Cloning And Functional Analysis Of Selected Cd Hyperaccumulation Related Genes From Sedum Alfredii Hance

Hyperaccmulating ecotype (HE) of Sedum alfredii Hance is a new Zn/Cd hyperaccumulator which is native in China. In this study, cDNA-AFLP technology was used to identify Cd-regulated genes in HE plants; two photosynthesis related genes were analysed by transgenic tobacco; three metal transporters were isolated using RACE method, and the function of these transporters were investigated through yeast complementation assay, real-time RT-PCR and transgenic plants. The main results are as follows:1. Gene expression differences between a hyperaccumulating ecotype (HE) and a non-hyperaccumulating ecotype (NHE) of Sedum alfredii Hance were quantified using the technique of cDNA-amplified fragment length polymorphism (cDNA-AFLP). HE and NHE plants were treated with Cd levels which were not toxic under long exposure. Cadmium contents in roots and shoots of both ecotypes of Sedum alfredii Hance were measured at three time points (0 h,24 h and 8 d). Approximately seventy genes were identified to be differentially expressed in the HE plants as compared with the NHE plants, of which fifty eight were successfully cloned and sequenced. Of the fifty eight transcript-derived fragments (TDFs) searched in the GenBank database,31 (53.5%) had significant similarity to functional-known genes, 6 (10.3%) were homologous to uncharacterized genes, and the rest 21 (36.2%) had no match in the database. The homologous genes could be classified into seven functional groups:stress responding, cellular metabolism and organization, photosynthetic process, transport facilitation, expression regulator, transcriptional factor, and protein binding. In comparison of gene expression in Cd exposure times (0 h,24 h,8 d),37 genes were found to be down-regulated and 21 genes were up-regulated with Cd exposure.2. The effects of Cd on the chlorophyll fluorescence parameters in hyperaccumulating ecotype and non-hyperaccumulating ecotype of Sedum were studied. Significant loss of photosystemⅡ(PSⅡ) photochemical efficiency and non-photochemical quenching (NPQ) values were found in non-hyperaccumulating ecotype (NHE) of Sedum following Cd treatment. The PSⅡphotochemical efficiency of hyperaccumulating ecotype (HE) of Sedum was not affected and the NPQ value was significantly increased. Both ecotypes of Sedum accumulated more than two-fold higher photosystemⅡsubunit S (PsbS) transcript level than control plants when exposed to Cd. Over-expression of PsbS gene from HE plants (SaPsbS) in tobacco enhanced plant growth and the transgenic tobacco possessed higher shoot biomass and greater NPQ capacity than the wild type plants in the presence of 100μM Cd. Two lines of transgenic tobacco plants (P8 and P10) accumulated significantly higher Cd in shoots than wild type plants.3. The Lhcb2 gene from HE plants was up-regulated more than three-fold while the non-hyperaccumulator accumulated one or two-fold higher amount of the mRNA than control plants under different concentrations of Cd2+ for 24 h. Lhcb2 expression was up-regulated more than five-fold in NHE plants when exposed to 2μM Cd2+ or 50μM Zn2+ for 8 d and the hyperaccumulator had over two-fold more mRNA abundance than the control plants. Over-expression of SaLhcb2 increased the shoot biomass by 14%-41% and the root biomass by 21%-57% without Cd2+ treatment. Four transgenic tobacco lines (L5, L7, L10 and L11) possessed higher shoot biomass than WT plants with Cd2+. Four transgenic lines (L7, L8, L10 and L11) accumulated 6% to 35% higher Cd2+ amounts in shoots than the wild type plants.4. Two genes encoding a member of plant P1B-type ATPase family were isolated from HE and NHE plants and they were named as SaHMA2 and SnHMA2, respectively. Subcellular localization analysis revealed that SaHMA2 and SnHMA2 localized to the plasma membrane. Heterologous expression of SaHMA2 inΔzrc1 mutant strain conferred Zn and Cd tolerance while the expression of SnHMA2 only partially restored tolerance to Zn and Cd. Both SaHMA2 and SnHMA2 proteins were shown to be localized to the plasma membrane and microsomal fraction inΔzrc1 mutant strain. TheΔzrc1 mutant strain expressing SaHMA2 or SnHMA2 accumulated significantly less amount of Zn and Cd than the yeast cells containing vector control. In planta, the HE plants showed higher expression levels both in roots and shoots than the NHE plants. The expression level in roots of HE plants was 4-fold higher than that of NHE plants and the shoots of HE plants possessed more than 10-fold higher expression level than that of NHE plants. The expression pattern was different between two ecotypes of plants when subjected to Cd. The expression level in roots of HE plants was up-regulated while it was reduced in NHE plants. The Cd and Zn concenctrations in shoot of transgenic tobacco over-expressing SaHMA2 (SaH1) were both 1.6 times of the WT plants; and the Cd and Zn concenctrations in root of SaHl were 66% and 44% of the WT plants. Over-expression of SnHMA2 reduced the Cd concenctration in root but did not enhance the Cd/Zn translocation. These results indicate that the SaHMA2 is responsible for the high efficiency of Cd/Zn translocation in HE plants.5. Microscopic imaging of Zn in leaf cross section from Zn/Cd hyperaccumulating ecotype of Sedum alfredii Hance revealed that the vacuoles stored Zn. Two genes encoding metal tolerance proteins (MTP1) were isolated from hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) of Sedum alfredii Hance. Subcellular localization analysis revealed that SaMTP1 and SnMTP1 localized to the tonoplast in plant. Heterologous expression of SaMTP1 from HE plants and SnMTP1 from NHE plants suppressed Zn hypersensitivity inΔzrc1 yeast mutant to the same level. The SaMTP1 and SnMTP1 proteins localized to the vacuolar compartment in yeast cells. Yeast transformants expressing SaMTP1 or SnMTP1 accumulated 1-fold higher concentration of Zn than the yeast cells containing the vector control. There were significant differences in the gene transcript level and expression pattern between two ecotypes of plants. The HE plants had more than 88-fold higher SaMTP1 expression level in shoot than the NHE plants. SaMTP1 mRNA in roots of HE plants was 6-fold higher than that of NHE plants. In shoot, the transcript level of SaMTP1 was up-regulated 1-fold by Zn while the expression of SnMTP1 was slightly inhibited. In root, SnMTP1 mRNA level was more abundant than SaMTP1 mRNA under Zn treatment. Cd treatment caused decrease in the transcript levels of SaMTPl and SnMTP1 in shoot. Over-expression of SaMTP1 or SnMTP1 significantly enhanced the Zn concentrations in shoot and root of transgenic tobacco. The Zn concecntrations in shoot and root of transgenic tobacco over-expressing SaMTP1 (SaM2) were 3.4 and 2.6 times of the WT plant. These data suggested that the SaMTP1 is responsible for Zn tolerance and accumulation in Zn/Cd HE plants.6. Microscopic imaging of Cd in leaf cross section from Zn/Cd hyperaccumulating ecotype of Sedum alfredii Hance revealed that vacuoles stored large amount of Cd. Two genes encoding CAX2-like proteins were isolated from hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) of Sedum, and they were named as SaCAX2 and SnCAX2. The deduced amino acid sequences of these two proteins shared 96% identities. Yeast complementation assay revealed that both SaCAX2 and SnCAX2 suppressed Ca hypersensitivity in yeast mutant K667. However, the transport ability of SaCAX2 was lower than that of SnCAX2. The expression levels of two genes in two ecotypes of Sedum were similar under control condition. In HE plants, Zn/Cd treatments reduced transcript level of SaCAX2 gene in root. The SaCAX2 transcript level in shoot was up-regulated by Cd treatment while it was reduced by Zn. In NHE plants, Zn did not induce changes in SnCAX2 transcript level both in root and shoot. When exposed to Cd, the root accumulated more SnCAX2 mRNA level than control plants and the SnCAX2 mRNA level in shoot was significantly reduced. Over-expression of SaCAX2 or SnCAX2 enhanced the Cd concenctration by 31% or 17% in root of tobacco, and also enhanced the Cd concenctration by 16% or 12% in shoot. These data indicated that SaCAX2 should be one of the transporters responsible for Cd sequestration in HE plants.