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The leaf protein pattern from drought-tolerant and drought-sensitive wheat varieties subjected to severe soil drought but with the possibility for recover from stress was studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots representing Rubisco large subunit (RLS) were identified using polyclonal antibodies against Rubisco and immunoblotting. Some qualitative and quantitative differences in the 2D-PAGE protein map of wheat varieties were revealed under drought conditions. Three days recovery of wheat plants were not enough for restoring RLS quantity to the level of controls after 7 days drought, especially in the drought-sensitive variety Miziya. There are contradictory data in the literature concerning increased or diminished RLS level in drought stressed plants. A comparison of RLS after SDSPAGE and 2D-PAGE was made. The revealed protein pattern depended on the presence or absence of protease inhibitors in the extraction buffer, on the procedure of extraction, and on the degree of stress.
Red (Trifolium pratense L., cv. ‘‘Start’’) and white clover varieties (Trifolium repens L., cv. ‘‘Debut’’ and cv. ‘‘Haifa’’) were waterlogged for 14 days and subsequently recovered for the period of 21 days. Physiological and biochemical responses of the clover varieties were distinctive, which suggested different sensitivity toward flooding. The comparative study of morphological and biochemical parameters such as stem length, leaflet area, dry weight, protein content, protein pattern and proteolytic degradation revealed prominent changes under waterlogging conditions. Protease activity in the stressed plants increased significantly, especially in red clover cv. ‘‘Start’’, which exhibited eightfold higher azocaseinolytic activity compared to the control. Changes in the protein profiles were detected by SDS-PAGE electrophoresis. The specific response of some proteins (Rubisco, Rubisco-binding protein, Rubisco activase, ClpA and ClpP protease subunits) toward the applied stress was assessed by immunoblotting. The results characterized the red clover cultivar ‘‘Start’’ as the most sensitive toward waterlogging, expressing reduced levels of Rubisco large and small subunits, high content of ClpP protease subunits and increased activity of protease isoforms.
Antioxidant response to drought in red (Trifolium pratense L., cv. ‘‘Start’’) and white clover (Trifolium repens L, cv. ‘‘Haifa’’ and cv. ‘‘Debut’’) grown as soil cultures was evaluated in water-deprived and recovered plants. Drought provoked oxidative stress in leaves confirmed by the considerable changes in electrolyte leakage, malondialdehyde, hydrogen peroxides and proline contents. Immunoblot of Δ-1-pyrroline-5-carboxylate synthetase (P5CS), which catalyzes the first two steps in proline biosynthesis, revealed strong induction of the enzyme in red clover plants submitted to drought. Water-deprived white clover plants exhibited distinct P5CS profiles. This was related to different drought tolerance of the studied T. repens cultivars. Isoenzyme analyses of superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) demonstrated certain differences in antioxidant defence among the tested varieties. It was confirmed that MnSOD (in both T. repens and T pratense) and FeSOD (in T. repens) isoforms were the most affected by drought. The red clover cultivar ‘‘Start’’ exhibited the lowest FeSOD and POX activities which could contribute to its poor performance under water deprivation.
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