Kyoko Higuchi

Metal movement within the plant: contribution of nicotianamine and yellow stripe 1-like transporters

Background Since the identification of the genes controlling the root acquisition of iron (Fe), the control of inter- and intracellular distribution has become an important challenge in understanding metal homeostasis. The identification of the yellow stripe-like (YSL) transporter family has paved the way to decipher the mechanisms of long-distance transport of Fe. Scope Once in the plant, Fe will systematically react with organic ligands whose identity is poorly known so far. Among potential ligands, nicotianamine has been identified as an important molecule for the circulation and delivery of metals since it participates in the loading of copper (Cu) and nickel in xylem and prevents Fe precipitation in leaves. Nicotianamine is a precursor of phytosiderophores, which are high-affinity Fe ligands exclusively synthesized by Poaceae species and excreted by roots for the chelation and acquisition of Fe. Maize YS1 is the founding member of a family of membrane transporters called YS1-like (YSL), which functions in root Fe-phytosiderophore uptake from the soil. Next to this well-known Fe acquisition role, most of the other YSL family members are likely to function in plant-wide distribution of metals since (a) they are produced in vascular tissues throughout the plant and (b) they are found in non-Poaceae species that do not synthesize phytosiderophores. The hypothesized activity as Fe-nicotianamine transporters of several YSL members has been demonstrated experimentally by heterologous expression in yeast or by electrophysiology in Xenopus oocytes but, despite numerous attempts, proof of the arabidopsis YSL substrate specificity is still lacking. Reverse genetics, however, has revealed a role for AtYSL members in the remobilization of Cu and zinc from senescing leaves, in the formation of pollen and in the Fe, zinc and Cu loading of seeds. Conclusions Preliminary data on the YSL family of transporters clearly argues in favour of its role in the long-distance transport of metals through and between vascular tissues to eventually support gametogenesis and embryo development.

Overexpression of the Barley Nicotianamine Synthase Gene HvNAS1 Increases Iron and Zinc Concentrations in Rice Grains

In humans, iron (Fe) and zinc (Zn) deficiencies result in major worldwide health problems. Transgenic technologies to produce Fe- and Zn-biofortified rice varieties offer a promising potential solution. Nicotianamine, the precursor of phytosiderophores, chelates Fe2+ and Zn2+ and plays an important role in transporting these metals to both vegetative and reproductive organs within the plant. Our objective was to increase Fe and Zn contents in rice grains by overexpressing the barley nicotianamine synthase gene HvNAS1. HvNAS1-overexpressing transgenic rice showed increased HvNAS1 expression and subsequent increases in endogenous nicotianamine and phytosiderophore content in shoots, roots, and seeds. Fe and Zn concentrations in polished T1 seeds from transgenic plants increased more than three and twofold, respectively; Fe and Zn concentrations also increased in both polished and brown T2 seeds. These results suggest that the overproduction of nicotianamine enhances the translocation of Fe and Zn into rice grains.

Three Nicotianamine Synthase Genes Isolated from Maize Are Differentially Regulated by Iron Nutritional Status

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30–38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.

Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition

To address the problem of iron-deficiency anemia, one of the most prevalent human micronutrient deficiencies globally, iron-biofortified rice was produced using three transgenic approaches: by enhancing iron storage in grains via expression of the iron storage protein ferritin using endosperm-specific promoters, enhancing iron translocation through overproduction of the natural metal chelator nicotianamine, and enhancing iron flux into the endosperm by means of iron(II)-nicotianamine transporter OsYSL2 expression under the control of an endosperm-specific promoter and sucrose transporter promoter. Our results indicate that the iron concentration in greenhouse-grown T2 polished seeds was sixfold higher and that in paddy field-grown T3 polished seeds was 4.4-fold higher than that in non-transgenic seeds, with no defect in yield. Moreover, the transgenic seeds accumulated zinc up to 1.6-times in the field. Our results demonstrate that introduction of multiple iron homeostasis genes is more effective for iron biofortification than the single introduction of individual genes.

Cloning of nicotianamine synthase genes from Arabidopsis thaliana

Abstract Nicotianamine synthase (NAS) catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine (NA). NA is present in all the plants; it chelates metal cations, and is considered to play a role in metal homeostasis in plants. Moreover, in graminaceous monocotyledonous plants, NA is an essential intermediate in the biosynthesis of mugineic acid family phytosiderophores (MAs). In order to identify the gene encoding NAS in dicotyledonous plants, Arabidopsis thaliana databases were searched using the nucleotide sequence of the NAS gene from barley (HvNAS), which we have recently isolated. We found several ESTs and three genomic sequences highly homologous to HvNAS in the databases. Based on these nucleotide sequences and that of HvNAS, we designed 2 sets of primers to isolate the NAS orthologues in Arabidopsis and succeeded in obtaining three DNA clones encoding AtNAS (AtNAS1, 2, and 3). These clones were expressed in Escherichia coli and their protein products displayed the N...

Remodeling of the Major Light-Harvesting Antenna Protein of PSII Protects the Young Leaves of Barley (Hordeum vulgare L.) from Photoinhibition under Prolonged Iron Deficiency

Because of the high demand for iron of the photosynthetic apparatus in thylakoid membranes, iron deficiency primarily affects the electron transfer between the two photosystems (PSI and PSII), resulting in photooxidative damage in plants. However, in barley, PSII is protected against photoinhibition, and the plant survives even with a low iron content in its chlorotic leaves. In this study, we report an adaptation mechanism of the photosynthetic apparatus in barley to iron deficiency, which is concomitant with the remodeling of a PSII antenna system. Transcriptome analysis revealed that long-term iron deficiency induced the expression of two genes of the major light-harvesting Chl a/b-binding protein of PSII (LHCII), namely HvLhcb1.11 and HvLhcb1.12. Chl fluorescence analysis of the wild type and Lhcb1-less chlorina mutants clearly showed that non-photochemical quenching (NPQ) of the wild type was increased by approximately 200% by iron deficiency, whereas NPQ of chlorina mutants did not change significantly under iron deficiency. The mutant showed severe photodamage in young leaves under prolonged iron deficiency, suggesting that the HvLhcb1 protein is essential for both thermal dissipation and photoprotection in iron-deficient barley. Analysis of thylakoid protein complexes revealed that the proportion of the monomeric form of Lhcb1 significantly increased in barley grown under iron-deficient conditions. We hypothesize that alteration of the HvLhcb1 subpopulations modifies the organization of LHCII in the thylakoid membranes, which is a key step for thermal dissipation to compensate for excess excitation energy and thereby protect the photosystems from serious damage in iron-deficient barley leaves.

Comparison of Iron Availability in Leaves of Barley and Rice

Iron (Fe) is an essential trace element in all eukaryotes. In higher plants, Fe deficiency causes interveinal chlorosis in young leaves. However, in barley and rice, both of which are “Strategy II” plants, the degree and the pattern of Fe-deficiency symptoms differ. In the present study, barley and rice plants were grown in the same container, i.e., by “coculturing,” to compensate for the amount of mugineic acids in rice in the nutrient solution. We examined the differential availability of Fe for distribution and retranslocation in shoots between barley and rice without considering the difference in the iron acquisition ability, which is affected by the differential mugineic acid secretion between barley and rice. Although the Fe concentration of young barley leaves had decreased under the coculture conditions, the SPAD value was similar to that in monocultured barley. In contrast, although there was an increase in the Fe concentration of the young leaves of cocultured rice, the SPAD value decreased, as in the case of monocultured rice. Rice accumulated Fe in old leaves, whereas in barley Fe was efficiently distributed to young leaves. Therefore, the SPAD value of the second leaf in rice remained constantly high. The Fe concentration of the second leaf in barley decreased under Fe-deficient coculture conditions, the SPAD value decreased and the senescence of the second leaf become accelerated. 59Fe pulse-labeling experiments suggested that in barley Fe was more efficiently retranslocated from old leaves to young leaves than that in rice. As a result, the level of Fe present in the fraction with a molecular weight lower than the 10,000/water-soluble Fe ratio was higher in the old leaves of barley than in the old leaves of rice under Fe-deficient conditions. Based on the results obtained, we suggest that the distribution and retranslocation characteristics of internal Fe in barley may be well adapted to Fe deficiency.

Difference in the distribution and speciation of cellular nickel between nickel‐tolerant and non‐tolerant Nicotiana tabacum L. cv. BY‐2 cells

To evaluate Ni dynamics at the subcellular level, the distribution and speciation of Ni were determined in wild-type (WT) and Ni-tolerant (NIT) tobacco BY-2 cell lines. When exposed to low but toxic levels of Ni, NIT cells were found to contain 2.5-fold more Ni (14% of whole-cell Ni values) in their cell walls than WT cells (6% of whole-cell Ni values). In addition to higher levels of Ni in the apoplast, a higher proportion (94%) of symplastic Ni was localized in the vacuoles of NIT cells than in the vacuoles of WT cells (81%). The concentration of cytosolic Ni in the NIT cells was significantly lower (18 nmol g(-1) FW) than that in the WT cells (85 nmol g(-1) FW). In silico simulation showed that 95% of vacuolar Ni was in the form of Ni-citrate complexes, and that free Ni(2+) was virtually absent in the NIT cells. On the other hand, the amount of free metal ions was markedly increased in WT cells because free citrate was depleted by chelation of Ni. A protoplast viability assay using BCECF-AM further demonstrated that the main mechanism that confers strong Ni tolerance was present in the symplast as opposed to the cell wall.

Allocation of Fe and ferric chelate reductase activities in mesophyll cells of barley and sorghum under Fe-deficient conditions

Although the photosynthetic apparatus requires large amounts of Fe, the adaptive mechanisms of mesophyll cells for Fe acquisition under Fe-deficient conditions are unknown. Barley and sorghum, which are tolerant and susceptible to Fe deficiency, respectively, have similar Fe and chlorophyll contents in their leaves. However, the Fe-deficient barley photosynthetic apparatus was functional while that of sorghum was not. We show that barley preferentially allocates Fe to thylakoid membranes under Fe-deficient conditions. On the other hand, in sorghum, the proportion of leaf Fe allocated to thylakoids was not altered by Fe deficiency. The relationship between the maintenance of photosynthesis and light-dependent ferric chelate reductase activity on plasma membranes and chloroplast envelopes is also discussed.

Iron deficiency causes zinc excess in Zea mays.

Abstract Iron deficiency stress causes a severe reduction in plant growth. Although Fe deficiency causes an imbalance in divalent heavy metal nutrients, the mechanisms underlying the growth reduction caused by this imbalance remain unclear. We investigated Zn uptake and accumulation in maize under Fe-deficient conditions. Under Fe-deficient conditions, Zn uptake was 15-fold higher and Zn accumulation was 16-fold higher than that under normal nutrient conditions. The Zn content of maize leaves under Fe-deficient conditions was >0.4 mg g−1 dry weight, which was higher than the content of plants grown in a nutrient solution containing 50 µM ZnCl2. Plant growth under conditions of both Fe and Zn deficiency was significantly higher than that under only Fe-deficient conditions. Moreover, Fe deficiency increased the thiol content of the plant. These results indicate that Fe deficiency causes excess uptake and accumulation of Zn, and that the stress resulting from the Zn overload accelerates growth reduction in maize.