Combined Effects Of Microcystins And Anatoxins On Growth And Hormone Balance In Lettuce

Eutrophication of water bodies is one of the most challenging environmental pollution problems facing humanity in the 21st century.The resulting cyanotoxins concentrations in water bodies are much higher than safe values,threatening human health.Cyanotoxins such as Microcystins（MCs）,Anatoxins（ANTXs）and Cylindrospermopsins（CYN）have been found to enter agricultural ecosystems with irrigation water,affecting not only the growth and yield of crops but also enriching the edible part of crops and threatening food safety along the Food safety is threatened by enrichment in the edible part of the crop and transmission along the food chain.Most studies have focused on the action characteristics of single cyanotoxins,while multiple cyanotoxins coexist in eutrophic water bodies,and the coexistence of MCs and ANTXs has been detected in 70%of water bodies,but little research has been conducted on the ecological safety risk of the coexistence of both toxins.Here,lettuce（Lactuca sativa L.）,a leafy vegetable with a high risk of accumulation of cyanotoxins,was studied to clarify the effects of MCs and ANTXs on the growth and food safety of lettuce,and common environmental concentrations of MCs and ANTXs in simulated water bodies(0.5,1,5,20,50and 100μg·L^-1)were treated with MCs and ANTXs,and the combined effects of two toxins were characterized and ecological risks were assessed based on the study of lettuce yield,hormone levels,reactive oxygen species（ROS）homeostasis and cyanotoxins enrichment risk under the combined stress of MCs and ANTXs using ELISA and UHPLC.The effects of MCs and ANTXs on endogenous hormone synthesis,metabolism,signaling pathways,and crosstalk ROS in lettuce under the combined stress of MCs and ANTXs were investigated by a combination of targeted metabolic analysis,LC-MS/MS and Real-time PCR,which explained the effects of MCs and ANTXs on crop growth and food safety at the metabolic and molecular levels.This will not only provide a theoretical basis for the aim evaluation of the ecological risk of combined cyanotoxins,but also provide a theoretical reference for guiding agricultural irrigation water safety.The main results are as follows:（1）The combined effects of MCs and ANTXs on lettuce growth,reactive oxygen species and hormone content showed that low concentration(0.5～20μg·L^-1)single MCs,single ANTXs and combined MCs+ANTXs treatments promoted lettuce growth,the increase range was 30%～94%which was associated with single or combined cyanotoxins treatments promoted indole-3-acetic（IAA）,zeatin（ZT）,gibberellin（GA₃）,abscisic acid（ABA）content and total antioxidant capacity（FRAP）.High concentration(50 and 100μg·L^-1)single MCs and MCs+ANTXs combination treatments inhibited lettuce growth,the decrease was0.5%～16.8%,which was caused by the decrease of IAA,ZT and GA₃ contents and FRAP under this treatment,while ABA content increased and induced excess ROS and lipid peroxidation.The combined MCs+ANTXs treatment showed antagonistic effects on lettuce growth,reactive oxygen species（ROS）and hormone content and the risk of lettuce yield safety were lower than single MCs but higher than single ANTXs treatment.The risk of yield reduction in lettuce by the combined treatment was higher than single ANTXs but lower than single MCs.In addition,the estimation of daily intake（EDI）of single MCs and MCs+ANTXs combined(20～100μg·L^-1)exceeded the tolerable daily intake（TDI）by 1.03-2.31 times,showed a higher health risk.The health risk of lettuce under MCs+ANTXs combination treatment was lower than single MCs treatment but higher than single ANTXs treatment.（2）Mediation of reactive oxygen species homeostasis by endogenous hormones in lettuce under the combined effects of MCs and ANTXs showed that low concentration(5μg·L^-1)single MCs or ANTXs treatment did not affect ROS homeostasis in lettuce,while the combined MCs+ANTXs treatment(5+5μg·L^-1)activated lettuce.The MCs+ANTXs treatment(5+5μg·L^-1)activated the expression of the gene encoding NADPH oxidase（Ls Rboh A）in lettuce to enhance NADPH oxidase activity and promote ROS production40.9%.High concentrations(100μg·L^-1)single MCs or ANTXs and MCs+ANTXs combination treatments up-regulated Ls Rboh A expression and NADPH oxidase activity in lettuce leaves,promoted SOD and CAT activities,and induced ROS overproduction60.5%～152.6%.Among them,the increase of ROS production and scavenging ability in lettuce by MCs+ANTXs combination treatment was higher than single ANTXs but lower than single MCs treatment,which was one reason for the antagonistic effect of MCs and ANTXs on ROS homeostasis in lettuce leaves.The addition of IAA inhibitor（NPA）resulted in increased ROS content in lettuce leaves under single MCs or ANTXs(5 and 100μg·L^-1)and combined MCs+ANTXs treatments(5+5 and 100+100μg·L^-1),mainly because of the promotion of ROS synthesis and inhibition in antioxidant enzyme scavenging ability.In contrast,the addition of an ABA inhibitor（Abamine）reduced the ROS content of lettuce leaves in the above treatment groups because of the inhibition of ROS production and the increase of antioxidant enzyme scavenging ability.The addition of IAA and ABA inhibitors revealed that hormones play a role in mediating the ROS production and scavenging pathways in lettuce leaves under the combined stress of MCs and ANTXs to regulate ROS homeostasis.（3）The combined effects of MCs and ANTXs on the anabolism and signal transduction pathways of lettuce hormones.The results showed that a single MCs or ANTXs at a low concentration(5μg·L^-1)promoted the level of IAA in lettuce about 96.8%和94.7%,which was related to the synthesis precursor L-tryptophan of lettuce.The contents of tryptophan acid（TRP）and tryptamine（TRA）as well as the activities of tryptophan decarboxylase（TDC）and tryptophan transaminase（TAAs）involved in IAA synthesis were increased,which was related to the promotion of IAA synthesis.Combined treatment of MCs+ANTXs(5+5μg·L^-1)promoted the synthesis of IAA,and promoted the content of IAA metabolite oxidative indole acetic acid（Ox IAA）and the activity of IAA oxidase,and accelerated the decomposition of IAA,which may be one of the reasons why low concentration(5+5μg·L^-1)MCs+ANTXs antagonized the IAA content in lettuce and increased by 66.1%.A high concentration(100μg·L^-1)of MCs+ANTXs combined treatment had higher IAA synthesis and decomposition ability than single ANTXs but lower than single MCs,resulting in the combined treatment still showing an antagonistic effect,the decrease was 25.6%～37.7%.Low and high concentrations(5 and 100μg·L^-1)of single MCs or combined treatments of ANTXs and MCs+ANTXs promoted ABA accumulation in lettuce leaves,the increase range was8.9%～632.8%,which was caused by the ABA metabolism phaseic acid（PA）,dihydrophaseic acid（DPA）and glucose ester of ABA（ABA-GE）increased,9’-cis-epoxycarotenoid dioxygenase（NCED）,which is key for ABA synthesis,and 8’-hydroxylase（8’-hydroxylase）,which is key for ABA decomposition.ABA 8’-OH)enzyme activity increased.Among them,MCs+ANTXs combined treatment increased the ABA synthesis and decomposition ability in lettuce higher than single ANTXs but lower than that of single MCs treatment,which was one reason that the combined treatment still showed an antagonistic effect.In addition,under single MCs or ANTXs and MCs+ANTXs combined treatment,changes in IAA and ABA content mediate ROS by activating the relative expression levels of key genes（RIC9,FERONIA,ABP19a,PYL9,ABI1,Sn RK2,ABI5 and PER1）in lettuce signaling.Generating and scavenging enzymatic activities to regulate ROS homeostasis in lettuce.In conclusion,the"antagonistic"effect between MCs and ANTXs was related to endogenous hormone synthesis,metabolism and their crosstalk with ROS homeostasis.This antagonism resulted in the coexistence of MCs and ANTXs in irrigation water with a lower risk to crop yield and food safety than single MCs,but higher than single ANTXs.Among them,the coexistence of medium concentration(20μg·L^-1)MCs and ANTXs in irrigation water may seriously threaten human health and safety while promoting crop yield.The above results can not only provide a better understanding of the complex action characteristics and mechanisms of complex cyanotoxins in terrestrial ecosystems,but also provide a theoretical reference for objectively evaluating the potential ecological risks brought by complex cyanotoxins in irrigation water.