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Mitochondria are the major organelles of energy production; however, active mitochondria can decline their energetic role and show a dysfunctional status. Mitochondrial dysfunction was induced by high non-physiological level of L-galactone-1,4-lactone (L-GalL), the precursor of ascorbate (AsA), in plant mitochondria. The dysfunction induced by L-GalL was associated with the fault in the mitochondrial electron partition and reactive oxygen species (ROS) over-production. Using mitochondria from RNAi-plant lines harbouring silenced L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activity, it was demonstrated that such dysfunction is dependent on this enzyme activity. The capacity of alternative respiration was strongly decreased by L-GalL, probably mediated by redox-inactivation of the alternative oxidase (AOX) enzyme. Although, alternative respiration was shown to be the key factor that helps support AsA synthesis in dysfunctional mitochondria. Experiments with respiratory inhibitors showed that ROS formation and mitochondrial dysfunction were more associated with the decline in the activities of COX (cytochrome oxidase) and particularly AOX than with the lower activities of respiratory complexes I and III. The application of high L-GalL concentrations induced proteomic changes that indicated alterations in proteins related to oxidative stress and energetic status. However, supra-optimal L-GalL concentration was not deleterious for plants. Instead, the L-GalLDH activity could be positive. Indeed, it was found that wild type plants performed better growth than L-GalLDH-RNAi plants in response to high non-physiological L-GalL concentrations., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH 2 Cl 2 fraction (CFD - Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC 50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH 2 Cl 2 : MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-β-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC 50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Iron is an essential nutrient but is toxic in excess mainly under acidic conditions. Yeasts have emerged as low cost, highly efficient soil inoculants for the decontamination of metal-polluted areas, harnessing an increasing understanding of their metal tolerance mechanisms. Here, we investigated the effects of extracellular iron and acid pH stress on the dimorphism of Yarrowia lipolytica. Its growth was unaffected by 1 or 2 mM FeSO4, while a strong cellular iron accumulation was detected. However, the iron treatments decreased the hyphal length and number, mainly at 2 mM FeSO4 and pH 4.5. Inward cell membrane H+ fluxes were found at pH 4.5 and 6.0 correlated with a pH increase at the cell surface and a conspicuous yeast-to-hypha transition activity. Conversely, a remarkable H+ efflux was detected at pH 3.0, related to the extracellular microenvironment acidification and inhibition of yeast-to-hypha transition. Iron treatments intensified H+ influxes at pH 4.5 and 6.0 and inhibited H+ efflux at pH 3.0. Moreover, iron treatments inhibited the expression and activities of the plasma membrane H+-ATPase, with the H+ transport inhibited to a greater extent than the ATP hydrolysis, suggesting an iron-induced uncoupling of the pump. Our data indicate that Y. lipolytica adaptations to high iron and acidic environments occur at the expense of remodelling the yeast morphogenesis through a cellular pH modulation by H+-ATPases and H+ coupled transporters, highlighting the capacity of this non-conventional yeast to accumulate high amounts of iron and its potential application for bioremediation.

Background: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers., Methods: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples., Findings: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence., Interpretation: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H + pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms., Funding: This work was supported by grants from CNPq and FAPERJ-Brazil., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Metastatic tumor cells have acidic extracellular pH and differential electrochemical H + gradients generated across their cell membranes by V-type H + -ATPases. This study shows that inhibition of the V-ATPases by the plant-derived monoterpene Myrtenal results in tumor cell death and decreased metastatic dissemination in mice., Methods: The Myrtenal anticancer toxicity was evaluated in vitro using murine (B16F0 and B16F10) and human (SkMel-5) melanoma cell lines, and in in vivo mouse metastatic dissemination model. Proton flux and extracellular acidification were directly evaluated at the surface of living cells using a non-invasive selective ion electrode approach., Results: The inhibition of V-ATPases by 100 μM Myrtenal disrupted the electrochemical H + gradient across the cell membranes, strongly induced cell death (4-5 fold), and decreased tumor cells migration and invasion in vitro. Myrtenal (15 mg/kg) also significantly reduced metastasis induced by B16F10 in vivo, further reinforcing that V-ATPase is a molecular target to halt the progression of cancers., Conclusions: These data revealed the therapeutic potential of Myrtenal as inhibitor of melanoma progression proposing a mechanism of action by which once inhibited by this monoterpene the proton pumps fail to activate cancer-related differential electrochemical gradients and H + fluxes across the tumor cell membranes, disrupting pH signatures inherent in tumor progression, resulting in reprogrammed cell death and metastasis inhibition., General Significance: The work represents a new mechanistic strategy for contention of melanoma, the most aggressive and deadly form of cutaneous neoplasm, and highlights Myrtenal, other related monoterpenes and derivatives as promising proton pump inhibitors with high chemotherapeutic potential., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H + pump affects cholesterol mobilization to the plasma membrane., Methods: Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H + transport in membrane vesicles, and intact cell H + fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET)., Results: Cholesterol depletion by 5mM MβCD was found to be inhibitory to the hydrolytic and H + pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H + efflux in live cells at the same extent of MβCD., Conclusions: We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin., General Significance: The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica , through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H + -ATPase. Morphological transition was followed microscopically, and the H + -ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA) induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H + -ATPase activity and H + efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status., Methods: In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface., Results: Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PP i ), probably by inducing disulfide bridge formation., Conclusions: Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface., General Significance: This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status., (Copyright © 2016 Elsevier B.V. All rights reserved.)

At relatively low concentrations, the element manganese (Mn) is essential for plant metabolism, especially for photosynthesis and as an enzyme antioxidant cofactor. However, industrial and agricultural activities have greatly increased Mn concentrations, and thereby contamination, in soils. We tested whether and how growth of Pisolithus tinctorius is influenced by Mn and glucose and compare the activities of oxidative stress enzymes as biochemical markers of Mn stress. We also compared nutrient accumulation, ecophysiology, and biochemical responses in Eucalyptus grandis which had been colonized by the ectomycorrhizal Pisolithus tinctorius with those which had not, when both were exposed to increasing Mn concentrations. In vitro experiments comprised six concentrations of Mn in three concentrations of glucose. In vivo experiments used plants colonized by Pisolithus tinctorius, or not colonized, grown with three concentrations of Mn (0, 200, and 1000 μM). We found that fungal growth and glucose concentration were correlated, but these were not influenced by Mn levels in the medium. The anti-oxidative enzymes catalase and glutathione S-transferase were both activated when the fungus was exposed to Mn. Also, mycorrhizal plants grew more and faster than non-mycorrhizal plants, whatever Mn exposure. Photosynthesis rate, intrinsic water use efficiency, and carboxylation efficiency were all inversely correlated with Mn concentration. Thus, we originally show that the ectomycorrhizal fungus provides protection for its host plants against varying and potentially toxic concentrations of Mn.

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro-embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal-bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H(+) -ATPase P- and V-types and H(+) -PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species., (Copyright © Physiologia Plantarum 2012.)

The present paper presents the partial characterization of a family I inorganic pyrophosphatase from the hard tick Rhipicephalus (Boophilus) microplus (BmPPase). The BmPPase gene was cloned from the tick embryo and sequenced. The deduced amino acid sequence shared high similarity with other eukaryotic PPases, on the other hand, BmPPase presented some cysteine residues non-conserved in other groups. This pyrophosphatase is inhibited by Ca(2+), and the inhibition is antagonized by Mg(2+), suggesting that the balance between free Ca(2+) and free Mg(2+) in the eggs could be involved in BmPPase activity control. We observed that the BmPPase transcripts are present in the fat body, midgut and ovary of ticks, in two developmental stages (partially and fully engorged females). However, higher transcription amounts were found in ovary from fully engorged females. BmPPase activity was considerably abolished by the thiol reagent dithionitrobenzoic acid (DTNB), suggesting that cysteine residues are exposed in its structure. Therefore, these cysteine residues play a critical role in the structural stability of BmPPase. Molecular dynamics simulation analysis indicates that BmPPase is the first Family I PPase that could promote disulfide bonds between cysteine residues 138-339 and 167-295. Finally, we believe that these cysteine residues exposed in the BmPPase structure can play an important controlling role regarding enzyme activity, which would be an interesting mechanism of redox control. The results presented here also indicate that this enzyme can be involved in embryogenesis of this arthropod, and may be useful as a target in the development of new tick control strategies., (Published by Elsevier B.V.)

We present a case of a 21-year-old woman sustaining a traumatic [fabellar] fracture following a motor vehicle accident. The fabellar fracture was confirmed on plain films, which prompted further evaluation of the knee with ultrasound and magnetic resonance imaging to evaluate other possible associated injuries. Fracture of the fabella is a rare condition. Clinically, patients present with posterolateral knee pain, edema, and limited knee extension. Occasionally these symptoms may be very subtle, delaying the correct diagnosis and patient management.

V H(+)-ATPase has an important role in a variety of key physiological processes. This enzyme is reversibly activated/partly inactivated by the addition/exhaustion of extracellular glucose. The current model of its regulation assumes the reversible disassembly/reassembly of ∼60-70% of the V1 and V0 membrane complexes, which are responsible for ATP hydrolysis and H(+) conductance, respectively. The number of assembled complexes determines the pump activity because disassembled complexes are inactive. The model predicts the identical catalytic properties for the activated and semi-active enzymes molecules. To verify the model predictions we have isolated total membranes from yeast spheroplasts that were pre-incubated either with or without glucose. Nitrate treatment of membranes revealed the similar ATPase inhibition for two enzyme states, suggesting that they have identical structures that are essential for ATP hydrolysis. However, H(+) transport was inhibited more than the ATPase activities, indicating a nitrate uncoupling action, which was significantly higher for the nonactivated enzyme. This finding suggests that the structure of the non-activated enzyme, which is essential for H(+) transport, is less stable than that of the activated enzyme. Moreover, the glucose activation of the pump increases i) its coupling capacity; ii) its K(M) for ATP hydrolysis and ATP affinity for H(+) transport; iii) the Vmax for H(+) transport in comparison with the Vmax for ATP hydrolysis and iv) the immune reactivity of catalytic subunit A and regulatory subunit B by 9.3 and 2.4 times, respectively. The protein content of subunits A and B was not changed by extracellular glucose. We propose that instead of the dissociation/reassociation of complexes V1 and V0, changes in the extracellular glucose concentration cause reversible and asymmetrical modulations in the immune reactivity of subunits A and B by their putative biochemical modifications. This response asymmetrically modulates H(+)-transport and ATP hydrolysis, exhibiting distinct properties for the activated versus non-activated enzymes.

This study establishes the role of P(5A)-type Cta4 ATPase in Ca(2+) sequestration in the endoplasmic reticulum by detecting an ATP-dependent, vanadate-sensitive and FCCP insensitive (45)Ca(2+)-transport in fission yeast membranes isolated by cellular fractionation. Specifically, the Ca(2+)-ATPase transport activity was decreased in ER membranes isolated from cells lacking a cta4(+) gene. Furthermore, a disruption of cta4(+) resulted in 6-fold increase of intracellular Ca(2+) levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca(2+) pumping. These data provide compelling biochemical evidence for a P(5A)-type Cta4 ATPase as an essential component of Ca(2+) transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast.

The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (P(i)) and this activation was much greater using polyP(3) than polyP(15). After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP(3) was 10 times stronger than that for polyP(15). Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5'-triphosphate synthesis in hard tick embryos.

It is widely reported that some humic substances behave as exogenous auxins influencing root growth by mechanisms that are not yet completely understood. This study explores the hypothesis that the humic acids' effects on root development involve a nitric oxide signaling. Maize seedlings were treated with HA 20 mg C L(-1), IAA 0.1 nM, and NO donors (SNP or GSNO), in combination with either the auxin-signaling inhibitor PCIB, the auxin efflux inhibitor TIBA, or the NO scavenger PTIO. H(+)-transport-competent plasma membrane vesicles were isolated from roots to investigate a possible link between NO-induced H(+)-pump and HA bioactivity. Plants treated with either HA or SNP stimulated similarly the lateral roots emergence even in the presence of the auxin inhibitors, whereas NO scavenger diminished this effect. These treatments induced H(+)-ATPase stimulation by threefold, which was abolished by PTIO and decreased by auxin inhibitors. HA-induced NO synthesis was also detected in the sites of lateral roots emergence. These data depict a new scenario where the root development stimulation and the H(+)-ATPase activation elicited by either HA or exogenous IAA depend essentially on mechanisms that use NO as a messenger induced site-specifically in the early stages of lateral root development.

Chemical reactions (hydrolysis, oxidation, reduction, methylation, alkyl compounds detachment) were applied to modify the structure of humic substances (HS) isolated from vermicompost. Structural and conformational changes of these humic derivatives were assessed by elemental analyses, size exclusion chromatography (HPSEC), solid-state nuclear magnetic resonance ((13)C CPMAS-NMR), and diffusion ordered spectroscopy (DOSY-NMR), whereas their bioactivity was evaluated by changes in root architecture and proton pump activation of tomato and maize. All humic derivatives exhibited a large bioactivity compared to original HS, both KMnO(4)-oxidized and methylated materials being the most effective. Whereas no general relationship was found between bioactivity and humic molecular sizes, the hydrophobicity index was significantly related with proton pump stimulation. It is suggested that the hydrophobic domain can preserve bioactive molecules such as auxins in the humic matter. In contact with root-exuded organic acids the hydrophobic weak forces could be disrupted, releasing bioactive compounds from humic aggregates. These findings were further supported by the fact that HS and all derivatives used in this study activated the auxin synthetic reporter DR5::GUS.

Preparative high performance size-exclusion chromatography (HPSEC) was applied to humic acids (HA) extracted from vermicompost in order to separate humic matter of different molecular dimension and evaluate the relationship between chemical properties of size-fractions (SF) and their effects on plant root growth. Molecular dimensions of components in humic SF was further achieved by diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY-NMR) based on diffusion coefficients (D), while carbon distribution was evaluated by solid state (CP/MAS) (13)C NMR. Seedlings of maize and Arabidopsis were treated with different concentrations of SF to evaluate root growth. Six different SF were obtained and their carbohydrate-like content and alkyl chain length decreased with decreasing molecular size. Progressive reduction of aromatic carbon was also observed with decreasing molecular size of separated fractions. Diffusion-ordered spectroscopy (DOSY) spectra showed that SF were composed of complex mixtures of aliphatic, aromatic and carbohydrates constituents that could be separated on the basis of their diffusion. All SF promoted root growth in Arabidopsis and maize seedlings but the effects differed according to molecular size and plant species. In Arabidopsis seedlings, the bulk HA and its SF revealed a classical large auxin-like exogenous response, i.e.: shortened the principal root axis and induced lateral roots, while the effects in maize corresponded to low auxin-like levels, as suggested by enhanced principal axis length and induction of lateral roots. The reduction of humic heterogeneity obtained in HPSEC separated size-fractions suggested that their physiological influence on root growth and architecture was less an effect of their size than their content of specific bioactive molecules. However, these molecules may be dynamically released from humic superstructures and exert their bioactivity when weaker is the humic conformational stability as that obtained in the separated size-fractions., (Copyright 2009 Elsevier Ltd. All rights reserved.)

H+ transport driven by V H+-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca2+-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H+ transport activity of P H+-ATPase by more than 75% while that of V H+-ATPase remained unchanged. ER H+ ATPase exhibits higher resistance to bafilomycin (I50=38.4 nM) than Golgi and vacuole pumps (I50=0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H+ transport mediated by V H+-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H+-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.

Roots undergo multiple changes as a consequence of arbuscular mycorrhizal (AM) interactions. One of the major alterations expected is the induction of membrane transport systems, including proton pumps. In this work, we investigated the changes in the activities of vacuolar and plasma membrane (PM) H(+) pumps from maize roots (Zea mays L.) in response to colonization by two species of AM fungi, Gigaspora margarita and Glomus clarum. Both the vacuolar and PM H(+)-ATPase activities were inhibited, while a concomitant strong stimulation of the vacuolar H(+)-PPase was found in the early stages of root colonization by G. clarum (30 days after inoculation), localized in the younger root regions. In contrast, roots colonized by G. margarita exhibited only stimulation of these enzymatic activities, suggesting a species-specific phenomenon. However, when the root surface H(+) effluxes were recorded using a noninvasive vibrating probe technique, a striking activation of the PM H(+)-ATPases was revealed specifically in the elongation zone of roots colonized with G. clarum. The data provide evidences for a coordinated regulation of the H(+) pumps, which depicts a mechanism underlying an activation of the root H(+)-PPase activity as an adaptative response to the energetic changes faced by the host root during the early stages of the AM interaction.

The present work evaluated polyphosphate (poly P) metabolism in nuclear and mitochondrial fractions during Rhipicephalus microplus embryogenesis. Nuclear poly P decreased and activity of exopolyphosphatase (PPX - polyphosphate-phosphohydrolases; EC 3.6.1.11) increased after embryo cellularization until the end of embryogenesis. The utilization of mitochondrial poly P content occurred between embryo cellularization and segmentation stages. Increasing amounts of total RNA extracted from eggs progressively enhanced nuclear PPX activity, whereas it exerted no effect on mitochondrial PPX activity. The decline in total poly P content after the 7th day of embryogenesis does not reflect the free P(i) increase and the total poly P chain length decrease after embryo cellularization. The Km(app) utilizing poly P(3), poly P(15) and poly P(65) as substrate was almost the same for the nuclear fraction (around 1muM), while the affinity for substrate in mitochondrial fraction was around 10 times higher for poly P(3) (Km(app) = 0.2muM) than for poly P(15) (Km(app) = 2.8muM) and poly P(65) (Km(app) = 3.6muM). PPX activity was stimulated by a factor of two by Mg2+ and Co2+ in the nuclear fraction and only by Mg2+ in the mitochondrial fraction. Heparin (20microg/mL) inhibited nuclear and mitochondrial PPX activity in about 90 and 95% respectively. Together, these data are consistent with the existence of two different PPX isoforms operating in the nuclei and mitochondria of the hard tick R. microplus with distinct metal dependence, inhibitor and activator sensitivities. The data also shed new light on poly P biochemistry during arthropod embryogenesis, opening new routes for future comparative studies on the physiological roles of different poly P pools distributed over cell compartments.

Environmental and developmental signals can elicit differential activation of membrane proton (H(+)) fluxes as one of the primary responses of plant and fungal cells. In recent work,1 we could determine that during the presymbiotic growth of arbuscular mycorrhizal (AM) fungi specific domains of H(+) flux are activated by clover root factors, namely host root exudates or whole root system. Consequently, activation on hyphal growth and branching were observed and the role of plasma membrane H(+)-ATPase was investigated. The specific inhibitors differentially abolished most of hyphal H(+) effluxes and fungal growth. As this enzyme can act in signal transduction pathways, we believe that spatial and temporal oscillations of the hyphal H(+) fluxes could represent a pH signature for both early events of the AM symbiosis and fungal ontogeny.

The electrochemical H(+) gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H(+)-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H(+)-ATPase (V-ATPase) even in the absence of the H(+) gradient in a water-Me2SO co-solvent mixture, resulting in net synthesis of [γ-(32)P]ATP from [(32)P]Pi and ADP. The water-Me2SO (5 to 20 %) media promoted inhibition of both PPi hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH4 (+)). This effect of Me2SO on the ATP⇔(32)P exchange reaction seems to be related to a decrease of the apparent K m of the V-ATPase for Pi. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.

Ion dynamics are important for cell nutrition and growth in fungi and plants. Here, the focus is on the relationship between the hyphal H(+) fluxes and the control of presymbiotic growth and host recognition by arbuscular mycorrhizal (AM) fungi. Fluxes of H(+) around azygopores and along lateral hyphae of Gigaspora margarita during presymbiotic growth, and their regulation by phosphate (P) and sucrose (Suc), were analyzed with an H(+)-specific vibrating probe. Changes in hyphal H(+) fluxes were followed after induction by root exudates (RE) or by the presence Trifolium repens roots. Differential sensitivity to P-type ATPase inhibitors (orthovanadate or erythrosin B) suggests an asymmetric distribution or activation of H(+)-pump isoforms along the hyphae of the AM fungi. Concentration of P and Suc affected the hyphal H(+) fluxes and growth rate. However, further increases in H+ efflux and growth rate were observed when the fungus was growing close to clover roots or pretreated with RE. The H(+) flux data correlate with those from polarized hyphal growth analyses, suggesting that spatial and temporal alterations of the hyphal H(+)fluxes are regulated by nutrient availability and might underlie a pH signaling elicitation by host RE during the early events of the AM symbiosis.

The effect of aluminum on dimorphic fungi Yarrowia lipolytica was investigated. High aluminum (0.5-1.0 mM AlK(SO(4))(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.

Increasing evidences have indicated that humic substances can induce plant growth and productivity by functioning as an environmental source of auxinic activity. Here we comparatively evaluate the effects of indole-3-acetic acid (IAA) and humic acids (HA) isolated from two different soils (Inseptsol and Ultisol) and two different organic residues (vermicompost and sewage sludge) on root development and on activities of plasmalemma and tonoplast H(+ )pumps from maize roots. The data show that HA isolated from these different sources as well as low IAA concentrations (10(-10) and 10(-15) M) improve root growth through a markedly proliferation of lateral roots along with a differential activation not only of the plasmalemma but also of vacuolar H(+)-ATPases and H(+)-pyrophosphatase. Further, the vacuolar H(+)-ATPase had a peak of stimulation in a range from 10(-8) to 10(-10) M IAA, whereas the H(+)-pyrophosphatase was sensitive to a much broader range of IAA concentrations from 10(-3) to 10(-15) M. It is proposed a complementary view of the acid growth mechanism in which a concerted activation of the plasmalemma and tonoplast H(+ )pumps plays a key role in the root cell expansion process driven by environment-derived molecules endowed with auxinic activity, such as that of humic substances.

The present work evaluates the kinetics of utilization of the main potential energy sources throughout the embryonic developmental stages of Boophilus microplus. The embryonic development of this arthropod is completed in 21 days. Cellularization of the blastoderm occurs on the 6th day and is rapidly followed by germ band extension and segmentation, whose first signs are visible on the 7th day. Cellularization is typically a maternal-driven process, carried out by molecular determinants deposited in the oocyte during oogenesis. On the other hand, segmentation is of zygotic nature, being the consequence of the synthesis of various components by the growing embryo. The enhancement in total B. microplus RNA was observed after cellularization, corroborating the replacement of maternal-driven processes by embryonic zygotic expression. An abrupt increase in oxygen consumption was observed from cellularization until the 8th day of development. The reduction in dry weight at the same period and the susceptibility of oxygen consumption to KCN suggest that the respiration process is activated during early embryonic development. A marked decrease in total lipid content occurred between the 5th and 7th days of development, suggesting this is the main energy source for cellularization. A major reduction in carbohydrate content occurred later, between the 7th and 9th days, and it could be assigned to the morphological segmentation of the embryo. Although the total amount of proteins remains unchanged from oviposition to hatching, a 15% reduction in vitellin (VT) content was observed before cellularization, up to the 4th day after egglaying. This observation was correlated to the synthesis of new proteins needed to support early embryo development. Additional 20% of VT was consumed thereafter, mainly at the end of embryogenesis, and in this case VT is probably used as energy source to the older embryo. Altogether, these data indicate different energy sources for maternal and zygotic driven processes.

In a previous report (Parasitology 116 (1998) 525) we isolated and characterized Boophilus Yolk pro-Cathepsin (BYC), an aspartic proteinase precursor from the eggs of the hard tick. The present study was designed to characterize the function of BYC in the consumption of vitellin (VT), the major yolk protein, during embryogenesis. Both purified BYC and total egg homogenate proteolytic activity showed a similar pH dependence profile with an acidic optimum. Purified BYC presented higher activity against VT as a substrate when compared to other proteins. The VT degradation pattern observed in vitro also showed a similar profile to that observed in vivo. Co-localization of BYC and acidic cortical yolk granules was performed by immunocytochemistry and confocal microscopy. Proton-pumping activity of yolk granules in vitro was higher in eggs collected 4 day after oviposition than in newly laid eggs. Taken together, our data suggest that BYC plays a major role in the degradation of VT and that its activity is controlled by acidification of yolk platelets localized at the cortical cytoplasm of the developing Boophilus microplus egg.

Earthworms (Eisenia foetida) produce humic substances that can influence plant growth by mechanisms that are not yet clear. In this work, we investigated the effects of humic acids (HAs) isolated from cattle manure earthworm compost on the earliest stages of lateral root development and on the plasma membrane H(+)-ATPase activity. These HAs enhance the root growth of maize (Zea mays) seedlings in conjunction with a marked proliferation of sites of lateral root emergence. They also stimulate the plasma membrane H(+)-ATPase activity, apparently associated with an ability to promote expression of this enzyme. In addition, structural analysis reveals the presence of exchangeable auxin groups in the macrostructure of the earthworm compost HA. These results may shed light on the hormonal activity that has been postulated for these humic substances.

F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlF(x)) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlF(x) reflects their activity as Pi analogs. For this purpose, (32)P-labeled phosphate uptake by excised roots and plasma membrane H(+)-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlF(x). In vitro, AlF(x) competitively inhibited the rate of root phosphate uptake as well as the H(+)-ATPase activity. Conversely, pretreatment of seedlings with AlF(x) in vivo promoted no effect on the H(+)-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlF(x) pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent (32)Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root (32)Pi uptake induced by AlF(x) pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF(3) and AlF(4)(-) among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed.