Molecular cloning of the plant plasma membrane H+-ATPase

Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H⁺-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H⁺-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.     

The Lmpma1 gene of Leptosphaeria maculans encodes a plasma membrane H+-ATPase isoform essential for pathogenicity towards oilseed rape

Following Agrobacterium tumefaciens-mediated mutagenesis in Leptosphaeria maculans, we identified the mutant 210, displaying total loss of pathogenicity towards its host plant (Brassica napus). Microscopic observations showed that m210 is unable to germinate on the host leaf surface and is thus blocked at the pre-penetration stage. The pathogenicity phenotype is linked with a single T-DNA insertion into the promoter region of a typical plasma membrane H⁺-ATPase-encoding gene, termed Lmpma1, thus leading to a twofold reduction in Lmpma1 expression. Since LmPMA1 is involved in intracellular pH homeostasis, we postulate that reduction in LmPMA1 activity disturbs the electrochemical transmembrane gradient in m210, thus leading to conidia defective in turgor pressure generation on leaf surface. Whole genome survey showed that L. maculans possesses a second plasma membrane H⁺-ATPase-encoding gene, termed Lmpma2. Silencing experiments, expression analyses and phylogenetic studies allowed us to highlight the essential role assumed by the Lmpma1 isoform in L. maculans pathogenicity.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

Dimorphism-associated changes in plasma membrane H+-ATPase activity of Candida albicans

In situ plasma membrane H⁺-ATPase activity was monitored during pH-regulated dimorphism of Candida albicans using permeabilized cells. ATPase activity was found to increase in both the bud and germ tube forming populations at 135 min which coincides with the time of evagination. Upon reaching the terminal phenotype the mycelial form exhibited higher H⁺-ATPase activity as compared to the yeast form. At the time of evagination H⁺-efflux exhibited an increase. K⁺ depletion resulted in attenuated ATPase activity and glucose induced H⁺-efflux. The results demonstrate that ATPase may play a regulatory role in dimorphism of C. albicans and K⁺ acts as a modulator.Abbreviations PM Plasma membrane - pHi intracellular pH - Pi inorganic phosphorus - TET Toluene: Ethanol: Triton X-100     

Localization of plasma membrane H+-ATPase in nodules of Phaseolus vulgaris L.

Legume nodules have specialized transport functions for the exchange of carbon and nitrogen compounds between bacteroids and root cells. Plasma membrane-type (vanadate-sensitive) H⁺-ATPase energizes secondary active transporters in plant cells and it could drive exchanges across peribacteroidal and plasmatic membranes. A nodule cDNA corresponding to a major isoform of Phaseolus vulgaris H⁺-ATPase (designated BHA1) has been cloned. BHA1 is a functional proton pump because after removal of its inhibitory domain and can complement a yeast mutant unable to synthesize a H⁺-ATPase. BHA1 is not nodule-specific, since it is also expressed in roots of uninfected plants. It belongs to the subfamily of plasma membrane H⁺-ATPases defined by the Arabidopsis AHA1, AHA2 and AHA3 genes and the tobacco PMA4 and corn MHA2 genes. In situ hybridization in nodule sections indicates high expression of BHA1 limited to uninfected cells. These results were confirmed by immunocytochemistry. The relatively low expression of plasma membrane-type H⁺-ATPase in Rhizobium-infected cells put a note of caution on the origin of the vanadate-sensitive ATPase described in preparations of peribacteroidal membranes. Also, our results indicate that active transport in symbiotic nodules is most intense at the plasma membrane of uninfected cells and support a specialized role of uninfected tissue for nitrogen transport.     

DNA methylation in the Alcohol dehydrogenase-1 gene of maize

Using a battery of methylation-sensitive restriction enzymes, cytosine methylation at 23 sites in a 7.6 kb region surrounding the Alcohol dehydrogenase-1 (Adh1) gene was measured in DNA prepared from immature maize cobs. Both the 5 upstream region and the entire coding region were hypomethylated in the two alleles examined. Methylation in Adh1 is independent of changes in Mutator transposable element methylation. The role of DNA methylation in Adh1 gene regulation is discussed.     

A DNA polymerase from maize axes: its purification and possible role

Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100–120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg²⁺, is stimulated by K⁺, has an optimum pH of 7.0 and its optimum temperature is 30–37 °C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28–40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase . A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase -antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme is discussed.     

Regulators of cell division in plant tissues

The cytokinins in certain fractions prepared from extracts of immature sweet-corn (Zea mays L.) kernels using polystyrene ion-exchange resins have been further investigated. Cytokinins active in the radish cotyledon bioassay were purified from these fractions and identified as 9--D-glucopyranosylzeatin, 9--D-glucopyranosyldihydrozeatin, O--D-glucopyranosylzeatin. and O--D-glucopyranosyl-9--D-ribofuranosylzeatin. In addition, compounds which resemble zeatin and its glycosides in chromatographic behaviour and in ultraviolet absorption characteristics were purified from extracts of the same material by high-performance liquid chromatography. In addition to zeatin and zeatin riboside, the following compounds were identified unambiguously: O--D-glucopyranosyl-9--D-ribofuranosyldihydrozeatin, O--D-glucopyranosyldihydrozeatin, and hihydrozeatin riboside. A further compound was tentatively identified as O--D-glucopyranosylzeatin, and at least two unidentified compounds appeared to be new derivatives of zeatin. In identifying the above compounds, chemical-ionization mass spectrometry proved to be an invaluable complementary technique, yielding spectra showing intense protonated-molecular-ion peaks and also prominent structure-related fragmentation that was either not evident or very minor in the electron-impact spectra. An assessment of the relative importance of the various possible mechanisms for cytokinin modification and inactivation in mature sweet-corn kernels was made by supplying [³H]zeatin and [³H]zeatin riboside to such kernels after excision. The principal metabolites of zeatin were adenine nucleotides, adenosine and adenine, while little of the metabolite radioactivity was attributable to known O-glucosides. Adenine nucleotides and adenine were the principal metabolites of zeatin riboside, while lesser metabolites were identified as adenosine, dihydrozeatin, and the O-glucosides of dihydrozeatin and dihydrozeatin riboside. Side-chain cleavage, rather than side-chain modification, appears to be the dominant form of cytokinin metabolism in mature sweet-corn kernels.Abbreviations CI-MS chemical-ionization mass spectrum - EIMS electron-impact mass spectrum - GC-MS combined gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - M⁺ molecular ion - MH⁺ protonated molecular ion - TLC thin-layer chromatography - TMS trimethylsilyl - UV ultraviolet XXVII=Letham et al. (1979)     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Polyamines as physiological regulators of 14-3-3 interaction with the plant plasma membrane H+-ATPase

Polyamines are abundant polycationic compounds involved in many plant physiological processes such as cell division, dormancy breaking, plant morphogenesis and response to environmental stresses. In this study, we investigated the possible role of these polycations in modulating the association of 14-3-3 proteins with the H(+)-ATPase. In vivo experiments demonstrate that, among the different polyamines, spermine brings about 2-fold stimulation of the H(+)-ATPase activity and this effect is due to an increase in 14-3-3 levels associated with the enzyme. In vivo administration of polyamine synthesis inhibitors causes a small but statistically significant decrease of the H(+)-ATPase phosphohydrolytic activity, demonstrating a physiological role for the polyamines in regulating the enzyme activity. Spermine stimulates the activity of the H(+)-ATPase AHA1 expressed in yeast, in the presence of exogenous 14-3-3 proteins, with a calculated S(50) of 70 microM. Moreover, spermine enhances the in vitro interaction of 14-3-3 proteins with the H(+)-ATPase and notably induces 14-3-3 association with the unphosphorylated C-terminal domain of the proton pump. Comparison of spermine with Mg(2+), necessary for binding of 14-3-3 proteins to different target proteins, shows that the polyamine effect is stronger than and additive to that of the divalent cation.     

Separation of tonoplast and plasma membrane H+-ATPase from Zucchini hypocotyls by consecutive sucrose and glycerol gradient centrifugation

Summary In order to isolate tonoplast and plasma membrane vesicles involved in ATP-dependent proton transport we devised a preparative procedure with two consecutive centrifugations. Three fractions were obtained on a sucrose step gradient: light microsomes, heavy microsomes, and a mitochondria-rich fraction. The light and heavy microsomal fractions were each recentrifuged on an isopycnic glycerol density gradient. Recentrifugation of light microsomes resulted in two fractions with H⁺-ATPase activity, one equilibrating at a density less than 1.11 g/cm³ and one equilibrating at a density of about 1.17g/cm³. Comparison with marker enzyme activities suggests that the upper fraction was enriched in tonoplast, and the dense fraction with plasma membrane. In addition to marker enzyme content, H⁺ transport in the H⁺-ATPase-containing fractions was further characterized with respect to pH dependence, cation and anion dependence, and uncouplers and inhibitors. H⁺ transport in all fractions was strongly dependent on the presence of halides but no specific stimulation by potassium or any other monovalent cation was found. Of the anions tested, malate and fumarate preferentially stimulated H⁺ transport in the tonoplast-enriched fraction. It is suggested that a Ca²⁺/H⁺ antiporter is present in all fractions. Only H⁺-ATPase in the plasma membrane-enriched fractions was sensitive to nystatin, an uncoupler, and to orthovanadate, an inhibitor. The tonoplast fraction was more sensitive to nitrate than the plasma membrane-enriched fraction, and all fractions showed some sensitivity to high concentrations of oligomycin. Oligomycin sensitivity was not due to the presence of mitochondria.     

Matrix-associated DNA from maize is enriched in repetitive sequences

In order to elucidate some features of the topological organization of DNA within the plant nucleus, DNA fragments involved in the attachment of the DNA loops to the nuclear matrix in maize were studied. The matrix-associated DNA from dry embryo and meristematic cells after extensive digestion with DNase I and high salt treatment was about 2% of the total DNA, sized within the range of 50 and 250 bp. This DNA was found to be enriched in repetitive DNA sequences, both for nuclei from dry embryo and meristematic cells. The loop size of the DNA in cells of Zea mays appeared to be between 5 and 25 kbp.Abbreviations EDTA Diamino-ethanetetraacetic acid - EtBr Ethidium bromide - LIS Lithium diiodosalicylate - PMSF Phenylmethylsulfonyl fluoride - SDS Sodium dodecyl sulfate     

Comparative analysis of nuclear and mitochondrial DNA topoisomerase I from <Emphasis Type="Italic">Zea mays</Emphasis>

The properties were compared for maize nuclear and mitochondrial DNA topoisomerases I (topo I). Some differences in their ability to bind to single-stranded DNA were revealed. Mitochondrial topo I was active only in the presence of Mg²⁺, whereas the activity of the nuclear enzyme did not completely depend on Mg²⁺, although being essentially stimulated in the presence of Mg²⁺. The mitochondrial enzyme covalently bound to the 5′ DNA end, as unique to prokaryotic topo I. The nuclear enzyme, like all eukaryotic topo I, covalently bound to the 3′ DNA end. A search for homologous sequences in several databases revealed genes probably encoding mitochondrial topo I in other higher plants. Using cDNA sequencing and in silico analysis, an orthologous gene was revealed in the maize genome. The gene was strongly homologous to the genes encoding prokaryotic topo I, which could explain the differences in properties between mitochondrial and nuclear topo I from maize. The presence of prokaryotic topo I in mitochondria of higher plants is interesting and important for studying the evolution of these plant organelles and the mechanisms of mitochondrial genome expression.     

Butyricin 7423 and the membrane H+-ATPase of Clostridium pasteurianum

The bacteriocin butyricin 7423 inhibited the activity of the membrane H⁺-ATPase (BF₀, F₁) of vegetative cells of Clostridium pasteurianum but not that of its soluble BF₁ component. In vitro studies with the H⁺-ATPases of mutant strains selected for diminished sensitivity (a) to butyricin 7423 and (b) to dicyclohexylcarbodi-imide, confirmed that butyricin 7423 interacts with the BF₀ component of this enzyme complex. Even so, certain other mutant strains displaying decreased sensitivity to butyricin 7423 possessed H⁺-ATPases which in vitro showed undiminished sensitivity to inhibition by the bacteriocin. Furthermore, from the changes in intracellular ATP concentration and in the rates and net extent of efflux of intracellular ⁸⁶Rb⁺ ions that were provoked by exposure of the parent and several of the mutant strains to butyricin 7423, it was concluded that its primary bactericidal action was not attributable to stoichiometric inhibition of the membrane H⁺-ATPase. High extracellular concentrations of K⁺ ions enabled Cl. pasteurianum to survive exposure to low concentrations of this membrane-active bacteriocin.Non-standard abbreviations H⁺-ATPase proton translocating adenosine 5-triphosphatase (EC 3.6.1.3) - DCCD dicyclohexylcarbodiimide     

The transposable element En/Spm-encoded TNPA protein contains a DNA binding and a dimerization domain

The En/Spm-encoded TNPA protein binds to 12-bp DNA sequence motifs that are present in the sub-termini of the transposable element. DNA binding of TNPA to monomeric and dimeric forms of the binding motif was analyzed by gel retardation and cross-linking studies. A DNA binding domain at the N-terminal and a dimerization domain at the C-terminal portion of TNPA were localized using deletion derivatives of TNPA. These domains are novel since no apparent homology has been found in the data bases. The stoichiometry of the TNPA-DNA complexes was analyzed. A special complex is formed with a tail-to-tail dimeric DNA binding motif, most probably involving two DNA-bound TNPA molecules that interact via their dimerization domains. In redox reactions the requirement for one or two disulfide bonds for DNA binding of TNPA was shown. The implications of these findings for the excision mechanism of En/Spm are discussed.     

The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.