Phytochelatin synthase OsPCS1 plays a crucial role in reducing arsenic levels in rice grains

Reduction of the level of arsenic (As) in rice grains is an important challenge for agriculture. A recent study reported that the OsABCC1 transporter prevents the accumulation of As in grains by sequestering As–phytochelatin complexes into vacuoles in the upper nodes. However, how phytochelatins are provided in response to As remains unclear. Here, we show that the phytochelatin synthase OsPCS1 plays a crucial role in reducing As levels in rice grains. Using a forward genetic approach, we isolated two rice mutants (has1 and has2) in which As levels were much higher in grains but significantly lower in node I compared with the wild type. Map‐based cloning identified the genes responsible as OsABCC1 in has1 and OsPCS1 in has2. The levels of As in grains and node I were similar between the two mutants, suggesting that OsABCC1 preferentially cooperates with OsPCS1 to sequester As, although rice has another phytochelatin synthase, OsPCS2. An in vitro phytochelatin synthesis assay indicated that OsPCS1 was more sensitive to activation by As than by cadmium, whereas OsPCS2 was more weakly activated by As than by cadmium. Transgenic plants highly expressing OsPCS1 showed significantly lower As levels in grains than did wild‐type plants. Our results provide new knowledge of the relative contribution of rice PCS paralogs to As sequestration and suggest a good candidate tool to reduce As levels in rice grains.     

A novel plant cysteine-rich peptide family conferring cadmium tolerance to yeast and plants

We have identified a novel cDNA clone, termed DcCDT1, from Digitaria ciliaris, that confers cadmium (Cd)-tolerance to yeast (Saccharomyces cerevisiae). The gene encodes a predicted peptide of 55 amino acid residues of which 15 (27.3%) are cysteine residues. We found that monocotyledonous plants possess multiple DcCDT1 homologues, for example rice contains five DcCDT1 homologues (designated OsCDT1∼5), whereas dicotyledonous plants, including Arabidopsis thaliana, Brassica rapa, poplar (Populus tremula × Populus alba) and Picea sitchensis, appear to possess only a single homologue. GFP fusion experiments demonstrate that DcCDT1 and OsCDT1 are targeted to both the plant cytoplasmic membranes and cell walls. Constitutive expression of DcCDT1 or OsCDT1 confers Cd-tolerance to transgenic A. thaliana plants by lowering the accumulation of Cd in the cells. The functions of the DcCDT1 family members are discussed in the light of these findings.Key words: cadmium, cysteine-rich peptide, hypoaccumulation, phytoremediation, toleranceCadmium (Cd) is a highly toxic transition metal, and is nonessential for almost all living organisms.¹ Therefore, Cd pollution of the earth''s environment could potentially cause serious problems for both the global ecosystem and human health.² Indeed, we have a tragic history brought about by Cd poisoning, with patients suffering kidney failure and unbearable pain in the joints and spine due to bone softening.^3,4 Despite the numerous laws that have been enacted to prevent further Cd contamination, yet pollution with heavy metals including Cd is still increasing on a global scale. As the control of Cd contamination of foodstuffs is expected to become extremely severe, remediation of Cd-contaminated soils is an issue that needs to be urgently addressed. One countermeasure strategy is the use of plants in phytoremediation⁵ which, despite its tremendous potential, still requires vast improvements before it can be promoted as an effective and established technology. Such improvements will necessitate the identification and development of novel and useful ‘molecular resources’. As a step towards this aim, we have screened cDNA libraries derived from natural habitat plants growing in a former mining site and isolated numerous candidate genes that could confer Cd tolerance to Cd-hypersensitive yeast mutant cells.⁶ Of these genes, we chose DcCDT1 (Digitaria ciliaris cadmium tolerance 1) for further analysis as it encodes a novel 55-amino acid-peptide product containing 15 cysteine (Cys) residues, and because several other Cys- rich peptides are known to function as heavy metal chelators.^7,8 Rice plants possess five DcCDT1 homologues, designated OsCDT1∼5, while other monocotyledonous plant species, such as maize and barley, also contain multiple DcCDT1 homologues. In contrast, Arabidopsis thaliana appears to contain only a single DcCDT1 homologue (accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_202281","term_id":"1063678830"}}NM_202281, At1g52827) with an open-reading frame (nucleotide positions 29–178) encoding a 49-amino acid peptide of sequence,MKAPPQQEMTYYDNVKKRQDEQGCLFATFYALFCCCCCYEKCKCCCCCV,whereas the database predicts a peptide (encoded by an alternative open-reading frame between nucleotides 63–251) consisting of 62-amino acids, MTM SRN GKT NKA AYS QRF TRC SVA VAA TRS ASV VAA AFD FYI CII IST LLS LIV SLA SQL LF, which is of unknown function and shows no similarity to DcCDT1. The 49-amino acid-peptide sequence (here termed AtCDT1) shares a high level of identity with DcCDT1 and OsCDT1, and these all contain 11 conserved Cys residues clustered in their carboxy-distal regions (Fig. 1A). Furthermore, as with DcCDT1 and OsCDT1, constitutive expression of AtCDT1 confers Cd-tolerance to S. cerevisiae (Fig. 1B), confirming that A. thaliana possesses a functional DcCDT1 counterpart. The question thus arises if other dicotyledonous plants also possess homologous gene(s). It appears that they do since we found two other examples, a Populus EST clone (accession number {"type":"entrez-nucleotide","attrs":{"text":"CU225257","term_id":"160955042"}}CU225257) and also a Brassica rapa subsp. pekinensis clone (accession number {"type":"entrez-nucleotide","attrs":{"text":"AC189268","term_id":"199579954"}}AC189268), that encode DcCDT1 homologues. However, further analyses will be required to determine whether these plants carry a single gene like Arabidopsis or a small gene family as in rice plants.Open in a separate windowFigure 1DcCDT1 and its homologues are a highly-conserved, Cys-rich family of proteins and the Arabidopsis homologue, AtCDT1, confers Cd-tolerant to yeast cells. (A) Amino acid alignment of DcCDT1, OsCDT1 and AtCDT1. OsCDT1 is one of the rice DcCDT1 homologues, and AtCDT1 is the Arabidopsis thaliana-derived 49-amino acid-peptide. Identical and conserved amino acid residues are highlighted in black and gray backgrounds, respectively. Conserved Cys residues are marked with an asterisk. (B) Constitutive expression of the nucleotide sequence encoding AtCDT1 confers Cd tolerance to yeast cells. S. cerevisiae strain, DTY165 (genotypic markers MATα, ura3-52, leu2-3,-112, his3-delta200, trp1-delta901, lys2-801, suc2-delta9), and its ycf1^6,17-mutant strain, DTY167 (genotypic markers MATα, ura3-52, leu2-3,-112, his3-delta200, trp1-delta901, lys2-801, suc2-delta9, ycf1::hisG), cells were transformed with the control vector p112A1NE or p112A1NE::AtCDT1.¹⁸ The transformants obtained were grown in yeast liquid culture media, and the cell density of the cultures then adjusted to an optical density of 1.0 at 600 nm (OD₆₀₀). Aliquots of 5 µl of the serial dilutions (1, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵) were then plated onto Synthetic Dropout (SD)-Trp media without CdCl₂ (left) or containing 60 µM CdCl₂ (right).Several different plant components contribute to heavy metal homeostasis and detoxification. Among these are the low molecular mass (4–14 kDa) metallothioneins that contain a high ratio of Cys residues, and the small peptides known as phytochelatins that have the general structure, (γ-Glu-Cys)_n-Gly.^7–10 Other Cys-rich plant proteins involved in Cd tolerance and detoxification have also been reported.^11,12 However, compared to all these, DcCDT1 and its homologues are unique and distinctive in both their peptide lengths (49–60 amino acids) and arrangement of Cys residues in the CL-(Y/F)-A-(C/T)-X5-CC-(F/C)-CCYE-(T/K)-C-(E/K)-C( CLDCL or delete)-CCCC consensus sequence.As with other non-essential heavy metals, Cd can be detoxified by a variety of mechanisms, including secretion, compartmentalization, or chelation by metal ligands.^13–16 DcCDT1 and OsCDT1 confer Cd-tolerance to both yeast and Arabidopsis via a reduction in their cellular Cd contents. Both proteins also appear to be localized to the plant cell surface, including the cell walls, as judged by our GFP-fusion experiments. Based on these findings, we propose several possible functions for this novel peptide family. In one mechanism, the DcCDT1 family proteins chelate Cd at the cellular surface and prevent further Cd entry into the cells. In an alternative mechanism, the intracellular-formed DcCDT1-Cd complex is secreted out from the cells via an unknown mechanism. Induced expression of DcCDT1 in ¹⁰⁹Cd-preloaded cells may well allow us to distinguish between these two possibilities.A final question that needs to be addressed is whether the genes encoding this family of proteins can be potentially useful genetic resources for phytoremediation. OsCDT1, one of the five rice DcCDT1 homologues, reduces the Cd contents of yeast and Arabidopsis cells. Therefore, from the viewpoint of food safety, this protein family may be useful since constitutive root-specific expression of the genes may contribute to reduced Cd accumulation in the edible plant parts. Alternatively, silencing of all DcCDT1 homologues in rice could plausibly result in the hyperaccumulation of Cd and the use of these plants in phytoremediation strategies.     

Arsenic biotransformation by Streptomyces sp. isolated from rice rhizosphere

Isolation and functional analysis of microbes mediating the methylation of arsenic (As) in paddy soils is important for understanding the origin of dimethylarsinic acid (DMA) in rice grains. Here, we isolated from the rice rhizosphere a unique bacterium responsible for As methylation. Strain GSRB54, which was isolated from the roots of rice plants grown in As‐contaminated paddy soil under anaerobic conditions, was classified into the genus Streptomyces by 16S ribosomal RNA sequencing. Sequence analysis of the arsenite S‐adenosylmethionine methyltransferase (arsM) gene revealed that GSRB54 arsM was phylogenetically different from known arsM genes in other bacteria. This strain produced DMA and monomethylarsonic acid when cultured in liquid medium containing arsenite [As(III)]. Heterologous expression of GSRB54 arsM in Escherichia coli promoted methylation of As(III) by converting it into DMA and trimethylarsine oxide. These results demonstrate that strain GSRB54 has a strong ability to methylate As. In addition, DMA was detected in the shoots of rice grown in liquid medium inoculated with GSRB54 and containing As(III). Since Streptomyces are generally aerobic bacteria, we speculate that strain GSRB54 inhabits the oxidative zone around roots of paddy rice and is associated with DMA accumulation in rice grains through As methylation in the rice rhizosphere.     

Citrinolactones A, B and C, and Sclerotinin C, plant growth regulators from Penicillium citrinum

New plant growth regulators, named citrinolactones A (1), B (2) and C (3) and sclerotinin C (4), were isolated from Penicillium citrinum and their structures established by spectroscopic methods including 2D NMR. Compounds 1 and 4 increased root growth in proportion to their concentration from 3 to 300 mg/l. In contrast, 2 completely inhibited root growth at a concentration of 300 mg/l and 3 did not show any effect on root growth in a concentration range of 3-300 mg/l.     

Novel Cysteine-Rich Peptides from Digitaria ciliaris and Oryza sativa Enhance Tolerance to Cadmium by Limiting its Cellular Accumulation

By means of functional screening using the cadmium (Cd)-sensitiveycf1 yeast mutant, we have isolated a novel cDNA clone, DcCDT1,from Digitaria ciliaris growing in a former mining area in northernJapan, and have shown that it confers Cd tolerance to the yeastcells, which accumulated almost 2-fold lower Cd levels thancontrol cells. The 521 bp DcCDT1 cDNA contains an open readingframe of 168 bp and encodes a deduced peptide, DcCDT1, thatis 55 amino acid residues in length, of which 15 (27.3%) arecysteine residues. Five DcCDT1 homologs (here termed OsCDT1–OsCDT5)have been identified in rice, and all of them were up-regulatedto varying degrees in the above-ground tissues by CdCl₂ treatment.Localization of green fluorescent protein fusions suggests thatDcCDT1 and OsCDT1 are targeted to both cytoplasmic membranesand cell walls of plant cells. Transgenic Arabidopsis thalianaplants overexpressing DcCDT1 or OsCDT1 displayed a Cd-tolerantphenotype and, consistent with our yeast data, accumulated loweramounts of Cd when grown on CdCl₂. Collectively, our data suggestthat DcCDT1 and OsCDT1 function to prevent entry of Cd intoyeast and plant cells and thereby enhance their Cd tolerance.