Silicon induces phytochelatin and ROS scavengers facilitating cadmium detoxification in rice

• Cadmium (Cd) is detrimental to crops and the environment. This work examines the natural mechanisms underlying silicon‐ (Si‐)directed Cd detoxification in rice plants.
• The addition of Si to plants under Cd stress caused significant improvements in morphological parameters, chlorophyll score, F_v/F_m and total soluble protein concentration compared to controls, confirming that Si is able to ameliorate Cd‐induced damage in rice plants. This morpho‐physiological evidence was correlated with decreased cell death and electrolyte leakage after Si application.
• The results showed no critical changes in root Cd concentration, while shoot Cd decreased significantly after Si supplementation in comparison with Cd‐stressed rice. Additionally, expression of Cd transporters (OsNRAMP5 and OsHMA2) was significantly down‐regulated while the concentration of phytochelatin, cysteine and glutathione, together with expression of OsPCS1 (phytochelatin synthase) in roots of Cd‐stressed rice was significantly induced when subjected to Si treatment. This confirms that the alleviation of Cd stress is not only limited to the down‐regulation of Cd transporters but also closely related to the phytochelatin‐driven vacuolar storage of Cd in rice roots.
• The enzymatic analysis further revealed the role of SOD and GR enzymes in protecting rice plants from Cd‐induced oxidative harm. These findings suggest a mechanistic basis in rice plants for Si‐mediated mitigation of Cd stress.

     

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.     

Calcium acetate‐induced reduction of cadmium accumulation in Oryza sativa: Expression of auto‐inhibited calcium‐ATPase and cadmium transporters

• Calcium (Ca) signalling has an essential role in regulating plant responses to various abiotic stresses.
• This study applied Ca in various forms (Ca acetate and CaCl₂) and concentrations to reduce cadmium (Cd) concentration in rice and propose a possible mechanism through which Ca acts to control the Cd concentration in rice.
• The results showed that supplementation of Cd‐contaminated soil with Ca acetate reduced the Cd concentration in rice after exposure for 7 days in both hydroponic and soil conditions. The possible involvement of the auto‐inhibited Ca²⁺‐ATPase gene (ACA) might act to control the primary signal of the Cd stress response. The messages from ACA3 and ACA13 tended to up‐regulate the low‐affinity cation transporter (OsLCT1) and down‐regulate Cd uptake and the Cd translocation transporter, including the genes, natural resistance‐associated macrophage protein 5 (Nramp5) and Zn/Cd‐transporting ATPase 2 (HMA2), which resulted in a reduction in the Cd concentration in rice. After cultivation for 120 days, the application of Ca acetate into Cd‐contaminated soil inhibited Cd uptake of rice.
• Increasing the Ca acetate concentration in the soil lowered the Cd concentration in rice shoots and grains. Moreover, Ca acetate maintained rice productivity and quality whereas both aspects decreased under Cd stress.

     

厚藤cDNA文库的构建和重金属镉耐受相关基因的筛选

厚藤（Ipomoea pes-caprae（L.） Sweet）是一种具有重要生态、观赏及药用价值的沙滩植物,对重金属镉（Cd）具有一定的富集能力,可作为Cd污染滨海地区的修复植物进行引种栽植和利用。本研究通过Gateway技术构建厚藤的cDNA文库,将该文库质粒转化酵母对Cd敏感的突变株ycf1△,采用全长cDNA过表达基因捕获系统（FOX）筛选厚藤重金属Cd胁迫耐受相关基因,并采用酵母互补实验进行基因的功能验证。本研究获得了2个能够恢复ycf1△对Cd敏感表型的重组质粒,经测序分析,该重组质粒包含的cDNA全长序列分别对应厚藤植物螯合肽合成酶基因（phytochelatin synthase）和金属硫蛋白基因（metallothionein）,分别将其命名为IpPCS和IpMT,通过功能分析,初步认定该基因为编码Cd耐受和解毒相关蛋白的候选基因。     

Expression of Caenorhabditis elegans PCS in the AtPCS1‐deficient Arabidopsis thaliana cad1‐3 mutant separates the metal tolerance and non‐host resistance functions of phytochelatin synthases

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.     

Semi‐dominant mutation in the cysteine‐rich receptor‐like kinase gene,ALS1, conducts constitutive defence response in rice

• Plants have evolved a sophisticated two‐branch defence system to prevent the growth and spread of pathogen infection. The novel Cys‐rich repeat (CRR) containing receptor‐like kinases, known as CRKs, were reported to mediate defence resistance in plants. For rice, there are only two reports of CRKs. A semi‐dominant lesion mimic mutant als1 (apoptosis leaf and sheath 1) in rice was identified to demonstrate spontaneous lesions on the leaf blade and sheath.
• A map‐based cloning strategy was used for fine mapping and cloning of ALS1, which was confirmed to be a typical CRK in rice. Functional studies of ALS1 were conducted, including phylogenetic analysis, expression analysis, subcellular location and blast resistance identification.
• Most pathogenesis‐related (PR) genes and other defence‐related genes were activated and up‐regulated to a high degree. ALS1 was expressed mainly in the leaf blade and sheath, in which further study revealed that ALS1 was present in the vascular bundles. ALS1 was located in the cell membrane of rice protoplasts, and its mutation did not change its subcellular location. Jasmonic acid (JA) and salicylic acid (SA) accumulation were observed in als1, and enhanced blast resistance was also observed.
• The mutation of ALS1 caused a constitutively activated defence response in als1. The results of our study imply that ALS1 participates in a defence response resembling the common SA‐, JA‐ and NH1‐mediated defence responses in rice.

     

A novel family of cys-rich membrane proteins mediates cadmium resistance in Arabidopsis

Cadmium (Cd) is a widespread pollutant that is toxic to plant growth. However, only a few genes that contribute to Cd resistance in plants have been identified. To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1. This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs). Database searches revealed that there are nine close homologs in Arabidopsis. Homologs were also found in other plants. Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1. AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis. Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II). AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd. Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants.     

Multi-tasking phytochelatin synthases

Phytochelatins are essential for cadmium and arsenic detoxification in plants, some fungi, and animals. It is mysterious, that the responsible enzymes, phytochelatin synthases (PCS), are constitutively expressed and so widespread in nature. Phylogenetic analysis indicates multiple horizontal transfers of PCS genes, but a bacterial origin appears unlikely. Differences between bacterial and eukaryotic PCS proteins in structure and activity had indicated bi-functionality of phytochelatin synthases as peptidases and transpeptidases. Recent observations indicate that PCS indeed serve physiological functions that most likely are much more prevalent than cadmium or arsenic detoxification. First, PCS-deficient Arabidopsis thaliana mutants are hypersensitive to zinc suggesting a role of phytochelatin synthesis, i.e. the formation of metal-binding peptides from glutathione in a transpeptidase reaction, in Zn homeostasis. Second, these mutants are also impaired in defense responses conferring resistance to incompatible pathogens (= nonhost resistance). The latter is hypothesized to be attributable to an involvement of PCS as a peptidase in indole glucosinolate metabolism. Possibly, micronutrient homeostasis and nonhost resistance are closely connected as PCS are not the only proteins involved in both processes.     

Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast.

Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of approximately 55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans. The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS-expressing cells accumulate more Cd2+ than controls. PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes.     

OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2 signalling in rice

Drought is one of the major abiotic stresses that directly implicate plant growth and crop productivity. Although many genes in response to drought stress have been identified, genetic improvement to drought resistance especially in food crops is showing relatively slow progress worldwide. Here, we reported the isolation of abscisic acid, stress and ripening (ASR) genes from upland rice variety, IRAT109 (Oryza sativa L. ssp. japonica), and demonstrated that overexpression of OsASR5 enhanced osmotic tolerance in Escherichia coli and drought tolerance in Arabidopsis and rice by regulating leaf water status under drought stress conditions. Moreover, overexpression of OsASR5 in rice increased endogenous ABA level and showed hypersensitive to exogenous ABA treatment at both germination and postgermination stages. The production of H₂O₂, a second messenger for the induction of stomatal closure in response to ABA, was activated in overexpression plants under drought stress conditions, consequently, increased stomatal closure and decreased stomatal conductance. In contrast, the loss‐of‐function mutant, osasr5, showed sensitivity to drought stress with lower relative water content under drought stress conditions. Further studies demonstrated that OsASR5 functioned as chaperone‐like protein and interacted with stress‐related HSP40 and 2OG‐Fe (II) oxygenase domain containing proteins in yeast and plants. Taken together, we suggest that OsASR5 plays multiple roles in response to drought stress by regulating ABA biosynthesis, promoting stomatal closure, as well as acting as chaperone‐like protein that possibly prevents drought stress‐related proteins from inactivation.     

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.     

Iron plaque decreases cadmium accumulation in Oryza sativa L. and serves as a source of iron

• Cadmium (Cd) contamination occurs in paddy soils; hence it is necessary to reduce Cd content of rice. Application and mode of action of ferrous sulphate in minimizing Cd in rice was monitored in the present study.
• Pot culture with Indian rice variety Swarna (MTU 7029) was maintained in Cd‐spiked soil containing ferrous sulphates, which is expected to reduce Cd accumulation in rice. Responses in rhizosphere pH, root surface, metal accumulation in plant and molecular physiological processes were monitored.
• Iron plaque was induced on root surfaces after FeSO4 application and the amount of Fe in plaque reduced with increases in Cd in the soil. Rhizosphere pH decreased during plaque formation and became more acidic due to secretion of organic acids from the roots under Cd treatment. Moreover, iron chelate reductase activity increased with Cd treatment, but in the absence of Cd, activity of this enzyme increased in plaque‐induced plants. Cd treatment caused expression of OsYSL18, whereas OsYSL15 was expressed only in roots without iron plaque. Fe content of plants increased during plaque formation, which protected plants from Cd‐induced Fe deficiency and metal toxicity. This was corroborated with increased biomass, chlorophyll content and quantum efficiency of photo‐synthesis among plaque‐induced plants.
• We conclude that ferrous sulphate‐induced iron plaque prevents Cd accumulation and Fe deficiency in rice. Iron released from plaque via organic acid mediated dissolution during Cd stress.

     

Melatonin mitigates cadmium and aluminium toxicity through modulation of antioxidant potential in Brassica napus L

• Melatonin has emerged as an essential molecule in plants, due to its role in defence against metal toxicity. Aluminium (Al) and cadmium (Cd) toxicity inhibit rapeseed seedling growth.
• In this study, we applied different doses of melatonin (50 and 100 µm ) to alleviate Al (25 µm ) and Cd (25 µm ) stress in rapeseed seedlings. Results show that Al and Cd caused toxicity in rapeseed seedling, as evidenced by a decrease in height, biomass and antioxidant enzyme activity.
• Melatonin increased the expression of melatonin biosynthesis‐related Brassica napus genes for caffeic acid O‐methyl transferase (BnCOMT) under Al and Cd stress. The genes BnCOMT‐1, BnCOMT‐5 and BnCOMT‐8 showed up‐regulated expression, while BnCOMT‐4 and BnCOMT‐6 were down‐regulated during incubation in water. Melatonin application increased the germination rate, shoot length, root length, fresh and dry weight of seedlings. Melatonin supplementation under Al and Cd stress increased superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, proline, chlorophyll and anthocyanin content, as well as photosynthesis rate. Both Cd and Al treatments significantly increased hydrogen peroxide and malondialdehyde levels in rapeseed seedlings, which were strictly counterbalanced by melatonin. Analysis of Cd and Al in different subcellular compartments showed that melatonin enhanced cell wall and soluble fractions, but reduced the vacuolar and organelle fractions in Al‐ and Cd‐treated seedlings.
• These results suggest that melatonin‐induced improvements in antioxidant potential, biomass, photosynthesis rate and successive Cd and Al sequestration play a pivotal role in plant tolerance to Al and Cd stress. This mechanism may have potential implications in safe food production.

     

The outer membrane Omp85‐like protein P39 influences metabolic homeostasis in mature Arabidopsis thaliana

• The Omp85 proteins form a large membrane protein family in bacteria and eukaryotes. Omp85 proteins are composed of a C‐terminal β‐barrel‐shaped membrane domain and one or more N‐terminal polypeptide transport‐associated (POTRA) domains. However, Arabidopsis thaliana contains two genes coding for Omp85 proteins without a POTRA domain. One gene is designated P39, according to the molecular weight of the encoded protein. The protein is targeted to plastids and it was established that p39 has electrophysiological properties similar to other Omp85 family members, particularly to that designated as Toc75V/Oep80.
• We analysed expression of the gene and characterised two T‐DNA insertion mutants, focusing on alterations in photosynthetic activity, plastid ultrastructure, global expression profile and metabolome.
• We observed pronounced expression of P39, especially in veins. Mutants of P39 show growth aberrations, reduced photosynthetic activity and changes in plastid ultrastructure, particularly in the leaf tip. Further, they display global alteration of gene expression and metabolite content in leaves of mature plants.
• We conclude that the function of the plastid‐localised and vein‐specific Omp85 family protein p39 is important, but not essential, for maintenance of metabolic homeostasis of full‐grown A. thaliana plants. Further, the function of p39 in veins influences the functionality of other plant tissues. The link connecting p39 function with metabolic regulation in mature A. thaliana is discussed.

     

Mitochondrial ribosomal protein S9M is involved in male gametogenesis and seed development in Arabidopsis

• Mitochondrial function is critical for cell vitality in all eukaryotes including plants. Although plant mitochondria contain many proteins, few have been studied in the context of plant development and physiology.
• We used knock‐down mutant RPS9M to study its important role in male gametogenesis and seed development in Arabidopsis thaliana.
• Knock‐down of RPS9M in the rps9m‐3 mutant led to abnormal pollen development and impaired pollen tube growth. In addition, both embryo and endosperm development were affected. Phenotype analysis revealed that the rps9m‐3 mutant contained a lower amount of endosperm and nuclear proteins, and both embryo cell division and embryo pattern were affected, resulting in an abnormal and defective embryo. Lowering the level of RPS9M in rps9m‐3 affects mitochondrial ribosome biogenesis, energy metabolism and production of ROS.
• Our data revealed that RPS9M plays important roles in normal gametophyte development and seed formation, possibly by sustaining mitochondrial function.

     

Genome‐wide association study identifies an NLR gene that confers partial resistance to Magnaporthe oryzae in rice

Because of the frequent breakdown of major resistance (R) genes, identification of new partial R genes against rice blast disease is an important goal of rice breeding. In this study, we used a core collection of the Rice Diversity Panel II (C‐RDP‐II), which contains 584 rice accessions and are genotyped with 700 000 single‐nucleotide polymorphism (SNP) markers. The C‐RDP‐II accessions were inoculated with three blast strains collected from different rice‐growing regions in China. Genome‐wide association study identified 27 loci associated with rice blast resistance (LABRs). Among them, 22 LABRs were not associated with any known blast R genes or QTLs. Interestingly, a nucleotide‐binding site leucine‐rich repeat (NLR) gene cluster exists in the LABR12 region on chromosome 4. One of the NLR genes is highly conserved in multiple partially resistant rice cultivars, and its expression is significantly up‐regulated at the early stages of rice blast infection. Knockout of this gene via CRISPR‐Cas9 in transgenic plants partially reduced blast resistance to four blast strains. The identification of this new non‐strain specific partial R gene, tentatively named rice blast Partial Resistance gene 1 (PiPR1), provides genetic material that will be useful for understanding the partial resistance mechanism and for breeding durably resistant cultivars against blast disease of rice.