Satoshi Mori

Thioredoxin h is one of the major proteins in rice phloem sap

Sieve tubes play important roles in the transfer of nutrients as well as signals. Hundreds of proteins were found in pure phloem sap collected from rice (Oryza sativa L. cv. Kantou) plants through the cut ends of insect stylets. These proteins may be involved in nutrient transfer and signal transduction. To characterize the nature of these proteins, the partial amino-acid sequence of a 13kDa protein, named RPP13-1, that was abundant in the pure phloem sap was determined. A cDNA clone of 687 bp, containing an open reading frame of 122 amino acids, was isolated using corresponding oligonucleotides as a probe. The deduced amino-acid sequence was very similar to that of the ubiquitous thiol redox protein, thioredoxin. The consensus sequences of thioredoxins are highly conserved. No putative signal peptide was identified. Antiserum against wheat thioredoxin h cross-reacted with RPP13-1 in the phloem sap of rice plants. RPP131 produced in Escherichia coli was reactive to antiserum against wheat thioredoxin h. Both E. coli-produced RPP13-1 and the phloem sap proteins catalyzed the reduction of the disulfide bonds of insulin in the presence of dithiothreitol. These results indicate that an active thioredoxin is a major protein translocating in rice sieve tubes.

Iron Inefficiency in Maize Mutant ys1 (Zea mays L. cv Yellow-Stripe) Is Caused by a Defect in Uptake of Iron Phytosiderophores

To determine the Fe inefficiency factors in the maize mutant ys1 (Zea mays L. cv Yellow Stripe), root exudates of Fe-inefficient ys1 and of two Fe-efficient maize cultivars (Alice, WF9) were collected in axenic nutrient solution cultures. Analysis by thin-layer chromatography and high-performance liquid chromatography revealed that under Fe deficiency ys1 released the phytosiderophore 2[prime]-deoxymugineic acid (DMA) in quantities similar to those of Alice and WF9. Under nonaxenic conditions, DMA released by plants of all three cultivars was rapidly decomposed by microorganisms in the nutrient solution. Uptake experiments with 59Fe-labeled DMA, purified from root exudates of either Fe-deficient Alice or ys1 plants, showed up to 20 times lower uptake and translocation of 59Fe in ys1 than in Alice or WF9 plants. The presence of microorganisms during preculture and short-term uptake experiments had no significant effect on uptake and translocation rates of 59Fe in Alice and ys1 plants. We conclude that Fe inefficiency in the maize mutant ys1 is the result of a defect in the uptake system for Fe-phytosiderophores.

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.

Biosynthetic pathway of phytosiderophores in iron-deficient graminaceous plants

Abstract Graminaceous plants secrete iron-chelators, called phytosiderophores, from the roots to solubilize the external insoluble iron, and the amount of the secreted phytosiderophores increases under iron deficiency stress (Takagi 1976). The only phytosiderophores known so far are mugineic acid-family (MAs). We developed a cell-free system derived from root tips of iron-deficient barley for the biosynthesis of 2′-deoxymugineic acid (DMA) from L-methionine (Mori and Nishizawa 1987; Shojima et al. 1990). At the same time, we showed that nicotianamine (NA) was converted to DMA, in vitro, in the presence of 2-oxoglutarate and NADH. We suggested that the synthesis of DMA from NA involves of the following two steps. At first, the amino group at the 3″-carbon of NA may be transferred to amino group acceptors (2-oxoglutarate, pyruvate, or oxalacetate), then the resulting 3″-oxo form may be reduced with reductants (NADH or NADPH) (Fig. 1). Since the putative intermediate of the 3″-oxo form was not available unti...

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).