The Physiological Response Mechanism Of Acetylcholine Regulating Cadmium Stress In Tobacco(Nicotiana Benthamiana)

In recent years,cadmium(Cd)pollution has become increasingly serious,owing to the industrial waste and misuse of chemical fertilizers.In particular,Cd is a nonessential element and posses highly toxic to plants.It poses a serious threat to the organism after entering the food chain.Due to its high mobility and easy absorption by plants,Cd eventually enters the food chain and poses a threat to animal and human health.At present,the application of exogenous substances is a rapid and effective way to alleviate heavy metal pollution in many soil remediation technologies.Studies have shown that acetylcholine,as an exogenous regulator,has been involved in alleviating salt,drought and osmotic stresses and other abiotic stresses,but the physiological mechanism of acetylcholine involved in Cd stress remains unclear.Therefore,in this study,the model plant tobacco(Nicotiana benthamiana)was selected as the experiment materials,a hydroponic experiment was conducted to clarify the effects of exogenous acetylcholine on the growth,photosynthetic performance,PSII photochemical characteristics,chloroplast ultrastructure,chemical form and distribution of Cd,and absorption and transportation of tobacco seedlings under Cd stress.This study aimed to provide the physiological regulatory mechanism of acetylcholine improving Cd tolerance of tobacco seedlings.The main results are as follows:1.Different concentrations of acetylcholine(5-150?M)alleviated the inhibition of growth in tobacco seedlings caused by Cd stress to a certain extent.Especially,application of acetylcholine(50?M)showed to be the most optimal concentration in alleviating photosynthetic and plant inhibition.Compared with Cd treatment alone,application of acetylcholine(50?M)treatment increased plant height,root length by27.38%and 12.50%,respectively.Acetylcholine alleviated the decrease of photosynthetic parameters induced by Cd toxicity and further improved photosynthetic activity by regulating stomatal aperture.2.Acetylcholine(50 and 100?M)treatment effectively reduced the lipid peroxidation and the accumulation of reactive oxygen species in tobacco seedlings under Cd stress.Especially 50?M acetylcholine effectively reduced ROS accumulation and cell membrane damage.Compared with Cd alone treatmnet,50?M acetylcholine significantly reduced the contents of H₂O₂ and O₂^·-by 50.23%and50.40%,respectively.In addition,acetylcholine enhanced the redox homeostasis to eliminate the accumulation of ROS in tobacco seedlings under Cd stress.On the one hand,the tolerance to Cd toxicity induced by acetylcholine was related to the increase of antioxidant enzymes activities(SOD,POD,CAT,APX).On the other hand,acetylcholine can play an important role in the function of glutathione ascorbic acid system by increasing GSH and As A contents as well as glutathione ascorbic acid system related enzymes to maintain redox homeostasis and reduce the accumulation of ROS and oxidative stress induced by Cd stress in tobacco seedlings.The results showed that the inhibition effect of Cd stress on plant growth was relieved.Combined with the growth index,photosynthetic index and related enzyme activity index under acetylcholine treatment,principal component analysis and fuzzy membership function method were used to evaluate the Cd tolerance in tobacco seedlings.The results showed that 50?M was the optimal concentration of acetylcholine to alleviate Cd toxicity.3.Cd induced oxidative stress which damaged chloroplast structure and degraded grana lamellae,thus inhibiting photosynthesis and reducing net photosynthetic rate.Exogenous acetylcholine effectively protected the ultrastructure of chloroplast,maintained the stability of photosynthetic apparatus and further enhanced the photosynthetic activity of tobacco seedlings by alleviating Cd induced oxidative damage and promoting chlorophyll synthesis.In addition,the distribution of light energy was further improved and the photosynthetic electron transfer was promoted by exogenous acetylcholine application.The addition of treatment acetylcholine optimized the energy flow parameters of tobacco leaves,made more energy used for electron transfer,reduced the energy consumption of antenna heat dissipation,improved the photochemical electron efficiency of reaction center,and then enhanced the light utilization ability,alleviated the damage of Cd to photosynthetic apparatus,thereby enhancing the resistance of tobacco seedlings to Cd stress.4.Exogenous acetylcholine attenuated Cd induced growth inhibition and root activity inhibition by inhibiting ROS accumulation and membrane lipid peroxidation.In addition,Cd stress increased the endogenous acetylcholine generation in tobacco seedlings through up-regulating the expression of acetylcholine synthetase and decomposition enzymes(Ch AT and ACh E),suggesting that acetylcholine involved in plants response to Cd stress.Meanwhile,exogenous acetylcholine alleviated Cd stress by regulating glyoxalase system and antioxidant system,maintaining the homeostasis of MG and ROS,and further regulating physiological and biochemical changes.Acetylcholine enhanced the ability of tobacco seedlings to resist osmotic stress under Cd stress mainly by increasing the accumulation of soluble sugar,soluble protein and betaine in tobacco seedlings.Exogenous acetylcholine alleviated Cd toxicity by regulating the concentration of divalent metal ions in tobacco plants to form Cd competition.Under Cd stress,acetylcholine induced the accumulation of endogenous hormones(ABA,IAA,GA3,ZR)to respond to Cd toxicity.Acetylcholine changed the content of organic acids components in the aboveground and underground parts of tobacco seedlings,and made Cd chelating with organic acid ligands to form Cd-organic acid chelate,which further reduced the toxic effect of Cd in plants.In addition,amount of free amino acids were induced under Cd stress,and the content of some amino acids increased after acetylcholine application.These amino acids were used as Cd binding ligands to bind with free Cd ions in tobacco,thus participating in the process of Cd detoxification.5.FTIR was used to analyze the wavelength changes of tobacco aboveground and root components to further explore the role of different parts of the plant components in improving tobacco Cd tolerance by acetylcholine application.FTIR analysis showed that the addition of exogenous acetylcholine increased the number of carboxyl and amino groups to combine with Cd²⁺through promoting the secretion of carbohydrate,protein and free amino acid groups,so as to reduce its toxic effect on tobacco seedlings.6.Appilcation of acetylcholine under Cd stress,the contents of glutathione(GSH),nonprotein mercaptan(NPTs)and phytochelatin(PCS)in shoots/roots were0.90/2.31,1.01/1.88 and 1.46/1.32 times higher than those in control group,respectively.Acetylcholine reduced the accumulation of Cd in roots by regulating the distribution of Cd in vacuoles and cell walls,thereby alleviating the damage of Cd stress to roots.In addition,under Cd stress,the application of acetylcholine significantly increased the expression of GSH2 and PCS1 genes,but decreased the transcription of NRAMP1 and HMA2,thus reducing the transfer of Cd from root to shoot.Those results suggested that acetylcholine reduced the Cd toxicity by enhancing the levels of glutathione and phytochelatin,improving the subcellular distribution and chemical forms of Cd,and inducing the expression of detoxification related genes.In conclusion,exogenous acetylcholine alleviated the photosynthesis and growth inhibition of tobacco caused by cadmium stress to a certain extent.It alleviated Cd toxicity mainly by regulating chemical forms,subcellular distribution,expression of Cd transport genes,and chelation of Cd with amino acids and organic ligands.The results provide a theoretical basis for the application of chemical regulation of acetylcholine in alleviating Cd toxicity.