Yasuhiro Ishimaru

Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese.

Rice (Oryza sativa) is indispensable in the diet of most of the worlds population. Thus, it is an important target in which to alter iron (Fe) uptake and homeostasis, so as to increase Fe accumulation in the grain. We previously isolated OsYSL2, a functional iron [Fe(II)]- and manganese [Mn(II)]-nicotianamine complex transporter that is expressed in phloem cells and developing seeds. We produced RNAi (OsYSL2i) and overexpression lines (OXOsYSL2) of OsYSL2. At the vegetative stage in an OsYSL2i line, the Fe and Mn concentrations were decreased in the shoots, and the Fe concentration was increased in the roots. At the reproductive stage, positron-emitting tracer imaging system analysis revealed that Fe translocation to the shoots and seeds was suppressed in OsYSL2i. The Fe and Mn concentrations were decreased in the seeds of OsYSL2i, especially in the endosperm. Moreover, the Fe concentration in OXOsYSL2 was lower in the seeds and shoots, but higher in the roots, compared with the wild type. Furthermore, when OsYSL2 expression was driven by the sucrose transporter promoter, the Fe concentration in the polished rice was up to 4.4-fold higher compared with the wild type. These results indicate that the altered expression of OsYSL2 changes the localization of Fe, and that OsYSL2 is a critical Fe-nicotianamine transporter important for Fe translocation, especially in the shoots and endosperm.

Characterizing the role of rice NRAMP5 in Manganese, Iron and Cadmium Transport

Metals like manganese (Mn) and iron (Fe) are essential for metabolism, while cadmium (Cd) is toxic for virtually all living organisms. Understanding the transport of these metals is important for breeding better crops. We have identified that OsNRAMP5 contributes to Mn, Fe and Cd transport in rice. OsNRAMP5 expression was restricted to roots epidermis, exodermis, and outer layers of the cortex as well as in tissues around the xylem. OsNRAMP5 localized to the plasma membrane, and complemented the growth of yeast strains defective in Mn, Fe, and Cd transport. OsNRAMP5 RNAi (OsNRAMP5i) plants accumulated less Mn in the roots, and less Mn and Fe in shoots, and xylem sap. The suppression of OsNRAMP5 promoted Cd translocation to shoots, highlighting the importance of this gene for Cd phytoremediation. These data reveal that OsNRAMP5 contributes to Mn, Cd, and Fe transport in rice and is important for plant growth and development.

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.

Iron deficiency enhances cadmium uptake and translocation mediated by the Fe2+ transporters OsIRT1 and OsIRT2 in rice

Abstract Cadmium (Cd) accumulation in rice grains is enhanced if ponded water is released from paddy fields during the reproductive stage (intermittent irrigation). The release of ponded water creates aerobic soil conditions under which Cd becomes soluble and iron (Fe) solubility decreases. We hypothesized that Fe shortage in rice induces Fe uptake and translocation and that Cd is also taken up and translocated throughout this process. Hydroponically cultured Fe-deficient rice absorbed more Cd than did Fe-sufficient rice, and the presence of Fe enhanced the translocation of Cd to the shoots. Yeast mutants expressing OsIRT1 and OsIRT2, which encode the rice Fe2+ transporter, became more sensitive to Cd, suggesting that Cd was absorbed by OsIRT1 and OsIRT2. We discuss the possibility that Cd accumulation in rice grains during the reproductive stage is mediated by the Fe transport system.

Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice

Rice (Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon ion-beam irradiation produced three rice mutants with <0.05 mg Cd⋅kg−1 in the grain compared with a mean of 1.73 mg Cd⋅kg−1 in the parent, Koshihikari. We identified the gene responsible for reduced Cd uptake and developed a strategy for marker-assisted selection of low-Cd cultivars. Sequence analysis revealed that these mutants have different mutations of the same gene (OsNRAMP5), which encodes a natural resistance-associated macrophage protein. Functional analysis revealed that the defective transporter protein encoded by the mutant osnramp5 greatly decreases Cd uptake by roots, resulting in decreased Cd in the straw and grain. In addition, we developed DNA markers to facilitate marker-assisted selection of cultivars carrying osnramp5. When grown in Cd-contaminated paddy fields, the mutants have nearly undetectable Cd in their grains and exhibit no agriculturally or economically adverse traits. Because mutants produced by ion-beam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide.

Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil

Iron (Fe) deficiency is a worldwide agricultural problem on calcareous soils with low-Fe availability due to high soil pH. Rice plants use a well documented phytosiderophore-based system (Strategy II) to take up Fe from the soil and also possess a direct Fe2+ transport system. Rice plants are extremely susceptible to low-Fe supply, however, because of low phytosiderophore secretion and low Fe3+ reduction activity. A yeast Fe3+ chelate-reductase gene refre1/372, selected for better performance at high pH, was fused to the promoter of the Fe-regulated transporter, OsIRT1, and introduced into rice plants. The transgene was expressed in response to a low-Fe nutritional status in roots of transformants. Transgenic rice plants expressing the refre1/372 gene showed higher Fe3+ chelate-reductase activity and a higher Fe-uptake rate than vector controls under Fe-deficient conditions. Consequently, transgenic rice plants exhibited an enhanced tolerance to low-Fe availability and 7.9× the grain yield of nontransformed plants in calcareous soils. This report shows that enhancing the Fe3+ chelate-reductase activity of rice plants that normally have low endogenous levels confers resistance to Fe deficiency.

Overexpression of the Barley Nicotianamine Synthase Gene HvNAS1 Increases Iron and Zinc Concentrations in Rice Grains

In humans, iron (Fe) and zinc (Zn) deficiencies result in major worldwide health problems. Transgenic technologies to produce Fe- and Zn-biofortified rice varieties offer a promising potential solution. Nicotianamine, the precursor of phytosiderophores, chelates Fe2+ and Zn2+ and plays an important role in transporting these metals to both vegetative and reproductive organs within the plant. Our objective was to increase Fe and Zn contents in rice grains by overexpressing the barley nicotianamine synthase gene HvNAS1. HvNAS1-overexpressing transgenic rice showed increased HvNAS1 expression and subsequent increases in endogenous nicotianamine and phytosiderophore content in shoots, roots, and seeds. Fe and Zn concentrations in polished T1 seeds from transgenic plants increased more than three and twofold, respectively; Fe and Zn concentrations also increased in both polished and brown T2 seeds. These results suggest that the overproduction of nicotianamine enhances the translocation of Fe and Zn into rice grains.

OsYSL18 is a rice iron(III)–deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints

Iron uptake and translocation in plants are important processes for both plant and human nutrition, whereas relatively little is known about the molecular mechanisms of iron transport within the plant body. Several reports have shown that yellow stripe 1 (YS1) and YS1-like (YSL) transporters mediate metal-phytosiderophore uptake and/or metal-nicotianamine translocation. Among the 18 YSL genes in rice (OsYSLs), OsYSL18 is predicted to encode a polypeptide of 679 amino acids containing 13 putative transmembrane domains. An OsYSL18-green fluorescent protein (GFP) fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL18 transports iron(III)–deoxymugineic acid, but not iron(II)–nicotianamine, zinc(II)–deoxymugineic acid, or zinc(II)–nicotianamine. Reverse transcriptase PCR analysis revealed more OsYSL18 transcripts in flowers than in shoots or roots. OsYSL18 promoter-β-glucuronidase (GUS) analysis revealed that OsYSL18 was expressed in reproductive organs including the pollen tube. In vegetative organs, OsYSL18 was specifically expressed in lamina joints, the inner cortex of crown roots, and phloem parenchyma and companion cells at the basal part of every leaf sheath. These results suggest that OsYSL18 is an iron-phytosiderophore transporter involved in the translocation of iron in reproductive organs and phloem in joints.

A rice phenolic efflux transporter is essential for solubilizing precipitated apoplasmic iron in the plant stele

Iron deficiency is one of the major agricultural problems, as 30% of the arable land of the world is too alkaline for optimal crop production, rendering plants short of available iron despite its abundance. To take up apoplasmic precipitated iron, plants secrete phenolics such as protocatechuic acid (PCA) and caffeic acid. The molecular pathways and genes of iron uptake strategies are already characterized, whereas the molecular mechanisms of phenolics synthesis and secretion have not been clarified, and no phenolics efflux transporters have been identified in plants yet. Here we describe the identification of a phenolics efflux transporter in rice. We identified a cadmium-accumulating rice mutant in which the amount of PCA and caffeic acid in the xylem sap was dramatically reduced and hence named it phenolics efflux zero 1 (pez1). PEZ1 localized to the plasma membrane and transported PCA when expressed in Xenopus laevis oocytes. PEZ1 localized mainly in the stele of roots. In the roots of pez1, precipitated apoplasmic iron increased. The growth of PEZ1 overexpression lines was severely restricted, and these lines accumulated more iron as a result of the high solubilization of precipitated apoplasmic iron in the stele. We show that PEZ1 is responsible for an increase of PCA concentration in the xylem sap and is essential for the utilization of apoplasmic precipitated iron in the stele.

Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition

To address the problem of iron-deficiency anemia, one of the most prevalent human micronutrient deficiencies globally, iron-biofortified rice was produced using three transgenic approaches: by enhancing iron storage in grains via expression of the iron storage protein ferritin using endosperm-specific promoters, enhancing iron translocation through overproduction of the natural metal chelator nicotianamine, and enhancing iron flux into the endosperm by means of iron(II)-nicotianamine transporter OsYSL2 expression under the control of an endosperm-specific promoter and sucrose transporter promoter. Our results indicate that the iron concentration in greenhouse-grown T2 polished seeds was sixfold higher and that in paddy field-grown T3 polished seeds was 4.4-fold higher than that in non-transgenic seeds, with no defect in yield. Moreover, the transgenic seeds accumulated zinc up to 1.6-times in the field. Our results demonstrate that introduction of multiple iron homeostasis genes is more effective for iron biofortification than the single introduction of individual genes.