Masayoshi Maeshima

Vacuolar H^+-pyrophosphatase

The H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) is a unique, electrogenic proton pump distributed among most land plants, but only some alga, protozoa, bacteria, and archaebacteria. This enzyme is a fine model for research on the coupling mechanism between the pyrophosphate hydrolysis and the active proton transport, since the enzyme consists of a single polypeptide with a calculated molecular mass of 71-80 kDa and its substrate is also simple. Cloning of the H(+)-PPase genes from several organisms has revealed the conserved regions that may be the catalytic site and/or participate in the enzymatic function. The primary sequences are reviewed with reference to biochemical properties of the enzyme, such as the requirement of Mg(2)(+) and K(+). In plant cells, H(+)-PPase coexists with H(+)-ATPase in a single vacuolar membrane. The physiological significance and the regulation of the gene expression of H(+)-PPase are also reviewed.

The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2.

Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.

The protein storage vacuole: a unique compound organelle

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.

Novel type aquaporin SIPs are mainly localized to the ER membrane and show cell-specific expression in Arabidopsis thaliana

We investigated the fourth subgroup of Arabidopsis aquaporin, small and basic intrinsic proteins (SIPs). When they were expressed in yeast, SIP1;1 and SIP1;2, but not SIP2;1, gave water‐channel activity. The transient expression of SIPs linked with green fluorescent protein in Arabidopsis cells and the subcellular fractionation of the tissue homogenate showed their ER localization. The SIP proteins were detected in all of the tissues, except for dry seeds. Histochemical analysis of promoter‐β‐glucuronidase fusions revealed the cell‐specific expression of SIPs. SIP1;1 and SIP1;2 may function as water channels in the ER, while SIP2;1 might act as an ER channel for other small molecules or ions.

CHARACTERIZATION OF MAJOR PROTEINS IN SWEET POTATO TUBEROUS ROOTS

The tuberous roots, but not other organs, of sweet potato contained large quantities of two proteins which accounted for more than 80% of the total proteins. The two proteins, tentatively named sporamins A and B, were monomeric forms with similar M,s (25 000). They were separated from each other by electrophoresis on polyacrylamide gels in a non-denaturing buffer or a buffer containing sodium dodecyl sulphate without being reduced by dithiothreitol. They were very similar to each other with respect to amino acid composition, peptide map and immunological properties. These proteins decreased in preference to other proteins during sprouting. The amino acid sequencing of the amino terminal part of sporamin A suggested that it consists of at least two molecular species with different combinations of a few amino acids.

NIP1;1, an Aquaporin Homolog, Determines the Arsenite Sensitivity of Arabidopsis thaliana *□

Arsenite [As(III)] is highly toxic to organisms, including plants. Very recently, transporters in rice responsible for As(III) transport have been described (Ma, J. F., Yamaji, N., Mitani, N., Xu, X. Y., Su, Y. H., McGrath, S. P., and Zhao, F. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 9931–9935), but little is known about As(III) tolerance. In this study, three independent As(III)-tolerant mutants were isolated from ethyl methanesulfonate-mutagenized M2 seeds of Arabidopsis thaliana. All three mutants carried independent mutations in Nodulin 26-like intrinsic protein 1;1 (NIP1;1), a homolog of an aquaporin. Two independent transgenic lines carrying T-DNA in NIP1;1 were highly tolerant to As(III), establishing that NIP1;1 is the causal gene of As(III) tolerance. Because an aquaglyceroporin is able to transport As(III), we measured As(III) transport activity. When expressed in Xenopus oocytes, NIP1;1 was capable of transporting As(III). As content in the mutant plants was 30% lower than in wild-type plants. Promoter β-glucuronidase and real-time PCR analysis showed that NIP1;1 is highly expressed in roots, and GFP-NIP1;1 is localized to the plasma membrane. These data show that NIP1;1 is involved in As(III) uptake into roots and that disruption of NIP1;1 function confers As(III) tolerance to plants. NIP1;2 and NIP5;1, closely related homologs of NIP1;1, were also permeable to As(III). Although the disruption of these genes reduced the As content in plants, As(III) tolerance was not observed in nip1;2 and nip5;1 mutants. This indicates that As(III) tolerance cannot be simply explained by decreased As contents in plants.

A Vacuolar-Type H+-ATPase in a Nonvacuolar Organelle Is Required for the Sorting of Soluble Vacuolar Protein Precursors in Tobacco Cells.

In plant cells, vacuolar matrix proteins are separated from the secretory proteins at the Golgi complex for transport to the vacuoles. To investigate the involvement of vacuolar-type ATPase (V-ATPase) in the vacuolar targeting of soluble proteins, we analyzed the effects of bafilomycin A1 and concanamycin A on the transport of vacuolar protein precursors in tobacco cells. Low concentrations of these inhibitors caused the missorting of several vacuolar protein precursors; sorting was more sensitive to concanamycin A than to bafilomycin A1. Secretion of soluble proteins from tobacco cells was also inhibited by bafilomycin A1 and concanamycin A. We next analyzed the subcellular localization of V-ATPase. V-ATPase was found in a wide variety of endomembrane organelles. Both ATPase activity and ATP-dependent proton-pumping activity in the Golgi-enriched fraction were more sensitive to concanamycin A than to bafilomycin A1, whereas these activities in the tonoplast fraction were almost equally sensitive to both reagents. Our observations indicate that the V-ATPase in the organelle that was recovered in the Golgi-enriched fraction is required for the transport of vacuolar protein precursors and that this V-ATPase is distinguishable from the tonoplast-associated V-ATPase.

Keep an Eye on PPi: The Vacuolar-Type H+-Pyrophosphatase Regulates Postgerminative Development in Arabidopsis

Oilseed germination requires gluconeogenesis, the conversion of storage lipids into carbohydrates, to sustain seedling heterotrophic growth. Pyrophosphate (PPi), a byproduct of ATP hydrolysis, is released by active metabolism in imbibed seeds. We report that the removal of PPi by plant vacuolar H+-pyrophosphatase is central for successful gluconeogenesis and resumption of postembryonic growth. Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H+-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1Pro:IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal β-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.

Increased Expression of Vacuolar Aquaporin and H+-ATPase Related to Motor Cell Function in Mimosa pudica L.

Mature motor cells of Mimosa pudica that exhibit large and rapid turgor variations in response to external stimuli are characterized by two distinct types of vacuoles, one containing large amounts of tannins (tannin vacuole) and one without tannins (colloidal or aqueous vacuole). In these highly specialized cells we measured the abundance of two tonoplast proteins, a putative water-channel protein (aquaporin belonging to the [gamma]-TIPs [tonoplast intrinsic proteins]) and the catalytic A-subunit of H+-ATPase, using either high-pressure freezing or chemical fixation and immunolocalization. [gamma]-TIP aquaporin was detected almost exclusively in the tonoplast of the colloidal vacuole, and the H+-ATPase was also mainly localized in the membrane of the same vacuole. Cortex cells of young pulvini cannot change shape rapidly. Development of the pulvinus into a motor organ was accompanied by a more than 3-fold increase per length unit of membrane in the abundance of both aquaporin and H+-ATPase cross-reacting protein. These results indicate that facilitated water fluxes across the vacuolar membrane and energization of the vacuole play a central role in these motor cells.

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+.