| Heavy metal pollution in soil has become a serious problem and needs to be solved urgently.Among those solutions,phytoremediation has the advantages of cost-effectiveness and is friendly to environments.However,low bioavailability and transfer coefficient of some metals in engineered plants become the main limiting factors for phytoremediation,which largely restricts the application of phytoremediation techniques for heavy metal contaminated soils.In recent years,researchers suggest that the application of "chelating-inducing" phytoremediation techniques can be helpful to overcome this obstacle.Natural low molecular weight organic acids such as citric acid,oxalic acid,tartaric acid,malic acid and succinic acid are important metabolites of plants,which are characterized by high degradability and low toxicity,and are more environmentally friendly.However,the effects of organic acids on phytoremediation vary from metal and type of engineering species.In this regard,it is necessary to deeply study the characteristics of target metals and engineering plants as well as their interaction mechanisms.Salix variegata Franch.,a perennial shrub of Salicaceae with strong abilities of Cd tolerance and accumulation,has great potential in the phytoremediation of Cd contaminated soils.In order to improve the ability of S.variegata to absorb and accumulate Cd,this study explored,using hydroponic culture,the effect of five exogenous organic acids addition(citric acid,oxalic acid,tartaric acid,malic acid and succinic acid)on Cd enrichment of S.variegata.Furthermore,three of the above organic acids with significant effects,citric acid,tartaric acid and malic acid,were intensively investigated in terms of growth ability,tolerance mechanism and gene expression.The main results were as follows:(1)The addition of citric acid,tartaric acid,malic acid and succinic acid had a significant effect on the increase of Cd accumulation in S.variegata.After treatment with citric acid,tartaric acid,succinic acid and malic acid,the accumulation of Cd in S.variegata plants was significantly increased,which was 210%,190%,190% and 178% of the Cd treatment group without organic acid.The biomass and Cd concentration of root was significantly increased by the addition of citric acid,thus increasing total Cd accumulation in S.variegata;the application of tartaric acid,succinic acid and malic acid significantly increased the accumulation of Cd in various organs of S.variegata,which raised the Cd extraction efficiency of S.variegata.Malic acid increased the total Cd accumulation by increasing the biomass of various organs of S.variegata.The tartaric acid and succinic acid increased the biomass and Cd content of S.variegata.Moreover,the contribution of tartaric acid to the Cd absorption capacity of S.variegata was greater than that of succinic acid.The addition of oxalic acid had no significant effect on the increase of biomass and Cd uptake capacity of S.variegata under Cd stress.(2)The application of citric acid,malic acid and tartaric acid enhanced the photosynthesis of S.variegata under Cd stress,which significantly promoted the growth ability of plants.The effect of tartaric acid and malic acid was more significant.Cd stress significantly reduced the photosynthesis of S.variegata by inhibiting the photosynthetic pigment content and the integrity of photosynthetic structure,which inhibited plant growth and significantly reduced biomass.Malic acid promoted the growth of plants by increasing the chlorophyll content and mitigating the inhibited photosynthetic structure due to Cd stress.As a result,the biomass of S.variegata was significantly increased.Tartaric acid had no significant effect in enhancing the photosynthetic pigment content of S.variegata,but it was a prominent role in relieving ultrastructural damage of plants under Cd stress.The addition of citric acid significantly increased the photosynthetic pigment content of S.variegata,and the number of chloroplasts in mesophyll cells also went up.The volume of mesophyll cells and chloroplasts raised.Citric acid treatment also alleviated the Cd damage to photosynthetic structure of S.variegata,but its effect was lower than that of tartaric acid and malic acid treatments.The results showed that application of tartaric acid and malic acid was more beneficial to increase the growth ability of S.variegata under Cd stress.(3)The addition of three organic acids significantly enhanced the resistance of S.variegata to oxidative stress,and the PC content of root increased dramatically under the supply of citric acid and malic acid.Under Cd stress,the activities of antioxidant enzymes in roots and leaves of plants were significantly enhanced,while the lipid peroxidation occurred severely,the MDA content in the roots and leaves of S.variegata increased by 103% and 69%,respectively,compared with the control group.The addition of three organic acids significantly enhanced the resistance of S.variegata to oxidative stress and reduced the lipid peroxidation induced by Cd stress.After citric acid,tartaric acid and malic acid were added,MDA content in S.variegata roots increased significantly,70%,49% and 32% in Cd treatment,respectively.MDA content in leaves decreased significantly only in malic acid treatment,which was 71% in Cd treatment.The addition of three organic acids also significantly increased the GSH content in the roots of S.variegata,further enhancing the antioxidant capacity and potential for synthesis of PC.The PC content in the roots of S.variegata increased significantly under the action of citric acid and malic acid,which was beneficial to improve Cd tolerance in plants.(4)The addition of citric acid,tartaric acid and malic acid had a significant effect on the expression of genes related to Cd accumulation and tolerance in S.variegata plants,especially in roots,and thus the Cd accumulation and tolerance of S.variegata was improved.Under Cd stress,the expression levels of P-type metal ATPase HMA1 and phytochelatin synthase family protein PCS1 in roots were significantly up-regulated,1.95 times and 30.45 times that of the control group,respectively.In leaves of S.variegata,the expression levels of metal-tolerant protein MTP4 and P-type metal ATPase HMA5 significantly increased,they were 1.64 times and 2.48 times of the control group,respectively.The addition of citric acid,tartaric acid and malic acid had different effects on the expression of candidate genes in roots and leaves of S.variegata.Under Cd stress,citric acid and tartaric acid promoted the Cd absorption of roots through the induction of NRAMP5 and MTP4.The Cd storage capacity of roots was raised by induction of PCS1.In the leaves of S.variegata,citric acid supply had a significant effect on increasing the expression levels of multiple candidate genes.In contrast,the addition of malic acid significantly increased the expression of NRAMP5 and PCS1 in leaves.Furthermore,the Cd tolerance of S.variegata was improved by the induction of HMA1,HMA3 and HMA5 by three organic acids.Under Cd stress,the candidate genes were mainly expressed in the roots.The supply of three organic acids had significant effects on the expression of genes related to Cd accumulation and tolerance in S.variegata,thus improving its Cd accumulation and tolerance capacity.The addition of citric acid,tartaric acid,malic acid and succinic acid showed a significant contribution to the total Cd accumulation in S.variegata.The application of tartaric acid,succinic acid and malic acid increased the Cd accumulation in the aerial part,which can effectively improve the extraction efficiency of S.variegata on Cd-contaminated soil.By studying the growth ability,tolerance mechanism and gene expression of S.variegata plants treated with citric acid,tartaric acid and malic acid under Cd stress,it was found that: The application of citric acid,malic acid and tartaric acid enhanced the photosynthesis of S.variegata under Cd stress,which significantly promoted the growth ability of plants.The effect of tartaric acid and malic acid was more significant.The addition of three organic acids significantly enhanced the resistance of S.variegata to oxidative stress and reduced the lipid peroxidation induced by Cd stress,and the effect of malic acid was the most obvious.The addition of three organic acids also significantly increased the GSH content in the roots of S.variegata,further enhancing the antioxidant capacity and the potential for synthesis of PC.The PC content in the roots of S.variegata increased significantly under the action of citric acid and malic acid,which was beneficial to improve the Cd tolerance in plants.The addition of three organic acids had a significant effect on the expression of genes related to Cd accumulation and tolerance in S.variegata,especially in roots,and thus the Cd accumulation and tolerance of S.variegata was improved. |
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