Glutathione Metabolisms In Cadmium Hyperaccumulator Sedum Alfredii Hance And Its Proteomics Analysis

Cadmium (Cd) is one of the particular dangerous environmental pollutants due to its high toxicity and great solubility character in water. Plant species differ significantly in uptake and tolerance to Cd and various other heavy metals. Metal-hyperaccumulating plants have the additional property of storing large amounts of metals in their aerial parts. This characteristic makes hyperaccumulators highly suitable for phytoremediation, a unique method in which plants are used for the cleanup of metal-polluted soils and wherein it is necessary to investigate the mechanisms responsible for tolerance and hyperaccumulation, using natural hyperaccumulators as model plant species. Sedum alfredii Hance, a natural inhabitant of old Pb/Zn mined site, has been identified as a Cd hyperaccumulator, with not only exceptional abilities to tolerate and accumulate high concentration of Cd, but also characteristics of relatively large biomass, fast growth, asexual propagation, and perennial. So, it is an ideal plant for studying mechanisms responsible for hyperaccumulation and for practical use in phytoremediation of contaminated soils.In the present work, we used hydroponically grown seedlings of hyperaccumulating ecotype (HE) and non- hyperaccumulating ecotype (NHE) of S. alfredii to check our hypothesis that both ascorbate-glutathione cycle and glutathione biosynthesis play important roles in countering Cd stress in the plant cultures. Moreover, we also took a primary study of the proteomic differences among the S.alfredii ecotypes and proteome changes in two ecotypes of S.alfredii upon exposure to Cd, and identified some proteins by MS. Main results were summarized as follows:1. It was confirmed that the S. alfredii ecotype collected from an old mined site (i.e. HE) has a greater ability to adapt Cd toxicity and Cd hyperaccumulation. Shoot Cd concentration and content of HE after 7 days' treatment with 100μM Cd²⁺ reached 4246 mg kg^-1 DW and 1370 ug plant^-1, respectively, which surpassed the generally accepted threshold concentration of 100 mg kg^-1 DW for Cd hyperaccumulators. NHE displayed significant toxicity symptoms such as stunted roots, thickened cuticle on root epidermis, cracked and brownish stem formation and wilted leaves upon exposure to Cd²⁺ over 10μM. A significant reduction in these morphological parameters i.e. length, diameter, surface area and volume and root activity was observed in NHE under excess Cd stress, while inhibition in HE was not sharp. In NHE shoot and root biomass production (expressed as dry weight) reduced significantly (up to 31.2%～46.8%) with≥10μM Cd²⁺. All these contrasting root morphological responses and root activity differences of the two ecotypes to Cd treatments may be partially responsible for their different abilities to tolerate and hyperaccumulate Cd.2. Cadmium induced severe ultrastructural changes in root meristematic and leaf palisade mesophyll cells of S. alfredii, but damage was more pronounced in NHE even when Cd concentrations were one tenth of those applied to HE. Maintenance of plant growth in HE may contribute to greater Cd uptake in plants and hence maybe beneficial for its translocation from root to shoot. Electron microscopic studies showed that root meristematic cells in both ecotypes exposed to different Cd concentrations exhibited severe ultrastructural changes over control. In root meristematic cells of NHE exposed to 10μM Cd²⁺, increased vacuolation, damaged membrane systems and swollen mitochondria were observed. The rough endoplasmic membrane appeared to have many attachments. Severely damaged cellular structures were noted in NHE roots exposed to 40μM Cd²⁺, where root epidermis was cracked with bacterial infection and integral cellular organization was lost. For HE at 100μM Cd²⁺, root cells still had a good cellular organization with distinct integrity of plasma membrane and cell wall. However, at 400μM Cd²⁺treatment, advanced vacuolation and rough endoplasmic membrane were the main toxicity symptoms in root cells. Nevertheless, the overall damage was much less compared to that of NHE at 10μM Cd²⁺. Ultrastructural studies revealed that chloroplast was an important site of damage induced by Cd stress. In NHE plants exposed high external Cd²⁺ concentrations of 10-40μM disorganization of chloroplasts was more common i.e. chloroplasts were rounded with a variable degree of internal structural disruption with a decrease in number of compact grana and light-colored thylakoid membranes. In case of HE at 100μM Cd²⁺, the chloroplasts and mitochondria were still relatively in better shape with clear and regular thylakoid membranes but showing a reduction in chloroplast size. For HE at 400μM Cd²⁺, the noted change in chloroplasts was only a considerable increase in the number and size of plastoglobuli. At larger magnification, swollen chloroplasts and loose thylakoid membranes were observed. These results indicated that Cd stress caused imbalanced synthesis of chloroplast lamellae leading to early leaf senescence. Other events that may take place during senescence included a reduction in the size of chloroplasts and loss of cytoplasmic components in the mesophyll cells of S. alfredii under high external Cd concentrations.3. Exposure to Cd elicited an oxidative busrt in NHE and HE indicated by the steady increase in H₂O₂ content and lipid peroxidation. Cadmium-stimulated distinct overproduction of H₂O₂ and O₂·- in leaves of both S. alfredii ecotypes, which was verified by a histochemical method with DAB and NBT, respectively. Accumulation of H₂O₂ or O₂·- could be eliminated by infiltration of AsA (a H₂O₂ scavenger) and TMP (an O₂·scavenger), respectively. Infiltration with DPI largely prevented H₂O₂ and O₂·- accumulation, revealing the involvement of NADPH oxidase in both NHE and HE. Treatment with BSO (a glutathione synthesis inhibitor) brought about significant (p<0.05) damage in leaves of HE with concomitant increases in H₂O₂ (31%) and O₂·- (13%) production suggesting that glutathione biosynthesis may contribute to counter the Cd-induced ROS production and help to adapt Cd toxicity. The oxidative deterioration is considered as an intrinsic feature of senescence process in plants. 4. Differences were noted in both HE and NHE for catalase (CAT), guaiacol peroxidase (G-POD), ascorbate peroxidase (APX) and glutathione reductase (GR) activities under various Cd stress levels. The SOD, G-POD and APX activities in NHE increased linearly upon≤5μM Cd²⁺ but conversely declined sharply at the higher Cd treatment. In the case of HE, increasing Cd treatments up to 100μM caused a marked reduction in CAT, G-POD, APX and GR activities, but recovered partly at 400μM Cd(2+). The following hypothetical framework may be suggested: Cd induces a transient loss in antioxidative capacity in HE, perhaps accompanied by a stimulation of oxidant producing enzymes, which resulted in intrinsic H₂O₂ accumulation. H₂O₂, then would act as a signaling molecule triggering secondary defenses. In addition, without Cd, on average, the ascorbe (AsA) contents in roots and leaves of NHE were 51% and 33%, respectively, higher than those in HE. Nevertheless, the oxidized form of ascorbate (DHA) concentration in roots and leaves of NHE was significantly higher than that of HE, by 2.06-folds and 1.11-folds, respectively. A strong rise in AsA contents in both NHE and HE occurred in 24 h after Cd application. Furthermore, the DHA/AsA ratio in leaves of NHE and HE with Cd application was maintained <1 (on average 0.44 and 0.33, respectively, in NHE leaves and HE leaves, a more reduced redox state of AsA) due to the persistent sharp increase in AsA contents with a simultaneously slight fluctuation in DHA concentrations. However, taking all observations together, we suggest that the plants response to Cd stress via activation of ascorbate-glutathione cycle for the removal of hydrogen peroxide were able to adopt a new metabolic equilibrium, allowing them to cope with Cd at enzymatic level. But when countering to excess Cd, loss of cellular redox homeostasis resulted in oxidative stress and toxicity.5. Application of Cd resulted in a significant (P < 0.05) enhancement in GSH pool in leaves and roots of HE; However, GSH concentration in NHE changed only a little and did not correlate to Cd tolerance. As compared to control plants, GSH contents in roots and leaves of HE were significantly elicited by Cd application in 48 h and afterwards, whereas GSH contents in roots of NHE were increased in 96 h. However, GSH in leaves of NHE with Cd treatments peaked at 48 h. BSO (a well known glutathione metabolism inhibitor) without Cd did not cause major changes in the H₂O₂ concentrations as well as in the growth of both NHE and HE. Treatment of BSO revealed a limited effect on GSH contents in roots and leaves of NHE, whereas BSO application reduced GSH pool of HE by 13% and 40% in roots and leaves, respectively, in comparison with the control plants. Generally, BSO combined with Cd treatment depleted GSH levels in the organs of both ecotypes, which were especially pronounced in HE at the end of experiments. Particularly, GSH levels in roots and leaves of HE grown with BSO combined with Cd for 8 d reduced more than 40 % of those treated with Cd only. Particularly, in Cd-treated HE plants, BSO resulted in a substantial enhancement in H₂O₂ accumulation relative to Cd treatment alone. The strong modulating effect of BSO on H₂O₂ accumulations and Cd concentrations in Cd-stressed HE plants may rule out the possibility that GSH biosynthesis play an important role as the signals for the stress regulation. A preferential Cd-stress response in leaves and roots of HE was related to changes in the glutathione redox state, whereas acclimation was marked by increased glutathione concentrations.6. Different extraction procedures were compared to screen the best one compatible with the high-reproducible 2-DE for S. alfredii. In particular, an extraction method of meaningful information about proteins from leaves and roots of S.alfredii was established. The remarkable characteristic of the protocol is combination of phenol extraction with TCA-acetone precipitation. Based on our optimized 2-DE patterns, protein preparation is free of interfering compounds with satisfactory and reproducible results and over 2000 spots were separated in individual gels.7. Based on this 2-DE platform with MS technology, we attempted to use proteomic profiling for exploring the molecular mechanisms related to Cd accumulation and tolerance in S.alfredii.The differential expression proteins were analyzed between two ecotypes of S.qfredii and from roots and leaves S.afredii exposure to Cd, respectively, using proteomic approach. 49 of these proteins were identified by MALDI-TOF-MS or ESI-MS including ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RubisCO), ABC transporter family protein, maturase K, root border cell-specific protein, actin, elongation factor 1-alph, histone H2A, Response regulator consisting of a Che Y-like receiver domain and a Fis-type HTH domain, auxin-responsive family protein, aspartyl protease family protein, transducin family protein, tryptophan synthase-related protein, amino acid transporter, leucine-rich repeat protein kinase, Alpha-glucan water dikinase, lipid-associated family protein, DNA binding/DNA-directed RNA polymerase, putative WD-40 repeat protein, SKP1 Interacting partner1, orf409, Os02g0439200 and Prp9 etc. These differential proteins were classified into the following functions: protein synthesis, signal transduction, transcription, metabolism, photosynthesis, cell structure, transporters, protein destination and strorage, unclear classification and unknown proteins.