, and peroxidase activity in Khajeh. Results: In the proteome analysis, a total of 188 spots, including 104 spots in the Synthetic II variety (salt-tolerant) and 84 spots in the Khajeh variety (salt-sensitive), were identified under salinity stress. Among the identified protein spots, 15 common spots had a significant change in expression between normal and salt stress conditions in leaves of the two alfalfa varieties. These proteins were grouped into nine functional categories: proteins involved in photosynthesis electron transfer (13.33%), energy metabolism (20%), transcription/translation (20%), stress and defense (13.33%), ROS scavenging and phytohormone signaling (6.67%), protein degradation (6.67%), ubiquitination (6.67%), biological and physiological processes (6.67%), and unknown (6.67%). Conclusion: Salinity stress reduced the yield of alfalfa, but the reduction was more pronounced in the sensitive variety of Khajeh. The salinity-tolerant variety, Synthetic II, responded better to salinity stress in terms of K+/Na+ ratio, soluble sugars, chlorophyll a and catalase activity as compared to Khajeh. Proteome analysis showed that the proteins involved in energy metabolism, transcription/- translation, photosynthesis, electron transfer, and defense were more important than other functional categories under salinity stress. These proteins mainly increased in the salt-tolerant variety, whereas they decreased in the salt-sensitive variety.