Transcriptomic analysis of cadmium stress response in the heavy metal hyperaccumulator Sedum alfredii Hance

Abstract

The Sedum alfredii Hance hyperaccumulating ecotype (HE) has the ability to hyperaccumulate cadmium (Cd), as well as zinc (Zn) and lead (Pb) in above-ground tissues. Although many physiological studies have been conducted with these plants, the molecular mechanisms underlying their hyper-tolerance to heavy metals are largely unknown. Here we report on the generation of 9.4 gigabases of adaptor-trimmed raw sequences and the assembly of 57,162 transcript contigs in S. alfredii Hance (HE) shoots by the combination of Roche 454 and Illumina/Solexa deep sequencing technologies. We also have functionally annotated the transcriptome and analyzed the transcriptome changes upon Cd hyperaccumulation in S. alfredii Hance (HE) shoots. There are 110 contigs and 123 contigs that were up-regulated (Fold Change ≥ 2.0) and down-regulated (Fold Change </=0.5) by chronic Cd treatment in S. alfredii Hance (HE) at q-value cutoff of 0.005, respectively. Quantitative RT-PCR was employed to compare gene expression patterns between S. alfredii Hance (HE) and non-hyperaccumulating ecotype (NHE). Our results demonstrated that several genes involved in cell wall modification, metal translocation and remobilization were more induced or constitutively expressed at higher levels in HE shoots than that in NHE shoots in response to Cd exposure. Together, our study provides large-scale expressed sequence information and genome-wide transcriptome profiling of Cd responses in S. alfredii Hance (HE) shoots.

Figure 1. Cd hyperaccumulation in S. alfredii Hance (HE).

(A) Growth of HE after exposure to 0 µM (Cont) or 100 µM Cd²⁺ (Cd) for 8 days. (B) Cd content (mg kg⁻¹ dry weight) in shoots and roots of HE. Bars depict SE (n = 3).

Figure 2. Gene Ontology (GO) analysis of S. alfredii Hance (HE) contigs.

GO distribution of (A) cellular component, (B) biological process and (C) molecular function.

Figure 3. Proportion of S. alfredii Hance (HE) contigs in different functional categories.

KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthology Groups (KOG) analysis was applied to the assembled contigs.

Figure 4. Homologous protein comparisons of S. alfredii Hance (HE) to other sequenced model plants.

(A) Distribution of S. alfredii homologous proteins identified in other plants using several BlastP E-value cutoffs. (B) Comparison of the number of homologs identified between S. alfredii and other plant proteomes with BlastP E-value cutoff of 1E-30. The number of contigs in each species is displayed in the same color as the species name.

Figure 5. Comparison of gene expression in S. alfredii Hance hyper-accumulation ecotype (HE) and non-hyperaccumulation ecotype (NHE).

(A) Transcriptional responses of two ecotypes to Cd exposure. Fold change of expression is the relative gene expression level in Cd treatment divided by the value in the control. (B) Relative gene expression levels of two ecotypes under normal growth condition. The relative gene expression is the expression level normalized to a constitutively expressed gene Actin. P-type metal ATPase HMA4: Sa_Contig11685; Zinc transporter ZIP1: Sa_Contig10290; Metal tolerance protein MTP3: Sa_Contig47062. The contig numbers for other genes are listed in Table 1. P values are indicated as follows: *P<0.05; **P<0.01; ***P<0.001. Bars depict SE (n = 3).