Naoko-Kishi Nishizawa

In vivo evidence that Ids3 from Hordeum vulgare encodes a dioxygenase that converts 2'-deoxymugineic acid to mugineic acid in transgenic rice

Abstract. 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.

Dynamic state of mugineic acid and analogous phytosiderophores in Fe‐deficient barley

Abstract An HPLC method for MAs (mugineic acid and its analogues) analysis was established. By this method, time course change of MAs secreted from the roots of Fe‐deficient barley were traced. After about one week of ‐Fe treatment, MAs secreted from the roots gradually increased and after 40 days it became plateau. The several MAs were synchronously secreted from the roots throughout a day; their amounts were maximum at noon. The pattern of MAs secretion was different from that of amino acids. MAI was found to be trace in roots, xylem sap and shoots of the control (+Fe) plant. However, MAs concentrations remarkably increased in those tissues when Fe‐deficiency chlorosis progressed due to ‐Fe treatment. From experiments of 14CO2‐fixation by photosynthesis, 14C‐MA1 secreted from the roots was found to be biosynthesized with a half‐life of 24 h.

A DIOXYGENASE GENE (IDS2) EXPRESSED UNDER IRON DEFICIENCY CONDITIONS IN THE ROOTS OF HORDEUM VULGARE

A λzapII cDNA library was constructed from mRNA isolated from Fe-deficient barley roots and screened with cDNA probes made from mRNA of Fe-deficient and Fe-sufficient (control) barley roots. Seven clones were selected. Among them a clone having the putative full-length mRNA of dioxygenase as judged by northern hybridization was selected and named Ids2 (iron deficiency-specific clone 2). Using a cDNA fragment as probe, two clones from the genomic library (λEMBL-III) were isolated and one was sequenced. The predicted amino acid sequence of Ids2 resembled that of 2-oxoglutarate-dependent dioxygenase. Ids2 is expressed in the Fe-deficient barley roots but is not in the leaves. The expression is repressed by the availability of Fe. Ids2 was also strongly expressed under Mn deficiency and weakly under Zn deficiency or excess NaCl (0.5%). The upstream 5′-flanking region of Ids2 has a root-specific cis element of the CaMV 35S promoter and a nodule-specific element of leghemoglobin, a metal regulatory element (MRE) and several Cu regulatory elements (UAS) of yeast metallothionein (CUP1).

Purification and characterization of nicotianamine synthase from Fe-deficient barley roots

Nicotianamine (NA), the key precursor of the mugineic acid family phytosiderophores (MAs), is synthesized from S-adenosylmethionine (SAM). The NA synthase was strongly induced by Fe-deficiency treatment, and the activity increased to the maximum level faster than the time of maximum level of MAs secretion and also before the appearance of severest chlorosis. The enzyme was mainly localized in the roots of barley. NA synthase had the optimum pH at 9.0, a molecular weight of about 40,000∼50,000 estimated by gel filtration or about 30,000 by SDS-PAGE. Using hydrophobic chromatography, hydroxylapatite chromatography, and preparative SDS-PAGE, NA synthase was purified as one band on SDS-PAGE.

The particular vesicle appearing in barley root cells and its relation to mugineic acid secretion

Abstract The changes in the ultrastructure of barley root cells induced by Fe‐deficiency was investigated in relation to secretion of mugineic acid and its analogues (MAS). We found that the particular vesicles were present in barley root cells under both conditions of sufficient and deficient iron. These particular vesicles were increased in number and volume under Fe‐deficient condition. These vesicles contained fibrous materials. Their limiting membrane was thinner than that of Golgi vesicles, and they had ribosomes around their surface. Their structure suggests that they originated from rough endoplasmic reticulum. The vesicle volume was expanded especially before sunrise when MAs were enriched in Fe‐deficient barley roots. In the midday, after secretion of MAs, the volume of the vesicles decreased.

The role of nicotianamine synthase in response to Fe nutrition status in Gramineae

Nicotianamine is an intermediate for the biosynthesis of mugineic acid-family phytosiderophores (MAs) in the Gramineae and a key substance for iron metabolism in dicots. Nicotianamine synthase catalyzes the formation of nicotianamine from S-adenosylmethionine. Nicotianamine synthase activity was induced in barley roots at the 3rd day after withholding Fe supply and declined within one day followmg the supply of Fe3+-epihydroxymugineic acid. The induction of nicotianamine synthase activity by Fe-deficiency was observed also in sorghum, maize, and rye, and the level of nicotianamine synthase activity was highly associated with the MAs secreted among graminaceous plant tested. Therefore, the nicotianamine synthase gene may be a suitable candidate for making a transgenic plant tolerant to Fe-deficiency.

Effect of iron deficiency on S‐adenosylmethionine synthetase in barley roots

Abstract S‐adenosyl‐L‐methionine (SAM) synthetase, which catalyzes methionine to SAM, is one of the enzymes involved in the biosynthesis of the mugineic acid‐family phytosiderophores (MAs). The MAs are secreted from grass roots under iron (Fe)‐deficient conditions. We compared the SAM synthetase activities between Fe‐deficient and Fe‐sufficient barley roots. However, no difference was observed. Then we performed Northern hybridization analysis between Fe‐deficient and Fe‐sufficient barley tissues by a cDNA fragment of SAM synthetase isolated from barley. No increase in the amount of mRNA was detected in the Fe‐deficient barley roots as compared to the Fe‐sufficient ones. It was suggested that the first step in the phytosiderophore biosynthesis induced under the Fe‐deficiency is not SAM synthetase, but NA synthase and the second induced step is NA aminotransferase.

Genotypic variation among Indian graminaceous species with respect to phytosiderophore secretion

Graminaceous plants can acquire Fe from sparingly soluble inorganic Fe (III) compounds through root secreted phytosiderophores by chelation.

Iron deficiency specific cDNA (Ids1) with two homologous cysteine rich MT domains from the roots of barley

Abstract For the purpose of gene cloning for HAs‐synthesis or Fe(III)‐MAs transporter a ? gt10 cDNA library was constructed from mRNA isolated from Fe‐deficient barley roots. The library was then differentially screened between cDNA probes made from mRNA isolated from barley roots treated with +Fe and ‐Fe. Seven clones which hybridized specifically to the probe of Fe‐deficiency were selected. Their inserts however were too short and not likely to include full length of mRNA. On the basis of these results we decided to screen a newly constructed ? zapII cDNA library with one of the seven clones as a probe and selected a clone presumably having the full length of mRNA compared with northern hybridization. We named this cone as Ids1. The sequenced Ids1 consists of 503 nucleotides containing a putative open reading frame of 222 bp. It encodes a protein of 74 residues (7500 Da) having two cysteine rich domains like animal MT (class I MT).

Inductions of two enzyme activities involved in the biosynthesis of mugineic acid in Fe-deficient barley roots

The temporal changes in two enzyme activities involved in the biosynthesis of mugineic acid-family phytosiderophores are described. Both nicotianamine synthase and nicotianamine aminotransferase are significantly induced by Fe-deficiency treatment. It took less than one week to induce the activities in primary root tips of young barley plants. Although the amount of secreted MAs was changed drastically during the day, these enzyme activities did not show daily changes.