Molecular Mechanisms Of Cd Tolerance And Accumulation In Metal Hyperaccumulator Sedum Alfredii

During the last decades, heavy metals has been becoming one of the most serious pollutants in soils due to industry and agriculture activities. Heavy metal polluted envrionment remediation by hyperaccumulator is the cutting edge of plant nutrition and environment bioremediation research. In present study, the molecular mechanisms of Cd hyperaccumulation in the chinese native Cd/Zn hyperaccumulator Sedum alfredii were studied by functionally characterizing Cd hyperaccumulation related genes by molecular biology technologies, such as RT-PCR, yeast heterologous expression, GFP fusion expression, and overexpression.The aim of the research is to provide theoretical foundation for breeding new plants tolerant to Cd pollution. The main results includes:1. In present study, we isolated and functionally characterized a P1B-type ATPase gene (SaHMA3) from S. alfredii Hance. The SaHMA3 alleles from hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) were characterized by constitutively expression in both shoot and root, and a tonoplast localized protein, but different in transport substrate specificity and expression level. SaHMA3h from HE plant is a Cd transporter. In contrast, SaHMA3n from NHE plants can transport both Zn and Cd. SaHMA3 showed significantly constitutively higher expression level in HE plants than NHE plants. Furthermore, the expression level of SaHMA3 in the shoots of HE was considerably higher than in the roots. Overexpression of SaHMA3h in tobacco plants significantly enhanced the Cd tolerance and accumulation, and especially increased the root sequestration of Cd. Taken together, our data postulate that SaHMA3 plays critical roles in Cd hyperaccumulation and hypertolerance in Cd hyperaccumulator S. alfredii Hance.2. In the present study, SaMT2, a type 2 metallothionein gene, was cloned from Cd/Zn co-hyperaccumulator S. alfredii Hance. SaMT2 encodes a putative peptide of 79 amino acid residues including two cysteine-rich domains. The transcript level of SaMT2 was higher in shoots than in roots of S. alfredii, and was significantly induced by Cd and Zn treatments. Yeast expression assay showed SaMT2 significantly enhanced Cd tolerance and accumulation in yeast. Ectopic expression of SaMT2 in tobacco enhanced Cd tolerance and accumulation in both shoots and roots of the transgenic plants. The transgenic plants had higher antioxidant enzyme activities and accumulated less H2O2 than wild-type plants under Cd treatment. Thus, SaMT2 could significantly enhance Cd tolerance and accumulation in transgenic tobacco plants by chelating metals and improving antioxidant system.3. Natural Resistance-Associated Macrophage Proteins (NRAMP) are a kind of Mn, Fe, Cd, Zn transporters that broadly distributed in plants, microbes and animals. These transporters play important roles in metal homostasis in plants. In the present study, five NRAMP transporters were idenfied from S. alfredii based on the transcriptome sequencing. Their full cDNA sequences were cloned by RACE technology and were analyzed using bioinformatics tools. RT-PCR, yeast complementation assay, GFP transient expression and overexpression were used to study the function NRAMP genes in S. alfredii. Their amino acid sequences showed considerable differences among each other. The similiarities among the NRAMP proteins were between 30% and 80%. The similiarity between SaNramp3 and SaNramp4 reached about 84%. As it is shown in NRAMPs of other plants, NRAMPs of S. alfredii showed 12 transmembrane motifs (TM) and a highly conservative transport motif which was between TM8 and TM9. The NRAMPs of S. alfredii could be divided into two group based on the phylogenic analysis. SaNrampl and SaNramp6 belonged to Group I. SaNramp2, SaNramp3 and SaNramp4 belonged to Group â…¡. The expression levels showed significant differences among the NRAMP genes. In S. aljredii, SaNramp3 showed the highest expression level. SaNrampl, SaNramp3 and SaNramp6 were selected for further studies due to their significant differences in the expression level between HE and NHE plants. SaNrampl, SaNramp3 and SaNramp6 were showed higher expression level in the shoots than in the roots of HE S. alfredii. In HE plants, the expression of these genes showed no significant changes under different metal treatments. In contrast, in NHE plants, SaNrampl could be significantly induced by Cd treatment and SaNramp3 could be induced by Zn, Cd and Fe deficience treatments. GFP transient expression in onion epidermal cells was used to study the subcellular localization of these genes. The results showed that SaNramp1 and SaNramp6 were localized at the plasma membrane and SaNramp3 was localized at the tonoplast. The function of NRAMP genes were analyzed using yeast complementation assay and plant overexpression assay. SaNrampl was a Cd, Mn and Zn transporter. SaNramp3 was a Cd, Mn and Fe transporter. SaNramp6 was a Cd and Mn transporter. However, these studies only elucidated the basic function of the NRAMP genes. Further studies were needed to clarify the roles of these genes in Cd hyperaccumulation of S. alfredii.