Physiological Mechanism In Alleviating The Damage Induced By Ca?No₃?₂ Stress In Pumpkin Rootstock-grafted Cucumer Seedlings

The increasing secondary stalinization of greenhouse cultivation resulted from the lack of rain and over fertilization,which severely limits the healthy and sustainable development of protected vegetable production.Studies have shown that soil stalinization characterized by accumulation of high levels of NO₃^-and Ca²⁺.Cucumber?Cucumis sativus L.?is one of the major horticultural crops in greenhouse cultivation.Cucumber maintains the relatively low absorption becasuse of the weak root system,and requires sufficient water and loose fertile soil,thus,cucumber is highly susceptible to soil salinity.Rootstock grafting is an efficient and environmentally friendly method to overcome salt stress in vegetable crops.However,a deeper functional mechanism of rootstock-grafting is limited.In this study,the salt-tolerant pumpkin 'Qingzhen No.1'?Cucurbita maxima×Cucurbita.moschata?was used as the rootstock and salt-sensitive cucumber 'Jinyou No.3' as the scion.The physiological metabolism?Proline and PAs?and signal transduction?NO and H₂O₂?were determinated to explore the physiological mechanisms between self-grafted and rootstock-grafted cucumber seedlings under 80 mM Ca?NO₃?₂ stress.The main results were as follows:1.The proline metabolism of self-grafted and grafted cucumber seedlings was investigated under 80 mM Ca?NO₃?₂ stress.The results showed that rootstock-grafting could obviously improve the growth of cucumber seedlings in comparison to self-grafted plants.Ca?NO₃?₂ stress could induce proline accumulation in the leaves of both types of grafted plants,while the rootstock-grafted plants maintained a higher proline content and reached its maximum at the 3rd day after salt treatment.In addition,under salt stress,compared to self-grafted seedlings,the pyrroline-5-carboxylate synthetase activity was significantly increased in leaves of rootstock-grafted plants and reached their maximum at the 7th day after salt teratment.Moreover,the ornithine aminotransferase activity was notably enhanced in the scion leaves of rootstock-grafted plants at the early stage and achieved its maximum at the 1st day of salt stress.However,the proline dehydrogenase activity was gradually decreased with time in rootstock-grafted plants under salt stress,and the values in rootstock-grafted plants were remarkably lower than that in self-grafted ones at the 5th and 7th day under salt stress.Furthermore,the gene expression trends of proline metabolism enzymes were broadly consistent with the variation of the enzymes activities.These results suggest that rootstock-grafting could regulate the proline metabolism,thereby enhancing the salt tolerance of cucumber plants.2.The PAs metabolism of self-grafted and grafted cucumber seedlings were investigated under 80 mM Ca?NO₃?₂ stress.Under Ca?NO₃?₂ stress,the contents of polyamine?PAs?was significantly increased,that is,free putrescine?Put?increased at the early stage of stress,and free spermidine?Spd?and spermine?Spm?increased with a time-dependent manner.Therein,the free PAs were maintained at a higher level in leaves of rootstock-grafted seedlings in comparision to self-grafted plant.In addition,the expression levels of ornithine decarboxylase?ODC?,arginine decarboxylase?ADC?,s-adenosine methionine decarboxylase?SAMDC?,spermidine synthase?SPDS?and spermine synthase?SPMS?were notably higher in leaves of rootstock-grafted cucumber seedlings compared with those in self-grafted plants under saline condition.Furthermore,compared with self-grafted plants,rootstock-grafted seedlings kept the relatively lower levels of polyamine oxidase?PAO?and diamine oxidase?DAO?activities and expressions.These results indicate that the rootstock-grafted seedlings could maintain a high level of PAs metabolism when exposed to salt stress.3.We investigated the role of NO and its interplay with H₂O₂ in self-grafted and salt-tolerant pumpkin rootstock-grafted cucumber seedlings in response to 80 mM Ca?NO₃?₂ stress.Endogenous NO and H₂O₂ production in self-grafted seedlings increased with a time-dependent manner and reached its maximum data after 24h of salt treatment,whereas in rootstock-grafted plants a transient increase in NO content was observed by salt stress for 2h accompanied with accumulation of H₂O₂.L-NAME,an inhibitor of nitric oxide synthase?NOS?,tungstate,an inhibitor of nitrate reductase?NR?,as well as cPTIO,a scavenger of NO,were found to significantly inhibit NO accumulation induced by salt stress in rootstock-grafted seedlings.However,there was no obvious influence on H₂O₂ production in these stress conditions.On the other hand,Ca?NO₃?₂ stress-induced NO accumulation was blocked by pre-treatment with a scavenger of H₂O₂?DMTU?and an inhibitor of H₂O₂ production by NADPH oxidase?DPI?.Meanwhile,the value of Fv/Fm,the activities and transcript levels of antioxidant enzymes were notably decreased in grafted seedlings after pre-treatment with these inhibitors under salt stress,while the rootstock-grafted plants maintained higher levels than that of self-grafted seedlings.Our results suggest that rootstock-grafting could alleviate the oxidative damage induced by Ca?NO₃?₂ stress of cucumber seedlings,which might be related to NO involved in H₂O₂ dependent-antioxidative metabolism.