Physiological And Molecular Mechanisms Of Exogenous ATP Inducing Salt Tolerance In Brassica Campestris L.

The salinization is a very serious environmental and agricultural problem in abiotic stress,which is one of the main factors that seriously restrict the growth,development and yield of crops.In order to survive under the stress,the plants form a set of high-efficiency defense mechanisms that recognizes the signals and regulate the growth and development of the plants,resisting the adverse environments.Reactive oxygen species?ROS?and calcium ions as second messengers play an important role in plant growth and development under abiotic stress.As an essential energy source for organisms,ATP regulates the second messenger(H₂O₂ and Ca²⁺)involved in the metabolism of substance and energy in cells,thereby enhancing the adaptability of plants in the environment of adversity.Some studies have shown that there may be some relationship between exogenous ATP and salt stress,but there are few reports have been reported on the growth and development of exogenous ATP in response to salt stress.In this experiment,the effects of exogenous ATP on Brassica campestris L.seedlings‘Longyou 8' under salt stress were studied by using histochemical staining,plant physiology and biochemistry,and biological experiment methods.To analyze the effects of exogenous ATP on active oxygen,antioxidant enzyme activity,membrane injury,osmotic adjustment of Brassica campestris seedlings under salt stress,and the effects of antioxidase genes,NADPH oxidase genes?RBOH?,P5CS1gene,MAPK kinase genes and salt-tolerant genes transcription level.The main results are as follows:1.The salt stress inhibited seed germination and biomass accumulation of Brassica campestris seeds.Exogenous ATP concentration of too low or too high inhibited Brassica campestris seed germination.25?mol/L exogenous ATP promoted seed germination and seedling biomass accumulation under salt stress.It is shown that exogenous ATP plays an important role in the process of seed germination and biomass accumulation of Brassica campestris.2.Exogenous ATP soaking significantly reduced the ROS and membrane damage,and increased the activity of antioxidant enzymes and osmotic regulators in Brassica campestris seedlings under salt stress.The results showed that seeds soaking with exogenous ATP could increase the activity of antioxidant enzymes to eliminate excess ROS,thereby reducing the oxidative damage of Brassica campestris seedlings induced salt stress,as well as improving the osmotic regulators,which enhancing the water retention of Brassica campestris seedlings.So as to reduce the damage to Brassica campestris under salt stress.3.Using the observe the coloration of Brassica campestris leaves,the results showed that the distribution and content of ROS and dead cells in leaves of Brassica campestris seedlings were different under different treatments.The ROS and death cells were widely distributed and accumulated under salt stress.Compared with NaCl,eATP+NaCl treatment decreased the ROS and death cells in Brassica campestris leaves.The results showed that exogenous ATP could alleviate the damage caused by salt stress by regulating the ROS content and the number of dead cells in Brassica campestris.4.Salt stress increased the contents of Na⁺,Ca²⁺,Cl^-and Na⁺/K⁺ratio in Brassica campestris leaves.Exogenous ATP decreased Na⁺/K⁺ratio and Na⁺,and increased Ca²⁺and K⁺contents in Brassica campestris leaves under NaCl stress.The results indicated that exogenous ATP could improve salt tolerance by affecting the ion content in the Brassica campestris leaves,and maintaining the steady state of sodium and potassium homeostasis.5.The NaCl stress increased the activities of antioxidant enzymes,osmotic regulators,membrane damage and decreased chlorophyll content in Brassica campestris leaves.Exogenous ATP decreased the damage of plasma membrane,enhanced antioxidant enzyme activity and chlorophyll synthesis,and increased proline and soluble sugar content in Brassica campestris leaves under NaCl stress.Compared with eATP+NaCl,the DPI+eATP+NaCl treatment,DMTU+eATP+NaCl treatment and EGTA+eATP+NaCl treatment reduced antioxidant enzyme activity,osmotic regulator content and chlorophyll,and increased the degree of membrane damage.The results showed that NADPH oxidase,H₂O₂ and Ca²⁺were involved in the regulation of the activity of antioxidant enzyme,membrane damage and osmosis by exogenous ATP under salt stress in Brassica campestris.6.The transcription levels of NADPH oxidase genes?RBOHD/RBOHF?,P5CS1gene,MAPK kinase genes?MAPK3/MAPK6?,salt tolerance genes?SOS1/NHX1?and antioxidant enzyme genes?CAT/SOD/APX/GR?were detected by real-time PCR.The results showed that the above genes were induced by salt stress,and exogenous ATP further promoted the expression of Brassica campestris genes under NaCl stress.Compared to the treatment of eATP+NaCl,the expression of genes were inhibited by DPI+eATP+NaCl treatment,DMTU+eATP+NaCl treatment and EGTA+eATP+NaCl treatment.The results indicated that NADPH oxidase,H₂O₂ and Ca²⁺were involved in the induction of salt-tolerance genes expression under salt stress by exogenous ATP.