Naoko K. Nishizawa

Iron Uptake, Translocation, and Regulation in Higher Plants

Iron is essential for the survival and proliferation of all plants. Higher plants have developed two distinct strategies to acquire iron, which is only slightly soluble, from the rhizosphere: the reduction strategy of nongraminaceous plants and the chelation strategy of graminaceous plants. Key molecular components-including transporters, enzymes, and chelators-have been clarified for both strategies, and many of these components are now thought to also function inside the plant to facilitate internal iron transport. Transporters for intracellular iron trafficking are also being clarified. A majority of genes encoding these components are transcriptionally regulated in response to iron availability. Recent research has uncovered central transcription factors, cis-acting elements, and molecular mechanisms regulating these genes. Manipulation of these molecular components has produced transgenic crops with enhanced tolerance to iron deficiency or with increased iron content in the edible parts.

Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes.

One of the widest ranging abiotic stresses in world agriculture arises from low iron (Fe) availability due to high soil pH, with 30% of arable land too alkaline for optimal crop production. Rice is especially susceptible to low iron supply, whereas other graminaceous crops such as barley are not. A barley genomic DNA fragment containing two naat genes, which encode crucial enzymes involved in the biosynthesis of phytosiderophores, was introduced into rice using Agrobacterium-mediated transformation and pBIGRZ1. Phytosiderophores are natural iron chelators that graminaceous plants secrete from their roots to solubilize iron in the soil. The two transgenes were expressed in response to low iron nutritional status in both the shoots and roots of rice transformants. Transgenic rice expressing the two genes showed a higher nicotianamine aminotransferase activity and secreted larger amounts of phytosiderophores than nontransformants under iron-deficient conditions. Consequently, the transgenic rice showed an enhanced tolerance to low iron availability and had 4.1 times greater grain yields than that of the nontransformant rice in an alkaline soil.

KORRIGAN, an Arabidopsis Endo-1,4-β-Glucanase, Localizes to the Cell Plate by Polarized Targeting and Is Essential for Cytokinesis

The formation of the cell plate, a unique structure in dividing plant cells, is pivotal for cytokinesis. A mutation in the Arabidopsis KORRIGAN (KOR) gene causes the formation of aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to severely abnormal seedling morphology. The mutant, designed kor1-2, was identified as a stronger allele than the previously identified kor1-1, which appears to be defective only in cell elongation. KOR1 encodes an endo-1,4-β-d-glucanase with a transmembrane domain and two putative polarized targeting signals in the cytosolic tail. When expressed in tobacco BY2 cells, a KOR1-GFP (green fluorescence protein) fusion protein was localized to growing cell plates. Substitution mutations in the polarized targeting motifs of KOR1 caused the fusion proteins to localize to the plasma membrane as well. Expression of these mutant genes in kor1-2 plants complemented only the cell elongation defect but not the cytokinesis defect, indicating that polarized targeting of KOR1 to forming cell plates is essential for cytokinesis. Our results suggest that KOR1 plays a critical role during cytokinesis.

Rice OsYSL15 Is an Iron-regulated Iron(III)-Deoxymugineic Acid Transporter Expressed in the Roots and Is Essential for Iron Uptake in Early Growth of the Seedlings

Graminaceous plants take up iron through YS1 (yellow stripe 1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in the epidermis/exodermis, cortex, and stele of rice roots. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-β-glucuronidase analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron(III)-deoxymugineic acid transporter responsible for iron uptake from the rhizosphere and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth.

Phytosiderophore Efflux Transporters Are Crucial for Iron Acquisition in Graminaceous Plants

Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released 14C-labeled deoxymugineic acid but not 14C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants.

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.

Cloning and Characterization of Deoxymugineic Acid Synthase Genes from Graminaceous Plants

Graminaceous plants have evolved a unique mechanism to acquire iron through the secretion of a family of small molecules, called mugineic acid family phytosiderophores (MAs). All MAs are synthesized from l-Met, sharing the same pathway from l-Met to 2′-deoxymugineic acid (DMA). DMA is synthesized through the reduction of a 3″-keto intermediate by deoxymugineic acid synthase (DMAS). We have isolated DMAS genes from rice (OsDMAS1), barley (HvDMAS1), wheat (TaD-MAS1), and maize (ZmDMAS1). Their nucleotide sequences indicate that OsDMAS1 encodes a predicted polypeptide of 318 amino acids, whereas the other three orthologs all encode predicted polypeptides of 314 amino acids and are highly homologous (82–97.5%) to each other. The DMAS proteins belong to the aldo-keto reductase superfamily 4 (AKR4) but do not fall within the existing subfamilies of AKR4 and appear to constitute a new subfamily within the AKR4 group. All of the proteins showed DMA synthesis activity in vitro. Their enzymatic activities were highest at pH 8–9, consistent with the hypothesis that DMA is synthesized in subcellular vesicles. Northern blot analysis revealed that the expression of each of the above DMAS genes is up-regulated under iron-deficient conditions in root tissue, and that of the genes OsDMAS1 and TaDMAS1 is up-regulated in shoot tissue. OsDMAS1 promoter-GUS analysis in iron-sufficient roots showed that its expression is restricted to cells participating in long distance transport and that it is highly up-regulated in the entire root under iron-deficient conditions. In shoot tissue, OsDMAS1 promoter drove expression in vascular bundles specifically under iron-deficient conditions.

Iron fortification of rice seeds through activation of the nicotianamine synthase gene

The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activation-tagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and OsNAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression.

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.