RFOs Metabolism During Cold Stress And Recovery Of Cucumber Leaves

In many plants, RFOs (Raffinose family oligosaccharides) accumulate in response to the imposition of environment stress. They are thought to be osmoprotectants, scavengers of oxygen free radical, dehydration protectants that can help plants to alleviate stress damage. Meanwhile, RFOs are also the major transport forms for photosynthetic carbon assimilates in some higher plants, which are commonly known as RFOs transporting species. At present, many studies on RFOs accumulation in sucrose transporting species'response to environment stress have been conducted, hower the RFOs metabolize in RFOs transportation species response to adversity stress is unclear.Cucumber (Cucumis sativus L.) is one of RFOs transportation species with important economic value, which often encounters various environment stress during its cultivation process, such as temperature stress and so on. Galactinol synthase (EC 2.4.1.123) plays as the key enzyme in catalyzing RFOs synthesis, a-galactosidase (EC 3.2.1.22) is the key enzyme of catalyzing RFOs hydrolysis. The genes encoding such two enzymes exist as multi-copies in cucumber genome. In cucumber, the RFOs has two main functions:stress protection and transport agent for photoassimilates. It is still not clear that the different functions and subcellular distributions among the genes which encoded galactinol synthase family and a-galactosidase family.Therefore, in this research, in order to exploring RFOs metabolic process in cucumber's response to cold stress, we investigated the changes of the genes expression and activities, and the subcellular distributions of relative enzymes in the accumulation and decomposition process of RFOs in cucumber leaves during cold stress and temperature recovery process. The results are as follows:1. After cold stress, the contents of sucrose, galactoside, raffinose, stachyose increase in the mesophyll cells of cucumber, the sugar contents gradually restore to normal level after recovering to normal temperature. The results of subcellular distribution of soluble sugars are as the follows:sucrose is mainly distributed in the cytoplasm and chloroplast, galactinol is mainly distributed in the cytoplasm, raffinose is distributed in cytoplasm, chloroplast and vacuole, and stachyose and galactose are mainly distributed in vacuole. Subcellular distributions of relative enzymes are as follows:acid a-galactosidase is mainly distributed in the vacuole, alkaline a-galactosidase is both distributed in the chloroplast and cytoplasm, and galactinol synthase, raffinose synthase and stachyose synthase are mainly distributed in the cytoplasm. The above-described subcellular distributions of sugars and enzymes show that raffinose in cucumber chloroplast, and raffinose and stachyose in vacuole are synthesized in the cytoplasm, and then transport to the chloroplast or vacuole after encountering cold stress.2. The enzymatic activity of galactinol synthase increase under cold stress, while decrease gradually after temperature recovery. In the four kinds of GolS genes, the cucumber leaves begin to express GolS ? and GolS ? in the third day of cold stress, and expression level significantly decrease in the middle of temperature recovery, while the expression levels of GolS ? and GolS ? have no obvious difference during the temperature treatment. These results show that cold stress induce the expression of GolS ? and GolS ?, and GolS ? and GolS ? play an important role resistance to cold stress in cucumber. The expression and activity of raffinose synthase gene increase after cold treatment, and decrease when temperature recovers, in consistent with raffinose content. However, expression and enzyme activity of stachyose synthase have no obvious changes during temperature treatment period.3. Expression of acid ?-galactosidase ? and ? does not change obviously during cold stress and temperature recovery; acid ?-galactosidase ? begins to express once temperature recovers. Detection for enzyme activity in subcellular indicates that enzyme activity of acid ?-galactosidase in the protoplast and vacuole both elevated significantly when the temperature recovers. Above results shows that ?-galactosidase ? of cucumber plays an important role during the process of vacuole RFOs decomposition after temperature recover.4. Expression of alkaline ?-galactosidase ? and ? decrease during cold stress and increase gradually when temperature recovering. Expression of alkaline ?-galactosidase ? does not change significantly during cold stress and temperature recovery. Detection for enzyme activity in subcellular indicates that enzyme activity of acid ?-galactosidase in the protoplasts and chloroplasts and increase when temperature recovering. These results demonstrate that ?-galactosidase ? and ? of cucumber are important for cytoplasm and chloroplast RFOs decomposition.5. Four kinds of cucumber ?-galactosidase-EGFP fusion expression vectors are successfully constructed by using the technologies such as RT-PCR, which can be applied to later subcellular localization experiment.