| In recent years, with the rapid development of protected horticulture in China, soil secondary salinization has been more and more severe. It has become the main obstacle of limiting the development of vegetable production under protected horticulture. Cucumber (Cucumis sativus L.) is the main vegetables under the protected cultivation conditions, and is sensitive to salt stress. It has been reported that grafting with salt-tolerant rootstock can improve cucumber tolerance to salt stress, but these researches are mainly about the mechanism of grafting on regulating the Na+and Cl- uptake and transport, little study has been down on the research of grafting on regulating the salt-induced oxidative stress. In our present study, the mechanism of grafting with salt-tolerant rootstock on regulating the H2O2-scavenging system under salt stress were evaluated, by investigating the response of H2O2-scavenging system to salt stress at grafted cucumber organic, chloroplast, and transcript levels. The salt-tolerant rootstock was clarified to play a primary important effect in determining the salt tolerance of cucumber seedlings under salt stress, according to the experiments of grafting with different genotype cultivars of cucumber scions and pumpkin rootstocks, and reciprocal grafting between cucumber and pumpkin. We explored the possible signal pathway of ABA in salt-tolerant rootstock roots on regulating the salt tolerance of cucumber, by investigating the ABA content and some signal molecules involved in ABA signal pathway (H2O2, NO and MAPK cascades) in grafted plant roots. The main contents and results in the present study are as follows:1. A salt-sensitive cultivar of cucumber (cv.'Jinchun No.2') was used. Plants pre-sprayed with H2O2 inhibitor (DPI,15μM) and scavenger (DMTU.5 mM) were exposed to 0 and 75 mM NaC1 stress. The changes in H2O2 content in cucumber seedlings and what this has to do with the salt-induced growth inhibition were investigated. The results showed that pre-spraying H2O2 inhibitor and scavenger decreased the H2O2 and MDA contents, while increased the root and shoot dry weights of cucumber seedling under NaCl stress. Correlation analysis showed that H2O2 content was negatively correlated with root and shoot dry weights. This result implied that the salt-induced H2O2 accumulation was an important reason for plant growth inhibition caused by salt stress.2. Cucumber plants (cv.'Jinchun No.2') were self-grafted or grafted onto salt-tolerant rootstock'Chaojiquanwang'(Cucurbita moschata Duch). The changes in plant growth and antioxidant defense system of non-grafted, self-grafted, and rootstock-grafted cucumber seedlings under 0 and 75 mM NaCl stress were investigated. The results showed that cucumber plants grafted onto salt-tolerant rootstock had higher activities of CAT, APX, GR, and MDAR, GSH/GSSG ratio, and the ferric reducing ability/antioxidant power, thereby decreasing the H2O2 and MDA contents in cucumber tissue. As a result, the salt-induced growth inhibition was alleviated.3. Chloroplast ultrastructure and the AsA-GSH cycle system in chloroplasts of self-grafted and rootstock-grafted cucumber leaves under 0,50, and 100 mM NaC1 stress were investigated. The results showed that the activities of APX, GR, and DHAR involved in AsA-GSH cycle and AsA/DHA ratio in the chloroplasts of rootstock-grafted plants under 50 and 100 mM NaCl, as well as the MDAR activity, GSH content, and GSH/GSSG ratio under 100 mM NaCl were all significant higher than those in self-grafted plants. This resulted in the lower H2O2 content in the chloroplast of rootstock-grafted plants. With increasing levels of NaC1 concentration, the chloroplast structure of self-grafted plants was visibly damaged. Particularly under 100 mM NaCl, the stacks of grana and intergranal lamellae were thin and obscure, even fractured, whereas the damage in the chloroplast of rootstock-grafted plants were less severe. The results suggest that rootstock grafting enhances the H2O2-scavenging capacity of the AsA-GSH cycle in the chloroplasts under salt stress, thereby protecting the chloroplast structure and improving the photosynthetic performance and growth of cucumber seedlings.4. Changes in the time course of H2O2 content and main antioxidant enzyme (CAT, APX, GR and MDAR) activities in the leaves of self-grafted and rootstock-grafted cucumber plants under 0 and 75 mM NaCl stress, as well as the mRNA levels of antioxidant enzyme genes in grafted cucumber plants under NaCl stress for 120 h were studied. The results showed that the H2O2 content in self-grafted leaves increased gradually during NaC1 stress from 24 h to 120 h, and showing a peak at 72 h, whereas the H2O2 content in rootstock-grafted increased more slowly and kept lower all the time. This could be associated with its higher antioxidant enzyme activities during NaCl stress from 48 h to 120 h. The changes in the mRNA level of CAT and cAPX in self-grafted plants leaves and roots, respectively, and the mRNA level of cAPX in rootstock-grafted plants leaves were consistent with the changes in their enzyme activity levels under NaCl stress. This result indicated that the activities of these enzymes were regulated at their mRNA levels.5. Two cucumber cultivars (cv.'Jinchun No.2', a relative salt-sensitive cultivar, and 'Jinyu No.1', a relative salt-tolerant cultivar) were grafted onto two rootstocks ('Figleaf Gourd', a relative salt-sensitive cultivar, and'Chaojiquanwang', a relative salt-tolerant cultivar), respectively. The effects of scion and rootstock on plant growth and the antioxidant defense systems of cucumber were assessed. The results showed that the salt injury index of plants grafted onto Chaojiquanwang was lower and the growth reduction was less severe under 100 mM NaCl stress. Correlation analysis showed that this could be partially attributed to its lower leaf Na+content and root H2O2 content, as well as the higher activities of SOD, POD, and CAT in the roots. This result implied that the salt-tolerant rootstock played a primary important effect in determining the salt tolerance of grafted cucumber seedlings under salt stress compared with the scion genotypes.6. A salt-sensitive cucumber cultivar (cv.'Jinchun No.2') and a salt-tolerant pumpkin cultivar ('Chaojiquanwang') were reciprocal grafted. Four types of grafted plants (scion/rootstock) were obtained, including cucumber/cucumber, pumpkin/pumpkin, cucumber/pumpkin, and pumpkin/cucumber. The effects of 0 and 75 mM NaC1 stress on H2O2 and MDA contents, antioxidant enzyme activities, ABA content and some signal molecules involved in ABA signal pathway (H2O2, NO and MAPK cascades) in grafted plant roots were investigated. The results showed that the ABA contents increased and the expression of signal regulatory genes such as MAPK1 and MAPK6 up-regulated significantly in the grafted plants roots under NaC1 stress when pumpkin was used as rootstock. The results implied that salinity induced the production of ABA and the response of MAPK cascades mediated by ABA in salt-tolerant rootstock roots. This resulted in the higher H2O2-scavenging enzyme activities in salt-tolerant rootstock roots for controlling the H2O2 and MDA contents at lower levels.In conclusion, the present study suggests that the salt-induced H2O2 production result in the plant growth inhibition caused by salt stress. Grafting with salt-tolerant rootstock can improve the salt tolerance of cucumber seedlings by regulating the H2O2-scavenging system in cucumber organic and chloroplast. The salt-tolerant rootstock plays a primary important effect in determining the salt tolerance of cucumber seedlings under salt stress compared with the scion genotypes. This could be attributed to the production of ABA and the regulation of ABA-mediated MAPK cascades in salt-tolerant rootstock roots under salt stress.
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