Kunihiro Kasamo

Isolation and characterization of cDNAs encoding vacuolar H(+)-pyrophosphatase isoforms from rice (Oryza sativa L.).

The vacuolar H+-pyrophosphatase (V-PPase) is an electrogenic H+ pump, which was found in the plant vacuolar membrane. Two cDNA clones (OVP1 and OVP2) encoding the V-PPase were isolated from cultured rice (Oryza sativa L.) cells and subsequently sequenced. The sequence analysis has revealed thatOVP1 contains 2316 nucleotides of open reading frame (ORF) and 362 nucleotides of the 3′-untranslated region, whereasOVP2 comprises 2304 nucleotides of ORF and 312 nucleotides of the 3′-untranslated region. The nucleotide sequences of ORF ofOVP1 andOVP2 are 80.7% identical, and their 5′- and 3′-untranslated regions have 39.4% and 48.4% identity, respectively. The polypeptides encoded by the ORF ofOVP1 andOVP2 contain 771 and 767 amino acids, respectively, and the sequences of the OVP proteins are very similar to those of other V-PPases, which are shown to have 85–91% homology. Chromosomal mapping by RFLP techniques demonstrates that OVP1 and OVP2 are isoforms encoded by different genes. BothOVP1 andOVP2 are mapped on the same chromosome (chromosome 6) to a distance of ca. 90 cM. Northern analysis indicates that theOVP1 andOVP2 are also expressed in intact rice plants andOVP2 shows higher expression in the calli than the roots and shoots, compared toOVP1. These results show that at least two genes encoding the V-PPases are present in rice genome and their expressions are probably regulated in a different manner.

A Novel Histidine-Rich CPx-ATPase from the Filamentous Cyanobacterium Oscillatoria brevis Related to Multiple-Heavy-Metal Cotolerance

A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium Oscillatoria brevis. Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases. The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix. However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site. Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu(+) and Ag(+)) and divalent (Zn(2+) and Cd(2+)) heavy-metal ions at similar levels. Experiments on heavy-metal tolerance in Escherichia coli with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals. This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.

Regulation of plasma membrane H^+-ATPase activity by the membrane environment

The plant plasma membrane H+-ATPase is a proton pump which plays a central role in physiological functions such as nutrient uptake and intracellular pH regulation. This pump belongs to the P3-type ATPase family and creates an electrochemical gradient across the plasma membrane. The generation of this gradient has a major role in providing the energy for secondary active transport across the plasma membrane. The activity of the proton pump is regulated by the transcriptional and post-translational levels and by membrane environmental factors such as membrane lipids. Several reviews have appeared during the last few years concerning the regulatory mechanism at transcriptional and post-translational levels. The plasma membrane H+-ATPase requires lipids for activity. This lipid dependency suggests a possible mode of regulation of the H+-ATPase via modification of its lipid environment. This review focuses on the regulation of plasma membrane H+-ATPase by membrane lipids surrounding H+-ATPase molecules.

The plasma membrane H+-ATPase from higher plants: functional reconstitution into liposomes and its regulation by phospholipids

Abstract In the past 6 years, molecular and genetic studies of plant plasma membrane H+-ATPase (PMHA) have been achieved to elucidate the relation between its gene structure and the expression of the gene. The reconstitution technique is a useful tool, so that functional reconstitution of the PMHA into liposomes has been successfully carried out to directly demonstrate the function of the membrane protein. During purification and reconstitution, PMHA activity and proton pumping were found to be regulated by phospholipids. This review summarizes currently available information concerning the physiological functions, molecular genetics, enzyme structure and functional reconstitution of the plant PMHA.

Expression of an aquaporin at night in relation to the growth and root water permeability in barley seedlings

Abstract Both root and shoot of barley seedlings showed continuous growth at night as well as in the daytime. Root hydraulic conductivity (LPr) was monitored, and high LPr values were recorded at night. For revealing the molecular mechanism, the presence of an aquaporin HvPIP2;1 in barley roots was investigated. The accumulation of the HvPIP2;1 transcript was mostly observed from the evening to the middle of the night. HvPIP2;1 protein was most abundant around midnight. As for tissue localization, HvPIP2;1 protein was abundant in cells involved in water transport. These results suggested the possible involvement of HvPIP2;1 in the mechanism of water transport in roots.

A metallothionein and CPx-ATPase handle heavy-metal tolerance in the filamentous cyanobacterium Oscillatoria brevis

A metallothionein (BmtA) and a CPx‐ATPase (Bxa1) have been identified and characterized from the cyanobacterium Oscillatoria brevis. Both bmtA and bxa1 expression can be markedly induced in vivo by Zn2+ or Cd2+. Over‐expression of bmtA or bxa1 in Escherichia coli enhances Zn2+ and Cd2+ tolerance in the transformant. Dynamic studies on the expression of two genes showed that the maximum expression of bxa1 induced by Zn2+ and Cd2+ was much quicker than that of bmtA, suggesting distinct physiological roles of metallothionein and CPx‐ATPase in the handling of surplus metal.

Transmembrane topography of plasma membrane constituents in mung bean (Vigna radiata L.) hypocotyl cells: I. Transmembrane distribution of phospholipids

The transmembrane distribution of phospholipids (PLs) in the plasma membrane (PM) of mung bean (Vigna radiata L.) hypocotyl cells was investigated using annexin V-fluorescein isothiocyanate, porcine pancreas phospholipase A(2), and (31)P-nuclear magnetic resonance (NMR) spectroscopy. Phosphatidylserine was not located on the cell surface of mung bean protoplasts. However, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid were found to be almost symmetrically distributed across right-side-out PM vesicles obtained by aqueous two-phase partitioning by porcine pancreas phospholipase A(2) assay. (31)P-NMR assay showed that the amount of PLs is about equal in the outer and the inner leaflets of the right-side-out PM vesicles. These results suggest that the topography of PM PLs might not contribute to well-known asymmetrical properties of the outer and inner surfaces of higher plant PMs. It is also indicated that inside-out PM vesicles created by Brij 58-treatment do not retain the native PL topography on dithionate reduction of 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled PLs incorporated in the PM vesicles.

Hydraulic conductivity and aquaporins of cortical cells in gravitropically bending roots of Pisum sativum L.

Abstract We examined the differential elongation of gravitropically bending roots of Pisum sativum L. in terms of cell enlargement and water uptake by cells in the growing tissue. Hydraulic conductivity between the elongating and mature tissues (Lp) was estimated from the equation G = A × Lp × Δψ, where G is the water-uptake rate, A is the surface area of a single cell and Δψ is the driving force. The rate of entry of water into a cell was estimated from the rate of increase in the volumes of cells in the outer cortex, which were calculated from longitudinal sections at given times. Gravitropic bending occurred 1 h after the application of gravi-stimulation and the curvature increased rapidly for the next 3 h. The biggest difference in the partial elongation rate between opposite sides of a root was found in the region 3 to 4 mm from the root tip at the start of stimulation. Cell enlargement rate was 2.8 to 3.8 times greater on the upper side of the root than on the lower side. The water potential and the osmotic potential, in both the elongating and mature tissues, were the same on both sides of the root. Therefore, there was no difference in the driving force for water flow. Hydraulic conductivity was 2.3 to 4.2 times greater on the upper side of the root than on the lower side. There was no difference between the upper and lower sides of the root in the amounts of 19-kD and 24-kD proteins in membrane fractions, which we assumed to be aquaporins (putative aquaporins), as estimated with two preparations of polyclonal antibodies. The differential elongation that occurred during root gravitropism was caused by a difference in Lp. However, the difference in Lp did not appear to be regulated by the concentration in cell membranes of the putative aquaporins.

Isolation of barley salT gene : Its relation to salt tolerance and to hormonal regulation by abscisic acid and jasmonic acid

Abstract A homologous gene to rice salT was isolated from barley and designated as Hv-salT. Since the expression of rice salT was regulated by plant hormones (abscisic acid and jasmonic acid) and salt stress, the effects of these plant hormones and salt stress on the growth of barley seedlings and the expression of Hv-salT were investigated. In contrast to rice salT, almost no changes in the expression of Hv-salT by hormones and salt stress were detected.

Functional reconstitution of the tonoplast proton-ATPase from higher plants

Tonoplast proton ATPase (V-ATPase) is the most widely spread H + pump in plants. The electrochemical gradient generated by the H + pump provides the driving force for the secondary transport of amino acids, ions, sugars, and several metabolites. The V-ATPase has an apparent functional mass of 400–600 kDa and comprises at least 9 or 10 different subunits, of which the catalytic 67–73 kDa, the neucleotide-binding 55–62 kDa, proteolipids 95–115 and 16–17 kDa, and 44–29 kDa required for activity and assembly are universal components. Reconstitution of the V-ATPase complex into liposome has been successful. Reconstitution is convenient to assess whether any set of subunits associated with the V-ATPase is sufficient to couple ATP hydrolysis to proton pumping. In this review, we describe practical approach of reconstitution of the V-ATPase from mung bean hypocotyls into asolectin liposomes.