CsABCG11.2 Mediating Theanine Absorption Alleviates Cadmium Toxicity In Camellia Sinensis

Theanine（Thea）is one of the most important health-promoting metabolites in tea plant.The increased biological activity of toxic elements such as cadmium（Cd）in cultivated soil leads to their accumulation in plant tissues,which in turn poses a serious threat to human health through the food chain.Thea has been confirmed to be a new regulatory substance involved in alleviating abiotic stresses such as salt and drought,but the physiological mechanism of Thea regulating Cd stress in plants remains unknown.In this study,transcriptome data analysis and yeast complementation assay were used to identify a transporter CsABCG11.2,which mediates uptake of Thea and Cd.Hydroponics experiments were used to simulate Cd stress treatment to reveal the function of CsABCG11.2 via Arabidopsis thaliana overexpression lines.This study aimed to clarify the effects of exogenous Thea on tea plant growth,photosynthetic performance,Cd absorption and accumulation,and Cd-induced antioxidant enzyme activities under Cd stress,evidencing that the underlined physiological mechanism regulated by CsABCG11.2-mediating Thea absorption to alleviate Cd toxicity.The main results are as follows:（1）Compared with those of pure Cd treatment(80μM Cd²⁺),the photosynthetic efficiency,malondialdehyde（MDA）content,superoxide dismutase（SOD）and catalase（CAT）activity of tea leaves were significantly increased when the tea trees were supplied with Thea and Cd(80μM Cd²⁺+50μM Thea),indicating that exogenous Thea could enhance the resistance of tea plants to Cd toxicity.When 80μM Cd²⁺and 50μM Thea were added,cadmium content in shoot of tea seedlings was significantly decreased.Under 80μM Cd²⁺Cadmium stress,the contents of free amino acids,tea polyphenols and caffeine in fresh leaves were significantly decreased.The co-treatment of 80μM Cd²⁺and 50μM Thea significantly increased the contents of free amino acids and theine in the young leaves of tea seedlings,suggesting that exogenous theanine can effectively alleviate the damage of cadmium stress on the growth of tea seedlings,reduce the absorption of Cd²⁺,improve the content of quality components,and ensure the normal growth of tea seedlings.Under 80μM Cd²⁺stress,some genes were significantly down-regulated in shoot and root compared with the control.The expression of theanine biosynthesis pathway genes Cs TS2,Cs GS1,Cs Ni R,Cs NR,Cs GDH1,CsALT and Cs NADH-GOGAT were significantly induced by80μM Cd²⁺+50μM Thea co-treatment.The results indicated that Cd stress reduced the expression of genes related to Thea synthesis and N assimilation pathway,while exogenous Thea could regulate the assimilation of N in tea leaves and roots,and induce the up-regulated expression of genes related to Thea synthesis in roots.Tea plants can regulate the expression levels of key genes in N assimilation and Thea synthesis pathway to establish a regulatory system to cope with Cd stress.（2）Transcriptome analysis showed that CsABCG11.2 expression level was significantly upregulated by low N,and yeast heterologous complementation test showed that CsABCG11.2 could absorb and transport heavy metals Cd and Thea.The q RT-PCR results showed that CsABCG11.2 was highly expressed in tea leaves and relatively low in roots,but its expression levels in roots was significantly up-regulated by Cd stress.Histochemical analysis showed that CsABCG11.2 was mainly expressed in leaves,root epidermis,petals and stigma of Arabidopsis thaliana under control treatment.The expression level of CsABCG11.2 was induced by Cd,Thea and low N treatment as indicated by the enhanced GUS activity in leaves,root tips and lateral roots.CsABCG11.2-overexpressing Arabidopsis lines were more sensitive to 40μM Cd²⁺treatment,while the treatment of 40μM Cd²⁺+50μM Thea significantly alleviated Cd toxicity,significantly reduced MDA content in Arabidopsis leaves,and significantly increased SOD,POD and CAT enzyme activities.Both tea plants and Arabidopsis assay results indicated that exogenous application of Thea alleviated Cd-induced oxidative stress.（3）Under the condition of 40μM Cd treatment,CsABCG11.2-overexpressing Arabidopsis thaliana showed significantly higher Cd content than the wild type,indicating that CsABCG11.2 promoted the uptake of Cd;under the condition of 40μM Cd²⁺+50μM,CsABCG11.2-overexpressing Arabidopsis lines had a significantly higher Cd content.The Cd content of each CsABCG11.2-overexpressing Arabidopsis lines was significantly lower than that of the wild type,indicating that the reduced Cd content of plants under co-treatment is likely caused by the competitive absorption between Cd²⁺and Thea mediated by CsABCG11.2.Further,the results of competitive absorption between gradient level of Thea and Cd treatment showed that the uptake of Cd by yeast heterologously expressing CsABCG11.2 decreased with the increase of Thea concentration,Thea acts as a substrate to competitively inhibit the Cd absorption of CsABCG11.2.Absorption,when the Thea concentration increased to 2 m M,the Cd concentration in the yeast maintained constant.Therefore,clarifying the mechanism related with substrate recognition and selective absorption of CsABCG11.2 will contribute to clean production of tea by improving the uptake of Thea through cultivation measures.