Mechanism Of Salt Tolerant Rootstock Raise The Salt Tolerance Of Cucumber Through Regulating The Uptake And Transportation Of Na~+

In recent years, with the development of protected culture, soil salinization has been more and more severe and has become the biggest obstacle of limiting the development of protected culture. Thus, the vegetable production in greenhouses has become a severe problem. Cucumber (Cucumis sativus L.) is one of the most popular vegetables under the protected cultivation conditions, and is sensitive to salt stress, but pumpkin (Cucurbita moschata Duch.) is salt-tolerant to salt stress. Previous studies suggested that the key reason is that pumpkin roots had higher capacity to limit the transport of Na+to the shoot than cucumber; however, the mechanism is not completely known. Cucumber plants ’Jinchun No.2’(Cucumis sativus L.), pumpkin ’Chaojiquanwang’(Cucurbita moschata Duch),’Jinchun No.2’grafted onto itself (self-grafted) and onto ’Chaojiquanwang’(rootstock-grafted) were used, X-ray microanalysis, non-invasive micro-test technique and gene expression were used to determine the differences of Na+uptake and transport between the self-grafted and rootstock-grafted plants, and to supply theoretic basis for clarify grafting enhance salt tolerance of cucumber. The main results are as follows:1. Experiments were conducted to investigate the relative content of ions in roots, stems below and above cotyledon of cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and rootstock-grafted cucumber under salt stress using X-ray microanalysis. The findings show that, the relative Na+content of rootstock-grafted and pumpkin plants were higher than cucumber in the roots, but lower than cucumber in the shoots, and the relative K+and Ca2+content were higher than cucumber. The relative Na+content in the cortex of root of pumpkin and grafted plants were higher, but the relative Na+content in the stele of root of pumpkin and grafted plants was lower than those of cucumber. The cortex of root of pumpkin and grafted plants had higher ability to restrict excess Na+loading in the xylem, resulting in a less accumulation of Na+in the shoot.2. Cucumber and pumpkin were used to investigate the Na+concentration in different parts of plants. This study showed that more Na+was stored in pumpkin roots compared with cucumber roots under200mM NaCl stress, resulting in the less accumulation of Na+in the shoot. The non-invasive micro-test technique (NMT) showed that the stem below cotyledon of pumpkin exhibited a smaller efflux of Na+from roots to shoots but a higher efflux of Na+from shoot to root, and pumpkin roots showed a higher capacity to extrude Na+, and a correspondingly increased H+influx. However, the200mM NaCl induced Na+/H+exchange in the root was inhibited by amiloride (a Na+/H+antiporter inhibitor) or vanadate (a plasma membrane H+-ATPase inhibitor). This result indicated that the Na+exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na+/H+antiporter across the plasma membrane. These results provide direct evidence of contrasting Na+transport ability from root to shoot in salt-sensitive cucumber and salt-tolerant pumpkin under NaCl stress.3. To assess the effects of salt-tolerant rootstock on plant growth, Na+concentration, the plasma membrane (PM) and vacuolar H+-ATPases and vacuolar H+-PPase systems and the expression of the encoding genes of these enzymes of grafted cucumber, cucumber were grafted onto itself and pumpkin rootstocks, respectively. Grafted plants were grown hydroponically and were exposed to0and90mM NaCl for5days. The results showed that NaCl induced significant decrease in growth parameters, increased Na+concentrations in roots and shoots of both types of plants, whereas smaller changes were observed in rootstock-grafted plants. NaCl distinctly stimulated the activities of plasma membrane H+-ATPase, vacuolar H+-ATPase and vacuolar H+-PPase, the expressions of plasma membrane H+-ATPase(PMA), vacuolar H+-ATPase (VHA) and vacuolar H+-PPase (VPP) encoding genes were also changed, but the rootstock-grafted plants maintained higher activity of these three enzymes in the roots. Results suggested that the higher salt tolerance of the rootstock-grafted cucumber seedlings could be partially attributed to the higher activities of PM H+-ATPase, vacuolar H+-ATPase and vacuolar H+-PPase under NaCl stress, and the regulation of the encoding PM H+-ATPase, vacuolar H+-ATPase genes may ocuured at post-transcriptional level, and the regulation of the encoding vacuolar H+-PPase may ocuured at transcriptional level.4. Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments:(1) CK (Control):nutrient solution alone;(2) Ca:10mM Ca(NO3)2+nutrient solution;(3) NaCl:90mM NaCl+nutrient solution; and (4) NaCl+Ca:90mM NaCl+10mM Ca(NO3)2+nutrient solution, to assess the effects of Ca2+on plant growth, ion concentration and the activity of plasma membrane H+-ATPases and the expression of the encoding genes. The results showed that, supplementary Ca(NO3)2ameliorated the negative effects of NaCl on plant dry mass, relative growth rate (RGR), as well as Ca+, K+, and Na+concentration, especially for pumpkin rootstock-grafted plants. The pumpkin rootstock-grafted plants had higher PM H+-ATPase activity as well as higher PMA and SOS1expression, higher ability to regulate the transport of Na+、K+, and Ca2+than the self-grafted plants under NaCl+Ca treatment through the highly sensitive Ca2+signal. Therefore, pumpkin rootstock grafting had higher ability to maintain ion balance, and showed higher salt tolerance.5. Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments:(1) CK (Control):nutrient solution alone;(2) K:10mM KNO3+nutrient solution;(3) NaCl:90mM NaCl+nutrient solution; and (4) NaCl+K:90mM NaCl+10mM KNO3+nutrient solution, to assess the effects of K+on plant growth, ion concentration. The results showed that, supplementary KNO3ameliorated the negative effects of NaCt on plant dry mass,as well as Ca2+, K+, and Na+concentration, increased K+/Na+and Ca2+/Na+, induced increased salt tolerance. Rootstock-grafted plants had higher K+/Na+selectivity and higher ability to maintain ion balance, showed higher salt tolerance than self-grafted plants.In conclusion, the present study suggests that, compared with the salt sensitive cucumber, pumpkin roots showed higher capacity to restrict the transport of Na+to steler and loading of Na+to xylem, the roots of the salt-tolerant rootstock also had higher ability to exclude Na+, which resulted in the lower Na+of the shoot and the higher salt tolerance of grafted plants. Na+efflux was regulated by the Na+/H+antiport of the plasma membrane, higher activity of proton pump and Na+/H+antiport on the plasma membrane could be attributed to the higher salt tolerance of grafted plants.