The Role Of Transglutaminase (TGase) In Cucumber And Tomato Plants In Response To Salt Stress

Soil salinization is one of the main abiotic stresses that limit plant production.Sodium chloride is the most abundant soluble salt in soil salinization and is the main type of salt stress.NaCl stress can destroy the ion balance in plants,causing ion toxicity,resulting in hindered plant growth,decreased yield and premature aging.In recent years,the protected horticulture has developed rapidly in China.However,due to the simplicity of horticultural infrastructure and the poorly ability to withstand natural disasters,the vegetables often encounter salt stress,which result in a decline in photosynthetic capacity,seriously restrict plant growth,vegetable yield and quality in our country.Additionally,In the long-term evolution process,plants have formed various defense mechanisms to deal with the external environment,in which protein cross-linking and transcriptional regulation play an important role in plant growth and development and stress response.TGase is a cross-linking enzyme widely present in animals and plants,involved in protein protection and transamination.At present,the study of TGase is primarily focused on animals,and our understanding of the function and mechanism of TGase in plants is scanty,especially in horticultural plants.Therefore,this experiment has deeply studied the function of TGase in horticultural crops in response to stress,which not only helps to improve our understanding of TGase,but also contributes to the cultivation of resistant vegetables and salt-tolerant varieties of facility vegetables.1.We demonstrated that TGase from cucumber played a critical role in the regulation of PA metabolism under salt stress.TGase expression was induced by salt stress in cucumber.The ectopic overexpression of cucumber TGase in tobacco conferred enhanced tolerance to salt stress based on both external symptoms and membrane integrity.The overexpression lines maintained high levels of PAs under salt stress,indicating that PAs play a vital role in TGase-induced salt tolerance.In contrast,the levels of Na+content in the wild-type(WT)plants increased,while the overexpression decreased.The expression levels of several genes related to ion exchange were enhanced,and the Na+/K+ ratio was decreased by increased TGase activity under salt stress.The activities of the proton-pump ATPase(H+-ATPase),vacuolar H+-ATPase(V-ATPase)and vacuolar H+-pyrophosphatase(PPase)were higher in the overexpression lines than those in the WT plants under salt stress.Meanwhile,the malondialdehyde(MDA)and H2O2 content in the overexpression lines was significantly lower than that in the WT plants,which was accompanied by increased antioxidant enzyme activity.Taken together,these findings demonstrate that TGase plays a critical role in salt tolerance,which may promote plant survival by regulating PAs metabolism and Na+/K+balance under salt stress.2.The transcription of cucumber TGase(CsTGase)was induced in response to light and during leaf development,and the CsTGase protein was expressed in the chloroplast and the cell wall.The overexpression of the CsTGase gene effectively ameliorated salt-induced photoinhibition in tobacco plants,increased the levels of chloroplast polyamines(PAs)and enhanced the abundance of D1 and D2 proteins.TGase also induced the expression of photosynthesis related genes and remodeling of thylakoids under normal conditions.However,salt stress treatment reduced the photosynthesis rate,PSII and PSI related genes expression,D1 and D2 proteins in wild-type(WT)plants,while these effects were alleviated in CsTGase overexpression plants.Taken together,our results indicate that TGase-dependent PA signaling protects the proteins of thylakoids,which plays a critical role in plant response to salt stress.Thus,overexpression of TGase may be an effective strategy for enhancing resistance to salt stress of salt-sensitive crops in agricultural production.3.We clarified the function of TGase in the regulation of photosynthesis by comparing wild type(WT),tgase mutants,generated by CRISPR/Cas9 system and TGase overexpressing(TGaseOE)plants in tomato.Our results showed that increasing the transcript level of TGase resulted in enhanced net photosynthetic rate(Pn),whereas tgase mutants significantly inhibited the Pn and CO2 assimilation as compared with WT.Overexpressing of TGase positively induced transcription levels of the Benson-Calvin cycle-related genes and increased activity of the cycle associated enzymes.In addition,the TGaseOE plants had higher protein levels of RbcL and RCA than that in WT plants and also showed a reduced redox status by enhancing the activity of dehydroascorbate reductase(DHAR)and glutathione reductase(GR),which was compromised in deficient plants.Taken together,TGase positively regulated photosynthesis by maintaining the activation states of Calvin cycle and inducing changes in cellular redox homeostasis in tomato.4.TGase can induce the formation of autophagosomes to delay leaf senescence and increase the salt tolerance of plants.Salt stress and dark treatment induced the expression of TGase.The tgase mutants increased the sensitivity of salt stress,while TGaseOE plants increased the salt tolerance.After salt stress,the chlorophyll content of TGaseOE plants was higher than that of the control plants,and the expressions of senescence-related and chlorophyll degradation-related genes were higher than that of the control plants,indicating that delaying senescence and inhibiting chlorophyll degradation are important factors for TGase to increase salt tolerance.On the other hand,the expression of TGase was significantly higher in the old leaves than in the mature leaves.After dark treatments,the chlorophyll content of TGaseOE plants was higher than that of the control plants,and the expressions of senescence-related and chlorophyll degradation-related genes were similar to those of salt stress treatments.In addition,the autophagosome content of TGaseOE was significantly higher than that of the control plants under the control conditions,and the autophagosome content of TGaseOE plants under salt stress was higher than that of the control plants,and the expression of ATGs gene was higher than that of WT plants.These results indicate that TGase can induce ATGs gene expression and autophagosome formation to delay leaf senescence and enhance plant salt tolerance.