The tonoplast H+-pyrophosphatase of radish seedlings: biochemical characteristics

The activity of the H⁺-pyrophosphatase (H⁺-PPase) was characterized in microsomes from 24-h-old radish ( Raphanus sativus L., ev. Tondo Rosso Quarantino) seedlings, which are virtually devoid of the tonoplast H⁺-ATPase. The H⁺-PPase was localized to membranes which roughly comigrated with the plasma membrane in a sucrose density gradient, but clearly separated from plasma membrane when microsomes were partitioned in an aqueous dextran-polyethylene glycol two-phase system. The H⁺-PPase activity was strictly dependent on Mg²⁺ and on the presence of a monovalent cation (K⁺=Rb⁺=NH₃⁺Cs⁺≫Na⁺Li⁺) and was insensitive to anions such as Cl−, Br−, NO₃− and SO₄^2-. It was inhibited by F−, imidodiphosphate and Ca²⁺. It had a pH optimum between pH 7.5 and 8.5 and was saturated by low concentrations of pyrophosphate (half saturation at 30 μ M pyrophosphate). All of these characteristics are identical to those reported for the tonoplast H⁺-PPase from various plant materials. The functional molecular weight of the H⁺-PPase, measured with the radiation-inactivation technique was 96 kDa.     

Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

Transport across the plasma membrane is driven by an electrochemical gradient of H⁺ ions generated by the plasma membrane proton pump (H⁺-ATPase). Random mutants of Arabidopsis H⁺-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H⁺-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H⁺ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H⁺ extrusion and in their ability to sustain an electrochemical H⁺ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H⁺-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H⁺ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.     

NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato

NaCl-induced changes in the accumulation of message for the 70 kDa subunit of the tonoplast H⁺-ATPase and plasma membrane H⁺-ATPase were studied in hydroponically grown plants of Lycopersicon esculentum Mill. cv. Large Cherry Red. There was increased accumulation of message for the 70 kDa (catalytic) subunit of the tonoplast H⁺-ATPase in expanded leaves of tomato plants 24 h after final NaCl concentrations were attained. This was a tissue-specific response; levels of this message were not elevated in roots or in young, unexpanded leaves. The NaCl-induced accumulation of this message was transient in the expanded leaves and returned to control levels within 7 days. The temporal and spatial patterns of NaCl-induced accumulation of message for the plasma membrane H⁺-ATPase differed from the patterns associated with the 70 kDa subunit of the tonoplast H⁺-ATPase. NaCl-induced accumulation of the plasma membrane H⁺-ATPase message occurred in both roots and expanded leaves. Initially accumulation of the plasma membrane H⁺-ATPase message was greater in root tissue than in expanded leaves, but increased to higher levels in expanded leaves after 7 days. These results suggest that increased expression of the tonoplast H⁺-ATPase is an early response to salinity stress and may be associated with survival mechanisms, rather than with long-term adaptive processes.     

Plasma membrane H-ATPase activity is involved in adaptation of tomato calli to NaCl

A tomato ( Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 m M NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 m M NaCl. Analysis of callus ion content showed a strong increase in Na⁺ and Cl⁻ both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H⁺ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H⁺ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H⁺-ATPase (EC 3.6.1.35) and ATP-dependent H⁺ transport, while the stimulation of H⁺ extrusion by cells tolerant to 50 m M NaCl was correlated with an increase in plasma membrane ATP-dependent H⁺ transport. The increase of ATP-dependent H⁺ extrusion in plasma membranes isolated from 50 m M NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H⁺. Relative to NaCl-sensitive calli, plasma membrane H⁺-ATPase from calli tolerant to 50 m M NaCl showed a lower K_m for Mg²⁺-ATP. Our results indicate that tolerance of tomato calli to 50 m M NaCl increases the affinity of plasma membrane H⁺-ATPase for the substrate ATP and stimulates the H⁺-pumping activity of this enzyme without modifying its phosphohydrolytic activity.     

Aluminum impairs morphogenic transition and stimulates H(+) transport mediated by the plasma membrane ATPase of Yarrowia lipolytica

The effect of aluminum on dimorphic fungi Yarrowia lipolytica was investigated. High aluminum (0.5–1.0 mM AlK(SO₄)₂) inhibits yeast–hypha transition. Both vanadate-sensitive H⁺ transport and ATPase activities were increased in total membranes isolated from aluminum-treated cells, indicating that a plasma membrane H⁺ pump was stimulated by aluminum. Furthermore, Al-treated cells showed a stronger H⁺ efflux in solid medium. The present results suggest that alterations in the plasma membrane H⁺ transport might underline a pH signaling required for yeast/hyphal development. The data point to the cell surface pH as a determinant of morphogenesis of Y. lipolytica and the plasma membrane H⁺-ATPase as a key factor of this process.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

Na+/H+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the salt-sensitive Plantago media

Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na⁺ ions. A salt-sensitive ( Plantago media L.) and a salt-tolerant ( P. maritima L.) species were compared with respect to Na⁺/H⁺ antiport activities at the tonoplast. After exposure of the plants to 50 m M NaCl for 6 days isolated tonoplast vesicles of P. maritima showed Na⁺/H⁺ antiport activity with saturation kinetics and a K_m of 2.4 m M Na⁺, NaCl-grown P. media and the control plants of both species showed no antiport activity. Selectivity of the antiport system for Na⁺ was high and was determined by adding different chloride salts after formation of a Δ pH in the vesicles. Specific tonoplast ATPase activities were similar in the two species and did not alter after exposure to NaCl stress.     

Proton pumping pyrophosphatase from higher plant mitochondria

In higher plant cells, there are some enzymes capable of utilizing pyrophosphate (PP_i) as an energy donor. Among these, membrane-bound proton pumping pyrophosphatases (H⁺-PP_iase) have been identified. In addition to the well-known vacuolar H⁺-PP_iase (V-PP_iase), there is evidence for the presence of a mitochondrial H⁺-PP_iase. This enzyme is localized on the inner surface of the inner membrane and catalyzes the specific hydrolysis of PP_i, coupled to proton transport, with a H⁺/PP_i stoichiometry of ca 2. This activity is Mg²⁺-requiring, is stimulated by monovalent cations, and is inhibited by Ca²⁺, F⁻ and diphosphonates. The H⁺-PP_iase contains a catalytic head which is constituted by a 35-kDa protein which is loosely bound to the inner membrane. This protein exhibits a PP_iase activity, stimulated by phospholipids, with characteristics very similar to the membrane-bound enzyme. The mitochondrial PP_iase is distinct from the V-PP_iase, because an antibody raised against the 35-kDa protein does not react with tonoplast membranes. The mitochondrial H⁺-PP_iase seems to have an F-type structure, similar to the F-ATP synthase and the membrane-bound PP_iases from mammalian and yeast mitochondria. It is suggested that, beside synthesizing PP_i, this enzyme may act as a buffer for the electrochemical proton gradient, by hydrolyzing PP_i, during conditions of oxygen deprivation.     

Enhanced H+/ATP coupling ratio of H+-ATPase and increased 14-3-3 protein content in plasma membrane of tomato cells upon osmotic shock

Modulation of proton extrusion and ATP-dependent H⁺ transport through the plasma membrane in relation to the presence of 14-3-3 proteins in this membrane in response to osmotic shock was studied in tomato ( Lycopersicon esculentum Mill. cv. Pera) cell cultures. In vivo H⁺ extrusion by cells was activated rapidly and significantly after adding 100 m M NaCl, 100 m M KCl, 50 m M Na₂SO₄, 1.6% sorbitol or 2 µ M fusicoccin to the medium. The increase in H⁺ extrusion by cells treated with 100 m M NaCl was correlated with an increase of H⁺ transport by the plasma membrane H⁺-ATPase (EC 3.6.1.35), but not with changes in ATP hydrolytic activity of this enzyme, suggesting an increased coupling ratio of the enzyme. Immunoblot experiments showed increased amounts of 14-3-3 proteins in plasma membrane fractions isolated from tomato cells treated with 100 m M NaCl as compared to control cells without changing the amount of plasma membrane H⁺-ATPase. Together, these data indicate that in tomato cells an osmotic shock could enhance coupling between ATP hydrolysis and proton transport at the plasma membrane through the formation of a membrane 14-3-3/H⁺-ATPase complex.     

Tonoplast energization: Two H+ pumps, one membrane

The vacuolar membrane (tonoplast) of plant cells contains two functionally and physically distinct phosphohydrolases, which catalyse electrogenic H⁺ -translocation: An ATPase (tp-ATPase; EC 3.6.1.3) and an inorganic pyrophosphatase (tp-PPase; 3.6.1.1). Neither enzyme belongs to the F₀F₁– or E₁E₂-categories of primary cation pumps, but instead belong to a third and fourth category of enzyme, respectively. Research priorities for the tp-ATPase are studies directed at understanding the roles of the 70 and 60 kDa subunits in catalysis and regulation; the involvement of the 16 kDa subunit in transmembrane H⁺ conduction; and investigations of F₀F₁- like structure/function partitioning. In the longer term, comparisons of sequence homology between the N,N'- dicyclohexylcarbodiimide -binding (16 kDa) proteins from different sources may enable elucidation of the evolutionary relationship of the tp-ATPase with other putative third-category H⁺– translocases. The tp-PPase, on the other hand, represents an exciting but largely unexplored biochemical entity, which necessitates a reconsideration of accepted views concerning the involvement of inorganic pyrophosphate (PPi) in transmembrane energy conservation. Just why the tonoplast should be endowed with two H⁺-translocases is a problem that can only be approached once consideration is given to the paramount question of H⁺/PPi stoichiometry. Once the stoichiometry is known, it should be possible to establish the physiological poise of the tp-PPase, and hence to speculate on its role in the metabolism of plant cells.     

Tolerance to NaCl induces changes in plasma membrane lipid composition,fluidity and H+-ATPase activity of tomato calli

Two tomato ( Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 m M NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H⁺-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 m M NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 m M NaCl, growth of calli tolerant to 100 m M NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 m M NaCl. Growth in the presence of 100 m M NaCl provoked an increase of Na⁺ and Cl⁻ content, but no significant changes in K⁺ and Ca²⁺. As compared with NaCl-sensitive and 50 m M NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 m M NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 m M NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 m M NaCl showed lower ATP hydrolysis and ATP-dependent H⁺-pumping activities, as well as a lower passive permeability to H⁺ than plasma membrane from NaCl-sensitive and 50 m M NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H⁺-ATPase activity in salt tolerance of tomato calli is discussed.     

The lysolipid sphingosine modulates pyrophosphatase activity in tonoplast vesicles and isolated vacuoles from a heterotrophic cell suspension culture of Chenopodium rubrum

The proton pumping activity of the tonoplast (vacuolar membrane) H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) has been studied on a tonoplast-enriched microsomal fraction and on intact vacuoles isolated from a heterotrophic cell suspension culture of Chenopodium rubrum L. in the presence of the lysosphingolipids D-sphingosine, psychosine (galactosylsphingosine) and lysosulfatide (sulfogalactosyl-sphingosine). Sphingosine strongly stimulates (K_a= 0.16 μ M ) the PPase activity, assayed both as ΔpH formation across the tonoplast vesicle membrane, and as reversible clamp current measured by the whole-vacuolar mode of the patch-clamp technique. Psychosine showed a minor, and lysosulfatide no stimulatory effect. No effect upon the ATPase activity has been observed. No sphingosine-induced change could be observed in the affinity of the PPase for its substrate (apparent K_m= 10 μ M MgPPi). We tentatively conclude that sphingosine, which is known as a potent inhibitor of the protein kinase C in animal cells, may be a regulator of the plant vacuolar PPase.     

In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots

The effect of boron excess and deficiency on H⁺ efflux from excised roots from sunflower ( Heliarahus annuus L. cv. Enano) seedlings and on plasma membrane H⁺-ATPase (EC 3.6.1.35) in isolated KI-washed microsomes has been investigated. When seedlings were grown in media with toxic levels of H₃BO₃ (5 m M ) or without added boron and exposed to light conditions, an inhibition of the capacity for external acidification by excised roots was observed as compared to roots from seedlings grown with optimal H₃BO₃ concentration (0.25 m M ). Toxic and deficient boron conditions also inhibited the vanadate-sensitive H⁺-ATPase of microsomes isolated from the roots. The mechanism of boron toxicity was investigated in vitro with microsorne vesicles. A strong effect of boron on the vanadate-sensitive, ATP-dependent H⁺ transport was found, but the vanadate-sensitive phospho-bydrolase activity was not affected. These results suggest that boron could exert an effect on the plasma membrane properties, directly or indirectly regulating, proton transport.     

Kinetics of net H+, Ca2+, K+, Na+, NH+4, and Cl- fluxes associated with post-chilling recovery of plasma membrane transporters in Zea mays leaf and root tissues

Although temperature-induced changes in membrane structure and activity seem to be central to chilling stress perception, the specific details of temperature effects on plant nutrient acquisition remain obscure. In this study, we have undertaken a comparative study of low temperature effects on the activity of plasma membrane transporters of different ions in corn ( Zea mays L.) leaf and root tissues by non-invasive measurements of net ion fluxes using ion-selective microelectrode (the MIFE) technique. Kinetics of net H⁺, Ca²⁺, K⁺, Na⁺,     and Cl^– fluxes were measured as plant tissues recovered after short-term (3 h) chilling stress. The major findings can be summarized as follows: (1) The critical temperatures, under which the recovery of the activity of plasma membrane transporters took place, were found to be the same for all ions measured and are likely to be associated with the phase transition of membrane lipids. (2) The most pronounced was the reduction in net uptake of K⁺ and     (3) Chilling treatment caused a significant net influx of Cl^– and Na⁺ in the leaf tissue. (4) For the same species, the critical temperatures for membrane-transport processes in roots were 2–2.5°C lower than in leaves. Possible physiological significance of these findings is discussed.     

Tonoplast H+ pumps are activated during callus formation of tuber tissues of Jerusalem artichoke (Helianthus tuberosus)

Changes in tonoplast H⁺-ATPase (EC 3.6.1.3) and H⁺–PPase (EC 3.6.1.1) activities were examined during the early period of callus formation in tuber tissues of Jerusalem artichoke ( Helianthus tuberosus L.). In callus-forming tissues cultured on a medium containing 2,4-D, the ATP-dependent H⁺-translocation activity of tonoplast vesicles increased 3-fold after a 2-day lag phase, while the ATP-hydrolytic activity and amount of tonoplast H⁺-ATPase protein were relatively constant after the lag phase. In the control tissue disks cultured on a medium free of 2,4-D, large declines in ATP-hydrolytic and ATP-dependent H⁺-translocation activities were observed. By contrast, the PP-dependent H⁺-translocation activity of tonoplast vesicles increased about 8-fold during the first 3 days of culture without any lag phase, and regardless of the presence of 2,4-D in the culture medium. However, the PP-hydrolytic activity and amount of H⁺-PPase protein did not change during the culture period, independently of callus formation. Transfer of the control tissue disks to the 2,4-D-containing medium, however, resulted in a further rapid stimulation of PP-dependent H⁺-translocation as well as an activation of ATP-dependent H⁺-translocation. These results suggest that both tonoplast H⁺ pumps are involved in callus formation of tuber tissues of Jerusalem artichoke.     

The hexose transporters at the plasma membrane and the tonoplast of transformed plant cells: kinetic characterization of two distinct carriers.

The plasma membrane hexose transporter and the tonoplast hexose transporter from heterotrophically grown transformed Nicotiana tabacum cells have been studied in vitro using membrane vesicles for trans-zero transport studies. In highly purified phase-partitioned outside-out plasma membrane vesicles (PMV) the hexose transporter showed an apparent Km value of 230 microM (substrate: 3-O-methyl-D-glucose (3-OMG); pHi 7.2/pHo 7.2), which was reduced to 120 microM when a pH gradient was imposed (pHo 5.7/pHi 7.2). However, the Vmax value was not affected indicating that no stable pH gradient was formed. Uptake experiments with 14C-labelled acetate supported this interpretation. Transport was insensitive to N-ethylmaleimide (NEM; up to 1 mM concentration) and p-chloromercuribenzene sulfonate (PCMBS; up to 500 microM), whereas the tonoplast hexose transporter (in mixed inside / out and outside / out vesicles) was inhibited by NEM in a substrate-protectable manner, and PCMBS was also inhibitory. Kinetically two components with apparent Km values of 6 and 20 mM could be distinguished for the tonoplast hexose transporter. Substrate specificities of both transporters were similar except for D-galactose and D-fructose. The results indicate structural differences between the tonoplast and plasma membrane hexose transporters in plants.