Suppression Of Cucumber Stachyose Synthase Gene (CsSTS) Inhibits Phloem Loading And Reduces Low Temperature Stress Tolerance

Cucumber (Cucumis sativus L.) is a stachyose-translocating plant. Stachyose synthase (STS, EC 2.4.1.67) is a key enzyme, it catalyzes raffinose and galactinol to produce stachyose and this synthase play a very important role in the physiological and biochemical processes of raffinose family oligosaccharides (RFOs). Although the stachyose synthase and stachyose synthase gens have been studied and reported, but few studies have been done about its functions of loading in leaves,carbohydrate distribution and the tolerance to abiotic stresses. In this paper, we cloned a stachyose synthase gene (CsSTS) from cucumber and then analyzed its gene expression, gene location and physiological functions. The main results we get are as follows:According to the genome database, CsSTS is only one copy in the cucumber. The spatiotemporal expression was analyzed by qRT-PCR and the results indicated that, although CsSTS was expressed in all detected tissues, the transcript level was the highest in mature leaves and was very low in other organs, such as roots, stems, young leaves, male and female flowers and fruits. As the different developing stages of leaf blades, the relative expression level of CsSTS increased gradually and reached the maximum in 12th day, then declined rapidly. In a day, the expression of CsSTS in cucumber showed a significant circadian rhythm and its expression level was the highest at 15:00 in the afternoon.Histochemical analysis indicated that CsSTS was expressed mainly in minor veins of the mature leaves and the results suggested that CsSTS was involved in phloem loading of carbohydrates. The subcellular localization showed that CsSTS was targeted to the plasma membrane,nucleus and cytosol.After CsSTS being interfered, the expression of CsSTS, STS activity and stachyose content were lower than the wild type, and accumulated a small amount of starch in leaves, suggesting that CsSTS was involved in phloem loading of cucumber leaves. Compared with wild type, the expression levels of CsSUT were obvious higher in CsSTS-RNAi lines, and the export ratios of sucrose and raffinose were somewhat elevated, but in CsSTS-OE lines, their expression levels were basically the similar to the wild types. As shown above, cucumber was symplastic phloem loading pathway (the main path), but there was another way coexisted simultaneously, viz. apoplastic pathway. From this point of view, cucumber was also a model plant of mixed loading pathway.Cucumber seedlings were selected and subjected to low temperature (6?, 72 h ) in order to further study the function of CsSTS. In the process, CsSTS expressions were up-regulated, enzyme activities,stachyose contents and antioxidant enzyme activities increased gradually in wild type plants and transgenic plants especially in the OE lines, but in the RNAi lines their elevated and increased levels were small. After low temperature treating for 72 hours, the leaves of CsSTS-RNAi lines appeared wilting and sagging obviously, but the growth of CsSTS-OE lines were affected slightly by low temperature. These results suggested that CsSTS was involved in response to low temperature stresses.Furthermore, the increased STS activity and stachyose content contributed to reduce the damage of low temperature for cucumber seedlings.