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.     

Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L. enhances cadmium and arsenic accumulation in tobacco

Phytochelatin synthase (PCS), the key enzyme involved in heavy metal detoxification and accumulation has been used from various sources to develop transgenic plants for the purpose of phytoremediation. However, some of the earlier studies provided contradictory results. Most of the PCS genes were isolated from plants that are not potential metal accumulators. In this study, we have isolated PCS gene from Ceratophyllum demersum cv. L. (CdPCS1), a submerged rootless aquatic macrophyte, which is considered as potential accumulator of heavy metals. The CdPCS1 cDNA of 1,757?bp encodes a polypeptide of 501 amino acid residues and differs from other known PCS with respect to the presence of a number of cysteine residues known for their interaction with heavy metals. Complementation of cad1-3 mutant of Arabidopsis deficient in PC (phytochelatin) biosynthesis by CdPCS1 suggests its role in the synthesis of PCs. Transgenic tobacco plants expressing CdPCS1 showed several-fold increased PC content and precursor non-protein thiols with enhanced accumulation of cadmium (Cd) and arsenic (As) without significant decrease in plant growth. We conclude that CdPCS1 encodes functional PCS and may be part of metal detoxification mechanism of the heavy metal accumulating plant C. demersum. KEY MESSAGE: Heterologous expression of PCS gene from C. demersum complements Arabidopsis cad1-3 mutant and leads to enhanced accumulation of Cd and As in transgenic tobacco.     

Functional characterisation of two phytochelatin synthases in rice (Oryza sativa cv. Milyang 117) that respond to cadmium stress

• Cadmium (Cd) is one of the most toxic heavy metals and a non‐essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised.
• To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl₂ using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15).
• The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector‐transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/‐15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/‐15 resulted in toxicity due to excess phytochelatin production in A. thaliana.
• These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.

     

Effects of Cadmium and Mixed Heavy Metals on Rice Growth in Liaoning,China

Joint effects of Cd and other heavy metals (Pb, Cu, Zn and As) on the growth and development of rice plants and the uptake of these heavy metals by rice were studied using the pot-culture method combined with chemical and statistical analyses. The results showed that the growth and development of rice plants were strongly influenced by the double-element combined pollution. There was an average decrease in the height of rice plants of 4.0–5.0 cm, and grain yield was decreased by 20.0–30.0%, compared with the control. The uptake of Cd by rice plants was promoted due to the interactions between Cd and the other heavy metals added to the soil. The Cd concentration in roots, stems/leaves and seeds increased 31.6–47.7, 16.7–61.5 and 19.6–78.6%, respectively. Due to interactions, uptake of Pb, Cu and Zn by roots and stems/leaves was inhibited, accumulation of Pb, Cu and Zn in seeds was increased, uptake of As by roots was promoted and uptake of As by stems/leaves was inhibited. In particular, the upward transporting ability of the heavy metals absorbed by rice plants was significantly increased.     

有机物料对污染土壤上水稻生长和重金属吸收的影响

采用盆栽试验,研究了施用有机碳源、菜籽饼和猪粪对污染土壤上水稻生长和重金属吸收特性的影响.结果表明： 施用菜籽饼和猪粪均能缓解重金属对水稻的毒害作用,促进水稻生长,显著增加地上部生物量和籽粒产量,降低糙米中重金属浓度;而有机碳源抑制水稻生长.与施用化肥相比,施用菜籽饼和猪粪处理的水稻籽粒产量分别增加128.3%和67.9%;施用菜籽饼处理的糙米Cd、Cu和Zn浓度分别降低47.6%、35.2%和21.5%,施用猪粪处理分别降低9.5%、21.2%和9.3%.土壤中DTPA提取态重金属浓度与水稻地上部生物量和重金属积累总量呈显著负相关.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

The OsNRAMP1 iron transporter is involved in Cd accumulation in rice

Cadmium (Cd) is a heavy metal toxic to humans and the accumulation of Cd in the rice grain is a major agricultural problem, particularly in Asia. The role of the iron transporter OsNRAMP1 in Cd uptake and transport in rice was investigated here. An OsNRAMP1:GFP fusion protein was localized to the plasma membrane in onion epidermal cells. The growth of yeast expressing OsNRAMP1 was impaired in the presence of Cd compared with yeast transformed with an empty vector. Moreover, the Cd content of OsNRAMP1-expressing yeast exceeded that of the vector control. The expression of OsNRAMP1 in the roots was higher in a high Cd-accumulating cultivar (Habataki) than a low Cd-accumulating cultivar (Sasanishiki) regardless of the presence of Cd, and the amino acid sequence of OsNRAMP1 showed 100% identity between Sasanishiki and Habataki. Over-expression of OsNRAMP1 in rice increased Cd accumulation in the leaves. These results suggest that OsNRAMP1 participates in cellular Cd uptake and Cd transport within plants, and the higher expression of OsNRAMP1 in the roots could lead to an increase in Cd accumulation in the shoots. Our results indicated that OsNRAMP1 is an important protein in high-level Cd accumulation in rice.     

碳酸钙对水稻吸收重金属(Pb、Cd、Zn)和As的影响

选用重金属(Pb、Cd、Zn)和As复合污染土壤进行水稻盆栽试验,结果表明,碳酸钙的添加显著提高了土壤pH值,显著降低了土壤中交换态Pb、Cd、Zn和As的含量,与对照相比,交换态Pb、Cd、Zn和As含量分别最多降低了98.35%,93.72%,98.52%和69.48%。碳酸钙对水稻根、稻谷干重和总生物量没有显著影响,添加量过高时显著降低了水稻分蘖数和茎叶干重,说明过量施用碳酸钙对水稻生长会产生负面作用。因为碳酸钙的添加,水稻植株各部位重金属Zn含量显著降低,糙米中Zn含量最多减少了34.95%;根、谷壳中Pb、Cd含量显著降低,但糙米中含量却未显著降低;水稻各部位As含量均没有显著降低。参照《食品中污染物限量》(GB2762—2012),试验糙米中Pb、Cd、无机As含量均未达到限量标准。显然,碳酸钙的添加降低了Pb、Cd、Zn的生物有效性(水稻根系对Pb、Cd、Zn的吸收累积减少),但并未有效地抑制Pb、Cd向糙米转运;碳酸钙显著降低了土壤的交换态As含量,但并未使土壤中As的生物有效性明显降低(水稻植株各部位的As含量并未显著减少)。     

Influence of Sulfur on Transcription of Genes Involved in Arsenic Accumulation in Rice Grains

Arsenic (As) contamination of rice grains affects millions of people worldwide. In this study, we found that sulfur application (20As+120S) decreased As concentration in rice grains by 44 % compared to grains without sulfur application (20As+0S). Importantly, sulfur application decreased arsenate [As(V)] and arsenite [As(III)] concentration in rice grains significantly, while there was no significant effect on dimethylarsenate (DMA) concentration. To elucidate the molecular basis of As accumulation in rice grains, we performed Illumina sequencing to acquire the differentially expressed genes induced by arsenate and sulfur treatments. By contrast with the control, the expression of 1,000 genes was found to be changed significantly, with 46 genes up-regulated and 954 genes down-regulated in grains grown in arsenate-contaminated soil (20As+0S). Between samples of control and arsenate together with sulfur treatment (20As+120S), 1,169 genes expressed significantly differently, with 16 genes up-regulated and 1,153 genes down-regulated. Sulfur application significantly changed the expression of genes involved in As metabolism in rice grains, significantly down-regulated phosphate transporter gene OsPT23 and aquaporin gene OsTIP4;2, while ABC transporter genes (OsABCG5, OsABCI7_2 and OsABC6) and phytochelatin synthase genes (OsPCS1, OsPCS3 and OsPCS13) were up-regulated. These results provide an insight into the molecular basis of how sulfur assimilation regulates As accumulation in rice grains.     

Molecular characterization of a rice metal tolerance protein, OsMTP1

Rice (Oryza sativa L. 'Nipponbare') cDNA subtractive suppression hybridization (SSH) libraries constructed using cadmium (Cd)-treated seedling roots were screened to isolate Cd-responsive genes. A cDNA clone, encoding the rice homolog of Metal Tolerance Protein (OsMTP1), was induced by Cd treatment. Plant MTPs belong to cation diffusion facilitator (CDF) protein family, which are widespread in bacteria, fungi, plants, and animals. OsMTP1 heterologous expression in yeast mutants showed that OsMTP1 was able to complement the mutant strains' hypersensitivity to Ni, Cd, and Zn, but not other metals including Co and Mn. OsMTP1 expression increased tolerance to Zn, Cd, and Ni in wild-type yeast BY4741 during the exponential growth phase. OsMTP1 fused to green fluorescent protein was localized in onion epidermal cell plasma membranes, consistent with an OsMTP1 function in heavy metal transporting. OsMTP1 dsRNAi mediated by transgenic assay in rice seedlings resulted in heavy metal sensitivity and changed the heavy metal accumulation in different organs of mature rice under low-concentration heavy metal stress. Taken together, our results show that OsMTP1 is a bivalent cation transporter localized in the cell membrane, which is necessary for efficient translocation of Zn, Cd and other heavy metals, and maintain ion homeostasis in plant.     

Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides

The dependence of phytochelatin synthase (gamma-glutamylcysteine dipeptidyltranspeptidase (PCS), EC ) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd(2+)- and Zn(2+)-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd.GS(2) or Zn.GS(2)) and free glutathione act as gamma-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both gamma-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.     

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl₂ over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.     

植物防御素调控水稻镉积累的新机制

镉是我国农产品的主要重金属污染物之一。随着我国土壤重金属污染问题日益突出, 包括稻米在内的农产品重金属超标时常发生。如何防控重金属在作物可食部位的积累, 在保证农产品安全的同时将农田重金属进行移除修复, 已成为我国农业生产急需解决的问题。最近, 中科院上海生命科学院植物生理生态所龚继明研究组和中国水稻所钱前研究组克隆到1个特异调控镉在水稻(Oryza sativa)叶片中积累的主效QTL基因CAL1。CAL1编码1个植物防御素类似蛋白, 通过与镉进行螯合, 将镉从维管束木质部薄壁细胞中分泌出来, 进入木质部参与长距离转运, 从而定向调控镉在水稻叶片等营养器官的积累而不影响籽粒镉的积累。该研究加深了人们对重金属镉在植物体内的转运和再分配机理的认识, 同时也为培育秸秆镉高积累而籽粒镉含量达标的“修复型”水稻品种提供有价值的新基因。研究成果具有重要的理论意义和应用价值。     

Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis

Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.     

The expression of heterologous Fe (III) phytosiderophore transporter HvYS1 in rice increases Fe uptake,translocation and seed loading and excludes heavy metals by selective Fe transport

Many metal transporters in plants are promiscuous, accommodating multiple divalent cations including some which are toxic to humans. Previous attempts to increase the iron (Fe) and zinc (Zn) content of rice endosperm by overexpressing different metal transporters have therefore led unintentionally to the accumulation of copper (Cu), manganese (Mn) and cadmium (Cd). Unlike other metal transporters, barley Yellow Stripe 1 (HvYS1) is specific for Fe. We investigated the mechanistic basis of this preference by constitutively expressing HvYS1 in rice under the control of the maize ubiquitin1 promoter and comparing the mobilization and loading of different metals. Plants expressing HvYS1 showed modest increases in Fe uptake, root‐to‐shoot translocation, seed accumulation and endosperm loading, but without any change in the uptake and root‐to‐shoot translocation of Zn, Mn or Cu, confirming the selective transport of Fe. The concentrations of Zn and Mn in the endosperm did not differ significantly between the wild‐type and HvYS1 lines, but the transgenic endosperm contained significantly lower concentrations of Cu. Furthermore, the transgenic lines showed a significantly reduced Cd uptake, root‐to‐shoot translocation and accumulation in the seeds. The underlying mechanism of metal uptake and translocation reflects the down‐regulation of promiscuous endogenous metal transporters revealing an internal feedback mechanism that limits seed loading with Fe. This promotes the preferential mobilization and loading of Fe, therefore displacing Cu and Cd in the seed.     

Role of the iron transporter OsNRAMP1 in cadmium uptake and accumulation in rice

The heavy metal cadmium (Cd) is toxic to humans, and its accumulation in rice grains is a major agricultural problem. Rice has seven putative metal transporter NRAMP genes, but microarray analysis showed that only OsNRAMP1 is highly up-regulated by iron (Fe) deficiency. OsNRAMP1 localized to the plasma membrane and transported Cd as well as Fe. OsNRAMP1 expression was observed mainly in roots and was higher in the roots of a high-Cd-accumulating cultivar (Habataki) than in those of a low-Cd-accumulating cultivar (Sasanishiki). The amino acid sequence of OsNRAMP1 in the Sasanishiki and Habataki cultivars was found to be 100% identical. These results suggest that OsNRAMP1 participates in cellular Cd uptake and that the differences observed in Cd accumulation among cultivars are because of differences in OsNRAMP1 expression levels in roots.     

The suppression of the glutelin storage protein gene in transgenic rice seeds results in a higher yield of recombinant protein

Glutelin is a major seed storage protein, accounting for 60?C80?% of the total endosperm protein content in rice. To test whether we could augment the expression of an introduced recombinant protein in rice by suppressing the glutelin gene, we generated transgenic glutelin RNAi (glu RNAi) rice seeds. RNA gel blot analyses confirmed that the endogenous glutelin gene was severely suppressed in these transgenic rice lines. RT-PCR analysis further revealed that all the members of glutelin multigene family were downregulated. Transgenic glu RNAi rice seeds expressing a recombinant red fluorescent protein (RFP) showed stronger fluorescence than seeds transformed with the RFP gene only. Western blot analysis further revealed that the relative accumulation of RFP in glu RNAi seeds was twofold higher than that in the RFP-only transgenic seeds. These results suggest that RNAi targeting of an endogenous storage protein could be of great utility in obtaining higher transgene expression in genetically engineered rice and other plant lines.     

The Effect of Bamboo Charcoal Application on Soil Nutrients and Heavy Metals in Rice

Soil heavy metal pollution is becoming more and more serious. Biomass charcoal application can play an important role in alleviating the toxicity of heavy metals in soils. Compared with other biochar, bamboo charcoal has more unique properties and may have a unique effect on heavy metal pollution. Zhejiang Province of Southeastern China is rich in bamboo resources. However, few studies related to bamboo charcoal application for heavy metal remediation in farmland were reported. In this study, four treatments with different amounts of bamboo charcoal application were set up through a field experiment, namely BC0 (no bamboo charcoal application), BC1 (2500 kg⋅ha⁻¹), BC2 (5000 kg⋅ha⁻¹), and BC3 (10000 kg⋅ha⁻¹), and each treatment was replicated three times. The results showed that (1) The application of bamboo charcoal significantly increased the soil pH and organic matter content. Compared with BC0, the pH and organic content of BC3 increased by 7.4% and 17.4% (P < 0.05) respectively. (2) The HCl-extractable Cd content of paddy soil in the BC1 treatment was significantly lower than other treatments (P < 0.05), and decreased by 15.3%, compared with BC0. The soil HCl-extractable Zn and Cu content did not differ significantly between treatments (P > 0.05). (3) With the increase of bamboo charcoal application, the Cd content in rice gradually decreased, the BC3 treatment significantly decreased by 39.0% (P < 0.05), and the Zn and Cu contents in rice did not differ significantly between treatments (P > 0.05), compared with BC0. (4) Soil pH, organic matter and Cd in rice seeds were significantly negatively correlated (P < 0.01). The heavy metal content in rice does not change with the change of heavy metal content with HCl-extractable state in soil. It means bamboo charcoal does not reduce heavy metal content in rice by simply declining the heavy metal content with HCl-extractable state. The mechanism of action is relatively complicated, and further study is needed.     

福建省水稻稻米重金属污染的对应分析

采用环境统计学方法布点采集福建省闽南、闽中和闽西北38个早晚稻稻谷样本,应用对应分析方法研究影响地区稻作类型的稻米Hg、As、Cd、Pb、Cu 5种重金属污染差异的主要因子,并通过主要因子揭示重金属与地区稻作类型间的关系.结果表明,福建省不同地区水稻稻米5种重金属含量与稻作类型存在较大差异,变异范围较大,其中超标率最高的是Pb,达100%,其次为Hg和Cd,分别为78.95%和50.0%,最低的是Cu,仅2.3%,而As无超标.Pb和Cd是导致地区稻作类型间差异的主要因子.聚类分析表明,38个稻谷样品可聚为7类,不同地区稻作类型,由于与污染源距离不一,受重金属污染的程度呈明显差异,体现不同的污染特征.可以认为,对不同地区稻作类型稻米重金属污染应采取地区控制与品种搭配相结合的策略,才能达到控制和消除稻米重金属污染的目的.