Effects Of Low Temperature On Antioxidant Enzymes And Leaf Proteome From Cucumis Sativus Of Different Genotypes

Cucumber (Cucumis sativus L.) cv. Xintaimici (a chilling-resistant cultivar) and cv. Jinyan no. 4 (a chilling-sensitive cultivar) were used to study the effects of low temperature on antioxidant enzymes and leaf proteome. The purpose of this project was to understand the relationship between ultrastructural changes and the antioxidant abilities caused by low temperature, and as well to check if higher contents of antioxidants, which are correlated with the differences in ultrastructure between low-temperature-stressed cucumber cultivars, can be used as biochemical indicators for breeding chilling-resistant cucumber cultivars in greenhouse. It is also our aim to separate protein spots induced by low temperature in cucumber leaves of the two genotypes, and threrby revealing the mechanism of cucumber responding to low temperature.The two cucumber cultivars were exposed to two temperatures (25/18 and 15/15℃) for 7 days. The fourth leaf was used to determine the activities of superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7), catalase (CAT, EC 1.11.1.6), and glutathione reductase (GR, EC 1.6.4.2). We also determinated the correlative data, such as the contents of malonaldehyde (MDA) and reduced glutathione (GSH). Using the transmission electron microscope, we observed the chloroplast, mitochondrion, endoplasmic reticulum (ER) and cell membrane. Meanwhile, the total proteins of the two cultivars were successively extracted by three-steps, separated the differential proteins by two-dimensional gel electrophoresis (2-DE) and analyzed through MALDI-TOF mass spectrometry. The results of this study were listed as the following:1. Effects of low temperature on antioxidant enzymesThe activities of SOD and GPX increased in low-temperature-stressed leaves of both cultivars, but the two enzymes were not responsible for the difference between cucumber cultivars. At 15/15℃, contents of GSH and GR activities increased more in leaves of'Xintaimici'than in those of'Jinyan no. 4', while CAT activities decreased less. At the 15/15℃treatment, the membranes of chloroplast, mitochondrion, ER and plasma were not significantly changed in'Xintaimici', whereas they were seriously affected in'Jinyan no. 4'. This result was consistent with the changes of MDA in low-temperature-stressed cucumber leaves. Therefore, the antioxidant activities were changed under low temperature according to cultivar-expected resistance, relating in part to the described ultrastructural changes. GSH, GR and CAT were affected by low temperature and cultivars and correlated with the difference in ultrastructure between low-temperature-stressed cucumber cultivars. We propose that the three antioxidants might be used as biochemical indicators to screen low-temperature-resistant cucumber cultivars.2. Effects of low temperature on leaf proteomeSDS-PAGE showed that few proteins were extracted by hydrophilic solutionⅠ. The extracts of solutionⅡandⅢwere separated through 2-DE. Compared with treatment of normal temperature, low temperature made'Xintaimici'cultivar produce 7 special protein spots, 2 up-regulated protein spots, 2 down-regulated protein spots and 5 unexpressed protein spots, and allowed'Jinyan no. 4'to produce 7 special protein spots, 1 up-regulated protein spot, 4 down-regulated protein spots and 9 unexpressed protein spots. Three special protein spots, which were highly expressed under low temperature, were identified to be the large subunit of ribulose 1,5-bisphosphate carboxylase. Therefore, there were few hydrophilic proteins in cucumber leaves. Low temperature influenced leaf proteome in two cucumber genotypes and different cultivars expressed different proteins. Ribulose 1,5-bisphosphate carboxylase may be related to low-temperature tolerance in cucumber leaves.