mugineic-acid and 2--deoxymugineic-acid

An organic chemistry approach to the mechanistic elucidation of iron acquisition in graminaceous plants is introduced here. To elucidate this detailed mechanism using phytosiderophores, the efficient synthesis of 2'-deoxymugineic acid (DMA), a phytosiderophore of rice, was established. The synthetic DMA was confirmed to have similar iron transport activity to that of natural mugineic acid (MA). It was also revealed that the addition of synthetic DMA, along with iron, to a rice hydroponic solution enabled the rice to grow well even under an alkaline condition, and DMA clearly showed its high potential as a fertilizer to improve food production. On the other hand, the 2'-hydroxy group of MA was confirmed to serve as a point of introduction for labeling, allowing the synthesis of various mugineic acid derivatives as molecular probes. The incorporation of fluorescent mugineic acid into cells allowed them to be clearly observed by fluorescence confocal analysis, and this provided the first direct experimental evidence of transporter-mediated internalization of mugineic acid into cells.

Topics: Azetidinecarboxylic Acid; Chemistry, Organic; Fertilizers; Hordeum; Iron; Iron Compounds; Membrane Transport Proteins; Organometallic Compounds; Oryza; Plant Proteins; Plant Roots; Siderophores; Staining and Labeling

To study the structure-activity relationship of mugineic acid (MA), a phytosiderophore isolated from Hordeum velugare L. var. Minorimugi, several 2-deoxymugineic acid (DMA) analogues were synthesized. 1H-NMR spectra of DMA analogues and their Co(III) complexes were first measured and analyzed to elucidate the structures of metal complexes. CD spectra of the Co(III) and Fe(III) complexes of DMA analogues were then measured and compared with those of MA. Furthermore, the interaction between the Fe(III) complexes of DMA analogues and the phytosiderophore-Fe(III) complex transporter found in maize was examined.

Topics: Azetidinecarboxylic Acid; Cobalt; Ferric Compounds; Hordeum; Membrane Transport Proteins; Nuclear Magnetic Resonance, Biomolecular; Siderophores; Structure-Activity Relationship; Zea mays

Nicotianamine (NA), 2'-deoxymugineic acid (DMA), and mugineic acid (MA) are chelators required for iron uptake and transport in plants. Nicotianamine aminotransferase (NAAT), 2'-deoxymugineic acid synthase (DMAS), transporter of MAs (TOM), and efflux transporter of NA (ENA) are involved in iron uptake and transport in rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare); however, these families have not been fully identified and comprehensively analyzed in maize (Zea mays L.).. Here, we identified 5 ZmNAAT, 9 ZmDMAS, 11 ZmTOM, and 2 ZmENA genes by genome mining. RNA-sequencing and quantitative real-time PCR analysis revealed that these genes are expressed in various tissues and respond differently to high and low iron conditions. In particular, iron deficiency stimulated the expression of ZmDMAS1, ZmTOM1, ZmTOM3, and ZmENA1. Furthermore, we determined protein subcellular localization by transient expression of green fluorescent protein fusions in maize mesophyll protoplasts. ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1 localized in the cytoplasm, whereas ZmTOMs and ZmENAs targeted to plasma and tonoplast membranes, endomembranes, and vesicles.. Our results suggest that the different gene expression profiles and subcellular localizations of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA family members may enable specific regulation of phytosiderophore metabolism in different tissues and under different external conditions, shedding light on iron homeostasis in maize and providing candidate genes for breeding iron-rich maize varieties.

Topics: Azetidinecarboxylic Acid; Biological Transport; Chromosomes, Plant; Gene Expression Regulation, Plant; Genes, Reporter; Genome, Plant; Homeostasis; Iron; Iron Deficiencies; Multigene Family; Phylogeny; Plant Proteins; Protein Transport; Recombinant Fusion Proteins; Siderophores; Transaminases; Zea mays

Iron (Fe) is an essential nutrient, but is poorly bioavailable because of its low solubility in alkaline soils; this leads to reduced agricultural productivity. To overcome this problem, we first showed that the soil application of synthetic 2'-deoxymugineic acid, a natural phytosiderophore from the Poaceae, can recover Fe deficiency in rice grown in calcareous soil. However, the high cost and poor stability of synthetic 2'-deoxymugineic acid preclude its agricultural use. In this work, we develop a more stable and less expensive analog, proline-2'-deoxymugineic acid, and demonstrate its practical synthesis and transport of its Fe-chelated form across the plasma membrane by Fe(III)•2'-deoxymugineic acid transporters. Possibility of its use as an iron fertilizer on alkaline soils is supported by promotion of rice growth in a calcareous soil by soil application of metal free proline-2'-deoxymugineic acid.

Topics: Azetidinecarboxylic Acid; Fertilizers; Iron; Siderophores; Soil

Essential metals, such as iron (Fe) and zinc (Zn), in grains are important sources for seed germination and nutritional requirements, but the molecular mechanisms underlying their loading into grains are poorly understood. Recently, nodes in rice (

Topics: Azetidinecarboxylic Acid; Biological Transport; Edible Grain; Gene Expression Regulation, Plant; Gene Knockout Techniques; Iron; Membrane Transport Proteins; Organ Specificity; Oryza; Plant Proteins; Plant Roots; Protein Transport; Siderophores; Vacuoles; Xylem; Zinc

The phytosiderophore 2'-deoxymugineic acid (DMA) is exuded via the root system by all grasses (including important crop plants like rice, wheat and barley) to mobilize Fe(III) from soil and improve plant Fe nutrition, crucial for high crop yields. Elucidation of the biogeochemistry of 2'-deoxymugineic acid in the rhizosphere requires its quantification in minute amounts. To this end, (13)C4-DMA was synthesized for the first time, from cheap isotopically labeled starting materials. The synthetic route utilizes L-allyl((13)C2)glycine and L-(2-(13)C)azetidine ((13)C)carboxylic acid as versatile labeled building blocks. The title compound was recently used as an internal standard for analysis of soil and plant samples allowing the first accurate quantification of DMA in these matrices by means of LC-MS/MS. It is furthermore used in tracer experiments investigating biodegradation of DMA in soil.

Topics: Azetidinecarboxylic Acid; Carbon Isotopes; Isotope Labeling; Soil

Phytosiderophores (PS) form stable complexes with various transition metals. These ligands are exuded by the roots of graminacous plants as a mechanism for mobilizing and acquiring soil iron. To investigate iron mobilization and transport, a novel LC method in combination with ESI-MS/MS for the determination of three Fe(III)-complexes with mugineic acid (MA), 2'-epi-MA and 2'-deoxymugineic acid (DMA) has been developed. Liquid chromatographic separation was realized using a silica-based mixed-mode reversed phase/weak-anion exchange type stationary phase and a 50 mM ammonium acetate buffer, pH 6.5. Baseline separation of the two complex diastereomers Fe(III)-MA and Fe(III)-epi-MA could be achieved. ESI-MS/MS detection allowed for simultaneous quantification of the complexes and the free ligands. Limits of detection were determined to be 0.001 and 0.05 μM for DMA and Fe(III)-DMA, respectively. The analytical figures of merit of the novel method were evaluated and compared with a CE-ESI-MS method that we had published earlier. The LC-ESI-MS/MS method has been successfully applied to real samples derived from preliminary extraction experiments.

Topics: Azetidinecarboxylic Acid; Chromatography, Liquid; Ferric Compounds; Siderophores; Soil; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Higher plants acquire iron (Fe) from the rhizosphere through two strategies. Strategy II, employed by graminaceous plants, involves secretion of phytosiderophores (e.g. deoxymugineic acid in rice [Oryza sativa]) by roots to solubilize Fe(III) in soil. In addition to taking up Fe in the form of Fe(III)-phytosiderophore, rice also possesses the strategy I-like system that may absorb Fe(II) directly. Through mutant screening, we isolated a rice mutant that could not grow with Fe(III)-citrate as the sole Fe source, but was able to grow when Fe(II)-EDTA was supplied. Surprisingly, the mutant accumulated more Fe and other divalent metals in roots and shoots than the wild type when both were supplied with EDTA-Fe(II) or grown under water-logged field conditions. Furthermore, the mutant had a significantly higher concentration of Fe in both unpolished and polished grains than the wild type. Using the map-based cloning method, we identified a point mutation in a gene encoding nicotianamine aminotransferase (NAAT1), which was responsible for the mutant phenotype. Because of the loss of function of NAAT1, the mutant failed to produce deoxymugineic acid and could not absorb Fe(III) efficiently. In contrast, nicotianamine, the substrate for NAAT1, accumulated markedly in roots and shoots of the mutant. Microarray analysis showed that the expression of a number of the genes involved in Fe(II) acquisition was greatly stimulated in the naat1 mutant. Our results demonstrate that disruption of deoxymugineic acid biosynthesis can stimulate Fe(II) acquisition and increase iron accumulation in rice.

Topics: Amino Acid Sequence; Azetidinecarboxylic Acid; Cations, Divalent; DNA Mutational Analysis; Genes, Plant; Iron; Molecular Sequence Data; Oryza; Point Mutation; Seedlings; Seeds; Transaminases; Up-Regulation

We proposed that an Fe-deficiency-induced gene, Ids3 (Iron deficiency specific clone no. 3), from barley (Hordeum vulgare L.) roots encodes a dioxygenase that catalyzes the hydroxylation step from 2'-deoxymugineic acid (DMA) to mugineic acid (MA). To prove this hypothesis, we introduced the Ids3 gene into rice (Oryza sativa L.), which lacks Ids3 homologues and secretes DMA, but not MA. Transgenic rice plants, carrying either Ids3 cDNA or a barley genomic DNA fragment (20 kb) containing Ids3, were obtained using Agrobacterium-mediated transformation. Ids3 cDNA under the control of the cauliflower mosaic virus 35S promoter was constitutively expressed in both the roots and the leaves of the transgenic rice, regardless of Fe nutrition status. In contrast, in the roots of transformants carrying a barley genomic fragment, transcripts of Ids3 were markedly increased in response to Fe deficiency. Slight expression of Ids3 was also observed in the leaves of the Fe-deficient plants. Western blot analysis confirmed the induction of Ids3 in response to Fe deficiency in the roots of the transformants carrying a genomic fragment. These expression patterns indicate that the 5'-flanking region of Ids3 works as a strong Fe-deficiency-inducible promoter in rice, as well as in barley. Both kinds of transgenic rice secreted MA in addition to DMA under Fe-deficient conditions, but wild-type rice secreted only DMA. This is in vivo evidence that IDS3 is the "MA synthase" that converts DMA to MA.

Topics: Agrobacterium tumefaciens; Alkyl and Aryl Transferases; Azetidinecarboxylic Acid; Blotting, Western; Chromosome Mapping; DNA Transposable Elements; Gene Expression Regulation, Plant; Glucuronidase; Hordeum; Iron; Mixed Function Oxygenases; Oryza; Plant Leaves; Plant Proteins; Plant Roots; Plants, Genetically Modified; Plasmids; Promoter Regions, Genetic; Siderophores; Transaminases

Topics: Azetidinecarboxylic Acid; Azetines; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Edible Grain; Hordeum