Seiji Nagasaka

Phytosiderophore Efflux Transporters Are Crucial for Iron Acquisition in Graminaceous Plants

Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released 14C-labeled deoxymugineic acid but not 14C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants.

Cloning and Characterization of Deoxymugineic Acid Synthase Genes from Graminaceous Plants

Graminaceous plants have evolved a unique mechanism to acquire iron through the secretion of a family of small molecules, called mugineic acid family phytosiderophores (MAs). All MAs are synthesized from l-Met, sharing the same pathway from l-Met to 2′-deoxymugineic acid (DMA). DMA is synthesized through the reduction of a 3″-keto intermediate by deoxymugineic acid synthase (DMAS). We have isolated DMAS genes from rice (OsDMAS1), barley (HvDMAS1), wheat (TaD-MAS1), and maize (ZmDMAS1). Their nucleotide sequences indicate that OsDMAS1 encodes a predicted polypeptide of 318 amino acids, whereas the other three orthologs all encode predicted polypeptides of 314 amino acids and are highly homologous (82–97.5%) to each other. The DMAS proteins belong to the aldo-keto reductase superfamily 4 (AKR4) but do not fall within the existing subfamilies of AKR4 and appear to constitute a new subfamily within the AKR4 group. All of the proteins showed DMA synthesis activity in vitro. Their enzymatic activities were highest at pH 8–9, consistent with the hypothesis that DMA is synthesized in subcellular vesicles. Northern blot analysis revealed that the expression of each of the above DMAS genes is up-regulated under iron-deficient conditions in root tissue, and that of the genes OsDMAS1 and TaDMAS1 is up-regulated in shoot tissue. OsDMAS1 promoter-GUS analysis in iron-sufficient roots showed that its expression is restricted to cells participating in long distance transport and that it is highly up-regulated in the entire root under iron-deficient conditions. In shoot tissue, OsDMAS1 promoter drove expression in vascular bundles specifically under iron-deficient conditions.

OsYSL18 is a rice iron(III)–deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints

Iron uptake and translocation in plants are important processes for both plant and human nutrition, whereas relatively little is known about the molecular mechanisms of iron transport within the plant body. Several reports have shown that yellow stripe 1 (YS1) and YS1-like (YSL) transporters mediate metal-phytosiderophore uptake and/or metal-nicotianamine translocation. Among the 18 YSL genes in rice (OsYSLs), OsYSL18 is predicted to encode a polypeptide of 679 amino acids containing 13 putative transmembrane domains. An OsYSL18-green fluorescent protein (GFP) fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL18 transports iron(III)–deoxymugineic acid, but not iron(II)–nicotianamine, zinc(II)–deoxymugineic acid, or zinc(II)–nicotianamine. Reverse transcriptase PCR analysis revealed more OsYSL18 transcripts in flowers than in shoots or roots. OsYSL18 promoter-β-glucuronidase (GUS) analysis revealed that OsYSL18 was expressed in reproductive organs including the pollen tube. In vegetative organs, OsYSL18 was specifically expressed in lamina joints, the inner cortex of crown roots, and phloem parenchyma and companion cells at the basal part of every leaf sheath. These results suggest that OsYSL18 is an iron-phytosiderophore transporter involved in the translocation of iron in reproductive organs and phloem in joints.

The rice mitochondrial iron transporter is essential for plant growth

In plants, iron (Fe) is essential for mitochondrial electron transport, heme, and Fe-Sulphur (Fe-S) cluster synthesis; however, plant mitochondrial Fe transporters have not been identified. Here we show, identify and characterize the rice mitochondrial Fe transporter (MIT). Based on a transfer DNA library screen, we identified a rice line showing symptoms of Fe deficiency while accumulating high shoot levels of Fe. Homozygous knockout of MIT in this line resulted in a lethal phenotype. MIT localized to the mitochondria and complemented the growth of Δmrs3Δmrs4 yeast defective in mitochondrial Fe transport. The growth of MIT-knockdown (mit-2) plants was also significantly impaired despite abundant Fe accumulation. Further, the decrease in the activity of the mitochondrial and cytosolic Fe-S enzyme, aconitase, indicated that Fe-S cluster synthesis is affected in mit-2 plants. These results indicate that MIT is a mitochondrial Fe transporter essential for rice growth and development.

Strigolactone signaling regulates rice leaf senescence in response to a phosphate deficiency

Strigolactones (SLs) act as plant hormones that inhibit shoot branching and stimulate secondary growth of the stem, primary root growth, and root hair elongation. In the moss Physcomitrella patens, SLs regulate branching of chloronemata and colony extension. In addition, SL-deficient and SL-insensitive mutants show delayed leaf senescence. To explore the effects of SLs on leaf senescence in rice (Oryza sativa L.), we treated leaf segments of rice dwarf mutants with a synthetic SL analogue, GR24, and evaluated their chlorophyll contents, ion leakage, and expression levels of senescence-associated genes. Exogenously applied GR24 restored normal leaf senescence in SL-deficient mutants, but not in SL-insensitive mutants. Most plants highly produce endogenous SLs in response to phosphate deficiency. Thus, we evaluated effects of GR24 under phosphate deficiency. Chlorophyll levels did not differ of in the wild-type between the sufficient and deficient phosphate conditions, but increased in the SL-deficient mutants under phosphate deficiency, leading in the strong promotion of leaf senescence by GR24 treatment. These results indicate that the mutants exhibited increased responsiveness to GR24 under phosphate deficiency. In addition, GR24 accelerated leaf senescence in the intact SL-deficient mutants under phosphate deficiency as well as dark-induced leaf senescence. The effects of GR24 were stronger in d10 compared to d17. Based on these results, we suggest that SLs regulate leaf senescence in response to phosphate deficiency.

Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation

Iron is essential for most living organisms. Plants transcriptionally induce genes involved in iron acquisition under conditions of low iron availability, but the nature of the deficiency signal and its sensors are unknown. Here we report the identification of new iron regulators in rice, designated Oryza sativa Haemerythrin motif-containing Really Interesting New Gene (RING)- and Zinc-finger protein 1 (OsHRZ1) and OsHRZ2. OsHRZ1, OsHRZ2 and their Arabidopsis homologue BRUTUS bind iron and zinc, and possess ubiquitination activity. OsHRZ1 and OsHRZ2 are susceptible to degradation in roots irrespective of iron conditions. OsHRZ-knockdown plants exhibit substantial tolerance to iron deficiency, and accumulate more iron in their shoots and grains irrespective of soil iron conditions. The expression of iron deficiency-inducible genes involved in iron utilization is enhanced in OsHRZ-knockdown plants, mostly under iron-sufficient conditions. These results suggest that OsHRZ1 and OsHRZ2 are iron-binding sensors that negatively regulate iron acquisition under conditions of iron sufficiency.

Glycolytic intermediates induce amorphous calcium carbonate formation in crustaceans

It has been thought that phosphorus in biominerals made of amorphous calcium carbonate (ACC) might be related to ACC formation, but no such phosphorus-containing compounds have ever been identified. Crustaceans use ACC biominerals in exoskeleton and gastroliths so that they will have easy access to calcium carbonate inside the body before and after molting. We have identified phosphoenolpyruvate and 3-phosphoglycerate, intermediates of the glycolytic pathway, in exoskeleton and gastroliths and found them important for stabilizing ACC.

Expression and enzyme activity of glutathione reductase is upregulated by Fe-deficiency in graminaceous plants.

Glutathione reductase (GR) plays an important role in the response to biotic and abiotic stresses in plants. We studied the expression patterns and enzyme activities of GR in graminaceous plants under Fe-sufficient and Fe-deficient conditions by isolating cDNA clones for chloroplastic GR (HvGR1) and cytosolic GR (HvGR2) from barley. We found that the sequences of GR1 and GR2 were highly conserved in graminaceous plants. Based on their nucleotide sequences, HvGR1 and HvGR2 were predicted to encode polypeptides of 550 and 497 amino acids, respectively. Both proteins showed in vitro GR activity, and the specific activity for HvGR1 was 3-fold that of HvGR2. Northern blot analyses were performed to examine the expression patterns of GR1 and GR2 in rice (Os), wheat (Ta), barley (Hv), and maize (Zm). HvGR1, HvGR2, and TaGR2 were upregulated in response to Fe-deficiency. Moreover, HvGR1 and TaGR1 were mainly expressed in shoot tissues, whereas HvGR2 and TaGR2 were primarily observed in root tissues. The GR activity increased in roots of barley, wheat, and maize and shoot tissues of rice, barley, and maize in response to Fe-deficiency. Furthermore, it appeared that GR was not post-transcriptionally regulated, at least in rice, wheat, and barley. These results suggest that GR may play a role in the Fe-deficiency response in graminaceous plants.

Diurnal changes in the expression of genes that participate im phytosiderophore synthesis in rice

Abstract The roots of graminaceous plants secrete mugineic acid family phytosiderophores (MAs), allowing the acquisition of sparingly soluble iron (Fe) from the soil. In barley, the secretion of MAs occurs in a distinct diurnal rhythm. Rice also secretes deoxymugineic acid (DMA) in response to Fe deficiency, but it is unclear whether the secretion also occurs in a diurnal rhythm. A time course analysis with Northern blots was used to examine the expression of genes encoding enzymes in the MA biosynthetic pathway. The transcriptional levels of the NAS and NAAT genes fluctuated diurnally in Fe-deficient rice roots. These results suggest that, as in barley, the secretion of DMA in rice changes in a diurnal rhythm. Furthermore, the transcriptional levels of genes encoding translation initiation factor 4A2 (elF4A2), ras-related small GTP-binding protein (GTPase), and ADP-ribosylation factor 1 (ARF1), which fluctuate diurnally in barley and are thought to be involved in the diurnal changes in MA secretion, also changed diurnally. As these genes are involved in intracellular vesicular transport, it is possible that secretion of DMA in rice is under the control of vesicular transport, as in barley root cells.

Nicotianamine synthase 2 localizes to the vesicles of iron‐deficient rice roots, and its mutation in the YXXφ or LL motif causes the disruption of vesicle formation or movement in rice

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.