Physiological And Biochemical Mechanisms In Alleviating Damage Of Ca?NO₃?₂ To Cucumber By Graft With Pumpkin

Protected cultivation is characteristiced by closed environment and overfertilization in pursuit of high production, and all these characteristics make soil salinization in greenhouse increasingly prominent and salinization, in turn, severely limits the healthy and sustainable development of vegetable industry. The major cation and anion that lead to salinizaition are Ca2+and NO3-. Recently, rootstock-grafting becomes an efficient way to overcome soil salinization for its advantages of easy-doing, low cost and environmentally friendly. Previous studies on the use of grafting to overcome soil salinization are more concentrated in the growth and physiological phenomena apparent, while a deeper functional mechanism of grafting is little explored. Therefore, the present study combines physiological performance (photosynthetic performance, leaf structure, osmotic adjustment) and antioxidant signaling both to explore the physiological and biochemical mechanisms of rootstock-grafting to improve Ca(N0s)2 stress tolerance of cucumber plants. The main results were presented as follows:1. Cucumber varieties 'Jinyou 3'was used as test material and cultured in Ca(NO3)2 solutions of various concentrations (0,20 mM,40 mM,60 mM,70 mM,80 mM and 90 mM). And then the influence of various concentrations on growth and photosynthetic capacity of cucumber seedlings were determined. Results indicated that Ca(NO3)2 stress of various concentrations designed in present experiment all inhibited the growth and photosynthetic capacity of cucumber plants and the suppression performance was more and more prominent as Ca(NO3)2 concentration increasing; however, there was no obvious difference between 90 mM and 80 mM Ca(NO3)2 treatment. Therefore, the concentration of 80 mM was selected as the best treatment concentration of Ca(NO3)2 to use in following experiments.2. Using 'Jinyou 3'cucumber as scion, then the difference of growth, photosynthetic capacity and leaf structure between self-grafted (S-G) and pumpkin-grafted (P-G) cucumbers under 80 mM Ca(NO3)2 stress were examined. Results showed that Ca(NO3)2 stress significantly inhibited the growth of two grafted seedlings, but the inhibition degree in pumpkin-grafted seedlings was significantly much lower at the 9th day of treatment; the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO2 concentration (Ci) of self-grafted cucumber seedlings were significantly inhibited by Ca(NO3)2 stress with reductions of 18.5%,90.8%,73.6% and 39.4%, however the reductions of these parameters in pumpkin-grafted seedlings were 8.1%,35.2%,30.3% and 19.9%, respectively; when exposure to Ca(NO3)2 stress, the palisade tissue, spongy tissue and leaf thickness of pumpkin-grafted seedlings were significantly induced to increase, nevertheless, these parameters in self-grafted cucumber seedlings seemed to have no change, indicating that grafting with pumpkin enhances photosynthetic efficiency of cucumber seedlings stress via alleviating stomatal and non-stomatal limitation and improving leaf structure under Ca(NO3)2.3. Self-grafted cucumber seedlings and pumpkin-grafted seedlings were used to study the changes of leaf membrane permeability, osmolytes and ion contents under Ca(NO3)2 treatment. The results showed that the membrane permeability of self-grafted cucumber seedlings was significantly enhanced and the increase degree in pumpkin-grafted seedlings was obviously lower; in leaves of self-grafted cucumber:the contents of free proline and soluble protein seemed to have no obvious change, the soluble sugar content increased significantly, grafting with pumpkin obviously increased the proline content and further induced the soluble sugar content; the changes of ion contents in leaves of pumpkin-grafted seedlings were not obvious, however the ion contents (except Na+) in leaves of self-grafted cucumber seedlings were obviously increased at 36h of Ca(NO3)2 treatment, the results indicated that grating with pumpkin may increase the contents of proline and soluble sugar to protect leaf membrane from injury of osmotic stress induced by Ca(NO3)2 stress.4. Self-grafted cucumber seedlings and pumpkin-grafted seedlings were used to study the changes of contents of ABA in roots and leaves and H2O2 in leaves and activities of antioxidant enzymes under Ca(NO3)2 stress. The results showed that the contents of ABA in roots and leaves of pumpkin-grafted seedlings reached the maximum at 3h, H2O2 in leaves of pumpkin-grafted seedlings reached the maximum at 6h under Ca(NO3)2 stress; the activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POD) in leaves varied as the Ca(NO3)2 stress continued and were maximized at 12h from the initial moment and then declined. However, these phenomena were not observed in the leaves of the self-grafted cucumber seedlings. Then we speculated that the more concentration of ABA and H2O2 and the higher antioxidant enzymes' activities in pumpkin-grafted seedlings are related with higher tolerance to Ca(NO3)2 stress of pumpkin-grafted cucumber seedlings.To clear the relationship among ABA, H2O2 and antioxidant enzymes'activities in grafting with pumpkin improving tolerance of cucumber seedlings, the pumpkin'Qingzhen 1'and cucumber 'Jinyou 3' were used as rootstock and scion respectively to investigate effects of ABA inhibitor sodium tungstate (T) on H2O2 production, antioxidant enzymes' activities and corresponding gene expression and H2O2 inhibitors (DPI, Tiron and DMTU) on antioxidant enzymes' activities and corresponding gene expression. Furthermore, in the leaves of pumpkin-grafted cucumber seedlings, H2O2 generation, antioxidant enzymes activities and gene expressions of SOD, POD and cAPX were almost inhibited by ABA inhibitor under Ca(NO3)2 stress, then this inhibition was significantly relieved with the addition of exogenous ABA. Meanwhile, pretreatment with DPI, Tiron and DMTU before Ca(NO3)2 exposure almost completely alleviated the activities and gene expressions of these antioxidant enzymes. Our results indicate that grafting-induced ABA production mediated H2O2 generation, which, in turn, resulted in the up-regulation of the activities and gene expressions of antioxidant enzymes in the leaves exposed to Ca(NO3)2 stress in ABA/ H2O2 signaling pathway.