Roles Of Phytochelatins And Phytochelatin Synthase In Plant Cadmium Tolerance And Accumulation

Heavy metal contamination is now becoming one of the most serious worldwide environmental problems. Phytoremediation is a technology that using plants to remove heavy metals from the soil or reduce their toxicity. This technology has made rapid progress because of its cost-effective and environmentally friendly manner. Genetic and molecular studies of plant heavy metal tolerance and accumulation mechanisms have been initiated to optimize and improve phytoremediation.Plants have developed a number of defense mechanisms for dealing with heavy metal stress. One mechanism involves the production of phytochelatins (PC) for detoxification or homeostasis of heavy metals. In this paper, the role of PC in cadmium (Cd) tolerance and accumulation was studied in two species of turfgrass creeping bentgrass [Agrostis stolonifera L. var. palustris (Huds) Farw cv. Princeville] and tall fescue (Festuca arundinacea schreb. cv. Vegas) and model plant tobacco, and the possible mechanisms involved were discussed.1. Cd tolerance and accumulation in turfgrass creeping bentgrass and tall fescueCd tolerance and accumulation ability were compared with two species of turfgrass creeping bentgrass and tall fescue. Different biochemical and physiological responses were observed, main results obtained were shown as follows:1.1 Effects of Cd on seedlings and suspension cells of two plants were examined, and the results suggested that creeping bentgrass has greater tolerance and accumulation ability than tall fescue. The half inhibited level of Cd for root growth of seedlings is 50μM for creeping bentgrass and 500μM for tall fescue, respectively. As far as relative cell viability is concerned, the lethal concentration of Cd is 2mM for creeping bentgrass and 500μM for tall fescue, respectively. When grown in 50 μM CdCl₂ contained agar medium for 1 month, Cd accumulation in seedlings of bentgrass is 1.68 and 1.93-fold of tall fescue in the presence of 50 and 500μM CdCl₂, respectively. The data of suspension cells is consistence with seedlings'. When 50μM CdCl₂ is applied, the Cd content of bentgrass is 6.71-fold of tall fescue. The Cd contents reached 2283.2 μg g-l DW in creeping bentgras and 797.5 μg g-l DW in tall fescue when external Cd concentration was 1000μm1.2 Toxic level of Cd treatment resulted in oxidative stress, and caused lipid peroxidation in tall fescue. The accumulation of MDA enhanced when external Cd concentration had been raised, and treatment of 200μM CdCl₂ for 7 days induced considerable MDA production in tall fescue.1.3 Both cells were capable of producing PC peptides in response to Cd, and the levels of PC increased when the external Cd concentration was raised. The content of PC is higher in bentgrasscell than in tall fescue, and level of PC in tall fescue was 11.22-fold of creeping bentgrass when 50uM CdCl2 was applied.1.4 Exogenous GSH treatment can decrease the sensitivity of cells to Cd. Application of 1 mM GSH and 2mM GSH increased 1.89 and 1.54-fold to cell viability in the presence of 2mM CdCl2) respectively. In contrast, exogenous GSH had no apparent effect on the cell viability of tall fescue cells in the presence of 150uM CdCl2. Pretreatment of y-ECS specific inhibitor-buthionine sulphoximine (BSO) could inhibit GSH biosynthesis and enhanced Cd-imposed inhibition of cell viability in both cells.1.5 The level of PC might not be responsible for the difference of tolerance and accumulation of two species. The more tolerant species creeping bentgrass accumulated more Cd, but its PC production level is lower than more sensitive species tall fescue whenever compared as same external or internal Cd concentration. The possible reasons might involve GSH content in cells, the efficiency of GSH biosynthesis and metal-imposed strain on different cells.2. Cd toleranceand accumulation in transgenic AtPCSl tobacco plantsPhytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. To investigate whether overexpression PC synthase (PCS) in transgenic plants can increase PC synthesis, metal tolerance and accumulation, Arabidopsis AtPCSl was introduced into tobacco (Nicotiana tabacum) by Agrobacterium tumefaciens-mediated transformation with a construct containing the AtPCSl cDNA under the control of cauliflower mosaic virus (CaMV) 35S promoter (35S/AtPCSl). Following screening, approximately 20 Tl lines were obtained and Southern blot and Northern blot analysis demonstrated the expression and inheritance of foreigh gene. The results of Cd exposure test showed that some transgenic pcs line exhibited significant different tolerant levels when compared with WT grown under 250|iM CdCl2 for 1 month. One AtPCSl overexpression line pcs-5 was significantly more sensitive than WT, and the root length of pcs-5 decreased to 57.8% that of WT. The results of Cd content assay showed that transgenic pcs line accumulated less Cd in seedlings than WT. When plants were subjected to 250uM CdCl2 for 3 days, total GSH> total acid soluable thiol (TAST) and PC levels exhibited significant difference between some transgenic seedlings and WT. Especially in pcs-5, total GSH level decreased to 70.58% that of WT, while PC production increased to 141% that of WT.Therefore, it was suggested that overexpression of PCS might be used to increase the level of PC in plants. However, overexpression of PCS might also lead to increased sensitivity to Cd. Critical levels of PC and GSH are important in heavy metal detoxification, and Cd tolerance and accumulation of plants are controlled by a complex network of homeostatic mechanisms.3. Agrobacterium tumefaciens-mtdiaXeA transformation of E. coli gshl in creeping bentgrassManipulating the expression of enzymes involved in glutathione and phytochelatins synthesis may be a good approach to enhance heavy-metal tolerance in plants. To develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, E. coli gshl encoding the rate-limiting enzyme of GSH biosynthesis was introduced into creeping bentgrass [Agrostis stolonifera L. var. palustris (Huds) Farw]. A protocol was developed for Agrobacterium /Mme/ac/ens-mediated transformation of creeping bentgrass. The transformation was performed using the vector pCAMBIA 1301 which contains gshl cDNA under control of 35S CaMV promoter. Embryogenic calli initiated from mature seeds were infected with A. tumefaciens strain EHA105 followed by hygromycin selection. 30 transgenic creeping bentgrass plants were obtained. Southern blot analysis demonstrated the integration of a single or two copies of foreign gene, and RT-PCR confirmed the transcription of the genes. Detailed study of the hereditary patterns of the transgenic plants is now in progress. This study will pave the way to enhance the tolerance and accumulation of creeping bentgrass which can be used in phytoremediation of toxic metals in soils.