Daisei Ueno

Gene limiting cadmium accumulation in rice

Intake of toxic cadmium (Cd) from rice caused Itai-itai disease in the past and it is still a threat for human health. Therefore, control of the accumulation of Cd from soil is an important food-safety issue, but the molecular mechanism for the control is unknown. Herein, we report a gene (OsHMA3) responsible for low Cd accumulation in rice that was isolated from a mapping population derived from a cross between a high and low Cd-accumulating cultivar. The gene encodes a transporter belonging to the P1B-type ATPase family, but shares low similarity with other members. Heterologous expression in yeast showed that the transporter from the low-Cd cultivar is functional, but the transporter from the high-Cd cultivar had lost its function, probably because of the single amino acid mutation. The transporter is mainly expressed in the tonoplast of root cells at a similar level in both the low and high Cd-accumulating cultivars. Overexpression of the functional gene from the low Cd-accumulating cultivar selectively decreased accumulation of Cd, but not other micronutrients in the grain. Our results indicated that OsHMA3 from the low Cd-accumulating cultivar limits translocation of Cd from the roots to the above-ground tissues by selectively sequestrating Cd into the root vacuoles.

OsFRDL1 is a citrate transporter required for efficient translocation of iron in rice.

Multidrug and toxic compound extrusion (MATE) transporters represent a large family in plants, but their functions are poorly understood. Here, we report the function of a rice (Oryza sativa) MATE gene (Os03g0216700, OsFRDL1), the closest homolog of barley (Hordeum vulgare) HvAACT1 (aluminum [Al]-activated citrate transporter 1), in terms of metal stress (iron [Fe] deficiency and Al toxicity). This gene was mainly expressed in the roots and the expression level was not affected by either Fe deficiency or Al toxicity. Knockout of this gene resulted in leaf chlorosis, lower leaf Fe concentration, higher accumulation of zinc and manganese concentration in the leaves, and precipitation of Fe in the roots stele. The concentration of citrate and ferric iron in the xylem sap was lower in the knockout line compared to the wild-type rice. Heterologous expression of OsFRDL1 in Xenopus oocytes showed transport activity for citrate. Immunostaining showed that OsFRDL1 was localized at the pericycle cells of the roots. On the other hand, there was no difference in the Al-induced secretion of citrate from the roots between the knockout line and the wild-type rice. Taken together, our results indicate that OsFRDL1 is a citrate transporter localized at the pericycle cells, which is necessary for efficient translocation of Fe to the shoot as a Fe-citrate complex.

Elevated expression of TcHMA3 plays a key role in the extreme Cd tolerance in a Cd-hyperaccumulating ecotype of Thlaspi caerulescens

Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.

Characterization of Cd Translocation and Identification of the Cd Form in Xylem Sap of the Cd-Hyperaccumulator Arabidopsis halleri

Arabidopsis halleri is a Cd hyperaccumulator; however, the mechanisms involved in the root to shoot translocation of Cd are not well understood. In this study, we characterized Cd transfer from the root medium to xylem in this species. Arabidopsis halleri accumulated 1,500 mg kg(-1) Cd in the shoot without growth inhibition. A time-course experiment showed that the release of Cd into the xylem was very rapid; by 2 h exposure to Cd, Cd concentration in the xylem sap was 5-fold higher than that in the external solution. The concentration of Cd in the xylem sap increased linearly with increasing Cd concentration in the external solution. Cd transfer to the xylem was completely inhibited by the metabolic inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cd concentration in the xylem sap was decreased by increasing the concentration of external Zn, but enhanced by Fe deficiency treatment. Analysis with 113Cd-nuclear magnetic resonance (NMR) showed that the chemical shift of 113Cd in the xylem sap was the same as that of Cd(NO3)2. Metal speciation with Geochem-PC also showed that Cd occurred mainly in the free ionic form in the xylem sap. These results suggest that Cd transfer from the root medium to the xylem in A. halleri is an energy-dependent process that is partly shared with Zn and/or Fe transport. Furthermore, Cd is translocated from roots to shoots in inorganic forms.

Mutation in Nicotianamine Aminotransferase Stimulated the Fe(II) Acquisition System and Led to Iron Accumulation in Rice

Higher plants acquire iron (Fe) from the rhizosphere through two strategies. Strategy II, employed by graminaceous plants, involves secretion of phytosiderophores (e.g. deoxymugineic acid in rice [Oryza sativa]) by roots to solubilize Fe(III) in soil. In addition to taking up Fe in the form of Fe(III)-phytosiderophore, rice also possesses the strategy I-like system that may absorb Fe(II) directly. Through mutant screening, we isolated a rice mutant that could not grow with Fe(III)-citrate as the sole Fe source, but was able to grow when Fe(II)-EDTA was supplied. Surprisingly, the mutant accumulated more Fe and other divalent metals in roots and shoots than the wild type when both were supplied with EDTA-Fe(II) or grown under water-logged field conditions. Furthermore, the mutant had a significantly higher concentration of Fe in both unpolished and polished grains than the wild type. Using the map-based cloning method, we identified a point mutation in a gene encoding nicotianamine aminotransferase (NAAT1), which was responsible for the mutant phenotype. Because of the loss of function of NAAT1, the mutant failed to produce deoxymugineic acid and could not absorb Fe(III) efficiently. In contrast, nicotianamine, the substrate for NAAT1, accumulated markedly in roots and shoots of the mutant. Microarray analysis showed that the expression of a number of the genes involved in Fe(II) acquisition was greatly stimulated in the naat1 mutant. Our results demonstrate that disruption of deoxymugineic acid biosynthesis can stimulate Fe(II) acquisition and increase iron accumulation in rice.

Variation in root‐to‐shoot translocation of cadmium and zinc among different accessions of the hyperaccumulators Thlaspi caerulescens and Thlaspi praecox

Efficient root-to-shoot translocation is a key trait of the zinc/cadmium hyperaccumulators Thlaspi caerulescens and Thlaspi praecox, but the extent of variation among different accessions and the underlying mechanisms remain unclear. Root-to-shoot translocation of Cd and Zn and apoplastic bypass flow were determined in 10 accessions of T. caerulescens and one of T. praecox, using radiolabels (109)Cd and (65)Zn. Two contrasting accessions (Pr and Ga) of T. caerulescens were further characterized for TcHMA4 expression and metal compartmentation in roots. Root-to-shoot translocation of (109)Cd and (65)Zn after 1 d exposure varied 4.4 to 5-fold among the 11 accessions, with a significant correlation between the two metals, but no significant correlation with uptake or the apoplastic bypass flow. The F(2) progeny from a cross between accessions from Prayon, Belgium (Pr) and Ganges, France (Ga) showed a continuous phenotype pattern and transgression. There was no significant difference in the TcHMA4 expression in roots between Pr and Ga. Compartmentation analysis showed a higher percentage of (109)Cd sequestered in the root vacuoles of Ga than Pr, the former being less efficient in translocation than the latter. Substantial natural variation exists in the root-to-shoot translocation of Cd and Zn, and root vacuolar sequestration may be an important factor related to this variation.

A major quantitative trait locus controlling cadmium translocation in rice (Oryza sativa).

The trait of low cadmium (Cd) accumulation in brown rice (Oryza sativa) is important for food safety. An effective way to reduce Cd accumulation in the grain is to control Cd transfer from the roots to the shoots. Here, we investigated genotypic variation in the shoot Cd concentration among 146 accessions from a rice core collection and performed a quantitative trait locus (QTL) analysis to determine the loci controlling shoot Cd accumulation. Furthermore, we physiologically characterized the two accessions used for QTL analysis. Large genotypic variation (13-fold) in the shoot Cd concentration was found. A major QTL was detected on chromosome 11 using a F2 population derived from Badari Dhan (a high-Cd accession) and Shwe War (a low-Cd accession). This QTL explained 16.1% of the phenotypic variation in Cd accumulation. Furthermore, this QTL was confirmed by analysis of advanced progeny. Physiological studies showed that Badari Dhan and Shwe War did not differ in uptake of Cd by the roots, but differed greatly in the translocation of Cd from the roots to the shoots. Taken together, our findings suggest that the major QTL detected is responsible for the translocation of Cd from the roots to the shoots.

Physiological, genetic, and molecular characterization of a high-Cd-accumulating rice cultivar, Jarjan

Cadmium (Cd) in rice is a major source of Cd intake for people on a staple rice diet. The mechanisms underlying Cd accumulation in rice plant are still poorly understood. Here, we characterized the physiology and genetics of Cd transport in a high-Cd-accumulating cultivar (Jarjan) of rice (Oryza sativa). Jarjan showed 5- to 34-fold higher Cd accumulation in the shoots and grains than the cultivar Nipponbare, when it was grown in either a non-Cd-contaminated or a Cd-contaminated soil. A short-term uptake experiment showed no significant difference in Cd uptake by the roots between the two cultivars. However, Jarjan translocated 49% of the total Cd taken up to the shoots, whereas Nipponbare retained most of the Cd in the roots. In both concentration- and time-dependent experiments, Jarjan showed a superior capacity for root-to-shoot translocation of Cd. These results indicate that the high-Cd-accumulation phenotype in Jarjan results from efficient translocation of Cd from roots to shoots. Genetic analysis using an F(2) population derived from Jarjan and Nipponbare revealed that plants showing high- and low-Cd-accumulation phenotypes segregated in a 1:3 ratio, indicating that high accumulation in Jarjan is controlled by a single recessive gene. Furthermore, we isolated OsHMA3, a gene encoding a tonoplast-localized Cd transporter from Jarjan. The OsHMA3 protein was localized in all roots cells, but the sequence has a mutation leading to loss of function. Therefore, failure to sequester Cd into the root vacuoles by OsHMA3 is probably responsible for high Cd accumulation in Jarjan.

Identification of a novel major quantitative trait locus controlling distribution of Cd between roots and shoots in rice.

Accumulation of Cd in rice grain is a serious concern of food safety since rice as a staple food is a major source of Cd intake in Asian countries. However, the mechanisms controlling Cd accumulation in rice are still poorly understood. Herein, we report both physiological and genetic analysis of two rice cultivars contrasting in Cd accumulation, which were screened from a core collection of rice cultivars. The cultivar Anjana Dhan (Indica) accumulated much higher levels of Cd than Nipponbare (Japonica) in the shoots and grains when grown in both soil and solution culture. A short-term uptake experiment (20 min) showed that Cd uptake by Nipponbare was higher than that by Anjana Dhan. However, the concentration of Cd in the shoot and xylem sap was much higher in Anjana Dhan than in Nipponbare. Of the Cd taken up by the roots, <4% was translocated to the shoots in Nipponbare, compared with 10-25% in Anjana Dhan, indicating a higher root-to-shoot translocation of Cd in the latter. A quantitative trait locus (QTL) analysis for Cd accumulation was performed using an F(2) population derived from Anjana Dhan and Nipponbare. A QTL with large effect for Cd accumulation was detected on the short arm of chromosome 7, explaining 85.6% of the phenotypic variance in the shoot Cd concentration of the F(2) population. High accumulation is likely to be controlled by a single recessive gene. A candidate genomic region was defined to <1.9 Mb by means of substitution mapping.

Mn tolerance in rice is mediated by MTP8.1, a member of the cation diffusion facilitator family

Manganese (Mn) is an essential micronutrient for plants, but is toxic when present in excess. The rice plant (Oryza sativa L.) accumulates high concentrations of Mn in the aerial parts; however, the molecular basis for Mn tolerance is poorly understood. In the present study, genes encoding Mn tolerance were screened for by expressing cDNAs of genes from rice shoots in Saccharomyces cerevisiae. A gene encoding a cation diffusion facilitator (CDF) family member, OsMTP8.1, was isolated, and its expression was found to enhance Mn accumulation and tolerance in S. cerevisiae. In plants, OsMTP8.1 and its transcript were mainly detected in shoots. High or low supply of Mn moderately induced an increase or decrease in the accumulation of OsMTP8.1, respectively. OsMTP8.1 was detected in all cells of leaf blades through immunohistochemistry. OsMTP8.1 fused to green fluorescent protein was localized to the tonoplast. Disruption of OsMTP8.1 resulted in decreased chlorophyll levels, growth inhibition in the presence of high concentrations of Mn, and decreased accumulation of Mn in shoots and roots. However, there was no difference in the accumulation of other metals, including Zn, Cu, Fe, Mg, Ca, and K. These results suggest that OsMTP8.1 is an Mn-specific transporter that sequesters Mn into vacuoles in rice and is required for Mn tolerance in shoots.