Boron deficiency energizes cyanide uptake and assimilation through activating plasma membrane H+-ATPase in rice plants.

The effect of boron (B) deficiency on mediating the contribution of H+-ATPase in the uptake and assimilation of exogenous cyanide (CN-) is investigated. Under CN- treatments, rice seedlings with B-deficient (-B) conditions exhibited significantly higher CN- uptake and assimilation rates than B-supplemented (+B) seedlings, whereas NH4+ uptake and assimilation rates were slightly higher in -B rice seedlings than in +B. In this connection, the expression pattern of genes encoding ß-CAS, ST, and H+-ATPase was assessed to unravel their role in the current scenario. The abundances of three ß-CAS isogenes (OsCYS-D1, OsCYS-D2, and OsCYS-C1) in rice tissues are upregulated from both "CN--B" and "CN-+B" treatments, however, only OsCYS-C1 in roots from the "CN--B" treatments was significantly upregulated than "CN-+B" treatments. Expression patterns of ST-related genes (OsStr9, OsStr22, and OsStr23) are tissue specific, in which significantly higher upregulation of ST-related genes was observed in shoots from "CN--B" treatments than "CN-+B" treatments. Expression pattern of 7 selected H+-ATPase isogenes, OsA1, OSA2, OsA3, OsA4, OsA7, OsA8, and OsA9 are quite tissue specific between "CN-+B" and "CN--B" treatments. Among these, OsA4 and OsA7 genes were highly activated in the uptake and assimilation of exogenous CN- in -B nutrient solution. These results indicated that B deficiency disturbs the pattern of N cycles in CN--treated rice seedlings, where activation of ST during CN- assimilation decreases the flux of the innate pool of NH4+ produced from CN- assimilation by the ß-CAS pathway in plants. Collectively, the B deficiency increased the uptake and assimilation of exogenous CN- through activating H+-ATPase.

Plasma Membrane H+-ATPase SmPHA4 Negatively Regulates the Biosynthesis of Tanshinones in Salvia miltiorrhiza.

Salvia miltiorrhiza Bunge has been widely used in the treatment of cardiovascular and cerebrovascular diseases, due to the pharmacological action of its active components such as the tanshinones. Plasma membrane (PM) H+-ATPase plays key roles in numerous physiological processes in plants. However, little is known about the PM H+-ATPase gene family in S. miltiorrhiza (Sm). Here, nine PM H+-ATPase isoforms were identified and named SmPHA1-SmPHA9. Phylogenetic tree analysis showed that the genetic distance of SmPHAs was relatively far in the S. miltiorrhiza PM H+-ATPase family. Moreover, the transmembrane structures were rich in SmPHA protein. In addition, SmPHA4 was found to be highly expressed in roots and flowers. HPLC revealed that accumulation of dihydrotanshinone (DT), cryptotanshinone (CT), and tanshinone I (TI) was significantly reduced in the SmPHA4-OE lines but was increased in the SmPHA4-RNAi lines, ranging from 2.54 to 3.52, 3.77 to 6.33, and 0.35 to 0.74 mg/g, respectively, suggesting that SmPHA4 is a candidate regulator of tanshinone metabolites. Moreover, qRT-PCR confirmed that the expression of tanshinone biosynthetic-related key enzymes was also upregulated in the SmPHA4-RNAi lines. In summary, this study highlighted PM H+-ATPase function and provided new insights into regulatory candidate genes for modulating secondary metabolism biosynthesis in S. miltiorrhiza.

Element content and expression of genes of interest in guard cells are connected to spatiotemporal variations in stomatal conductance.

Element content and expression of genes of interest on single cell types, such as stomata, provide valuable insights into their specific physiology, improving our understanding of leaf gas exchange regulation. We investigated how far differences in stomatal conductance (gs ) can be ascribed to changes in guard cells functioning in amphistomateous leaves. gs was measured during the day on both leaf sides, on well-watered and drought-stressed trees (two Populus euramericana Moench and two Populus nigra L. genotypes). In parallel, guard cells were dissected for element content and gene expressions analyses. Both were strongly arranged according to genotype, and drought had the lowest impact overall. Normalizing the data by genotype highlighted a structure on the basis of leaf sides and time of day both for element content and gene expression. Guard cells magnesium, phosphorus, and chlorine were the most abundant on the abaxial side in the morning, where gs was at the highest. In contrast, genes encoding H+ -ATPase and aquaporins were usually more abundant in the afternoon, whereas genes encoding Ca2+ -vacuolar antiporters, K+ channels, and ABA-related genes were in general more abundant on the adaxial side. Our work highlights the unique physiology of each leaf side and their analogous rhythmicity through the day.

Exacerbated effects of prorenin on hypothalamic magnocellular neuronal activity and vasopressin plasma levels during salt-sensitive hypertension.

Accumulating evidence supports that the brain renin-angiotensin system (RAS), including prorenin (PR) and its receptor (PRR), two newly discovered RAS players, contribute to sympathoexcitation in salt-sensitive hypertension. Still, whether PR also contributed to elevated circulating levels of neurohormones such as vasopressin (VP) during salt-sensitive hypertension, and if so, what are the precise underlying mechanisms, remains to be determined. To address these questions, we obtained patch-clamp recordings from hypothalamic magnocellular neurosecretory neurons (MNNs) that synthesize the neurohormones oxytocin and VP in acute hypothalamic slices obtained from sham and deoxycorticosterone acetate (DOCA)-salt-treated hypertensive rats. We found that focal application of PR markedly increased membrane excitability and firing responses in MNNs of DOCA-salt, compared with sham rats. This effect included a shorter latency to spike initiation and increased numbers of spikes in response to depolarizing stimuli and was mediated by a more robust inhibition of A-type K+ channels in DOCA-salt compared with sham rats. On the other hand, the afterhyperpolarizing potential mediated by the activation of Ca2+-dependent K+ channel was not affected by PR. mRNA expression of PRR, VP, and the Kv4.3 K+ channel subunit in the supraoptic nucleus of DOCA-salt hypertensive rats was increased compared with sham rats. Finally, we report a significant decrease of plasma VP levels in neuron-selective PRR knockdown mice treated with DOCA-salt, compared with wild-type DOCA-salt-treated mice. Together, these results support that activation of PRR contributes to increased excitability and firing discharge of MNNs and increased plasma levels of VP in DOCA-salt hypertension.NEW & NOTEWORTHY Our studies support that prorenin (PR) and its receptor (PRR) within the hypothalamus contribute to elevated plasma vasopressin levels in deoxycorticosterone acetate-salt hypertension, in part because of an exacerbated effect of PR on magnocellular neurosecretory neuron excitability; Moreover, our study implicates A-type K+ channels as key underlying molecular targets mediating these effects. Thus, PR/PRR stands as a novel therapeutic target for the treatment of neurohumoral activation in salt-sensitive hypertension.

Effect of exogenous calcium on growth, nutrients uptake and plasma membrane H+-ATPase and Ca2+-ATPase activities in soybean (Glycine max) seedlings under simulated acid rain stress.

Calcium (Ca) is one of essential elements for plant growth and development, and also plays a role in regulating plant cell physiology and cellular response to the environment. Here, we studied whether calcium played a role in enhancing tolerance of plants to acid rain stress by hydroponics and simulating acid rain stress. Our results show that acid rain (pH 4.5/pH 3.0) caused decreases in dry weight biomass, chlorophyll content and uptake of nutrients elements (NO3-, P, K, Mg, Zn and Mo) and an increase in membrane permeability of root. However, all parameters in soybean treated with exogenous calcium (5 mM) and acid rain at pH 4.5 were closed to the control levels. In addition, exogenous calcium (5 mM) alleviated the inhibition induced by pH 3.0 acid rain on the activity of plasma membranes H+-ATPase and the expression of GmPHA1 at transcriptional level, being benefiting to maintaining uptake of nutrients (NO3-, P, K, Mg, and Zn), and then lower the decrease in dry weight biomass and chlorophyll content. After a 5-day recovery (without acid rain stress), all parameters in soybean treated with acid rain at pH 3.0 and exogenous calcium were still worse than those of the control, but obviously better than those treated with acid rain at pH 3.0. Higher activity of plasma membrane H+-ATPase in soybean treated with acid rain at pH 3.0 and exogenous calcium was good to uptake of nutrients and promoted the recovery of soybean growth, compared with soybean treated with acid rain at pH 3.0. In conclusion, exogenous calcium could alleviate the inhibition caused by acid rain on soybean growth by increasing the activity of plasma membrane H+-ATPase for providing driving force to nutrient absorption, and its regulating effect was limited by intensity of acid rain. Furthermore, the application of exogenous calcium can be one of ways to alleviate the damage caused by acid rain to plants.

ATP synthesis in the energy metabolism pathway: a new perspective for manipulating CdSe quantum dots biosynthesized in Saccharomyces cerevisiae.

Due to a growing trend in their biomedical application, biosynthesized nanomaterials are of great interest to researchers nowadays with their biocompatible, low-energy consumption, economic, and tunable characteristics. It is important to understand the mechanism of biosynthesis in order to achieve more efficient applications. Since there are only rare studies on the influences of cellular energy levels on biosynthesis, the influence of energy is often overlooked. Through determination of the intracellular ATP concentrations during the biosynthesis process, significant changes were observed. In addition, ATP synthesis deficiency caused great decreases in quantum dots (QDs) biosynthesis in the Δatp1, Δatp2, Δatp14, and Δatp17 strains. With inductively coupled plasma-atomic emission spectrometry and atomic absorption spectroscopy analyses, it was found that ATP affected the accumulation of the seleno-precursor and helped with the uptake of Cd and the formation of QDs. We successfully enhanced the fluorescence intensity 1.5 or 2 times through genetic modification to increase ATP or SeAM (the seleno analog of S-adenosylmethionine, the product that would accumulate when ATP is accrued). This work explains the mechanism for the correlation of the cellular energy level and QDs biosynthesis in living cells, demonstrates control of the biosynthesis using this mechanism, and thus provides a new manipulation strategy for the biosynthesis of other nanomaterials to widen their applications.

Arabidopsis plasma membrane H+-ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress.

Phosphorus deficiency in soil is one of the major limiting factors for plant growth. Plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the plant response to low-phosphorus stress (LP). However, few details are known regarding the action of PM H+-ATPase in mediating root proton (H+) flux and root growth under LP. In this study, we investigated the involvement and function of different Arabidopsis PM H+-ATPase genes in root H+ flux in response to LP. First, we examined the expressions of all Arabidopsis PM H+-ATPase gene family members (AHA1-AHA11) under LP. Expression of AHA2 and AHA7 in roots was enhanced under this condition. When the two genes were deficient in their respective Arabidopsis mutant plants, root growth and responses of the mutants to LP were highly inhibited compared with the wild-type plant. AHA2-deficient plants exhibited reduced primary root elongation and lower H+ efflux in the root elongation zone. AHA7-deficient plants exhibited reduced root hair density and lower H+ efflux in the root hair zone. The modulation of H+ efflux by AHA2 or AHA7 was affected by the action of 14-3-3 proteins and/or auxin regulatory pathways in the context of root growth and response to LP. Our results suggest that under LP conditions, AHA2 acts mainly to modulate primary root elongation by mediating H+ efflux in the root elongation zone, whereas AHA7 plays an important role in root hair formation by mediating H+ efflux in the root hair zone.

Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae.

Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton-pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H(+) -ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531 and 595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.

Alterations of mitochondrial protein assembly and jasmonic acid biosynthesis pathway in Honglian (HL)-type cytoplasmic male sterility rice.

It has been suggested that the mitochondrial chimeric gene orfH79 is the cause for abortion of microspores in Honglian cytoplasmic male sterile rice, yet little is known regarding its mechanism of action. In this study, we used a mass spectrometry-based quantitative proteomics strategy to compare the mitochondrial proteome between the sterile line Yuetai A and its fertile near-isogenic line Yuetai B. We discovered a reduced quantity of specific proteins in mitochondrial complexes in Yuetai A compared with Yuetai B, indicating a defect in mitochondrial complex assembly in the sterile line. Western blotting showed that ORFH79 protein and ATP1 protein, an F(1) sector component of complex V, are both associated with large protein complexes of similar size. Respiratory complex activity assays and transmission electron microscopy revealed functional and morphological defects in the mitochondria of Yuetai A when compared with Yuetai B. In addition, we identified one sex determination TASSELSEED2-like protein increased in Yuetai A, leading to the discovery of an aberrant variation of the jasmonic acid pathway during the development of microspores.

Catalytic subunits atpα and atpß from the Pacific white shrimp Litopenaeus vannamei F(O)F (1) ATP-synthase complex: cDNA sequences, phylogenies, and mRNA quantification during hypoxia.

In the mitochondrial F(O)F(1) ATP-synthase/ATPase complex, subunits α and ß are part of the extrinsic portion that catalyses ATP synthesis. Since there are no reports about genes and proteins from these subunits in crustaceans, we analyzed the cDNA sequences of both subunits in the whiteleg shrimp Litopenaeus vannamei and their phylogenetic relationships. We also investigated the effect of hypoxia on shrimp by measuring changes in the mRNA amounts of atpα and atpß. Our results confirmed highly conserved regions for both subunits and underlined unique features among others. The ATPß deduced protein of shrimp was less conserved in size and sequence than ATPα. The relative mRNA amounts of atpα and atpß changed in shrimp pleopods; hypoxia at 1.5 mg/L caused an increase in atpß transcripts and a subsequent decrease when shrimp were re-oxygenated. Results confirm that changes in the mRNAs of the ATP-synthase subunits are part of the mechanisms allowing shrimp to deal with the metabolic adjustment displayed to tolerate hypoxia.

A yeast cell-based system for screening Candida glabrata multidrug resistance reversal agents and selection of loss-of-function pdr1 mutants.

In the pathogenic yeast Candida glabrata, multidrug resistance is associated with the overexpression of drug efflux pumps caused by gain-of-function mutations in the CgPDR1 gene. CgPdr1p transcription factor, which activates the expression of several drug efflux transporter genes, is considered to be a promising target for compounds sensitizing the multidrug-resistant yeast cells. Here, we describe a cell-based screening system for detecting the inhibitory activity of compounds interfering with the CgPdr1p function in a heterologous genetic background of the hypersensitive Saccharomyces cerevisiae mutant strain. The screening is based on the ability to abrogate the growth defect of cells suffering from the galactose-induced and CgPdr1p-driven overexpression of a dominant lethal pma1(D378N) allele placed under the control of the ScPDR5 promoter. The system allows rapid identification of multidrug resistance reversal agents inhibiting the CgPdr1p activity or loss-of-function Cgpdr1 mutations, and is amenable to high-throughput screening on solid or liquid media.

Cd2+, Mn2+, Ni2+ and Se2+ toxicity to Saccharomyces cerevisiae lacking YPK9p the orthologue of human ATP13A2.

The Saccharomyces cerevisiae gene YPK9 encodes a putative integral membrane protein which is 58% similar and 38% identical in amino acid sequence to the human lysosomal P(5B) ATPase ATP13A2. Mutations in ATP13A2 have been found in patients with Kufor-Rakeb syndrome, a form of juvenile Parkinsonism. We report that Ypk9p localizes to the yeast vacuole and that deletion of YPK9 confers sensitivity for growth for cadmium, manganese, nickel or selenium. These results suggest that Ypk9p may play a role in sequestration of divalent heavy metal ions. Further studies on the function of Ypk9p/ATP13A2 may help to define the molecular basis of Kufor-Rakeb syndrome and provide a potential link to environmental factors such as heavy metals contributing to some forms of Parkinsonism.

The concerted nature between three catalytic subunits driving the F1 rotary motor.

F(1), a rotational molecular motor, shows strong cooperativity during ATP catalysis when driving the rotation of the central gamma subunit surrounded by the alpha(3)beta(3) subunits. To understand how the three catalytic beta subunits cooperate to drive rotation, we made a hybrid F(1) containing one or two mutant beta subunits with altered catalytic kinetics and observed its rotations. Analysis of the asymmetric stepwise rotations elucidated a concerted nature inside the F(1) complex where all three beta subunits participate to rotate the gamma subunit with a 120 degrees phase. In addition, observing hybrid F(1) rotations at various solution conditions, such as ADP, P(i) and the ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) provides additional information for each elementary event. This novel experimental system, which combines single molecule observations and biochemical methods, enables us to dynamically visualize the catalytic coordination inside active enzymes and shed light on how biological machines provide unidirectional functions and rectify information from stochastic reactions.

Kidney collecting duct acid-base "regulon".

Kidneys are essential for acid-base homeostasis, especially when organisms cope with changes in acid or base dietary intake. Because collecting ducts constitute the final site for regulating urine acid-base balance, we undertook to identify the gene network involved in acid-base transport and regulation in the mouse outer medullary collecting duct (OMCD). For this purpose, we combined kidney functional studies and quantitative analysis of gene expression in OMCDs, by transcriptome and candidate gene approaches, during metabolic acidosis. Furthermore, to better delineate the set of genes concerned with acid-base disturbance, the OMCD transcriptome of acidotic mice was compared with that of both normal mice and mice undergoing an adaptative response through potassium depletion. Metabolic acidosis, achieved through an NH4Cl-supplemented diet for 3 days, not only induced acid secretion but also stimulated the aldosterone and vasopressin systems and triggered cell proliferation. Accordingly, metabolic acidosis increased the expression of genes involved in acid-base transport, sodium transport, water transport, and cell proliferation. In particular, >25 transcripts encoding proteins involved in urine acidification (subunits of H-ATPase, kidney anion exchanger, chloride channel Clcka, carbonic anhydrase-2, aldolase) were co-regulated during acidosis. These transcripts, which cooperate to achieve a similar function and are co-regulated during acidosis, constitute a functional unit that we propose to call a "regulon".

Sodium, potassium-atpases in algae and oomycetes.

We have investigated the presence of K(+)-transporting ATPases that belong to the phylogenetic group of animal Na(+),K(+)-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H(+)- and Na(+),K(+)-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells.

Starch biosynthesis during pollen maturation is associated with altered patterns of gene expression in maize.

Starch biosynthesis during pollen maturation is not well understood in terms of genes/proteins and intracellular controls that regulate it in developing pollen. We have studied two specific developmental stages: "early," characterized by the lack of starch, before or during pollen mitosis I; and "late," an actively starch-filling post-pollen mitosis I phase in S-type cytoplasmic male-sterile (S-CMS) and two related male-fertile genotypes. The male-fertile starch-positive, but not the CMS starch-deficient, genotypes showed changes in the expression patterns of a large number of genes during this metabolic transition. In addition to a battery of housekeeping genes of carbohydrate metabolism, we observed changes in hexose transporter, plasma membrane H(+)-ATPase, ZmMADS1, and 14-3-3 proteins. Reduction or deficiency in 14-3-3 protein levels in all three major cellular sites (amyloplasts [starch], mitochondria, and cytosol) in male-sterile relative to male-fertile genotypes are of potential interest because of interorganellar communication in this CMS system. Further, the levels of hexose sugars were significantly reduced in male-sterile as compared with male-fertile tissues, not only at "early" and "late" stages but also at an earlier point during meiosis. Collectively, these data suggest that combined effects of both reduced sugars and their reduced flux in starch biosynthesis along with a strong possibility for altered redox passage may lead to the observed temporal changes in gene expressions, and ultimately pollen sterility.

First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts.

Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H(+) ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all 'patch-catch' experiments and could be detected even in 2560-fold dilutions of the pollen contents. H(+) ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT-PCR experiments, a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts.

The activity of plasma membrane H(+)-ATPase is strongly stimulated during Saccharomyces cerevisiae adaptation to growth under high copper stress, accompanying intracellular acidification.

For the adaptation of cells of Saccharomyces cerevisiae, a period of latency is necessary before exponential growth is resumed in a medium supplemented with a highly inhibitory concentration of copper. In this work, we have examined some physiological responses occurring during this period of adaptation. The results revealed that plasma membrane H(+)-ATPase (PM-ATPase) activity is strongly stimulated (up to 24-fold) during copper-induced latency in growth medium with glucose, reaching maximal levels when the cells were about to start inhibited exponential growth. This in vivo activation of the ATPase activity by copper was accompanied by the stimulation of the H(+)-pumping activity of the enzyme in vivo and was essentially due to the increase of the apparent V(max) for MgATP. Although the exact molecular basis of the reported plasma membrane ATPase activation was not clarified, no increase in the mRNA levels from the encoding genes PMA1 and PMA2 was apparently detected during copper-induced latency. The physiological response reported here may allow the cells to cope with copper-induced lipid peroxidation and consequent decrease in plasma membrane lipid ordering and increase in the non-specific permeability to protons. The consequences of these copper deleterious effects were revealed by the decrease of the intracellular pH (pH(i)) of the yeast population, from approximately pH(i) 6 to pH(i) 5, during copper-induced latency in growth medium at pH 4.3. The time-dependent patterns of plasma membrane ATPase activation and of the decrease of pH(i) during the period of adaptation to growth with copper correlate, suggesting that the regulation of this membrane enzyme activity may be triggered by intracellular acidification. Consistent with this idea, when exponential growth under copper stress was resumed and the pH(i) of the yeast population recovered up to physiological values, plasma membrane ATPase activity simultaneously decreased from the highly stimulated level attained during the adaptation period of latency.

Blue light activates the plasma membrane H(+)-ATPase by phosphorylation of the C-terminus in stomatal guard cells.

The opening of stomata, which is driven by the accumulation of K(+) salt in guard cells, is induced by blue light (BL). The BL activates the H(+) pump; however, the mechanism by which the perception of BL is transduced into the pump activation remains unknown. We present evidence that the pump is the plasma membrane H(+)-ATPase and that BL activates the H(+)-ATPase via phosphorylation. A pulse of BL (30 s, 100 micromol/m(2)/s) increased ATP hydrolysis by the plasma membrane H(+)-ATPase and H(+) pumping in Vicia guard cell protoplasts with a similar time course. The H(+)-ATPase was phosphorylated reversibly by BL, and the phosphorylation levels paralleled the ATP hydrolytic activity. The phosphorylation occurred exclusively in the C-termini of H(+)-ATPases on both serine and threonine residues in two isoproteins of H(+)-ATPase in guard cells. An endogenous 14-3-3 protein was co-precipitated with H(+)-ATPase, and the recombinant 14-3-3 protein bound to the phosphorylated C-termini of H(+)-ATPases. These findings demonstrate that BL activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus by a serine/threonine protein kinase, and that the 14-3-3 protein has a key role in the activation.

ATP synthase 5' untranslated regions are specifically bound by chloroplast polypeptides.

Expression of the large ATP synthase gene cluster in spinach (Spinacia oleracea) chloroplasts is regulated at the post-transcriptional level. RNA stability and the translational efficiency of some chloroplast transcripts have been shown to be regulated through RNA-protein interactions in the 5' untranslated region (5' UTR). In this report we show that spinach chloroplast extracts contain polypeptides that specifically interact with the 5' UTRs of three of the four genes in the large ATP synthase gene cluster. A subset of binding polypeptides may be gene-specific, although at least one appears to be a more general chloroplast RNA-binding protein. We hypothesize that these RNA-protein interactions may affect the expression of this gene cluster from two perspectives. The first would be at a gene-specific level, which could serve to control the stoichiometry of ATP synthase subunits. The second would be a more global effect, which may adjust the abundance of the entire ATP synthase complex in response to environmental or developmental cues.