Response Of Glutathione In Lettuce To Microcystins And Anatoxins Exposure

Due to climate change and human activities,eutrophication has intensified,leading to frequent outbreaks of cyanobacterial blooms and causing the concentration of cyanobacterial toxins in water bodies,such as microcystins（MCs）and anatoxins（ANTXs）,to exceed safe levels.When water bodies contaminated with cyanobacterial toxins enter agricultural ecosystems through irrigation,it not only has adverse effects on crop growth and yield but also accumulates in the edible parts of crops,posing a threat to human health.Existing studies have mainly focused on the ecological risk assessment of a single type of cyanobacterial toxin,and there is little research on the ecological risk of the coexistence of two common cyanobacterial toxins（MCs and ANTXs）in water bodies.Therefore,this paper aims to explore characteristics of the combined effects of MCs and ANTXs and their ecological risks.Lettuce（Lactuca sativa L.）was used as the experimental material and a combination of chlorophyll fluorometer,real-time quantitative PCR,enzyme-linked immunosorbent assay,and ultra-high-performance liquid chromatography,the paper identifies the compound effects of MCs and ANTXs on lettuce growth and chlorophyll fluorescence.By externally applying glutathione（GSH）or GSH synthetase inhibitors（BSO）to clarify the mechanism of the role of lettuce’s ascorbic acid（As A）-GSH cycle and GSH biocompatibility in adapting to the combined stress of MCs and ANTXs.This study not only provides new basic data for objectively evaluating the ecological risk of cyanobacterial toxins but also provides new ideas for taking measures to reduce the harm of cyanobacterial toxins to plants.Main results are as follows:（1）The combined effects of microcystins（MCs）and anatoxins（ANTXs）on lettuce growth and chlorophyll fluorescence characteristics:The inhibition ratio（RI value）of lettuce growth and chlorophyll fluorescence by different concentrations(5+5,20+20,50+50,and100+100μg·L^-1)of MCs and ANTXs was less than 1,indicating an antagonistic effect between MCs and ANTXs,this suggests that the presence of ANTXs in the water can mitigate the adverse effects of MCs on lettuce growth.Even after 7 days of recovery（under non-toxic stress conditions）,the combined effects of MCs and ANTXs still showed an antagonistic effect,which may be related to the accumulation of MCs or ANTXs in lettuce tissues.（2）Effects of GSH/BSO application on lettuce growth and chlorophyll fluorescence under MCs and ANTXs treatments were investigated.Compared with the control group（CK）,the low-concentration(5μg·L^-1)toxin treatment group[MCs（5M）,ANTXs（5A）,and MCs+ANTXs（5M+5A）]promoted the above-ground growth of lettuce,with the growth-promoting effect in the order of 5A>5M>5M+5A,which was related to the increase in lettuce photosynthetic efficiency（Fv/Fm）and electron transfer rate（ETR）.However,the high-concentration(100μg·L^-1)toxin treatment group（100M,100A,and 100M+100A）inhibited lettuce growth,with the growth-inhibiting effect in the order of100M>100M+100A>100A,possibly because lettuce Fv/Fm,ETR,and fluorescence decay rate（Rfd）were inhibited.After the application of GSH(1 mmol·L^-1),the growth of lettuce in the GSH+low-concentration toxin treatment group（GSH+5M,GSH+5A,or GSH+5M+5A）was higher than that in CK,but there was no significant difference compared to the low-concentration toxin treatment group.After the application of BSO(0.5 mmol·L^-1),the growth of lettuce in the BSO+low-concentration toxin treatment group（BSO+5M,BSO+5A,or BSO+5M+5A）was lower than that in CK and the low-concentration toxin treatment group.These results indicated that the endogenous GSH in lettuce had a strong regulatory ability under low-concentration MCs and ANTXs treatments,which could promote lettuce growth,so the application of GSH did not supplement the endogenous GSH.However,the GSH+high-concentration toxin treatment group（G+100M,G+100A,and G+100M+100A）was significantly lower than CK but higher than the high-concentration toxin treatment group,while the growth of lettuce in the BSO+high-concentration toxin treatment group was lower than CK and the high-concentration toxin treatment group,indicating that the application of GSH could alleviate the inhibition of high-concentration MCs and ANTXs on lettuce growth.After 7 days of recovery,the above-ground growth of lettuce in the GSH+high-concentration toxin treatment group was higher than CK,indicating that the application of GSH continued to alleviate the inhibition of high-concentration MCs and ANTXs on lettuce growth and that the above-ground growth recovered to the normal level,which was related to the alleviation of inhibition of Fv/Fm,ETR,photochemical quenching（q N）,non-photochemical quenching（q P）,and Rfd.（3）Effects of exogenous GSH/BSO on the As A-GSH cycle in lettuce under MCs and ANTXs treatments were investigated:Compared to the control group,treatment with 5M and5A reduced the content of hydrogen peroxide（H₂O₂）in lettuce,while treatment with 5M+5A increased the content of H₂O₂in lettuce,which was related to the inhibition of GSH and As A/DHA in lettuce.High toxin concentration treatment increased the content of H₂O₂in lettuce,which was related to the inhibition of GSH,GSH/oxidized glutathione（GSSG）,Ls GR,Ls APX1（cytosolic）and Ls APX2 expression,as well as glutathione reductase（GR）and ascorbate peroxidase（APX）activity in lettuce.Exogenous GSH had no significant effect on the content of H₂O₂in lettuce compared to low toxin concentration treatment group.However,in the GSH+high toxin concentration treatment group,the content of H₂O₂in lettuce was higher than in the control group but lower than in the high toxin concentration treatment group,indicating that exogenous GSH could alleviate oxidative damage to lettuce caused by high toxin concentration treatment,by promoting the increase of endogenous GSH and GSH/GSSG,enhancing the expression of Ls GR,Ls APX1（cytosolic）and Ls APX2,and increasing the activity of GR and APX,thus alleviating oxidative damage.In the BSO treatment group,the content of H₂O₂in lettuce was higher than in the control group,and BSO increased the content of H₂O₂in both low and high toxin concentration treatment groups,indicating that low toxin concentration treatment caused oxidative damage to lettuce when GSH synthesis was inhibited,which was related to the inhibition of GSH,As A,cellular redox level,and MDHAR.After a recovery period of 7 days,the content of H₂O₂in the BSO+5M treatment group was not significantly different from that in the control group,while the content of H₂O₂in the BSO+5A or BSO+5M+5A treatment groups remained higher than that in the control group.Furthermore,exogenous BSO still increased the content of H₂O₂in lettuce,indicating that the oxidative damage to lettuce caused by exogenous BSO was irreversible,which was related to the decrease of GSH and cellular redox level.The results suggest that endogenous GSH can maintain reactive oxygen species（ROS）balance under toxin stress,which is an important reason for the promotion of lettuce growth under low toxin concentration treatment with MCs and ANTXs.（4）Effects of exogenous GSH/BSO on the bioaccumulation of GSH in lettuce under MCs and ANTXs treatments were investigated:MCs and ANTXs accumulated in the low concentration toxin treatment group,with a higher accumulation of MCs in the 5M+5A group compared to 5M alone,while ANTXs accumulated less in the 5M+5A group compared to 5A alone.This suggests that the coexistence of low concentrations of MCs and ANTXs increases MCs accumulation and decreases ANTXs accumulation in lettuce.In the high concentration toxin treatment group,the accumulation of both MCs and ANTXs in lettuce was higher than that in the low concentration group,with a lower accumulation of MCs in the 100M+100A group compared to 100M alone,and a lower accumulation of ANTXs in the 100M+100A group compared to 100A alone.This indicates that the coexistence of high concentrations of MCs and ANTXs reduces the accumulation of both toxins in lettuce,which is related to the regulation of Ls GST expression and GST activity in lettuce.Compared with the group without external application,exogenous GSH reduced the accumulation of MCs or ANTXs in all treatment groups,indicating that exogenous GSH can detoxify MCs and ANTXs through GST induction of GSH bioaccumulation,thus reducing the ecological risk of coexistence of MCs and ANTXs.However,exogenous GSH cannot alleviate the human health risks posed by high concentrations of MCs and MCs+ANTXs.Compared with the group without external application,exogenous BSO increased the accumulation of MCs or ANTXs in all treatment groups.After inhibition of GSH generation by exogenous BSO,the pathway of GSH detoxification of MCs and ANTXs through GST was inhibited,resulting in excessive accumulation of MCs and ANTXs.After 7 days of recovery,the alleviating effect of exogenous GSH on the high concentration toxin treatment group still persisted.After exogenous BSO treatment,human health risks appeared in the low concentration MCs+ANTXs treatment group,but the risk decreased to a safe level after a recovery period.In summary,the effects of MCs and ANTXs on lettuce growth exhibit an"antagonistic"relationship,indicating that the presence of ANTXs(≥50μg·L^-1)in water can alleviate the adverse effects of MCs(≥50μg·L^-1)on lettuce growth.Positive and negative verification of the role of GSH in lettuce under MCs and ANTXs stress was carried out through the external application of GSH/BSO.It was found that GSH in lettuce can regulate the ability of lettuce to adapt to cyanobacterial toxin stress by maintaining the effectiveness of the As A-GSH cycle to regulate intracellular reactive oxygen species balance,and detoxifying MCs and ANTXs through GSH bioconjugation.Moreover,the external application of GSH can mitigate the damage caused by high concentrations of MCs and ANTXs to lettuce,which can be an effective way to alleviate cyanobacterial toxin stress.