A γ-glutamyl cyclotransferase protects Arabidopsis plants from heavy metal toxicity by recycling glutamate to maintain glutathione homeostasis.

Plants detoxify toxic metals through a GSH-dependent pathway. GSH homeostasis is maintained by the γ-glutamyl cycle, which involves GSH synthesis and degradation and the recycling of component amino acids. The enzyme γ-glutamyl cyclotransferase (GGCT) is involved in Glu recycling, but the gene(s) encoding GGCT has not been identified in plants. Here, we report that an Arabidopsis thaliana protein with a cation transport regulator-like domain, hereafter referred to as GGCT2;1, functions as γ-glutamyl cyclotransferase. Heterologous expression of GGCT2;1 in Saccharomyces cerevisiae produced phenotypes that were consistent with decreased GSH content attributable to either GSH degradation or the diversion of γ-glutamyl peptides to produce 5-oxoproline (5-OP). 5-OP levels were further increased by the addition of arsenite and GSH to the medium, indicating that GGCT2;1 participates in the cellular response to arsenic (As) via GSH degradation. Recombinant GGCT2;1 converted both GSH and γ-glutamyl Ala to 5-OP in vitro. GGCT2;1 transcripts were upregulated in As-treated Arabidopsis, and ggct2;1 knockout mutants were more tolerant to As and cadmium than the wild type. Overexpression of GGCT2;1 in Arabidopsis resulted in the accumulation of 5-OP. Under As toxicity, the overexpression lines showed minimal changes in de novo Glu synthesis, while the ggct2;1 mutant increased nitrogen assimilation by severalfold, resulting in a very low As/N ratio in tissue. Thus, our results suggest that GGCT2;1 ensures sufficient GSH turnover during abiotic stress by recycling Glu.

Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells.

BACKGROUND: Taxol(®) (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control. RESULTS: Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. CONCLUSIONS: This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

Expression profiling of Crambe abyssinica under arsenate stress identifies genes and gene networks involved in arsenic metabolism and detoxification.

BACKGROUND: Arsenic contamination is widespread throughout the world and this toxic metalloid is known to cause cancers of organs such as liver, kidney, skin, and lung in human. In spite of a recent surge in arsenic related studies, we are still far from a comprehensive understanding of arsenic uptake, detoxification, and sequestration in plants. Crambe abyssinica, commonly known as 'abyssinian mustard', is a non-food, high biomass oil seed crop that is naturally tolerant to heavy metals. Moreover, it accumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae family. Thus, C. abyssinica has great potential to be utilized as an ideal inedible crop for phytoremediation of heavy metals and metalloids. However, the mechanism of arsenic metabolism in higher plants, including C. abyssinica, remains elusive. RESULTS: To identify the differentially expressed transcripts and the pathways involved in arsenic metabolism and detoxification, C. abyssinica plants were subjected to arsenate stress and a PCR-Select Suppression Subtraction Hybridization (SSH) approach was employed. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The transcripts corresponding to the subtracted cDNAs showed strong upregulation by arsenate stress as confirmed by the semi-quantitative RT-PCR. CONCLUSIONS: Our study revealed novel insights into the plant defense mechanisms and the regulation of genes and gene networks in response to arsenate toxicity. The differential expression of transcripts encoding glutathione-S-transferases, antioxidants, sulfur metabolism, heat-shock proteins, metal transporters, and enzymes in the ubiquitination pathway of protein degradation as well as several unknown novel proteins serve as molecular evidence for the physiological responses to arsenate stress in plants. Additionally, many of these cDNA clones showing strong upregulation due to arsenate stress could be used as valuable markers. Further characterization of these differentially expressed genes would be useful to develop novel strategies for efficient phytoremediation as well as for engineering arsenic tolerant crops with reduced arsenic translocation to the edible parts of plants.

Understanding the physiological and molecular mechanism of persistent organic pollutant uptake and detoxification in cucurbit species (zucchini and squash).

Cucurbita pepo ssp pepo (zucchini) roots phytoextract significant amounts of persistent organic pollutants (POPs) from soil, followed by effective translocation to aboveground tissues. The closely related C. pepo ssp ovifera (squash) does not have this ability. In a DDE-contaminated field soil, zucchini roots and stems contained 3.6 and 6.6-fold greater contaminant than did squash tissues, respectively, and zucchini phytoextracted 12-times more DDE from soil than squash. In batch hydroponics, squash was significantly more sensitive to DDE (2-20 mg/L) exposure; 4 mg/L DDE significantly reduced squash biomass (14%) whereas for zucchini, biomass reductions were observed at 20 mg/L (20%). PCR select Suppression Subtraction Hybridization was used to identify differentially expressed genes in DDE treated zucchini relative to DDE treated squash or non-treated zucchini. After differential screening to eliminate false positives, unique cDNA clones were sequenced. Out of 40 shoot cDNA sequences, 34 cDNAs have homology to parts of phloem filament protein 1 (PP1). Out of 6 cDNAs from the root tissue, two cDNAs are similar to cytochrome P450 like proteins, and one cDNA matches a putative senescence associated protein. From the DDE exposed zucchini seedlings cDNA library, out of 22 differentially expressed genes, 14 cDNAs were found to have homology with genes involved in abiotic stresses, signaling, lipid metabolism, and photosynthesis. A large number of cDNA sequences were found to encode novel unknown proteins that may be involved in uncharacterized pathways of DDE metabolism in plants. A semiquantitative RT-PCR analysis of isolated genes confirmed up-regulation in response to DDE exposure.

Effect of amendments on the extractability, retention and plant uptake of metals on a sewage-irrigated soil.

Laboratory and greenhouse experiments were conducted to evaluate the effects of farmyard manure (FYM), CaCO(3) and single superphosphate (SSP) on retention and availability of Zn, Cu and Ni in sewage-irrigated soil. We also assessed the suitability of 0.05M EDTA for predicting the effectiveness of these amendments in reducing the phytoavailability of metals. Results indicated that EDTA could successfully predict the phytoavailability of Zn and Ni in amended soil, whereas it failed in case of Cu. By and large, application of CaCO(3), either alone or in combination with FYM had a positive effect on the retention of Zn, Cu and Ni in soil. Application of CaCO(3) alone or in combination with FYM was equally effective in reducing the Zn content in lettuce, whereas sole application of CaCO(3) significantly reduced Ni content. However, only SSP was found to be effective in reducing the Cu content in lettuce.

Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds.

BACKGROUND: Camelina sativa is an emerging dedicated oilseed crop designed for biofuel and biodiesel applications as well as a source for edible and general-purpose oils. Such valuable oilseed crop is subjected to plant breeding programs and is suggested for large-scale production of better seed and oil quality. To accomplish this objective and to further enhance its oil content, a better understanding of lipid metabolism at the molecular level in this plant is critical. Here, we applied tissue transcriptomics and lipid composition analysis to identify and profile the genes and gene networks associated with triacylglycerol (TAG) biosynthesis, and to investigate how those genes are interacting to determine the quantity and quality of Camelina oil during seed development. RESULTS: Our Camelina transcriptome data analysis revealed an approximate of 57,854 and 57,973 genes actively expressing in developing seeds (RPKM ≥ 0.1) at 10-15 (Cs-14) and 16-21 (Cs-21) days after flowering (DAF), respectively. Of these, 7932 genes showed temporal and differential gene expression during the seed development (log2 fold change ≥1.5 or ≤-1.5; P ≤ 0.05). The differentially expressed genes (DEGs) were annotated and were found to be involved in distinct functional categories and metabolic pathways. Furthermore, performing quantitative real-time PCR for selected candidate genes associated with TAG biosynthesis validated RNA-seq data. Our results showed strong positive correlations between the expression abundance measured using both qPCR and RNA-Seq technologies. Furthermore, the analysis of fatty-acid content and composition revealed major changes throughout seed development, with the amount of oil accumulate rapidly at early mid seed development stages (from 16-28 DAF onwards), while no important changes were observed in the fatty-acid profile between seeds at 28 DAF and mature seeds. CONCLUSIONS: This study is highly useful for understanding the regulation of TAG biosynthesis and identifying the rate-limiting steps in TAG pathways at seed development stages, providing a precise selection of candidate genes for developing Camelina varieties with improved seed and oil yields.

Identifying genes and gene networks involved in chromium metabolism and detoxification in Crambe abyssinica.

Chromium pollution is a serious environmental problem with few cost-effective remediation strategies available. Crambe abyssinica (a member of Brassicaseae), a non-food, fast growing high biomass crop, is an ideal candidate for phytoremediation of heavy metals contaminated soils. The present study used a PCR-Select Suppression Subtraction Hybridization approach in C. abyssinica to isolate differentially expressed genes in response to Cr exposure. A total of 72 differentially expressed subtracted cDNAs were sequenced and found to represent 43 genes. The subtracted cDNAs suggest that Cr stress significantly affects pathways related to stress/defense, ion transporters, sulfur assimilation, cell signaling, protein degradation, photosynthesis and cell metabolism. The regulation of these genes in response to Cr exposure was further confirmed by semi-quantitative RT-PCR. Characterization of these differentially expressed genes may enable the engineering of non-food, high-biomass plants, including C. abyssinica, for phytoremediation of Cr-contaminated soils and sediments.