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A highly sensitive quantitative method for assaying nicotianamine (NA) and 2'-deoxymugineic acid (DMA) using liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) was developed. This method requires fewer sample preparation steps than high-performance liquid chromatography (HPLC). The amino and hydroxyl groups of NA and DMA were derivatized using 9-fluorenylmethoxycarbonyl chloride, which improved their retention on a C18 reversed-phase column and facilitated separation. The amounts of NA and DMA in 10 μl of xylem sap from rice cultivated under Fe-sufficient and Fe-deficient conditions were quantified without concentration. In Fe-sufficient plants, the concentrations of NA and DMA were almost equal to that of Fe. In Fe-deficient plants, the concentration of NA did not change significantly, whereas that of DMA increased markedly.

Iron (Fe) is essential for most living organisms, and thus Fe deficiency poses a major abiotic stress in crop production. Plants induce Fe utilization systems under conditions of low-Fe availability, but the molecular mechanisms of gene regulation under Fe deficiency remain largely unknown. We identified a novel transcription factor in rice and barley, IDEF2, which specifically binds to the Fe deficiency-responsive cis-acting element 2 (IDE2), using yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets (CASTing) experiment revealed that IDEF2 predominantly recognizes CA[A/C]G[T/C][T/C/A][T/C/A] within IDE2 as the core binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by RNA interference (RNAi) and chimeric repressor gene-silencing technology (CRES-T) caused aberrant Fe distribution between shoots and roots in rice grown hydroponically under Fe-sufficient or deficient conditions. These results reveal novel cis-element/trans-factor interactions that are functionally associated with iron homeostasis.

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]

A highly sensitive quantitative method for assaying nicotianamine (NA) and 2′-deoxymugineic acid (DMA) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) was developed. The amino and hydroxyl groups of NA and DMA were derivatized using 9-fluorenylmethoxycarbonyl chloride. The amounts of NA and DMA in 10 μl of xylem sap from rice cultivated under iron (Fe)-sufficient and Fe-deficient conditions were quantified without concentration. In Fe-sufficient plants, the concentrations of NA and DMA were almost equal to that of Fe. In Fe-deficient plants, the concentration of NA did not change significantly, whereas that of DMA increased markedly. [ABSTRACT FROM PUBLISHER]

Nicotianamine (NA), a metal chelator ubiquitous in higher plants, serves as an antihypertensive substance in humans. To engineer a novel antihypertensive rice that contains larger amounts of NA, the barley NA synthase gene, HvNAS1, was introduced into rice via Agrobacterium-mediated transformation. The introduced HvNAS1 was driven by pGluB-1, which induces strong gene expression in the endosperm of rice seeds. The NA content in transgenic rice seeds was up to fourfold greater than that in non-transgenic rice seeds. The Cre/ loxP DNA excision (CLX) system was used to remove the selectable marker gene for antibiotic resistance. Furthermore, the transgenic rice was crossed with a cleistogamous mutant to prevent gene transfer via pollen dispersal. These two modifications may minimize public concern with regard to the use of this transgenic rice. [ABSTRACT FROM AUTHOR]

Zinc (Zn) excess has significant toxicity to biological systems through metal-based cytotoxic reactions. Nicotianamine (NA) and deoxymugineic acid (DMA) are low-molecular-weight, high-affinity transition metal chelators. Studies have shown that NA may have a role in the tolerance of excess Zn. We show that a gene coding the iron (Fe)-regulated DNA-binding transcription factor (OsIRO2) and the downstream genes of OsIRO2, such as NA synthase, DMA synthase and the DMA-Fe3+ transporter, were induced in rice roots by excess Zn. Consistent with the expression of these genes, the amounts of endogenous NA, endogenous DMA and DMA secretion increased in the excess Zn roots. Although the Fe concentration in the excess Zn roots was much higher than that in the control, rice ferritin gene, OsFer1, was downregulated in Zn excess roots. OsIRT1, which is upregulated by Fe deficiency, was not induced in Zn excess roots, suggesting that OsIRO2 may not be induced simply by the Fe deficiency caused by excess Zn. The data indicate that the induction of OsIRO2 by excess Zn is responsible for the production of NA and DMA, which may play a role in maintaining cellular Zn availability. [ABSTRACT FROM AUTHOR]

The article focuses on a highly sensitive quantitative technique for determining nicotianamine by liquid chromatography/electrospray ionization time-of-flight mass spectrometry. The authors note that fluorenylmethoxycarbonylation of nicotianamine reduced its polarity and enabled its retention in a reversed-phase column. They stress that the high sensitivity of the analytical method allowed them to measure the amount of nicotianamine.