| Salinity stress is one of the major abiotic stress factors affecting plant growth and development.Recent studies have reported that the application of exogenous factor is a simple and effective method to improve plant tolerance and crop yield under salt stress.Acetylcholine?ACh?is one of the important neurotransmitters,involved in signal transduction function in human and animal brain.Studies on the physiological effects and mechanism of ACh have also been carried out in plants,especially how external application of ACh can improve the physiological effects of stress resistance in plants.Therefore,it is of great significance to understand the mechanisms of ACh on improving plant stress resistance for the improvement of crop tolerance to broad stress factors and the rational utilization of ACh.In present research,Nicotiana benthamiana,grown hydroponically,was chosen as experimental plant material to study the differences?times and parts?on the ACh concentration of seedlings under salinity stress.The effects of exogenous ACh on the growth,photosynthetic characteristics,water metabolism,chlorophyll compound metabolism and transcriptome analysis of N.benthamiana seedlings under salt stress were studied.The research may help to understand the mechanism for ACh-mediated tolerance to salt stress in plants,which provides a theoretical basis for the application of acetylcholine in strengthening the salinity stress tolerance in crops and enhance the yield productivity.Main results of the study are as follows:1.The enzyme-linked immunosorbent assay?ELl SA?was used to detect the roots,stems,old leaves and young leaves of N.benthamiana in different periods under salt stress treatment.The results showed that the existence of ACh in various parts of N.benthamiana which showed the trend as young leaf>root>stem>old leaf.ACh was firstly accumulated in roots under salt stress.The concentration of ACh reached the maximum value in stem and young leaves after 72 h of salt stress,the difference was significant compared with control.Among them,the concentration in young leaves after 72 h of salt stress treatment increased by 74.34%over the control.It showed that roots of N.benthamiana seedlings were first to exhibit the response to salt stress,and young leaves of the aerial part responded mainly to ACh in response to the external salinity stress,and this effect was related to treatment duration as well.These results showed that the distribution of ACh in the N.benthamiana and the variation in response to salt stress.2.Effect of ACh application through roots or leaf spraying with different concentrations of 1,10,50,and 100???M was also investigated,and the effects on plant growth and key physiological parameters of N.benthamiana seedlings under salinity stress was studied.The results showed that exogenous application of 10???M of ACh is more effective in promoting the plant growth and dry matter accumulation of N.benthamiana seedlings under salt stress.The dry weight of the above-ground parts of N.benthamiana seedlings increased by 10.54%compared with the salt stressed seedlings.In addition,leaf water content,root activity and accumulation of proline and soluble sugar increased by exogenous application of 10???M ACh under salt stress.And it results in the dramatically reduced in malondialdehyde content,relative conductivity,and water saturation deficit of N.benthamiana seedlings.It reflects that the appropriate concentration of ACh could effectively alleviate the salinity induced damage to membrane lipid peroxidation thereby maintaining the stability of cellular membrane and promoting osmotic regulation of plants,hence maintaining the growth of plants and enhancing the salt tolerance of N.benthamiana seedlings.3.Application of 10???M ACh noticeably improved root hydraulic conductance and leaf relative water content under salinity stress,respectively increased by 65.45%and 15.46%compared with salt stress.Exogenously supplied ACh significantly decreased the Na+/K+of roots,stems and leaves under Na Cl treatment by 67.00%,34.98%and 26.14%respectively over the salt-stressed plants without ACh supplementation.Leaf staining?Evans blue,nitrogen blue tetrazole and diaminobenzidine?showed that the reduction of reactive oxygen species was induced by ACh under salinity stress.And there was a significant reduction in O2·-,H2O2and MDA content of ACh and Na Cl+ACh treated plants through quantitative detection.Relative to the Na Cl stressed counterparts,a decline of 29.96%,24.87%and 46.7%in Na Cl+ACh seedlings after five days of salt treatment was observed.Meanwhile,exogenous ACh significantly increased leaf SOD and POD activities under salt stress.ACh concentration and the activities of choline acetyltransferase activity of N.benthamiana were increased by 17.36%and 23.6%in Na Cl+ACh treatment,respectively compared with salt stress.Exogenous ACh can alleviate the loss of water under salt stress to certain extent,improve ion imbalance,improve antioxidant capacity,ACh synthesis and metabolism as well as other processes,thereby alleviating the salt stress damage of plants.4.The accumulation of chlorophyll biosynthetic intermediates,such as porphobilinogen?PBG?and uroporphyrin III?URO III?were largely accumulated in ACh treated seedlings under normal as well as salt stressed conditions.In addition,the downstream intermediates?Proto-IX,Mg-Proto IX and Pchl?were significantly promoted in the by ACh treatment under salt stress.This influence of ACh on chlorophyll synthesis was confirmed by up-regulation of HEMA1,CHLH,CAO and POR genes.Relative to Na Cl-stressed counterparts,ameliorations of 54.56%for A,86.47%for gs,13.79%for Ci and 31.36%for E were observed after 15 days of stress exposure,and the Fo and Fm value of the leaves also significantly increased by the exogenous supplementation with ACh under salinity stress.Therefore,exogenous ACh relieved the leaves chlorosis of seedling caused by salt stress,promoted the process of chlorophyll synthesis,improved the chlorophyll fluorescence parameters and photosynthetic performance,and thus improved the salt tolerance of plants.5.Transcriptome studies showed that salt stress significantly regulated the expression of many metabolic pathways and stress-responsive functional genes.Under salt stress conditions,ACh has a significant effect on tobacco transcriptome,resulting in significant downregulation and upregulation of 131 genes and 527 genes;KEGG pathway analysis showed that differentially expressed genes in N.benthamiana were significantly enriched in 16 pathways after salt stress induced by ACh.Among them,a high proportion of specific differentially expressed genes are enriched in DNA replication,microbial metabolism in multiple environments,and endoplasmic reticulum protein processing and plant hormone signal transduction pathways in response to salt stress.The significant differential expression of encoding osmoregulation adjustment,photosynthetic and antioxidant-related pathway could induce by ACh resist to salt stress.Exogenous ACh can enhance the expression of brassinolide,auxin,gibberellin and salicylic acid signal transduction pathways and some transcription factor genes;meanwhile,the modification of cell wall synthesis plays an important role in the process of ACh improve the tolerance for salt stress.Exogenous ACh resulted in the changes of transcriptome tend to return to the control level under salt stress.These results suggest that ACh may play an elicitor-like triggering role and participate in the crosstalk network of salt toxicity alleviation in tobacco.These observations confirm the role of applied ACh in regulation of molecular mechanisms of salinity tolerance in plants.In conclusion,ACh improved the salt tolerance of tobacco seedlings by alleviating oxidative damage,enhancing plant water uptake,regulating photosynthetic ability and chlorophyll metabolism.It was confirmed by transcriptome sequencing technique that exogenously applied ACh differentially induces the expression of genes and molecular regulatory pathways involved in alleviating salt stress.However,the molecular mechanisms underpinnings the actual mechanisms are still unknown,therefore further studies can be worthwhile.These finding provide evidence for the physiological role of ACh and serve as a platform for its possible applications in improving the tolerance of plant exposed to salt stress. |
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