Miki Kawachi

Vacuolar Nicotianamine Has Critical and Distinct Roles under Iron Deficiency and for Zinc Sequestration in Arabidopsis

This work implicates the Arabidopsis membrane protein ZIF1 in the vacuolar accumulation of the low molecular mass metal chelator nicotianamine. Precise regulation of intracellular nicotianamine distribution is critical for both Zn and Fe homeostasis as well as for the selective discrimination between these chemically similar micronutrients along the pathway of their movement inside the plant. The essential micronutrients Fe and Zn often limit plant growth but are toxic in excess. Arabidopsis thaliana ZINC-INDUCED FACILITATOR1 (ZIF1) is a vacuolar membrane major facilitator superfamily protein required for basal Zn tolerance. Here, we show that overexpression of ZIF1 enhances the partitioning into vacuoles of the low molecular mass metal chelator nicotianamine and leads to pronounced nicotianamine accumulation in roots, accompanied by vacuolar buildup of Zn. Heterologous ZIF1 protein localizes to vacuolar membranes and enhances nicotianamine contents of yeast cells engineered to synthesize nicotianamine, without complementing a Zn-hypersensitive mutant that additionally lacks vacuolar membrane Zn2+/H+ antiport activity. Retention in roots of Zn, but not of Fe, is enhanced in ZIF1 overexpressors at the expense of the shoots. Furthermore, these lines exhibit impaired intercellular Fe movement in leaves and constitutive Fe deficiency symptoms, thus phenocopying nicotianamine biosynthesis mutants. Hence, perturbing the subcellular distribution of the chelator nicotianamine has profound, yet distinct, effects on Zn and Fe with respect to their subcellular and interorgan partitioning. The zif1 mutant is also hypersensitive to Fe deficiency, even in media lacking added Zn. Therefore, accurate levels of ZIF1 expression are critical for both Zn and Fe homeostasis. This will help to advance the biofortification of crops.

Deletion of a Histidine-rich Loop of AtMTP1, a Vacuolar Zn2+/H+ Antiporter of Arabidopsis thaliana, Stimulates the Transport Activity

Arabidopsis thaliana AtMTP1 belongs to the cation diffusion facilitator family and is localized on the vacuolar membrane. We investigated the enzymatic kinetics of AtMTP1 by a heterologous expression system in the yeast Saccharomyces cerevisiae, which lacked genes for vacuolar membrane zinc transporters ZRC1 and COT1. The yeast mutant expressing AtMTP1 heterologously was tolerant to 10 mm ZnCl2. Active transport of zinc into vacuoles of living yeast cells expressing AtMTP1 was confirmed by the fluorescent zinc indicator FuraZin-1. Zinc transport was quantitatively analyzed by using vacuolar membrane vesicles prepared from AtMTP1-expressing yeast cells and radioisotope 65Zn2+. Active zinc uptake depended on a pH gradient generated by endogenous vacuolar H+-ATPase. The activity was inhibited by bafilomycin A1, an inhibitor of the H+-ATPase. The Km for Zn2+ and Vmax of AtMTP1 were determined to be 0.30 μm and 1.22 nmol/min/mg, respectively. We prepared a mutant AtMTP1 that lacked the major part (32 residues from 185 to 216) of a long histidine-rich hydrophilic loop in the central part of AtMTP1. Yeast cells expressing the mutant became hyperresistant to high concentrations of Zn2+ and resistant to Co2+. The Km and Vmax values were increased 2–11-fold. These results indicate that AtMTP1 functions as a Zn2+/H+ antiporter in vacuoles and that a histidine-rich region is not essential for zinc transport. We propose that a histidine-rich loop functions as a buffering pocket of Zn2+ and a sensor of the zinc level at the cytoplasmic surface. This loop may be involved in the maintenance of the level of cytoplasmic Zn2+.

A Mutant Strain Arabidopsis thaliana that Lacks Vacuolar Membrane Zinc Transporter MTP1 Revealed the Latent Tolerance to Excessive Zinc

A mutant line of Arabidopsis thaliana that lacks a vacuolar membrane Zn(2+)/H(+) antiporter MTP1 is sensitive to zinc. We examined the physiological changes in this loss-of-function mutant under high-Zn conditions to gain an understanding of the mechanism of adaptation to Zn stress. When grown in excessive Zn and observed using energy-dispersive X-ray analysis, wild-type roots were found to accumulate Zn in vacuolar-like organelles but mutant roots did not. The Zn content of mutant roots, determined by chemical analysis, was one-third that of wild-type roots grown in high-Zn medium. Severe inhibition of root growth was observed in mtp1-1 seedlings in 500 muM ZnSO(4). Suppression of cell division and elongation by excessive Zn was reversible and the cells resumed growth in normal medium. In mutant roots, a marked formation of reactive oxygen species (ROS) appeared in the meristematic zone, where the MTP1 gene was highly expressed. Zn treatment enhanced the expression of several genes involved in Zn tolerance: namely, the plasma membrane Zn(2+)-export ATPase, HMA4, and plasma and vacuolar membrane proton pumps. CuZn-superoxide dismutases, involved in the detoxification of ROS, were also induced. The expression of plasma membrane Zn-uptake transporter, ZIP1, was suppressed. The up- or down-regulation of these genes might confer the resistance to Zn toxicity. These results indicate an essential role of MTP1 in detoxification of excessive Zn and provide novel information on the latent adaptation mechanism to Zn stress, which is hidden by MTP1.

Synchrony between flower opening and petal-color change from red to blue in morning glory, Ipomoea tricolor cv. Heavenly Blue

Petal color change in morning glory Ipomoea tricolor cv. Heavenly Blue, from red to blue, during the flower-opening period is due to an unusual increase in vacuolar pH (pHv) from 6.6 to 7.7 in colored epidermal cells. We clarified that this pHv increase is involved in tonoplast-localized Na+/H+ exchanger (NHX). However, the mechanism of pHv increase and the physiological role of NHX1 in petal cells have remained obscure. In this study, synchrony of petal-color change from red to blue, pHv increase, K+ accumulation, and cell expansion growth during flower-opening period were examined with special reference to ItNHX1. We concluded that ItNHX1 exchanges K+, but not Na+, with H+ to accumulate an ionic osmoticum in the vacuole, which is then followed by cell expansion growth. This function may lead to full opening of petals with a characteristic blue color.

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1.

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn2+/H+ antiporter of the cation diffusion facilitator family. Here we present a structure–function analysis of AtMTP1‐mediated transport and its remarkable Zn2+ selectivity by functional complementation tests of more than 50 mutant variants in metal‐sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad‐specificity divalent cation transporter YiiP. The Zn2+‐binding sites of EcYiiP in the cytoplasmic C‐terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N‐terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn‐sensitive yeast strain. On the cytosolic side of the active Zn2+‐binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non‐selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn2+ into the intra‐membrane Zn2+‐binding site of AtMTP1. A conformational change in the immediately preceding His‐rich cytosolic loop may displace Asn258 and permit Zn2+ entry into the pore. This would allow dynamic coupling of Zn2+ transport to the His‐rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn2+ levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N‐terminal and His‐rich intra‐molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra‐ or inter‐molecular domain interactions, all of which are not conserved in the non‐selective EcYiiP.

A high molecular mass zinc transporter MTP12 forms a functional heteromeric complex with MTP5 in the Golgi in Arabidopsis thaliana.

Zinc (Zn) is an essential micronutrient required for plant growth and development. In Arabidopsis thaliana, several families of Zn transporters engaged in Zn import, export and intracellular compartmentalization play important roles in Zn homeostasis. We describe a novel Zn transporter, A. thaliana metal tolerance protein 12 (AtMTP12), which belongs to the cation diffusion facilitator family. AtMTP12 is predicted to consist of 798 amino acids and have 14 transmembrane segments. The expression of AtMTP12 in suspension‐cultured cells was not affected by Zn deficiency or excess. Heterologous expression in a mutant of budding yeast (Saccharomyces cerevisiae) that lacks Msc2p, an orthologue of AtMTP12, revealed that AtMTP12 complements the growth phenotype of the msc2 mutant when AtMTP5t1, one of the splicing variants of AtMTP5, is coexpressed. Transient expression of AtMTP12‐fused green fluorescent protein in A. thaliana mesophyll protoplasts demonstrated that AtMTP12 is localized to the Golgi apparatus. Moreover, AtMTP12 and AtMTP5t1 interact in the Golgi, as determined by a bimolecular fluorescence complementation assay. These results suggest that AtMTP12 forms a functional complex with AtMTP5t1 to transport Zn into the Golgi.

Zinc‐binding and structural properties of the histidine‐rich loop of Arabidopsis thaliana vacuolar membrane zinc transporter MTP1

The vacuolar Zn2+/H+ antiporter of Arabidopsis thaliana, AtMTP1, has a cytosolic histidine‐rich loop (His‐loop). We characterized the structures and Zn2+‐binding properties of the His‐loop and other domains. Circular dichroism analyses revealed that the His‐loop partly consists of a polyproline type II structure and that its conformational change is induced by Zn2+ as well as the C‐terminal domain. Isothermal titration calorimetry of the His‐loop revealed a binding number of four Zn2+ per molecule. Numbers of Ni and Co associated with the His‐loop were approximately one ion per molecule and the thermodynamic parameters of the association with these ions were different from that of Zn2+. These results suggest the involvement of the His‐loop in sensing cytosolic Zn2+ and in the regulation of zinc transport activity through Zn2+‐induced structural change.

Characterization of the histidine-rich loop of Arabidopsis vacuolar membrane zinc transporter AtMTP1 as a sensor of zinc level in the cytosol.

The vacuolar Zn(2+)/H(+) antiporter of Arabidopsis thaliana, AtMTP1, has a long cytosolic histidine-rich loop. A mutated AtMTP1 in which the first half of the loop (His-half) was deleted exhibited a 11-fold higher transport velocity in yeast cells. Transgenic lines overexpressing the His-half-deleted AtMTP1 in the loss-of-function mutant were evaluated for growth and metal content in the presence of various zinc concentrations. These overexpressing lines (35S-AtMTP1 and 35S-His-half lines) showed high tolerance to excess concentrations of zinc at 150 µM, as did the wild type, compared with the loss-of-function line. The His-half AtMTP1 transported cobalt in a heterologous expression assay in yeast, but the cumulative amount of cobalt in 35S-His-half plants was not increased. Moreover, the accumulation of calcium and iron was not changed in plants. Under zinc-deficient conditions, growth of 35S-His-half lines was markedly suppressed. Under the same conditions, the 35S-His-half lines accumulated larger amounts of zinc in roots and smaller amounts of zinc in shoots compared with the other lines, suggesting an abnormal accumulation of zinc in the roots of 35S-His-half lines. As a result, the shoots may exhibit zinc deficiency. Taken together, these results suggest that the His-loop acts as a sensor of cytosolic zinc to maintain an essential level in the cytosol and that the dysfunction of the loop results in an uncontrolled accumulation of zinc in the vacuoles of root cells.

Up-regulation of genes involved in N -acetylglucosamine uptake and metabolism suggests a recycling mode of chitin in intraradical mycelium of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi colonize roots and form two kinds of mycelium, intraradical mycelium (IRM) and extraradical mycelium (ERM). Arbuscules are characteristic IRM structures that highly branch within host cells in order to mediate resource exchange between the symbionts. They are ephemeral structures and at the end of their life span, arbuscular branches collapse from the tip, fungal cytoplasm withdraws, and the whole arbuscule shrinks into fungal clumps. The exoskeleton of an arbuscule contains structured chitin, which is a polymer of N-acetylglucosamine (GlcNAc), whereas a collapsed arbuscule does not. The molecular mechanisms underlying the turnover of chitin in AM fungi remain unknown. Here, a GlcNAc transporter, RiNGT, was identified from the AM fungus Rhizophagus irregularis. Yeast mutants defective in endogenous GlcNAc uptake and expressing RiNGT took up 14C-GlcNAc, and the optimum uptake was at acidic pH values (pH 4.0–4.5). The transcript levels of RiNGT in IRM in mycorrhizal Lotus japonicus roots were over 1000 times higher than those in ERM. GlcNAc-6-phosphate deacetylase (DAC1) and glucosamine-6-phosphate isomerase (NAG1) genes, which are related to the GlcNAc catabolism pathway, were also induced in IRM. Altogether, data suggest the existence of an enhanced recycling mode of GlcNAc in IRM of AM fungi.

The Role of Membrane Transport in the Detoxification and Accumulation of Zinc in Plants

Zinc (Zn) is an essential micronutrient having various cellular functions. It is an important component of the functional structure of numerous enzymes and regulatory proteins. Zn is important in the functional regulation of enzymes and membrane transport systems as a messenger in intracellular signaling. However, in plants, excess Zn is toxic and causes chlorosis and growth disorders. Thus, to ensure Zn homeostasis, transporters act in coordination to mediate cellular import and export of Zn and distribution between cell organelles. The transport machinery responsible for uptake and export of Zn has been identified and its intracellular localization, such as the plasma and vacuolar membranes and the chloroplast envelope, has been studied. Uptake and export are required to ensure that Zn reaches its target proteins and also for detoxification or sequestration of Zn in the cell. Transporters implicated in Zn transport include members of the metal tolerance protein (MTP), ZRT1/IRT1-like protein (ZIP), and heavy metal ATPase (HMA) families. Their roles in the acquisition, distribution, homeostasis, and signaling of Zn are described.