Mechanism Of Grafting Cucumber Onto Momordica Rootstock To Alleviate Photoinhibition And Oxidative Stress Under Heat Stress

The increased global temperature and frequency of extreme high-temperature cause the metabolism disorders,abnormal development,and reduced economic yield of crops,which has become a major obstacle to the sustainable development of the agricultural industry.Cucumber(Cucumis sativus L.)is a vegetable crop with high economic benefits whose roots are thin and heat-sensitive.In the afternoon of summer and autumn in China,the temperature inside the facility far exceeds the optimum temperature for cucumber growth,and even causes thermal damage to the plant.Studies have shown that grafting onto stress-resistant rootstocks significantly increases plant tolerance to abiotic stresses.However,the current research on the mechanism of how heat-resistant rootstock alleviates plant injury induced by high temperature is far from sufficient.Therefore,in this study,the heat-sensitive cucumber variety ’Jinchun NO.2’ was used as the scion,and the heat-resistant Momordica(Momordica charantia L.)variety ’Changlv’ was used as the rootstock.The substrate cultivation and the ’cleft grafting’ methods were used for obtaining materials.The effects of Momordica rootstock on photosynthesis,the metabolism of reactive oxygen species and thylakoid membrane proteome of cucumber leaves under heat stress were studied to clarify the Momordica rootstock induced alleviation mechanism of the photoinhibition and oxidative stress caused by high temperature.1.High-temperature caused the growth inhibition of the cucumber seedlings grafted onto Momordica rootstocks and cucumbers,and the inhibition of the self-rooted seedlings was significantly more serious than that of the Momordica-grafted seedlings.Under heat stress,the root activity of grafted seedlings decreased,the content of osmotic substances increased,while the roots of Momordica maintained high vigor,the relative water content of their leaves was higher,and more proline and total soluble sugar were accumulated.In addition,compared with self-grafted plants at preference temperature,the photosynthetic pigment content,net photosynthetic rate(Pn).PSⅡ maximum photochemical efficiency(Fv/Fm)and non-photochemical quenching(NPQ)in self-rooted grafted seedlings were significant decreased after heat stress,and the carotenoid(Car)content,NPQ and its main component,the quenching depending on the proton gradient across the thylakoid membrane(qE)were increased in the Momordica grafted cucumbers,in which the Pn and Fv/Fm also maintained high levels.Results indicate that the cucumbers grafted onto Momordica rootstocks have higher water holding capacity,photosynthetic pigment content,photochemical efficiency,and heat dissipation capacity under heat stress,thus having higher heat resistance.2.Heat stress induced membrane lipid peroxidation,electrolyte extravasation and the abundantly production of ROS were observed in the leaves of grafted seedlings.Momordica grafting can increase the activity of antioxidant enzymes,promote the expression of enzyme-encoding genes,increase the content and proportion of reduced states of AsA and GSH,and reduce the production rate of O2·-,the accumulation of H2O2,malondialdehyde(MDA)content and relative electrolyte permeability(REL)in leaves of cucumbers under heat stress.Results indicate that Momordica-grafting can increase the reducing potential of cell by regulating the ROS scavenging system including AsA-GSH cycle,thereby maintaining the intracellular redox homeostasis and reducing the oxidative damage of membrane of cucumber leaves under heat stress.3.Twenty-five significant differentially accumulated thylakoid membrane proteins were identified by Blue-Native-SDS(BN-SDS)two-dimensional gel electrophoresis(2-DE)coupled tandem time-of-flight mass spectrometry technique(MALDI-TOF/TOF-MS).The accumulation of 13 proteins was down-regulated in self-grafted plants,and Momordica grafting promoted/stabilized the accumulation of 12 proteins and significantly upregulated the abundances of PSⅡ subunit PsbS and micro-light-harvesting antenna proteins(CP24,CP26 and CP29.1).Transcriptional level analysis and Western blotting further indicated that grafting onto Momordica rootstock can protect the photosynthetic apparatus from redundant energy damage by effectively regulating the expression levels of PSⅡ light-harvesting proteins,heat-dissipation related proteins,and their genes,thereby stabilizing the thylakoid membrane.The stability of the protein library relieves light inhibition under heat stress and improves the heat resistance of the plants.