Establishment Of Root Transformation System In Cucurbit Crops And Functional Characterization Of Pumpkin CmoPIP1-4 Under Salinity And Drought Stress

Drought and salinity are the two most prevalent abiotic factors that hinder agricultural productivity and pose a serious threat to food security.Pumpkin（Cucurbita moschata Duch.）is a common rootstock for other Cucurbitaceae crops due to its strong resistance against abiotic stresses.It has a crucial role in enhancing the scions resistance to abiotic stress.However,the mechanism remains unknown.Several studies have demonstrated that plasma membrane intrinsic proteins（PIPs）are important to regulate salt and drought stress responses.CmoPIP1-4 knockout enhances pumpkin sensitivity to salt stress in previous experiments,and it is speculated that it may be involved in the regulation of salt tolerance in pumpkin.However,its specific function has not been determined,and it is unclear if it functions under drought stress.Although stable transformation systems in watermelon（Citrullus lanatus）and cucumber（Cucumis sativus）have been established,pumpkin still lacks a stable transformation system.In this study,first an Agrobacterium rhizogenes（A.rhizogenes）-mediated hairy root transient transformation system for cucurbit crops was developed,and then it was further utilized to identify the biological function of CmoPIP1-4 in pumpkin under salinity and drought stress.The findings of this research will not only provide a basis for the function of this gene in pumpkin rootstock,but also provide opportunities for gene mining and stress resistance research.The main findings of the research are as follows:1.A.rhizogenes-mediated transformation system of the main cucurbit crops was established.Pumpkin,cucumber,melon,bottle gourd,and luffa gourd may be transformed transiently using A.rhizogenes,and gene editing or overexpression in the roots could be performed.The study clarified the impact of A.rhizogenes strains,Agrobacterium solution OD₆₀₀ concentration,infected seedling age,co-cultivation time,hypocotyl length,cotyledon length,MS（AS）medium sugar concentration,and hypocotyl cutting method on transformation efficiency of hairy roots.An efficient root transformation system suitable for a variety of cucurbit crops was optimized and established,where transformation efficiencies of pumpkin,cucumber,melon,bottle gourd,and luffa gourd were achieved up to 84.31%,73.33%,68.9%,73.9%,and 71.4%,respectively.2.The CmoPIP1-4 gene was knocked out using the established pumpkin hairy root transformation system,and its function in response to salt stress in pumpkin was validated.Twenty-one CmoPIP genes were identified in the whole pumpkin genome by bioinformatics analysis and further divided into two subgroups,CmoPIP1s and CmoPIP2s.Overexpression of CmoPIP1-4 in yeast cells enhanced its tolerance against salt stress.However,knockout of the CmoPIP1-4 gene in pumpkin roots significantly increased its salt sensitivity,thereby reducing the activities of enzymatic antioxidants（SOD,POD,and CAT）,along with their corresponding gene expression.The knockout of CmoPIP1-4 knockout increased the photosynthetic damage by decreasing chlorophyll content and chlorophyll fluorescence（F_v/F_m）,hence increasing the Na⁺/K⁺ratio and ROS damage in knockout plants.Moreover,the expression levels of key salt tolerance genes（Cmo SOS1,Cmo HKT1;1,and Cmo NHX4）were also downregulated in CmoPIP1-4 knockout plants.Taken together,the overall results demonstrate that CmoPIP1-4 is essential for maintaining photosynthetic integrity and sodium-potassium homeostasis under salt stress by regulating the expression of salt tolerance genes in pumpkin.3.The CmoPIP1-4 is a plasma membrane localized protein that has been identified in pumpkin plants as a drought responsive gene.Overexpression of CmoPIP1-4 enhances drought resistance in yeast.Under drought stress,knockout of CmoPIP1-4 increased the drought sensitivity by reducing H₂S and abscisic acid（ABA）levels and exhibiting excessive water loss by increasing the transpiration rates and stomatal conductance,as well as increasing the stomatal density and aperture in pumpkin plants.In contrast,wild-type empty-vector plants displayed resistance against drought by increasing the H₂S and ABA levels,lowering transpiration rates and stomatal conductance,which reduced the water loss while maintaining the relative water contents.The knockout of CmoPIP1-4 induced physiological damage to the photosynthetic apparatus and thus influenced the sugar metabolism by reducing the sugar content under drought stress.Furthermore,the expression profiling of drought-responsive genes（Cmo HSP70s,Cmo NCED3 and 4）,and key genes involved in sugar metabolism（Cmo Glu and Cmo Suc）were significantly reduced in knockout plants under drought stress,indicating that knockout of CmoPIP1-4 increased the drought sensitivity of pumpkin plants.Moreover,drought stress also affects root functionality by altering cell water permeability and thus affects root growth by altering the root architecture in CmoPIP1-4CRISPR plants under drought stress.However,in empty vector plants,the PIPs might stabilize the root hydraulics and reduce the damage to the root architectural system.Additionally,we also found that the expression level of Cmo DCD was significantly increased while interacting with CmoPIP1-4 to activate the drought signaling response,validating the role of H₂S in the drought tolerance mechanism of pumpkin plants.Altogether,the results suggest that CmoPIP1-4 regulates the H₂S-mediated signaling pathway to modulate stomatal density and aperture in pumpkin plants,thus enhancing drought stress tolerance and adaptability in pumpkin plants.In conclusion,in this study we have developed a hairy root transient transformation system suitable for a variety of cucurbit crops,and further,the established transformation system was utilized to elucidate the functions of the pumpkin PIP1-4 gene（CmoPIP1-4）under salt and drought stress conditions.Under salt stress,CmoPIP1-4 improved pumpkin resistance to salt stress by maintaining photosynthesis performance,activating antioxidant enzymes,and increasing key salt tolerance gene expression.Moreover,under drought stress,CmoPIP1-4 activates the H₂S-mediated signaling pathway to regulate photosynthetic machinery,thereby enhancing drought stress tolerance and adaptability.