Separation and Identification of Specific Proteins in Leaves of Grafted Cucumber under NaCl Stress

Using the rootstock Shintosa Supreme pumpkin (Cucurbita maxima x C moschata) which is a salt tolerant cultivar from Japan, and a scion of cucumber (Cucumis sativus L.) cv. Jinyan No. 4 from China, grafting was made to compare the differences in leaf soluble protein components by isoelectrofocusing- sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (IEF-SDS-PAGE) analysis between grafted and scion own-rooted plants under 100 mM NaCl stress. The gels were scanned by the densitometer VersaDoc 3000, which generated the digitalized images. The computer analyses including automatic spot detection, quantification of protein spots, alignment and matching between the different two-dimensional electrophoresis (2-DE) gels, were carried out by using PDQuest software. The results showed that 16 specific proteins were identified to be related to salt stress in leaves of grafted cucumber after 2 d of exposure to NaCl stress. Their molecular weights (M-w) were 110.6, 89.7, 76.5, 56.4, 49.4, 39.6, 36.2, 35.8, 34.7, 34.4, 34.3, 33.4, 22.7, 19.8, 15.9, and 12.9 kDa, respectively. Three protein spots were selected by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS), but only one of them was successfully identified. By searching SwissProt 45.2 protein database, the map was matched with O-methyltransferase (with accession number P47917, theoretical M-w 39.58 kDa, and theoretical pI 5.45). O-methyltransferase was reported to be related to salt tolerance. In addition, the synthesis of other proteins in the leaves of grafted plants was up-regulated compared with that of the own-rooted plants. These results indicated that specific proteins were synthesized and some of which were up-regulated in the leaves of grafted plants under NaCl stress, resulting in the increase of the salt tolerance of grafted plants.