Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein     

The possible role of redox-associated protons in growth of plant cells

The protons excreted by plant cells may arise by two different mechanisms: (1) by the action of the plasma membrane H⁺-ATPase and (2) by plasma membrane redox reactions. The exact proportion from each source is not known, but the plasma membrane H⁺-ATPase is, by far, the major contributor to proton efflux. There is still some question of whether the redox-associated protons produced by NADH oxidation on the inner side of the plasma membrane traverse the membrane in a 1 : 1 relationship with electrons generated in the redox reactions. Membrane depolarization observed in the presence of ferricyanide reduction by plasma membranes of whole cells or tissues or the lag period between ferricyanide reduction and medium acidification argue that only scalar protons may be involved. The other major argument against tight coupling between protons and electrons involves the concept of strong charge compensation. When ferricyanide is reduced to ferrocyanide on the outside of cells or tissues, an extra negative charge arises, which is compensated for by the release of H⁺ or K⁺, so that the total ratio of increased H⁺ plus K⁺ equals the electrons transferred by transmembrane electron transport. These are strong arguments against a tight coupling between electrons and protons excreted by the plasma membrane. On the other hand, there is no question that inhibitor studies provide evidence for two mechanisms of proton generation by plasma membranes. When the H⁺-ATPase activity is totally inhibited, the addition of ferricyanide induces a burst of extra proton excretion, orvice versa, when plasma membrane redox reactions are inhibited, the H⁺-ATPase can function normally. Since plasma membrane redox reactions and associated H⁺ excretion are related to growth, it is possible that in plants the ATPase-generated protons have a different function from redox-associated protons. The H⁺-ATPase-generated protons have been considered for many years to be necessary for cell wall expansion, allowing elongation to take place. A special function of the redox-generated protons may be in initiating proliferative cell growth, based on the presence of a hormone-stimulated NADH oxidase in membranes of soybean hypocotyls and stimulation of root growth by low concentrations of oxidants. Here we propose that this NADH oxidase and the redox protons released by its action control growth. The mechanism for this may be the evolution of protons into a special membrane domain, from which a signal to initiate cell proliferation may originate, independent of the action of the H⁺-ATPase-generated protons. It is also possible that both expansion and proliferative growth are controlled by redox-generated protons.     

Plasma membrane ATPase and H+ transport activities in microsomal membranes from mycorrhizal tomato roots

ATPase activity, ATP-dependent H⁺ transport and the amount of antigenic tomato plasma membrane H⁺-APTase have been analysed in membrane vesicles isolated from Glomus mosseae- or Glomus intraradices-colonized roots and from non-mycorrhizal tomato roots. Microsomal protein content was higher in mycorrhizal than in control roots. The specific activity of the plasma membrane H⁺-ATPase was not affected by mycorrhizal colonization, although this activity increased in membranes isolated from mycorrhizal roots when expressed on a fresh weight basis. Western blot analysis of microsomal proteins using antibodies raised against the Arabidopsis thaliana plasma membrane H⁺ - ATPase showed that mycorrhizal colonization did not change the relative amount of tomato plasma membrane ATPase in the microsomes. However, on a fresh weight basis, there was a greater amount of this protein in roots of mycorrhizal plants. In addition, mycorrhizal membranes showed a higher specific activity of the vanadate-sensitive ATP-dependant H⁺ transport than membranes isolated from control roots. These results suggest that mycorrhiza might regulate the plasma membrane ATPase by increasing the coupling efficiency between H⁺ transport and ATP hydrolysis. The observed effects of mycorrhizal colonization on plasma membrane H⁺-ATPase were independent of the AM fungal species colonizing the root system.     

H-ATPase Activity from Storage Tissue of Beta vulgaris: IV. N,N'-Dicyclohexylcarbodiimide Binding and Inhibition of the Plasma Membrane H-ATPase

The molecular weight and isoelectric point of the plasma membrane H⁺-ATPase from red beet storage tissue were determined using N,N′-dicyclohexylcarbodiimide (DCCD) and a H⁺-ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [¹⁴C]-DCCD at 0°C, a single 97,000 dalton protein was visualized on a fluorograph of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [¹⁴C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H⁺-ATPase. An antibody raised against the plasma membrane H⁺-ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H⁺-ATPase to be 6.5.     

Characterisation of the water expulsion vacuole inPhytophthora nicotianae zoospores

Summary The water expulsion vacuole (WEV) in zoospores ofPhytophthora nicotianae and other members of the Oomycetes is believed to function in cell osmoregulation. We have used videomicroscopy to analyse the behaviour of the WEV during zoospore development, motility and encystment inP. nicotianae. After cleavage of multinucleate sporangia, the WEV begins to pulse slowly but soon attains a rate similar to that seen in motile zoospores. In zoospores, the WEV has a mean cycle time of 5.7 ± 0.71 s. The WEV continues to pulse at this rate until approximately 4 min after the onset of encystment. At this stage, pulsing slows progressively until it becomes undetectable. The commencement of WEV operation in sporangia coincides with the reduction of zoospore volume prior to release from the sporangium. Disappearance of the WEV during encystment occurs as formation of a cell wall allows the generation of turgor pressure in the cyst. As in other organisms, the WEV inP. nicotianae zoospores consists of a central bladder surrounded by a vesicular and tubular spongiome. Immunolabelling with a monoclonal antibody directed towards vacuolar H⁺-ATPase reveals that this enzyme is confined to membranes of the spongiome and is absent from the bladder membrane or zoospore plasma membrane. An antibody directed towards plasma membrane H⁺-ATPase shows the presence of this ATPase in both the bladder membrane and the plasma membrane over the cell body but not the flagella. Analysis of ATPase activity in microsomal fractions fromP. nicotianae zoospores has provided information on the biochemical properties of the ATPases in these cells and has shown that they are similar to those in true fungi. Inhibition of the vacuolar H⁺-ATPase by potassium nitrate causes a reduction in the pulse rate of the WEV in zoospores and leads to premature encystment. These results give support to the idea that the vacuolar H⁺-ATPase plays an important role in water accumulation by the spongiome in oomycete zoospores, as it does in other protists.Abbreviations BMM butyl methylmethacrylate - F fix 4% formaldehyde fixation - GF fix 4% formaldehyde and 0.2% glutaraldehyde fixation - V-ATPase vacuolar H⁺-ATPase - WEV water expulsion vacuole     

Immunocytochemical localization of Na+/K+-ATPase and V-H+-ATPase in the salivary glands of the cockroach,Periplaneta americana

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na⁺/K⁺-ATPase (sodium pump) and a vacuolar-type H⁺-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na⁺/K⁺-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H⁺-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na⁺/K⁺-ATPase in the peripheral cells is probably directly involved in the formation of the Na⁺-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H⁺-ATPase.     

Immunolocalization of H+-ATPases in the plasma membrane of pollen grains and pollen tubes ofLilium longiflorum

Summary A heterogeneous distribution of H⁺-ATPase was visualized in germinated pollen ofLilium longiflorum using monoclonal antibodies raised against plasma membrane H⁺-ATPase. Immunolocalization studies of protoplasts and subprotoplasts derived from pollen tubes and sectioned pollen grains and pollen tubes show that H⁺-ATPases are abundant in the plasma membrane of pollen grains but are absent or sparsely distributed in the plasma membrane of pollen tubes. This polar distribution of H⁺-ATPases is probably the basis of the endogenous current pattern measured in growing lily pollen and involved in pollen tube tip growth.Abbreviations BSA bovine serum albumine - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - Tris 2-amino-2-hydroxymethyl-1,3-propandiol     

Growth cycle stage-dependent NaCl induction of plasma membrane H+-ATPase mRNA accumulation in de-adapted tobacco cells

A cDNA clone encoding an isoform of the plasma membrane H⁺-ATPase was isolated from Nicotiana tabacum. The steady-state plasma membrane H⁺-ATPase message levels were the same in unadapted tobacco cells and tobacco cells adapted to 428 mol m⁻³ NaCl. When cells adapted to 428 mol m⁻³ NaCl maintained in the absence of NaCl (deadapted) for an excess of 100 passages were exposed to 400 mol m⁻³ NaCl for 24 h, there was an increased accumulation of plasma membrane H⁺-ATPase message. The NaCl responsiveness of the deadapted cells was dependent upon the growth cycle stage. Alterations in the levels of plasma membrane FT-ATPase message during the growth cycle support a role for the H⁺-ATPase in cell growth. These results document the induction by NaCl of plasma membrane FT-ATPase message accumulation in tobacco cells, and suggest that enhanced expression of the plasma membrane FT-ATPase has a role in the short term response of cells of NaCl, but is not necessarily involved in long-term adaptation.     

Salinity adaptation of plasma membrane H+-ATPase in the salt marsh plant Spartina patens: ATP hydrolysis and enzyme kinetics

Spartina patens, an intertidal C4 grass, grows in the upper salt marsh and tolerates coastal seawater salinity. The regulation of ion movement across the plasma membrane (PM) for plant salt tolerance is thought to be achieved by an electrochemical gradient generated by plasma membrane H⁺-ATPase. In this study, the change of PM H⁺-ATPase in response to NaCl was characterized for S. patens callus. Callus was cultured for 10 weeks under salinity levels of 0 mM, 170 mM, 340 mM, and 510 mM NaCl. Plasma membrane was isolated from a Dextran/PEG aqueous polymer two-phase system and the purity was demonstrated with membrane enzyme markers. There was a significant increase (up to 2-3 fold) of PM H⁺-ATPase activity when callus was grown on media containing NaCl. The incremental activation of PM H⁺-ATPase activity would enable the cell to tolerate higher cytoplasmic NaCl concentrations. PM H⁺-ATPase appeared to have a higher Vmax and a lower substrate concentration (Km to reach Vmax. When growth medium salinity increased from 0 mM to 170 and 340 mM, the Vmax of H⁺-ATPase increased from 0.64 to 1.00 and 1.73, respectively, while the Km decreased from 3.58 to 2.07 and 2.44 mM, respectively. In vitro NaCl inhibition kinetic data revealed a pattern of non-competitive inhibition by NaCl on PM H⁺-ATPase. The response of PM H⁺-ATPase in S. patens callus suggests that this species has evolved mechanisms that can regulate this important enzyme when cells are exposed to NaCl.     

A mutant of Arabidopsis thaliana partially resistant to fusicoccin has reduced plasma membrane H+-ATPase

5-2 is a mutant of Arabidopsis thaliana which is partially resistant to fusicoccin in vivo. We have analysed fusicoccin binding and the activity and amount of H⁺-ATPase in plasma membrane isolated from mature leaves of the wild type and of mutant 5-2. Fusicoccin binding was similar in plasma membrane from the two genotypes, while H⁺-ATPase activity was markedly (c. 50%) lower in plasma membrane from mutant 5-2 than in that from the wild type. The H⁺-ATPase of mutant 5-2 was activated by fusicoccin as much as that of the wild type. In plasma membrane from mutant 5-2, the amount of immunodetectable H⁺-ATPase, quantified by densitometry of Western blots, was about half that in the wild type. These results indicate that the major defect of mutant 5-2 detectable at the plasma membrane level is a reduction in the amount of H⁺-ATPase.     

Role of the plasma membrane H<Superscript>+</Superscript>-ATPase in auxin-induced elongation growth: historical and new aspects

This article will cover historical and recent aspects of reactions and mechanisms involved in the auxin-induced signalling cascade that terminates in the dramatic elongation growth of cells and plant organs. Massive evidence has accumulated that the final target of auxin action is the plasma membrane H⁺-ATPase, which excretes H⁺ ions into the cell wall compartment and, in an antiport, takes up K⁺ ions through an inwardly rectifying K⁺ channel. The auxin-enhanced H⁺ pumping lowers the cell wall pH, activates pH-sensitive enzymes and proteins within the wall, and initiates cell-wall loosening and extension growth. These processes, induced by auxin or by the "super-auxin" fusicoccin, can be blocked instantly and specifically by a voltage inhibition of the H⁺-ATPase due to removal of K⁺ ions or the addition of K⁺-channel blockers. Vice versa, H⁺ pumping and growth are immediately switched on by addition of K⁺ ions. Furthermore, the treatment of segments either with auxin or with fusicoccin (which activates the H⁺-ATPase irreversibly) or with acid buffers (from outside) causes an identical transformation and degradation pattern of cell wall constituents during cell-wall loosening and growth. These and other results described below are in agreement with the acid-growth theory of elongation growth. However, objections to this theory are also discussed.     

Immunocytochemical localization of H+?K+-ATPase in the rat colon

Summary The presence and distribution of gastric-type H⁺−K⁺-ATPase were investigated in the rat colon using a monoclonal antibody raised against hog gastric H⁺−K⁺-ATPase. Rat stomach was used as positive control. Rat kidney and ileum, in both of which H⁺−K⁺-ATPase has been reported in the past, were also studied. In stomach, very strong staining was found confined to the parietal cells, and a strong band atM _r∼94 kDa on the immunoblots. In colon a moderate staining was found in the supranuclear region of the epithelial cells, with similar intensity and distribution of staining of the surface and deep mucosa of the crypts, throughout the length of the colon. Another monoclonal antibody, specific to the 31 kDa subunit of H⁺-ATPase, used as a negative control, or omission of the primary antibody, resulted in lack of any staining in either colon or stomach. On immunoblots of homogenates of colonic mucosa, no specific band could be identified, either due to very low expression of the H⁺−K⁺-ATPase or loss of antigenicity of the epitope during the processing steps. No positive staining was observed in rat kidney and ileum, suggesting that they contain isoforms that are structurally different.     

Distribution of Plasma Membrane H+-ATPase and Polar Current Patterns in Leaves and Stems of Elodea canadensis*

The proton pumping ATPase in the plasma membrane of Elodea canadensis is believed to play a major role in inorganic carbon acquisition. To investigate potentially different carbon uptake strategies within the same plant, plasma membrane H⁺-ATPase distribution and polar current patterns were investigated in Elodea leaves and stems. Specific activity of plasma membrane H⁺-ATPase in leaf microsomal fractions was tenfold higher than in stem derived microsomes. Probing western blots with a monoclonal antibody specific for plasma membrane H⁺-ATPase, yielded strongly visible double bands at 100 kDa in leaf microsome preparations, whereas little antigen was detected in analogous stem microsome preparations. Using the same plasma membrane H⁺-ATPase specific antibody on tissue sections, the enzyme was found almost exclusively localized at the border of cells at the lower leaf surface. A positive ion current leaving the lower leaf surface was measured, using a vibrating probe device. Part of this current entered the upper leaf surface and part of it the internodes of the stem. The experimental results support the view, that Elodea leaves have different means of inorganic carbon uptake than stem internodes.     

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H⁺-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H⁺-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized -subunit of the H⁺-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H⁺-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.This paper is No. 61 in the seriesBiogenesis of Mitochondria. For paper No. 60, see Novitskiet al. (1984).     

Absence of plasma membrane H+-ATPase in plasmodesmata located in pit-fields of the young reactive pulvinus ofMimosa pudica L.

Summary Immunocytochemical techniques were employed to study the spatial distribution of the plasma membrane H⁺-ATPase within various cell types of the young reactive primary pulvinus ofMimosa pudica L. These cells were interconnected by large numbers of plasmodesmata, being concentrated within pit-fields. Although we could routinely detect evidence of the H⁺-ATPase along the plasma membrane, immunolabelling was rarely, if ever, observed along the plasma membranes of the plasmodesmata. This finding is discussed with respect to the likely specialized supramolecular structure of the plasmodesma.Abbreviations SEL size exclusion limit of plasmodesmata     

Plasma membrane K+/H+-ATPase From leishmania donovani

Leishmania donovani has an active ^K+/H⁺ exchange system on the surface membrane. Modulation of external K⁺ concentration resulted in a corresponding change in internal pH (pH_i) suggesting a link between proton and potassium transport. Although a Na⁺/H⁺ antiporter is present on the plasma membrane, its sensitivity to amiloride suggests that it operates independent of K⁺/H⁺ exchange. Reduction of cellular ATP with NaN₃ and KCN inhibits K⁺/H⁺ exchange showing thereby that the process is energy dependent. The K⁺/H⁺ exchange is sensitive to inhibitors of the gastric K⁺/H⁺-ATPase. It is concluded that the H⁺-ATPase previously reported on the plasma membrane of L. donovani is in fact a K⁺/H⁺-ATPase. © 1994 wiley-Liss, Inc.     

Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H⁺-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H⁺-ATPase antibody clones, raised against the PM H⁺-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H⁺-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K⁺ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes. Received: 3 July 2000 / Accepted: 12 October 2000     

Properties of plasma membrane H<Superscript>+</Superscript>-ATPase in salt-treated <Emphasis Type="Italic">Populus euphratica</Emphasis> callus

The plasma membrane (PM) vesicles from Populus euphratica (P. euphratica) callus were isolated to investigate the properties of the PM H⁺-ATPase. An enrichment of sealed and oriented right-side-out PM vesicles was demonstrated by measurement of the purity and orientation of membrane vesicles in the upper phase fraction. Analysis of pH optimum, temperature effects and kinetic properties showed that the properties of the PM H⁺-ATPase from woody plant P. euphratica callus were consistent with those from herbaceous species. Application of various thiol reagents to the reaction revealed that reduced thiol groups were essential to maintain the PM H⁺-ATPase activity. In addition, there was increased H⁺-ATPase activity in the PM vesicles when callus was exposed to NaCl. Western blotting analysis demonstrated an enhancement of H⁺-ATPase content in NaCl-treated P. euphratica callus compared with the control.     

Regulation of H+Pumping by Plant Plasmalemma under Osmotic Stress: The Role of 14-3-3 Proteins

All higher plants have high-specific sites for binding fusicoccin (FCBS), a metabolite of the fungus Fusicoccum amygdaliDel. These sites are localized on the plasmalemma and produced by the association of the dimers of 14-3-3 proteins with the C-terminal autoinhibitory domain of H⁺-ATPase. Considering the fusicoccin binding to the plasmalemma as an index characterizing the formation of this complex, we studied the influence of osmotic stress on the interaction between 14-3-3 proteins and H⁺-ATPase in the suspension-cultured sugar beet cells and protoplasts obtained from them. An increase in the osmolarity of the extracellular medium up to 0.3 Osm was shown to enhance proton efflux from the cells by several times. The number of FCBS in isolated plasma membranes increased in parallel, whereas 14-3-3 proteins accumulated in this membrane to a lesser degree. The amount of H⁺-ATPase molecules did not change, and the ATP-hydrolase activity changed insignificantly. The data obtained indicate that osmotic stress affects H⁺-ATPase pumping in the plasmalemma through its influence on the coupling between H⁺-transport and ATP hydrolysis; 14-3-3 proteins are involved in this coupling. The interaction between the plasmalemma and the cell wall is suggested to be very important in this process.