Nicotianamine Synthesis by OsNAS3 Is Important for Mitigating Iron Excess Stress in Rice.

Iron (Fe) toxicity in plants causes tissue damage and cellular homeostasis disorders, thereby affecting plant growth and development. Nicotianamine (NA) is a ubiquitous chelator of metal cations and is responsible for metal homeostasis. Rice has three NA synthase (NAS) genes, of which the expression of OsNAS1 and OsNAS2 but not of OsNAS3 is strongly induced in response to Fe deficiency. Recently, we found that OsNAS3 expression is strongly induced with excess Fe in most rice tissues, particularly old leaves, suggesting that it may play a vital role under excess Fe conditions. However, the mechanism by which OsNAS3 responds to excess Fe in rice remains poorly understood. In this study, we clarified the physiological response of OsNAS3 expression to excess Fe and the role of NA synthesis in this condition. Promoter GUS analyses revealed that OsNAS3 was widely expressed in roots, especially in vascular bundle, epidermis, exodermis, stem, and old leaf tissues under Fe excess compared to control plants. Nicotianamine and deoxymugineic acid (DMA; a type of phytosiderophore synthesized by Strategy II species) were present in roots and shoots under Fe excess likewise under control conditions. In addition, OsNAS3 knockout plants were sensitive to excess Fe, exhibiting inferior growth, reduced dry weight, severer leaf bronzing, and greater Fe accumulation in their leaves than non-transformants with excess Fe. We also observed that NA-overproducing rice was tolerant of excess Fe. These results show that NA synthesized by OsNAS3 under Fe excess condition is to mitigate excess Fe whereas NA synthesized by OsNAS1 and OsNAS2 under normal Fe condition is to enhance Fe translocation, suggesting the different roles and functions of the NA existence between these two conditions. Overall, these findings suggest that rice synthesizes NA with OsNAS3 under Fe excess in roots and shoots, and that NA and DMA within the plant body are important for mitigating excess Fe stress and alleviating other metal deficiencies in rice. This report will be important for the development of tolerant rice adapted to Fe-contaminated soils.

Characterization of the Nicotianamine Exporter ENA1 in Rice.

Under iron (Fe) deficiency, graminaceous plants produce and secrete Fe-chelating phytosiderophores of the mugineic acid (MA) family into the rhizosphere to solubilize and mediate uptake of sparingly soluble Fe in the soil. MAs and their biosynthetic intermediate, nicotianamine (NA), are also important for the translocation of divalent metals such as Fe and zinc (Zn) throughout the plant body. In this study, the physiological role of the efflux transporter EFFLUX TRANSPORTER OF NA (ENA1), which exports NA out of cells, was analyzed in rice. Promoter-GUS analysis showed that ENA1 was mainly expressed in roots, and strongly upregulated under Fe-deficient conditions. In epidermal onion cells and rice roots, green fluorescent protein-tagged ENA1 localized mainly to the plasma membrane, while a part of the fluorescence was observed in vesicular structures in the cytoplasm. In the younger stage after germination, ENA1-overexpressing rice plants exhibited truncated roots with many root hairs compared to wild-type plants, while these phenotype were not observed in high Zn-containing medium. In Arabidopsis, which use a different strategy for Fe uptake from rice, ENA1 overexpression did not show any apparent phenotypes. Oligo DNA microarray analysis in rice showed that ENA1 knockout affects the response to stress, especially in root plastids. These results suggest that ENA1 might be recycling between the plasma membrane and cellular compartments by vesicular transport, playing an important role in the transport of NA, which is important for the physiological response to Fe deficiency.

Iron transport and its regulation in plants.

Iron is an essential element for plants as well as other organisms, functioning in various cellular processes, including respiration, chlorophyll biosynthesis, and photosynthesis. Plants take up iron from soil in which iron solubility is extremely low especially under aerobic conditions at high-pH range. Therefore, plants have evolved efficient iron-uptake mechanisms. Because iron is prone to being precipitated and excess ionic iron is cytotoxic, plants also have sophisticated internal iron-transport mechanisms. These transport mechanisms comprise iron chelators including nicotianamine, mugineic acid family phytosiderophores and citrate, and various types of transporters of these chelators, iron-chelate complexes, or free iron ions. To maintain iron homeostasis, plants have developed mechanisms for regulating gene expression in response to iron availability. Expression of various genes involved in iron uptake and translocation is induced under iron deficiency by transcription factor networks and is negatively regulated by the ubiquitin ligase HRZ/BTS. This response is deduced to be mediated by cellular iron sensing as well as long-distance iron signaling. The ubiquitin ligase HRZ/BTS is a candidate intracellular iron sensor because it binds to iron and zinc, and its activity is affected by iron availability. The iron-excess response of plants is thought to be partially independent of the iron-deficiency response. In this review, we summarize and discuss extant knowledge of plant iron transport and its regulation.

The Nicotianamine Synthase Gene Is a Useful Candidate for Improving the Nutritional Qualities and Fe-Deficiency Tolerance of Various Crops.

With the global population predicted to grow by at least 25% by the year 2050, the sustainable production of nutritious foods will be necessary for human health and the environment. Iron (Fe) is an essential nutrient for both plants and humans. Fe is poorly soluble, especially at high pH levels, at which it is difficult for living organisms to accumulate sufficient Fe. In plants, Fe deficiency leads to low yield and poor nutritional quality, as it significantly affects chlorophyll synthesis. Fe deficiency is a worldwide agricultural problem that is especially serious in soils with a high pH, such as calcareous soils, which comprise approximately 30% of cultivated soils worldwide. Genetic improvements in crops that can tolerate Fe deficiency will be required to meet the demands for crop production and could ultimately contribute to the amelioration of global warming. Nicotianamine (NA) is an Fe chelator in plants that is involved in metal translocation in the plant body. In mammals, NA inhibits angiotensin I-converting enzyme, which plays a key role in blood pressure control. It was recently shown that the enhancement of NA production using nicotianamine synthase is useful for increasing not only NA but also Fe and Zn levels in crops such as rice, soybean, and sweet potato. Additionally, these plants showed Fe-deficiency tolerance in calcareous soil. These results suggested that NAS overexpression simultaneously improves food quality and increases plant production. This review summarizes progress in generating crops overexpressing NAS.

Paralogs and mutants show that one DMA synthase functions in iron homeostasis in rice.

Rice (Oryza sativa) secretes 2'-deoxymugineic acid (DMA) to acquire insoluble iron (Fe) from the rhizosphere. In rice, DMA is synthesized by DMA synthase 1 (OsDMAS1), a member of the aldo-keto reductase super family. We screened OsDMAS1 paralogs for DMA synthesis. None of these paralogs displayed in vitro DMA synthesis activity, suggesting that rice only harbors one functional DMAS. We further characterized OsDMAS1 mutant plants. We failed to screen homozygous knock-out plants (dmas-1), so we characterized DMAS knock-down plants (dmas-kd1 and dmas-kd2). Under Fe-deficient conditions, dmas-kd1 plants were more chlorotic compared to the wild-type (WT) plants, and the expression of OsNAS3, OsYSL2, OsIRT1, and OsIRO2 was significantly up-regulated in the dmas-kd1 mutant, indicating that metal homeostasis was significantly disturbed. The secretion of DMA in dmas-kd1 was not significantly reduced. The dmas-kd1 plants accumulated less Fe in their roots compared to WT plants when grown with 10 µM FeSO4. The dmas-kd1 plants accumulated more Zn in their roots compared to WT plants under Fe-deficient, Fe-EDTA, and FeSO4 conditions. In both dehusked rice seeds (brown rice) and polished rice, no differences were observed for Fe, Cu, or Mn accumulation, whereas dmas-kd1 seeds significantly accumulated more Zn in brown rice. Our data suggests that rice only harbors one functional gene for DMA synthesis. In addition, the knock-down of OsDMAS1 significantly up-regulates the genes involved in Fe uptake and homeostasis.

The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice.

Iron is an essential metal element for all living organisms. Graminaceous plants produce and secrete mugineic acid family phytosiderophores from their roots to acquire iron in the soil. Phytosiderophores chelate and solubilize insoluble iron hydroxide in the soil. Subsequently, plants take up iron-phytosiderophore complexes through specific transporters on the root cell membrane. Phytosiderophores are also thought to be important for the internal transport of various transition metals, including iron. In this study, we analyzed TOM2 and TOM3, rice homologs of transporter of mugineic acid family phytosiderophores 1 (TOM1), a crucial efflux transporter directly involved in phytosiderophore secretion into the soil. Transgenic rice analysis using promoter-ß-glucuronidase revealed that TOM2 was expressed in tissues involved in metal translocation, whereas TOM3 was expressed only in restricted parts of the plant. Strong TOM2 expression was observed in developing tissues during seed maturation and germination, whereas TOM3 expression was weak during seed maturation. Transgenic rice in which TOM2 expression was repressed by RNA interference showed growth defects compared with non-transformants and TOM3-repressed rice. Xenopus laevis oocytes expressing TOM2 released (14)C-labeled deoxymugineic acid, the initial phytosiderophore compound in the biosynthetic pathway in rice. In onion epidermal and rice root cells, the TOM2-GFP fusion protein localized to the cell membrane, indicating that the TOM2 protein is a transporter for phytosiderophore efflux to the cell exterior. Our results indicate that TOM2 is involved in the internal transport of deoxymugineic acid, which is required for normal plant growth.

Transcriptomic analyses of maize ys1 and ys3 mutants reveal maize iron homeostasis.

To acquire iron (Fe), graminaceous plants secrete mugineic acid family phytosiderophores (MAs) (Takagi, 1976 [1]) through the MAs efflux transporter TOM1 (Nozoye et al., 2011 [2]) and take up Fe in the form of Fe(III)-MAs complexes through the Fe(III)-MAs transporter YS1 (Curie et al., 2001 [3]). Yellow stripe 1 (ys1) and ys3 are recessive mutants of maize (Zea mays L.) that result in symptoms typical of Fe deficiency, i.e., interveinal chlorosis of the leaves. The ys1 mutant is defective in the YS1 transporter and is therefore unable to take up Fe(III)-MAs complexes. While the ys3 mutant has been shown to be defective in MA release, the causative gene has not been identified. The objective of the present work was to identify the genes responsible for the ys1 and ys3 phenotypes, so as to extend our understanding of Fe homeostasis in maize by qRT-PCR. In agreement with previous reports, the expression level of YS1 was decreased in the ys1 mutant. Moreover, we identified that the expression level of a homolog of TOM1 in maize (ZmTOM1) was significantly decreased in the ys3 mutant. Here described the quality control and analysis that were performed on the dataset. The data is publicly available through the GEO database with accession number GSE44557. The interpretation and description of these data are included in a manuscript (Nozoye et al., 2013 [4]).

Strigolactone regulates anthocyanin accumulation, acid phosphatases production and plant growth under low phosphate condition in Arabidopsis.

Phosphate is an essential macronutrient in plant growth and development; however, the concentration of inorganic phosphate (Pi) in soil is often suboptimal for crop performance. Accordingly, plants have developed physiological strategies to adapt to low Pi availability. Here, we report that typical Pi starvation responses in Arabidopsis are partially dependent on the strigolactone (SL) signaling pathway. SL treatment induced root hair elongation, anthocyanin accumulation, activation of acid phosphatase, and reduced plant weight, which are characteristic responses to phosphate starvation. Furthermore, the expression profile of SL-response genes correlated with the expression of genes induced by Pi starvation. These results suggest a potential overlap between SL signaling and Pi starvation signaling pathways in plants.

Enhanced levels of nicotianamine promote iron accumulation and tolerance to calcareous soil in soybean.

Iron (Fe) is an essential nutrient in both plants and humans. Fe deficiency on calcareous soil with low Fe availability is a major agricultural problem. Nicotianamine (NA) is one of the Fe chelator in plants, which is involved in metal translocation into seeds, and serves as an antihypertensive substance in humans. In this study, soybean plants overexpressing the barley NA synthase 1 (HvNAS1) gene driven by the constitutive CaMV 35S promoter were produced using Agrobacterium-mediated transformation. The transgenic soybean showed no growth defect and grew normally. The NA content of transgenic soybean seeds was up to four-fold greater than that of non-transgenic (NT) soybean seeds. The level of HvNAS1 expression was positively correlated with the amount of NA, and a high concentration of NA was maintained in the seeds in succeeding generations. The Fe concentration was approximately two-fold greater in transgenic soybean seeds than in NT soybean seeds. Furthermore, the transgenic soybeans showed tolerance to low Fe availability in calcareous soil. Our results suggested that increasing the NA content in soybean seeds by the overexpression of HvNAS1 offers potential benefits for both human health and agricultural productivity.

Rice nicotianamine synthase localizes to particular vesicles for proper function.

Graminaceous plants release mugineic acid family phytosiderophores to acquire iron from the soil. Recently, we reported that particular vesicles are involved in deoxymugineic acid (DMA) and nicotianamine (NA) biosynthesis and in DMA secretion from rice roots. A fusion protein of rice NA synthase 2 (OsNAS2) and synthetic green fluorescent protein (sGFP) was observed in a dot-like pattern, moving dynamically within the cell. OsNAS2 mutated in the tyrosine motif or di-leucine motif, which was reported to be involved in cellular transport, caused a disruption in vesicular movement and vesicular localization, respectively. Unlike OsNAS2, Arabidopsis NA synthases AtNAS1-4 were distributed uniformly in the cytoplasm with no localization in dot-like structures when transiently expressed in tobacco BY-2 cells. Interestingly, Fe deficiency-inducible genes were upregulated in the OsNAS2-sGFP plants, and the amounts of NA and DMA produced and DMA secreted by the OsNAS2-sGFP plants were significantly higher than in those by the non-transformants and domain-mutated lines. We propose a model for OsNAS2-localized vesicles in rice, and discuss why the introduction of OsNAS2-sGFP caused a disturbance in Fe homeostasis.

Expression of peanut Iron Regulated Transporter 1 in tobacco and rice plants confers improved iron nutrition.

Iron (Fe) limitation is a widespread agricultural problem in calcareous soils and severely limits crop production. Iron Regulated Transporter 1 (IRT1) is a key component for Fe uptake from the soil in dicot plants. In this study, the peanut (Arachis hypogaea L.) AhIRT1 was introduced into tobacco and rice plants using an Fe-deficiency-inducible artificial promoter. Induced expression of AhIRT1 in tobacco plants resulted in accumulation of Fe in young leaves under Fe deficient conditions. Even under Fe-excess conditions, the Fe concentration was also markedly enhanced, suggesting that the Fe status did not affect the uptake and translocation of Fe by AhIRT1 in the transgenic plants. Most importantly, the transgenic tobacco plants showed improved tolerance to Fe limitation in culture in two types of calcareous soils. Additionally, the induced expression of AhIRT1 in rice plants also resulted in high tolerance to low Fe availability in calcareous soils.

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.

Exploiting new tools for iron bio-fortification of rice.

Rice is one of the most important staple crops and efficient iron (Fe) adsorption during growth not only improves rice yield, but also enriches this essential micronutrient in rice grains to address Fe deficiency in humans. In this article, we review updates on research into the molecular mechanisms regulating Fe uptake from soil and its transport from roots to shoots to seeds in rice plants. Understanding the regulation and expression of genes involved in Fe homeostasis will benefit the development of variants with enhanced Fe utilization to improve rice output and quality.

Characterizing the crucial components of iron homeostasis in the maize mutants ys1 and ys3.

To acquire iron (Fe), graminaceous plants secrete mugineic acid family phytosiderophores through the phytosiderophore efflux transporter TOM1 and take up Fe in the form of Fe(III)-phytosiderophore complexes. Yellow stripe 1 (ys1) and ys3 are recessive mutants of maize (Zea mays L.) that show typical symptoms of Fe deficiency, i.e., interveinal chlorosis of the leaves. The ys1 mutant is defective in the Fe(III)-phytosiderophore transporter YS1 and is therefore unable to take up Fe(III)-phytosiderophore complexes. While the ys3 mutant has been shown to be defective in phytosiderophores release, the causative gene has not been identified. The present study was performed to characterize the expression profiles of the genes in ys1 and ys3 mutants to extend our understanding of Fe homeostasis in maize. Using quantitative real-time polymerase chain reaction, we assessed changes in the levels of gene expression in response to Fe deficiency of genes involved in Fe homeostasis, such as those related to phytosiderophore biosynthesis and Fe transport. As with other crops, these Fe deficiency-inducible genes were also upregulated in maize. In addition, these Fe deficiency-inducible genes were upregulated in both the ys1 and ys3 mutants, even under Fe-sufficient conditions. Indeed, the Fe concentrations in the roots of ys1 and ys3 plants were lower than that of wild-type controls. These results suggest that ys1 and ys3 are Fe-deficient during growth in the presence of Fe. In agreement with previous reports, the level of YS1 expression decreased in the ys1 mutant. Moreover, the expression level of a homolog of TOM1 in maize decreased significantly in the ys3 mutant. Unspliced introns of ZmTOM1 were detected only in ys3, and not in YS1YS3 or ys1, suggesting that ZmTOM1 may be involved in the ys3 phenotype.

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 (14)C-labeled deoxymugineic acid but not (14)C-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.

The spatial expression and regulation of transcription factors IDEF1 and IDEF2.

BACKGROUND AND AIMS: Under conditions of low iron availability, rice plants induce genes involved in iron uptake and utilization. The iron deficiency-responsive cis-acting element binding factors 1 and 2 (IDEF1 and IDEF2) regulate transcriptional response to iron deficiency in rice roots. Clarification of the functions of IDEF1 and IDEF2 could uncover the gene regulation mechanism. METHODS: Spatial patterns of IDEF1 and IDEF2 expression were analysed by histochemical staining of IDEF1 and IDEF2 promoter-GUS transgenic rice lines. Expression patterns of the target genes of IDEF1 and IDEF2 were analysed using transformants with induced or repressed expression of IDEF1 or IDEF2 grown in iron-rich or in iron-deficient solutions for 1 d. KEY RESULTS: IDEF1 and IDEF2 were highly expressed in the basal parts of the lateral roots and vascular bundles. IDEF1 and IDEF2 expression was dominant in leaf mesophyll and vascular cells, respectively. These expression patterns were similar under both iron-deficient and iron-sufficient conditions. IDEF1 was strongly expressed in pollen, ovaries, the aleurone layer and embryo. IDEF2 was expressed in pollen, ovaries and the dorsal vascular region of the endosperm. During seed germination, IDEF1 and IDEF2 were expressed in the endosperm and embryo. Expression of IDEF1 target genes was regulated in iron-rich roots similar to early iron-deficiency stages. In addition, the expression patterns of IDEF2 target genes were similar between iron-rich conditions and early or subsequent iron deficiency. CONCLUSIONS: IDEF1 and IDEF2 are constitutively expressed during both vegetative and reproductive stages. The spatial expression patterns of IDEF1 and IDEF2 overlap with their target genes in restricted cell types, but not in all cells. The spatial expression patterns and gene regulation of IDEF1 and IDEF2 in roots are generally conserved under conditions of iron sufficiency and deficiency, suggesting complicated interactions with unknown factors for sensing and transmitting iron-deficiency signals.

Production of Ascaris suum As14 protein and its fusion protein with cholera toxin B subunit in rice seeds.

Plants are attractive vaccine production and oral delivery systems. Cereals are excellent candidate for edible vaccines, which can express and store high levels of proteins for extended periods of time without degradation. In this study, we produced a 14-kDa protective surface antigen of Ascaris suum L3 larvae and its fusion chimera with a mucosal carrier molecule cholera toxin B subunit (CTB) in rice (Oryza sativa L.) under the control of the endosperm-specific glutelin-B promoter. We found that the recombinant protein expression levels reached 1.5 microg per seed, a comparably high amount as compared to previously reported transgenic rice expression experiments. Potentials of transgenic rice plants as a source of oral vaccines against swine roundworm are discussed.

Oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (As16) of Ascaris suum fused with cholera toxin B subunit.

Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated the rice Oryza sativa for production of As16-an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the endosperm reached 50 microg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic edible vaccine for controlling parasitic infection in animals.