Characterization Of The Involvement Of Cytokinin Receptor Genes SlHK2 And SlHK3 In Plant Responses To Drought,Heat And Oxidative Stresses In Tomato

Drought,heat,and other natural environmental stress factors can alter normal physiological and biochemical processes of plants,induce endogenous oxidative stress,and severely affect plant growth and development.Simultaneous occurrence of drought and heat stresses,especially often in summer,usually results in abnormal growth of many plants.Tomato is one of the most widely produced and consumed vegetables in the world.Drought,heat and other environmental stresses can affect the growth,flowering and fruit development of tomato plants,and further lead to the reduction of fruit production and quality.Therefore,it is of significant importance to investigate molecular mechanisms of the resistance to environmental stress in tomato.Cytokinins are a type of multifaceted plant hormone that plays critical roles in plants.Cytokinins participate in various plant growth and development processes,such as cell division and proliferation,organogenesis,leaf senescence,through their his-kinase receptors and signaling pathway.Previous studies show that cytokinin receptor genes likely play negative roles in plant responses to abiotic stresses like drought.There are three cytokinin receptor genes in tomato,but their detailed roles in abiotic stress responses of tomato are unclear.In this study,we used the cultivar‘Micro-Tom’as the wild type（WT）tomato plants,and specifically downregulated the expression of cytokinin receptor genes SlHK2 and SlHK3in the transgenic lines through RNAi technology.We treated the RNAi and WT tomato plants with drought,heat,drought and heat,and methyl viologen（a chemical generating oxidative stress）,and demonstrated the negative regulatory roles of SlHK2 and SlHK3 in the responses to these stresses in tomato.Detailed results are as follows:1.We constructed the single RNAi（SlHK2 RNAi,SlHK3 RNAi）and double RNAi（SlHK2-3 RNAi）vectors,and obtained SlHK2 RNAi,SlHK3 RNAi and SlHK2-3 RNAi lines through Agrobacterium tumefaciens mediated transformation method.Compared with the WT plants,the SlHK2 RNAi and SlHK3 RNAi plants do not have significant phenotypic changes,while the shoots of the SlHK2-3 RNAi plants were largely dwarf and the roots were signicantly enhanced in length and number.2.First,we treated the SlHK2 RNAi and WT plants with drought,heat,and combined（drought+heat）stresses.We found that the decrease of photosynthesis rate,stomatal conductance,and CO₂assimilation decreased obviously more slowly in the SlHK2 RNAi plants than in the WT plants.Further,the maximum photochemical yield of photosystem II（F_v/F_m）,electron transport rate（ETR）,photosystem II（PSII）efficiency(Φ_PSII),and other photosynthesis related parameters in the SlHK2 RNAi plants were significantly higher than those in the WT plants under all the stressed conditions.In addition,the SlHK2 RNAi plants exhibited significantly lower malondialdehyde（MDA）and hydrogen peroxide （H₂O₂）contents,while significantly higher activity of the enzymes scavenging reactive oxygen species（ROS）,which include superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,than the WT plants.These results indicate that the SlHK2 RNAi plants have stronger photoprotective and antioxidant capabilities than the WT plants to avoid excessive damages under drought and heat conditions.3.Next,we treated the SlHK2 RNAi,SlHK3 RNAi,and SlHK2-3 RNAi and WT plants with drought stress.The results demonstrated that the survival rate of the SlHK2-3 RNAi plants was highest,and that of the SlHK2 and SlHK3 RNAi plants was sequentially lower,while that of the WT plants was lowest.Further,the SlHK2-3 RNAi plants could maintain higher photosynthesis and CO₂assimilation,while lower ROS accumulation than the other plants under drought conditions.Besides,the SlHK2-3 RNAi plants showed significantly higher expression levels of abscisic acid（ABA）biosynthesis genes SlNCEDs,and significantly higher ABA sensitivity than the WT plants.These results indicate that the SlHK2-3 RNAi plants have stronger tolerance to drought stress than the WT plants,and this stronger tolerance is regulated by the biosynthesis and response to ABA,and the ROS scavenging activity in tomato plants.4.We further treated the SlHK2-3 RNAi and WT plants with 100μM MV to induce oxidative stress.The results showed that the photosynthetic system of the WT plants was severely damaged by the MV induced oxidative stress.In comparison,the SlHK2-3 RNAi plants had better photoprotective capability,less ROS content,and higher levels of endogenous antioxidants,ROS scavenging enzymetic activity,as well as expression of related genes,than the WT plants.Moreover,under oxidative stress,the expression levels of proline and sucrose synthesis genes were significantly higher in the SlHK2-3 RNAi than the WT plants.These results demonstrate that the SlHK2-3 RNAi plants have higher tolerance to oxidative stress than the WT plants.In conclusion,our study indicates a negative regulatory role of the cytokinin receptor genes SlHK2 and SlHK3 in the responses of tomato to drought,heat and oxidative stresses.The negative regulatory effects of SlHK2 are stronger than that of SlHK3,and the effects of these two genes are additive.The reduced expression of SlHK2 and SlHK3 enhances ABA biosynthesis and sensitivity,as well as oxidative stress tolerance in plants,and further improves plant tolerance to abiotic stresses.Our study provides basis for future analysis on the regulatory roles of cytokinins and cytokinin receptor genes in abiotic stress responses,and provides theoretical supports for the breeding of drought-and heat-tolerant tomato plants.