Functional Analysis Of Pumpkin RbohD Under Salt Stress And Its Mechanism Of Synergistic Regulation Of Salt Tolerance With CIPK1

The increasingly serious soil salinization has hindered the normal production of crops.Grafting with resistant rootstocks is an effective and convenient method to deal with soil salinization.The salt tolerance of resistant rootstock roots can protect scions from salt stress.Signal perception and transduction under salt stress is the basis of a series of salt tolerance responses in plants.H₂O₂ signal and Ca²⁺signal play an important role in the early stage of salt stress,and the two signals are cross-linked.The Rboh family is the main source of reactive oxygen species in plants and plays an important role in plant growth and development and response to stress.CIPK is an important effector protein in Ca²⁺signal transduction,and its function under salt stress has been widely reported.Our previous study showed that pumpkin had a root H₂O₂ burst when treated with salt for 3 h,and CmoRbohD2 could improve the salt tolerance of pumpkin.Alternatively,we identified CmoCIPK1 protein interacting with both pumpkin CmoRbohD1 and CmoRbohD2.However,the function of CmoRbohD1 under salt stress and the relationship of CmoRbohD1 between CmoRbohD2 are unclear,Furthermore,the mechanism of CmoCIPK1-mediated Ca²⁺signal and CmoRbohD1/D2-mediated H₂O₂signal synergistically regulating salt tolerance of pumpkin needs to be further explored.Therefore,this study mainly focused on the functional verification of two RbohDs and CIPK1 interacting with them in pumpkin under salt stress and the regulation of RbohD1and RbohD2.The results obtained are as follows:1.Our previous study found that at 3 h of salt treatment,pumpkin had the phenomenon of root H₂O₂ burst.The pumpkin seedlings were treated with DPI and EGTA,and we found that the root H₂O₂ burst was significantly inhibited in the early stage of salt stress.Further analysis of the expression levels of CmoRbohD1、CmoRbohD2 and CmoCIPK1 genes in the early stage of salt stress showed that the three genes responded to early salt stress,indicating that the early signal transduction of pumpkin under salt stress was mainly mediated by H₂O₂ signal and Ca²⁺signal,and RbohD and CIPK1 protein were involved in the early response process of salt stress.2.We generated CmoRbohD1 and CmoRbohD2 root overexpression and knockout plants using root transformation method.Compared with EV,the salt tolerance of CmoRbohD1 and CmoRbohD2 root overexpression plants increased,the relative electronic conductivity（REC）and malondialdehyde（MDA）of leaves and roots were lower than EV,Na⁺accumulation was significantly lower than EV,while the K⁺content was significantly higher than EV.The salt sensitivity of CmoRbohD1 and CmoRbohD2root single mutants increased,and their damage under salt stress was more significant than that of EV,in addition,the salt tolerance of CmoRbohD1 and CmoRbohD2 root double mutant was lower than CmoRbohD1/D2 single mutant,indicating that the gene function of CmoRbohD1 and CmoRbohD2 was probably redundant.The results showed that CmoRbohD1 and CmoRbohD2 could affect the production of H₂O₂ in pumpkin roots at the early stage of salt stress,indicating that CmoRbohD1 and CmoRbohD2 were involved in the regulation of salt tolerance of pumpkin by mediating H₂O₂ signal at the early stage of salt stress.3.We generated CmoCIPK1 root overexpression and knockout plants using root transformation method,and conducted a salt stress treatment.The results showed that CmoCIPK1 can positively regulate the salt tolerance of pumpkin,the salt tolerance of CmoCIPK1 root overexpression plants increased,while the CmoCIPK1 root knockout plants had higher salt sensitivity.The determination of H₂O₂ content at 3 h of salt treatment showed that CmoCIPK1 could also affect the production of H₂O₂ in pumpkin roots at the early stage of salt stress,which may be achieved by regulating the activity of CmoRbohD1 and CmoRbohD2.4.In order to further prove the mechanism of CIPK1 and RbohD regulating the production of reactive oxygen species,we further screened CBLs interacting with CIPK1by Co-IP assay,and selected CBL8 with higher relative expression in the early stage of salt stress,we transiently transformed CBL8-CIPK1 complex and RbohD gene in tabacco leaves.The results showed that both Ca²⁺and CmoCBL8-CmoCIPK1 complex could enhance the activity of CmoRbohD1 and CmoRbohD2 and affect the production of ROS in tabacco,and Ca²⁺could also enhance the regulation of CmoCBL8-CmoCIPK1complex on CmoRbohD1 and CmoRbohD2.