We identified 18 putative yellow stripe 1 (YS1)-like genes (OsYSLs) in the rice genome that exhibited 36-76% sequence similarity to maize iron(III)-phytosiderophore transporter YS1. Of particular interest was OsYSL2, the transcripts of which were not detected in the roots of either iron-sufficient or iron-deficient plants, but dramatic expression was induced in the leaves by iron deficiency. Based on the nucleotide sequence, OsYSL2 was predicted to encode a polypeptide of 674 amino acids containing 14 putative transmembrane domains. OsYSL2:green fluorescent protein (GFP) was localized in the plasma membrane of onion epidermal cells. Promoter:b-glucuronidase (GUS) analysis revealed that OsYSL2 was expressed in companion cells in ironsufficient roots. GUS activity was increased in companion cells, but no GUS staining was observed in epidermal or cortex cells, even in iron-deficient roots. In the leaves and leaf sheaths of iron-sufficient rice, GUS staining was observed in phloem cells of the vascular bundles. In iron-deficient leaves, the OsYSL2 promoter was active in all tissues with particularly strong GUS activity evident in companion cells. The phloem-specific expression of the OsYSL2 promoter suggests that OsYSL2 is involved in the phloem transport of iron. Strong OsYSL2 promoter activity was also detected in developing seeds. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL2 transported iron(II)-nicotianamine (NA) and manganese(II)-NA, but did not transport iron(III)-phyosiderophore. These results suggest that OsYSL2 is a rice metal-NA transporter that is responsible for the phloem transport of iron and manganese, including the translocation of iron and manganese into the grain.

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.To acquire Fe, graminaceous plants secrete Fe chelators, known as mugineic-acid family phytosiderophores (MAs). MAs dissolve Fe in the rhizosphere, followed by reabsorption of the Fe(III)-MA complexes through YS1 transporters in the plasma membrane (Takagi, 1976; Curie et al., 2001). Only graminaceous plants use the MA mechanism of acquiring Fe(III), classified as the Strategy II mechanism (Marschner et al., 1986). Fe deficiency is a problem in crop production worldwide, especially in calcareous soils, where Fe is sparingly soluble due to the high soil pH. The ability of graminaceous plants to tolerate Fe deficiency is thought to depend on the quantity of MAs secreted during Fe deficiency (Takagi, 1976;Mori et al., , 1988Rö mheld, 1987;Kawai et al., 1988;Mihashi and Mori, 1989;Singh et al., 1993). The biosynthetic pathways of MAs (Fig. 1A) have been determined Kawai et al., 1988;Shojima et al., 1990;Ma et al., 1999), and almost all the genes involved have been isolated in our laboratory (Higuchi et al., 1999b;Takahashi et al., 1999;Nakanishi et al., 2000;Kobayashi et al., 2001). NAS is a key enzyme in MA biosynthesis, catalyzing the trimerization of SAM into one molecule of NA (Higuchi et al., 1999b). NAS activity in graminaceous plants is well correlated with tolerance to Fe deficiency. In maize (Zea mays), a plant susceptible to Fe deficiency, NAS activity is very low (Higuchi et al., 1996a), and maize secretes lower amounts of MAs than barley or oat (Avena sativa), cereals that are tolerant of low Fe supply (Rö mheld, 1987;Lytle and Jolley, 1991). NAS is also important for growth in nongraminaceous plants, which do not ...

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