Application Of γ-aminobutyric Acid In Promoting Tomato Pesticide Metabolism And Alleviating Phytotoxicity

Tomato is one of the three major world trade vegetables and occupies an important position in the global vegetable trade.However,in the production process,continuous or excessive application of pesticides often occurs,which will cause serious tomato phytotoxicity and become a major obstacle to the export of vegetables in my country.As a signal molecule,γ-aminobutyric acid（GABA）can regulate many physiological processes,including growth,development and stress response.Therefore,it is of great significance to study the physiological mechanism and application effect of γ-aminobutyric acid（GABA）on tomato phytotoxicity mitigation and metabolic regulation,which is of great significance to realize the safe production of vegetables and promote the development of my country’s agricultural export trade.In this paper,wild-type tomato（Solanum lycopersicum cv.Ailsa Craig）was used as the test material,and chlorothalonil,which is commonly used in production,was used as the test pesticide,and its effect on GABA homeostasis was studied.The wild type（WT）of tomato GABA was set as the control,and the application effect of GABA in alleviating the phytotoxicity and the effects of GABA were tested by exogenous spraying of GABA（WT+GABA）combined with the materials of hyperaccumulating mutants（GABA-2 and GABA-3）.Research on the mechanism of action to promote the degradation of pesticides.In addition,in order to further explore the key genes of chlorothalonil metabolism and clarify the molecular mechanism of GABA promoting pesticide degradation,we performed transcriptome analysis.The main findings are as follows:1.Exogenous or endogenous increase of GABA in tomato strains can effectively reduce the residues of chlorothalonil and promote pesticide metabolism.By analyzing the changes of malondialdehyde（MDA）concentration,the maximum PSII quantum yield（Fv/Fm）and the physiological state of leaves of wild-type tomato treated with different concentrations of chlorothalonil,it was found that with the increase of chlorothalonil concentration The damage to tomato leaves was enhanced,and the application of 3mM GABA and GABA-2 and GABA-3 leaves could significantly inhibit this adverse symptom,indicating that GABA can significantly reduce the pesticide damage to tomato plants.2.Analysis of GABA synthesis and metabolism pathways,it was found that after chlorothalonil treatment,plants positively regulated the GABA shunting pathway,which significantly increased the activity of GAD and significantly decreased the activity of GABA-TP,which directly led to the decrease of Glu and the accumulation of GABA,which was resistant to chlorothalonil stress..At the same time,both exogenous application of GABA and genetic manipulation of GABA-TP could significantly increase the level of endogenous GABA,make GAD activity exhibit typical feedback inhibition,and activate chlorothalonil stress response by regulating the GABA shunt pathway.Analyzing the metabolism of PAs,another pathway of GABA biosynthesis,it was found that chlorothalonil treatment significantly increased the activities of arginine decarboxylase（ADC）,polyamine oxidase（PAO）and diamine oxidase（DAO）,but ornithine The decarboxylase（ODC）activity did not change significantly,resulting in significant differences in the concentrations of Put,Spd,Spm and total PAs in the early and late stages of chlorothalonil stress.The（Spd+Spm）/Put ratio continued to decline.These results indicated that the biosynthesis and metabolism of PAs were accelerated under chlorothalonil stress.When the endogenous GABA content increased,the effect of GABA on PAO activity showed an opposite trend,promoting the total accumulation of PAs and the change from Put to Spd and Spm,thereby increasing the（Spd+Spm）/Put ratio.This suggests that when stress occurs,GABA sends a feedback signal to promote the accumulation of PAs,thereby enhancing the chlorothalonil stress response.3.Chlorothalonil treatment significantly inhibited the net photosynthetic rate（PN）of wild-type plants.After 1 day treatment with chlorothalonil,the stomatal conductance（Gs）and intercellular CO2 concentration（Ci）of tomato leaves showed a downward trend,while the stomatal limit value（Ls）showed an upward trend.Compared with wild-type tomato,exogenous administration of GABA and GABA hyperaccumulator mutants can restore the suppressed PN.reduce Gs,Ci,and increase Ls.The results suggest that GABA-regulated stomatal movement may be a resistance mechanism to reduce pesticide absorption.After treatment with chlorothalonil,the maximum PSII quantum yield（Fv/Fm）,the actual photochemical efficiency of PSII（ΦPSII）and the photochemical quenching coefficient（qP）decreased.However,the Fv/Fm,ΦPSII and qP of exogenous administration of 3 mM GABA and GABA hyperaccumulator mutants were significantly alleviated.Compared with the above three parameters,non-photochemical quenching（NPQ）,PSII light-harvesting antenna（Ex）etc.show the opposite trend.This indicated that GABA enhanced stress resistance by regulating the changes of chlorophyll fluorescence parameters.Compared with the wild type,WT+GABA,GABA-2 and GABA-3 plants had higher levels of superoxide dismutase（SOD）,ascorbate peroxidase（APX）,catalase（CAT）and guaiacol peroxidase（GPX）activities were significantly increased,and the levels of O2·-,H2O2 and MDA were significantly decreased.This indicated that GABA-induced reactive oxygen species could provide an early warning response to chlorothalonil stress.At 0 days,both exogenous and endogenous GABA can promote the accumulation of O2·-and H2O2 but the concentration has not reached the level of cell damage,while superoxide dismutase（SOD）,ascorbate peroxidase（APX）,peroxidase Catalase（CAT）and guaiacol peroxidase（GPX）activities were both activated by exogenous and endogenous GABA.Compared with wild type,the activities of SOD,APX,CAT and GPX in WT+GABA,GABA-2 and GABA-3 plants increased significantly,and the levels of O2·-,H2O2 and MDA decreased significantly within 0-10 days.These results suggest that GABA-induced reactive oxygen species can provide an early warning response to chlorothalonil stress.4.The detoxification process of plants was analyzed,and it was found that in wild-type plants,the activity of glutathione S-transferase（GST）continued to increase with the increase of chlorothalonil stress time.After 6 days of treatment,the total glutathione level was significantly increased,the reduced glutathione（GSH）was depleted and its concentration was significantly decreased within 1-6 days,and the redox state of glutathione remained at a low level.These results suggest that GST takes GSH as a substrate and actively participates in the detoxification process of chlorothalonil.Both exogenous spraying and endogenous hyperaccumulation of GABA significantly increased GST activity,GSH and total glutathione concentrations compared to wild type,resulting in high levels of glutathione redox state.This means that GABA has a powerful effect in improving de novo synthesis and regeneration of GSH and can be used in combination with GST for chlorothalonil detoxification.Then,transcriptome analysis was performed to explore the key genes of chlorothalonil metabolism,and 16 key pesticide metabolism genes mediated by GABA were successfully identified in tomato.