Study On The Mechanism Of Different Tolerance To Metolachlor In Maize

Maize is a main crop in China and rational herbicide usage is the gurantee of food security.Metolachlaor is a widely used chloroacetanilide herbicide in maize filed and caused phytotoxicity and reduced the production to susceptible maize cultivars under certain circumstance.As an important detoxification enzyme,Glutathione S-transferases are reported to be involved in the metabolism of various xenobiotic compounds in plants.In this study,we compared the metabolic system and antioxidant system to explore the mechanism of differential tolerance to chloroacetanilide herbicides in maize cultivars and different tissues.And exploring the detoxify function of GST34 by heterologous expression.The results could be helpful in genetics breeding of herbicide-tolerant maize and provide a good sight to herbicide detoxification in plant.1.Differential metolachlor tolerance between maize cultivarsTwo maize cultivars,Nongdan86(tolerant)and Zhengdan 958(susceptible),with 4.1-fold tolerance difference to metolachlor were screened by bioassays.The metabolism experiment result suggested that the metabolism rate of metolachlor was more rapid in Nongda86 cultivar than that in Zhengda958 cultivar.Synergist experiment indicated GSTs inhibitor(ETA)increased the toxicity of metolachlor to maize cultivars and the different metabolism rate could be elimated by ETA,which suggested that GSTs was involved in the differential detoxification of metolachlor between the two maize cultivarsThe expression profiles of GST isozymes with or without metolachlor treatment were measured by quantitative RT-PCR.The results showed that there was no difference on the expression of half GST genes between Nongda86 and Zhengdan958 without metolachlor treatment,and 7 GST genes had significant higher expression in Nongda 86,9 GST genes had significant higher expression in Zhengdan 958.However,most GST genes were slightly up-regulated in Nongda86,but severely inhibited in Zhengdan958 after 24 h metolachlor treatment.As a result,the expression level of most GST genes in Nongda 86 were higher than that in Zhengdan 958.The result of GST activity was consistent with that in gene expression profiles.2.Differential metolachlor tolerance between maize shoots and rootsThe bioassay result in the tissues of Zhengdan 958 cultivar indicated that the tolerance level of maize root to metolachlor was more than 5-fold than that in maize shoots.The synergist experiment indicated that the GSTs inhibitor ETA can increase the toxicity of metolachlor to roots and eliminated the difference tolerance between maize shoots and roots.The expression analysis of the GST genes family between roots and shoots revealed that the expression level of most GST genes in roots are 5-to 10-fold,and even more than 100-fold than that in shoots both in control and metolachlor-treated plants,except for GST10,GST24 and GST33 were lower in roots than that in shoots.GST activity in roots was found to be 8-fold higher than that in shoots,which was well correlated with the gene expression profile between shoots and roots.3.Expression characterization and heterologous expression of mazie GST34The quantitative RT-PCR results indicated that GST34 expressed in the second-leaf stage of Nongda86 but not in Zhengdan958.And the expression level of ZmGST34 increased rapidly with growth.Chloroacetanilide herbicides or phytohormones treatments had no influence on the expression level of GST34.The protokaryon expression vector pET-26b(+)/GST34 was constructed and expressed successfully in E.coli.The extracted recombinant protein showed metabolism activity towards chloroacetanilide herbicides(excepet for alachlor),atrazine and nicosulfuron.The eukaryon expression vector pCAMBIA1304/GST34 was constructed and expressed successfully in Arabidopsis thaliana.The bioassay results indicated that the tolerance to chloroacetanilide herbicides in transgenic plants were higher than that in the wild line.4.Comparation of antioxidant system between differential metolachlor tolerance maize cultivarsComparation on the antioxidant system between Nongda 86 and Zhengdan 958 maize cultivars showed that metolachlor treatment had no influence on the activities of antioxidant enzymes(SOD,CAT and POD),which showed no significant difference between maize cultivars.The total antioxidant capacity and the content of flavonoids were both higher in Zhengdan 958 after metolachlor treatment when compared with that in Nongda86.The content of MDA showed no significant change after metolachlor treatment,which indicated that no oxidative damage was caused by metolachlor treatment.Anthocyanin,an antioxidate compound in plant,was found to be accumulated higher in Nongda 86 than that in Zhengdan 958,and decreased significantly in both cultivars after metolachlor treatment.The expression analysis of anthocyanin biosynthesis genes by RT-qPCR showed that the down-regulation of PAL,CHS,DFR,ANS,F3'H and BZ2 were speculated to be the inhibition sites of anthocyanin biosynthsis by metolachlor.The higher expression of DFR,ANS and BZ2,key genes of anthocyanin sysnthesis,were deduced to be responsible for the higher level of anthocyanins in Nongda86 compared to Zhengdan958.