Isolation and expression analysis of cadmium-induced genes from Cd/Mn hyperaccumulator Phytolacca americana in response to high Cd exposure.

Phytolacca americana is recognised as a hyperaccumulator that accumulates cadmium (Cd) and manganese (Mn). Although most studies have provided abundant physiological evidence, little is known about the molecular mechanisms of Cd accumulation in P. americana. In this study, Cd-induced genes were isolated using suppression subtractive hybridisation (SSH) library construction, and gene expression patterns under Cd stress were quantified using real-time quantitative PCR. The functions of PaGST, PaMT and PaFe-SOD were confirmed in transformant yeast. Reactive oxygen species (ROS) formation and cell death in root tips were detected, and SOD and POD activities in leaf tissue were also analysed. There were about 447 expressed sequence tags (ESTs) identified and confirmed. GO analysis showed those genes were mainly involved in metabolism, cell stress and defence, transcription and translation, signal transduction, transport, energy and ion transport, which formed the basis for a molecular understanding of P. americana Cd tolerance mechanisms. Cd also stimulated ROS formation and modified the antioxidant systems. Taken together, our results indicate that ROS formation and Cd-induced gene expression favour P. americana tolerance by activating the defence system and permitting subsequent adaptation to Cd toxicity.

Transcriptome analysis of Phytolacca americana L. in response to cadmium stress.

Phytolacca americana L. (pokeweed) has metal phytoremediation potential, but little is known about its metal accumulation-related genes. In this study, the de novo sequencing of total RNA produced 53.15 million reads covering 10.63 gigabases of transcriptome raw data in cadmium (Cd)-treated and untreated pokeweed. Of the 97,502 assembled unigenes, 42,197 had significant matches in a public database and were annotated accordingly. An expression level comparison between the samples revealed 1515 differentially expressed genes (DEGs), 923 down- and 592 up-regulated under Cd treatment. A KEGG pathway enrichment analysis of DEGs revealed that they were involved in 72 metabolism pathways, with photosynthesis, phenylalanine metabolism, ribosome, phenylpropanoid biosynthesis, flavonoid biosynthesis and carbon fixation in photosynthetic organisms containing 24, 18, 72, 14, 7 and 15 genes, respectively. Genes related to heavy metal tolerance, absorption, transport and accumulation were also identified, including 11 expansins, 8 nicotianamine synthases, 6 aquaporins, 4 ZRT/IRT-like proteins, 3 ABC transporters and 3 metallothioneins. The gene expression results of 12 randomly selected DEGs were validated using quantitative real-time PCR, and showed different response patterns to Cd in their roots, stems and leaves. These results may be helpful in increasing our understanding of heavy metal hyperaccumulators and in future phytoremediation applications.

A small RNA targets pokeweed antiviral protein transcript.

Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.

[Antioxidative enzymes play key roles in cadmium tolerance of Phytolacca americana].

Phytolacca americana L. has the capacity to take up and accumulate to very high levels heavy metals such as Mn and Cd, and is used for phytoextraction of heavy metal contaminated soils. The role of antioxidative enzyme of Phytolacca americana in response to Cd stress is unknown. The 6-week-old seedlings of Phytolacca americana were exposed to half strength Hoagland solution with 200 micromol/L CdCl2 or 400 micromol/L CdCl2 for 4 days. The content of H2O2 and MDA, and electrolyte leakage increased, while the photosynthetic rate decreased, indicated that the oxidative damage induced by Cd stress in Phytolacca americana was one of the metal toxicity mechanism. The activities of SOD and POD increased rapidly with elevated Cd concentration and exposure time, CAT activity was stable in response to 200 micromol/L CdCl2 stress, and increased only at 3 d later upon 400 micromol/L CdCl2, treatment. Suggested that the enzymatic antioxidation capacity played important role in Cd tolerance of hyperaccumulator plant.

Cadmium accumulation and distribution in populations of Phytolacca americana L. and the role of transpiration.

The concentrations of heavy metals in Phytolacca americana L. and corresponding soil samples from three contaminated sites and an uncontaminated site were studied. Hydroponic experiments were also conducted to investigate the Cd uptake ability and mechanism of P. americana. The field results showed that the average Cd concentration was 42 mg kg(-1) in P. americana leaves, with the highest concentration of 402 mg kg(-1) found at Datianwan. A significant relationship was observed between the concentrations of Cd in leaves and those of corresponding soils on a logarithmic scale. Under laboratory hydroponic conditions, the maximum Cd concentration in aerial tissues of P. americana was 637 mg kg(-1), under treatment with 100 microM Cd. The population from the uncontaminated site (Zijinshan) also had a remarkable ability to accumulate Cd in shoots to concentrations well in excess of 100 microM in the hydroponic experiment, similar to the population from contaminated site, suggesting that Cd accumulation is a constitutive trait of P. americana. In the presence of 100 microM Cd, the addition of polyethylene glycol decreased leaf transpiration, the shoot Cd concentration, and the shoot/root Cd concentration ratio. There was a significantly positive relationship between the shoot Cd concentration and the leaf transpiration of P. americana. A similar significant positive correlation was also obtained between the shoot/root Cd concentration and leaf transpiration. Moreover, pretreatment with 5 microM abscisic acid or 5 microM HgCl(2) significantly decreased the Cd concentration in P. americana shoots. These results suggest that transpiration has an important role in Cd accumulation in shoots of P. americana.