Mechanism Of Root-Sourced H₂O₂ Signal Mediated Elevated Salt Tolerance In Cucumber Grafted Onto Pumpkin Rootstock

Grafting is utilized as a reliable technique to improve the salt tolerance of plants.Previous studies of our research group have studied the mechanisms involved in this process such as ions transport,antioxidant system and photosynthesis system by grafting cucumber onto pumpkin.During recent years,the reactive oxygen species?ROS?has been widely considered as a key signaling substance.Many studies have proved that H₂O₂plays an important role in response to biotic and abiotic stresses.The mechanism defining how H₂O₂ enhance the salt tolerance of plants has been reported in different species.However,the information regarding the relationship between H₂O₂ and salt tolerance in grafted plants is limited.In this study,we developed two grafting combinations by using plant material having contrasting salt tolerance capacity and examined the functions of H₂O₂ in response to salinity.The main results are as follows:1.Low NaCl concentration treatment did not cause significant damage to the plasma membrane but can be used as a reliable trait for evaluating the salt tolerance of different grafted transplants.For screening a suitable concentration of NaCl used in the H₂O₂signal studying,pumpkin-grafted cucumber?C/P?and self-grafted cucumber?C/C?were treated with various concentrations of NaCl?35 mM,75 mM,100 mM,150 mM,200mM?.The growth parameters?biomass,leaf area,number of leaves?,Fv/Fm,H₂O₂ and relative conductivity?REC?were determined after five days.Finally,a moderate concentration of 75 mM was selected for the further studies.Under this concentration of NaCl,the C/P has a higher biomass,leaf area,leaf number and Fv/Fm compared with C/C;however,both grafting combinations showed no difference in the root REC and H₂O₂compared with the control.This indicated that 75 mM was a suitable NaCl concentration to avoid plasma membrane damage.2.The root-induced H₂O₂ signal was produced at an early stage of salt stress and transmitted rapidly to the shoot in pumpkin-grafted plants.The Na⁺,K⁺and H₂O₂ levels in roots and leaves were determined for C/P and C/C plants at different intervals after NaCl treatment?1 h,3 h,12 h,24 h,48 h and 120 h?.The Fv/Fm and two Na⁺transport-related genes?PMA and SOS1?expression levels were also measured.Results showed that the roots H₂O₂ levels of the two grafting combinations were at peak after 3 h of NaCl treatment.However,the rapid increase of H₂O₂ was only observed in leaves of C/P but not in the leaves of C/C.Besides that,the accumulation Na⁺was faster than the H₂O₂ in the leaves of C/P.This indicated that enhanced H₂O₂ level in leaves of C/P was not the result of Na⁺accumulation but a signal substance.The SOS1 was up-regulated within 3 h after NaCl treatment in both C/C and C/P plants.By comparison,the up-regulation of PMA was only detected in C/P.Thus,it can be concluded that Na⁺transport was mediated via H₂O₂ in C/P plants based on the function of PMA but not the SOS1.The process of H₂O₂ formation in the root tips were monitored by fluorescent dye with a NaCl transient treatment.Result showed that H₂O₂ signal are produced after two minutes of NaCl treatment and shifted to the upper part of the root rapidly.3.The root-induced H₂O₂ signal mediated the Na⁺/H⁺anti-transport system,which is produced by the NADPH oxidase located in plasma membrane.To study the function of H₂O₂ involved in the process of Na⁺transport from root to shoot,an experiment was conducted with a NADPH oxidase inhibitor DPI.The Na⁺and K⁺contents were detected with or without the DPI pretreatment after 24 h of NaCl.Results showed that DPI pretreatment can significantly increase the amount of Na⁺accumulated in the leaves of C/P plants.After that,the ions fluxes of the two grafting combinations were monitored with the Non-invasive Micro-test Technique?NMT?in the roots?Na⁺,H⁺,K⁺?or stems?Na⁺,K⁺?.Results showed that the increased Na⁺level in the leaves of C/P caused by an impaired Na⁺/H⁺anti-transport system in roots that is maintained by H₂O₂ signal.The activity of H^+-ATPase and the expression level of PMA was also decreased under the DPI pretreatment.4.The root-induced H₂O₂ signal participated in the activation process of the antioxidant system in shoot under salt stress.To study the function of H₂O₂ in antioxidant system under salt stress,activities of four key enzymes?APX,CAT,POD and SOD?were measured with different treatments.Results showed that C/P has a more rapid increment in four enzymes.DPI pretreatment with roots could significantly inhibit the activities of the four enzymes in leaves,particularly in C/P which imply that the root-induced H₂O₂was not only control the antioxidant system in root but also has effects on the shoot.In order to reveal the mechanism of root-shoot communications,the ABA inhibitor fluridone were applied with DPI,and the ABA concentrations in xylem sap were detected in two grafting combinations.Results showed that the ABA was significantly decreased after pretreatment with fluridone or DPI in both grafting combinations.This indicates that the ABA might be a signal involved in long distance signaling and activated the stomatal closure and antioxidant enzymes.5.There were more Rboh members in pumpkin compared with cucumber,which could be a possiblereason that pumpkin is more tolerant than cucumber under salt stress conditions.The phylogenetic tree of respiratory burst oxidase homologs in pumpkin and cucumber were constructed by the members of Rboh from Arabidopsis thaliana,Cucurbita moschata,and Cucumis sativus.Compared with Cucumis sativus,there were more Rboh members found in Cucurbita moschata.Besides that,genes expression pattern with the 10 Rbohs in Cucurbita moschata showed the different members of Rboh response to different treatment?ABA,NaCl,Sorbitol and H₂O₂?.This information can help to explain why the pumpkin has higher tolerance under various stress conditions compared with cucumber.Subcellular localization of 3 Rboh genes from pumpkin or cucumber in tobacco epidemic cells proved that the Rbohs were located in plasma membrane.In conclusion,the root H₂O₂ signal enhances the salt tolerance of pumpkin-grafted cucumber by mediating the root Na⁺/H⁺anti transport system to maintain Na⁺efflux in roots,inducing early stomatal closure and triggering the activities of antioxidant enzymes in the shoots.