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Graminaceous plants release mugineic acid family phytosiderophores ( MAs) to acquire iron from the soil. Here, we show that deoxymugineic acid ( DMA) secretion from rice roots fluctuates throughout the day, and that vesicles accumulate in roots before MAs secretion. We developed transgenic rice plants that express rice nicotianamine ( NA) synthase ( NAS) 2 ( OsNAS2) fused to synthetic green fluorescent protein ( sGFP) under the control of its own promoter. In root cells, OsNAS2- sGFP fluorescence was observed in a dot-like pattern, moving dynamically within the cell. This suggests that these vesicles are involved in NA and DMA biosynthesis. A tyrosine motif and a di-leucine motif, which have been reported to be involved in cellular transport, are conserved in all identified NAS proteins in plants. OsNAS2 mutated in the tyrosine motif showed NAS activity and was localized to the vesicles; however, these vesicles stuck together and did not move. On the other hand, OsNAS2 mutated in the di-leucine motif lost NAS activity and did not localize to these vesicles. The amounts of NA and DMA produced and the amount of DMA secreted by OsNAS2- sGFP plants were significantly higher than in non-transformants and domain-mutated lines, suggesting that OsNAS2- sGFP, but not the mutated forms, was functional in vivo. Overall, the localization of NAS to vesicles and the transport of these vesicles are crucial steps in NA synthesis, leading to DMA synthesis and secretion in rice. [ABSTRACT FROM AUTHOR]

Rice ( Oryza sativa) is indispensable in the diet of most of the world’s 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 (OX OsYSL2) 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 OX OsYSL2 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. [ABSTRACT FROM AUTHOR]

Higher plants maintain iron homeostasis by regulating the expression of iron (Fe)-related genes in accordance with Fe availability. The transcription factor IDEF1 regulates the response to Fe deficiency in Oryza sativa (rice) by recognizing CATGC sequences within the Fe deficiency-responsive cis-acting element IDE1. To investigate the function of IDEF1 in detail, we analyzed the response to Fe deficiency in transgenic rice plants exhibiting induced or repressed IDEF1 expression. Fe-deficiency treatment in hydroponic culture revealed that IDEF1 knock-down plants are susceptible to early-stage Fe deficiency, in contrast to IDEF1-induced plants. Time-course expression analyses using quantitative reverse-transcriptase PCR revealed that the IDEF1 expression level was positively correlated with the level of induction of the Fe utilization-related genes OsIRO2, OsYSL15, OsIRT1, OsYSL2, OsNAS1, OsNAS2, OsNAS3 and OsDMAS1, just after the onset of Fe starvation. However, this overall transactivation mediated by IDEF1 became less evident in subsequent stages. Microarray and in-silico analyses revealed that genes positively regulated by IDEF1, especially at the early stage, exhibit over-representation of CATGC and IDE1-like elements within the proximal promoter regions. These results indicate the existence of early and subsequent responses to Fe deficiency, with the former requiring IDEF1 more specifically. Proximal regions of IDEF1-regulated gene promoters also showed enrichment of RY elements (CATGCA), which regulate gene expression during seed maturation. The expression of several genes encoding late embryogenesis abundant proteins, including Osem, was induced in Fe-deficient roots and/or leaves in an IDEF1-dependent manner, suggesting a possible function of seed maturation-related genes in Fe-deficient vegetative organs. [ABSTRACT FROM AUTHOR]

Iron (Fe) deficiency is a major abiotic stress in crop production. Although responses to Fe deficiency in graminaceous plants, such as increased production and secretion of mugineic acid family phytosiderophores (MAs), have been described, the gene regulation mechanisms related to these responses are largely unknown. To elucidate the regulation mechanisms of the genes related to Fe acquisition in graminaceous plants, we characterized the Fe-deficiency-inducible basic helix-loop-helix transcription factor OsIRO2 in rice. In yeast cells, OsIRO2 functioned as a transcriptional activator. In rice, overexpression of OsIRO2 resulted in increased MAs secretion, whereas repression of OsIRO2 resulted in lower MAs secretion and hypersensitivity to Fe deficiency. Northern blots revealed that the expression of the genes involved in the Fe(III)–MAs transport system was dependent on OsIRO2. The expression of the genes for nicotianamine synthase, a key enzyme in MAs synthesis, was notably affected by the level of OsIRO2 expression. Microarray analysis demonstrated that OsIRO2 regulates 59 Fe-deficiency-induced genes in roots. Some of these genes, including two transcription factors upregulated by Fe deficiency, possessed the OsIRO2 binding sequence in their upstream regions. OsIRO2 possesses a homologous sequence of the Fe-deficiency-responsive cis-acting elements (IDEs) in its upstream region. We propose a novel gene regulation network for Fe-deficiency responses, including OsIRO2, IDEs and the two transcription factors. [ABSTRACT FROM AUTHOR]

Mugineic acid family phytosiderophores (MAs) are metal chelators that are produced in graminaceous plants in response to iron (Fe) deficiency, but current evidence regarding secretion of MAs during zinc (Zn) deficiency is contradictory. Our studies using HPLC analysis showed that Zn deficiency induces the synthesis and secretion of MAs in barley plants. The levels of the HvNAS1, HvNAAT-A, HvNAAT-B, HvIDS2 and HvIDS3 transcripts, which encode the enzymes involved in the synthesis of MAs, were increased in Zn-deficient roots. Studies of the genes involved in the methionine cycle using microarray analysis showed that the transcripts of these genes were increased in both Zn-deficient and Fe-deficient barley roots, probably allowing the plant to meet its demand for methionine, a precursor in the synthesis of MAs. In addition, HvNAAT-B transcripts were detected in Zn-deficient shoots, but not in those that were deficient in Fe. Increased synthesis of MAs in Zn-deficient barley was not due to a deficiency of Fe, because Zn-deficient barley accumulated more Fe than did the control plants, ferritin transcripts were increased in Zn-deficient plants, and Zn deficiency promoted Fe transport from root to shoot. Moreover, analysis using the positron-emitting tracer imaging system (PETIS) confirmed that more 62Zn(II)-MAs than 62Zn2+ were absorbed by the roots of Zn-deficient barley plants. These data suggest that the increased biosynthesis and secretion of MAs arising from a shortage of Zn are not due to an induced Fe deficiency, and that secreted MAs are effective in absorbing Zn from the soil. [ABSTRACT FROM AUTHOR]

Only graminaceous monocots possess the Strategy II iron (Fe)-uptake system in which Fe is absorbed by roots as an Fe3+-phytosiderophore. In spite of being a Strategy II plant, however, rice ( Oryza sativa) contains the previously identified Fe2+ transporter OsIRT1 . In this study, we isolated the OsIRT2 gene from rice, which is highly homologous to OsIRT1. Real-time PCR analysis revealed that OsIRT1 and OsIRT2 are expressed predominantly in roots, and these transporters are induced by low-Fe conditions. When expressed in yeast ( Saccharomyces cerevisiae) cells, OsIRT2 cDNA reversed the growth defects of a yeast Fe-uptake mutant. This was similar to the effect of OsIRT1 cDNA. OsIRT1– and OsIRT2–green fluorescent protein fusion proteins localized to the plasma membrane when transiently expressed in onion ( Allium cepa L.) epidermal cells. OsIRT1 promoter– GUS analysis revealed that OsIRT1 is expressed in the epidermis and exodermis of the elongating zone and in the inner layer of the cortex of the mature zone of Fe-deficient roots. OsIRT1 expression was also detected in the ccompanion cells. Analysis using the positron-emitting tracer imaging system showed that rice plants are able to take up both an Fe3+-phytosiderophore and Fe2+. This result indicates that, in addition to absorbing an Fe3+-phytosiderophore, rice possesses a novel Fe-uptake system that directly absorbs the Fe2+, a strategy that is advantageous for growth in submerged conditions. [ABSTRACT FROM AUTHOR]

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: β-glucuronidase (GUS) analysis revealed that OsYSL2 was expressed in companion cells in iron-sufficient 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. [ABSTRACT FROM AUTHOR]

Nicotianamine (NA), a chelator of metals, is ubiquitously present in higher plants. In graminaceous plants, NA is a biosynthetic precursor of phytosiderophores and is thus a crucial component for iron (Fe) acquisition. Here, we show that three rice NA synthase ( NAS) genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long-distance transport of Fe and that the three genes are differentially regulated by Fe. OsNAS1 and OsNAS2 transcripts were detected in Fe-sufficient roots but not in leaves, and levels of both increased markedly in both roots and leaves in response to Fe deficiency. In contrast, the OsNAS3 transcript was present in leaves but was very low in roots of Fe-sufficient plants. Further, OsNAS3 expression was induced in roots but was suppressed in leaves in response to Fe deficiency. Promoter–GUS analysis revealed that OsNAS1 and OsNAS2 were expressed in Fe-sufficient roots in companion cells and pericycle cells adjacent to the protoxylem. With Fe deficiency, OsNAS1 and OsNAS2 expression extended to all root cells along with an increase in phytosiderophore secretion. In Fe-deficient plants, OsNAS1 and OsNAS2 were expressed in the vascular bundles of green leaves and in all cells of leaves showing severe chlorosis. OsNAS3 expression was restricted to the pericycle and companion cells of the roots, and in companion cells of leaves irrespective of Fe status. These results strongly suggested that NAS and NA play an important role in long-distance transport of Fe in rice plants, in addition to their roles in phytosiderophore secretion from roots. [ABSTRACT FROM AUTHOR]

Examines the nicotianamine NA (NA) synthase gene expression in barley and rice in iron (Fe) deficient condition. Alteration of genes during adaptation to Fe deficiency; Role of NA in metal cation homeostasis; Trimerization of S-adenosylmethionine.

Only graminaceous monocots possess the Strategy II iron (Fe)-uptake system in which Fe is absorbed by roots as an Fe3+-phytosiderophore. In spite of being a Strategy II plant, however, rice ( Oryza sativa) contains the previously identified Fe2+ transporter OsIRT1 . In this study, we isolated the OsIRT2 gene from rice, which is highly homologous to OsIRT1. Real-time PCR analysis revealed that OsIRT1 and OsIRT2 are expressed predominantly in roots, and these transporters are induced by low-Fe conditions. When expressed in yeast ( Saccharomyces cerevisiae) cells, OsIRT2 cDNA reversed the growth defects of a yeast Fe-uptake mutant. This was similar to the effect of OsIRT1 cDNA. OsIRT1– and OsIRT2–green fluorescent protein fusion proteins localized to the plasma membrane when transiently expressed in onion ( Allium cepa L.) epidermal cells. OsIRT1 promoter– GUS analysis revealed that OsIRT1 is expressed in the epidermis and exodermis of the elongating zone and in the inner layer of the cortex of the mature zone of Fe-deficient roots. OsIRT1 expression was also detected in the ccompanion cells. Analysis using the positron-emitting tracer imaging system showed that rice plants are able to take up both an Fe3+-phytosiderophore and Fe2+. This result indicates that, in addition to absorbing an Fe3+-phytosiderophore, rice possesses a novel Fe-uptake system that directly absorbs the Fe2+, a strategy that is advantageous for growth in submerged conditions. [ABSTRACT FROM AUTHOR]