Acidocalcisomes

Acidocalcisomes are acidic organelles containing calcium and a high concentration of phosphorus in the form of pyrophosphate (PP_i) and polyphosphate (poly P). Organelles with these characteristics have been found from bacteria to human cells implying an early appearance and persistence over evolutionary time or their appearance by convergent evolution. Acidification of the organelles is driven by the presence of vacuolar proton pumps, one of which, the vacuolar proton pyrophosphatase, is absent in animals, where it is substituted by a vacuolar proton ATPase. A number of other pumps, antiporters, and channels have been described in acidocalcisomes of different species and are responsible for their internal content. Enzymes involved in the synthesis and degradation of PP_i and poly P are present within the organelle. Acidocalcisomes function as storage sites for cations and phosphorus, and participate in PP_i and poly P metabolism, calcium homeostasis, maintenance of intracellular pH, and osmoregulation. Experiments in which the acidocalcisome Ca²⁺-ATPase of different parasites were downregulated or eliminated, or acidocalcisome Ca²⁺ was depleted revealed the importance of this store in Ca²⁺ signaling needed for host invasion and virulence. Acidocalcisomes interact with other organelles in a number of organisms suggesting their association with the endosomal/lysosomal pathway, and are considered part of the lysosome-related group of organelles.     

Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca²⁺-ATPase, V-H⁺-ATPase, H⁺-PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes.     

Proteomic Analysis of the Acidocalcisome,an Organelle Conserved from Bacteria to Human Cells

Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H⁺-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate.     

Acidocalcisomes - conserved from bacteria to man

Recent work has shown that acidocalcisomes, which are electron-dense acidic organelles rich in calcium and polyphosphate, are the only organelles that have been conserved during evolution from prokaryotes to eukaryotes. Acidocalcisomes were first described in trypanosomatids and have been characterized in most detail in these species. Acidocalcisomes have been linked with several functions, including storage of cations and phosphorus, polyphosphate metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis and osmoregulation. Here, we review acidocalcisome ultrastructure, composition and function in different trypanosomatids and other organisms.     

Calcium‐ and polyphosphate‐containing acidocalcisomes in chicken egg yolk

Background information. Poly P (inorganic polyphosphate) is a polymer formed by P_i residues linked by high‐energy phosphoanhydride bonds. The presence of poly P in bacteria, fungi, algae and protists has been widely recognized, but the distribution of poly P in more complex eukaryotes has been poorly studied. Poly P accumulates, together with calcium, in acidic vesicles or acidocalcisomes in a number of organisms and possesses a diverse array of functions, including roles in stress response, blood clotting, inflammation, calcification, cell proliferation and apoptosis. Results. We report here that a considerable amount of phosphorus in the yolk of chicken eggs is in the form of poly P. DAPI (4′,6‐diamidino‐2‐phenylindole) staining showed that poly P is localized mainly in electron‐dense vesicles located inside larger vacuoles (compound organelles) that are randomly distributed in the yolk. These internal vesicles were shown to contain calcium, potassium, sodium, magnesium, phosphorus, chlorine, iron and zinc, as detected by X‐ray microanalysis and elemental mapping. These vesicles stain with the acidophilic dye Acridine Orange. The presence of poly P in organellar fractions of the egg yolk was evident in agarose gels stained with Toluidine Blue and DAPI. Of the total phosphate (P_i) of yolk organelles, 16% is present in the form of poly P. Total poly P content was not altered during the first 4 days of embryogenesis, but poly P chain length decreased after 1 day of development. Conclusions. The results of the present study identify a novel organelle in chicken egg yolk comprising acidic vesicles with a morphology, physiology and composition similar to those of acidocalcisomes, within larger acidic vacuoles. The elemental composition of these acidocalcisomes is proportionally similar to the elemental composition of the yolk, suggesting that most of these elements are located in these organelles, which might be an important storage compartment in eggs.     

Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplaneta americana

Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H⁺-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H⁺-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg²⁺ and inhibited by NaF, as well as by IDP and Ca²⁺. Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H⁺-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.     

PPiase-Activated ATP-Dependent H+ Transport at the Tonoplast of Mesophyll Cells of the CAM Plant Kalanchoë daigremontiana*

The co-ordinated action of the two proton-transporting enzymes at the tonoplast of the CAM plants. daigremontiana, viz. the ATPase and the PP_iase, was studied by measuring fluorescent dye quenching. The initial rates of ATP and PP_i-dependent H⁺ transport into tonoplast vesicles were additive, i.e. the sum of the rates obtained with each substrate alone was in the range obtained with both substrates added together at the same time. Conversely, the activities of the two H⁺ pumps were non-additive in establishing the steady-state level, indicating that the final steady state was under thermodynamic control of a maximal attainable proton gradient. The initial rates of ATP-dependent H⁺ transport were stimulated enormously if ATP was added a few minutes after pre-energization of the vesicles with PP_i. This stimulation was observed only when the PP_iase was active. A similar effect was not found for PP_i-dependent H⁺ transport after pre-energization with ATP. Hence, a PP_iase-activated ATP-dependent H⁺ transport can be distinguished from the basic ATP- and the basic PP_i-dependent H⁺ transport. In parallel a PP_i-dependent stimulation of ATP hydrolysis in the absence of ionophores was measured, which can only be attributed to the activity of the PP_iase. PP_iase-activated ATP-dependent H⁺ transport depends on the presence of permeant anions. It shows properties of both H⁺ transport activities, i.e. the chloride and malate stimulation and the DCCD inhibition of the ATP-dependent H⁺ transport activity, the nitrate stimulation and the KF inhibition of the PP_i-dependent H⁺ transport activity. Only MgPP_i and MgATP were effective as the respective substrates. The PP_iase-activated ATP-dependent H⁺ transport had a half life of about 5–9 minutes. It is concluded that the PP_iase may play an important role in kinetic regulation of the ATPase, and implications for CAM metabolism are discussed.     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

A contractile vacuole complex is involved in osmoregulation in Trypanosoma cruzi

Acidocalcisomes are dense, acidic organelles with a high concentration of phosphorus present as pyrophosphate and polyphosphate complexed with calcium and other cations. Acidocalcisomes have been linked to the contractile vacuole complex in Chlamydomonas reinhardtii, Dictyostelium discoideum, and Trypanosoma cruzi. A microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex in T. cruzi results in translocation of aquaporin and the resulting water movement which, in addition to swelling of acidocalcisomes, is responsible for the volume reversal not accounted for by efflux of osmolytes. Polyphosphate hydrolysis occurs during hyposmotic stress, probably increasing the osmotic pressure of the contractile vacuole and facilitating water movement.     

Catalysis by isolated β-subunits of the ATP Synthase/ATPase from Thermophilic bacillus PS3. Hydrolysis of Pyrophosphate

Although the capacity of isolated β-subunits of the ATP synthase/ATPase to perform catalysis has been extensively studied, the results have not conclusively shown that the subunits are catalytically active. Since soluble F₁ of mitochondrial H⁺-ATPase can bind inorganic pyrophosphate (PP_i) and synthesize PP_i from medium phosphate, we examined if purified His-tagged β-subunits from Thermophilic bacillus PS3 can hydrolyze PP_i. The difference spectra in the near UV CD of β-subunits with and without PP_i show that PP_i binds to the subunits. Other studies show that β-subunits hydrolyze [³²P] PP_i through a Mg²⁺-dependent process with an optimal pH of 8.3. Free Mg²⁺ is required for maximal hydrolytic rates. The Km for PP_i is 75 μM and the Vmax is 800 pmol/min/mg. ATP is a weak inhibitor of the reaction, it diminishes the Vmax and increases the Km for PP_i. Thus, isolated β-subunits are catalytically competent with PP_i as substrate; apparently, the assembly of β-subunits into the ATPase complex changes substrate specificity, and leads to an increase in catalytic rates.     

Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+)/H(+) countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Effects of destruxins on free calcium and hydrogen ions in insect hemocytes

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the ento mopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects ofdestruxins on changes in free calcium and hydrogen ions in the hemocytes ofExolontha serrulata, Bombyx mori and the Spodoptera litura SL1 cell line were detected using laser scanning confocal mi croscopy （LSCM）. An instant Ca2＋ influx of hemocytes induced by destruxins A and B （DA and DB） was recorded. The DA/DBdependent Ca2＋ influx was not influenced by the Ca2＋ channel inhibitors 2aminoethoxydiphenyl borane （2APB） and U73122. It also had an apparently different LSCM profile from that of the ionomycindependent Ca2＋ influx. However, the instant Ca2＋ influx was not seen in the SL1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2＋ was observed. Meanwhile, an instant intracellular free H＋ decrease aroused by DA and DB was found. DB at 20/zmol/L and DA at 690/zmol/L significantly reduced intracellular free H＋ levels. Furthermore, the vacuolar H＋ATPase （VATPase） inhibitor bafilomycin A1 had obvious effects on the decreases ofintracellular free H＋ in hemocytes. These results suggest that the mechanism of DA/DBdependent Ca2＋ influx is perhaps not related to Ca2＋ channels and ionophores; rather, the intracellular free H＋ decrease might be due to VATPase inhibition.     

Sodium sensing induces different changes in free cytosolic calcium concentration and pH in salt-tolerant and -sensitive rice (Oryza sativa) cultivars

Perception of salt stress in plant cells induces a change in the free cytosolic Ca²⁺, [Ca²⁺]_cyt, which transfers downstream reactions toward salt tolerance. Changes in cytosolic H⁺ concentration, [H⁺]_cyt, are closely linked to the [Ca²⁺]_cyt dynamics under various stress signals. In this study, salt‐induced changes in [Ca²⁺]_cyt, and [H⁺]_cyt and vacuolar [H⁺] concentrations were monitored in single protoplasts of rice (Oryza sativa L. indica cvs. Pokkali and BRRI Dhan29) by fluorescence microscopy. Changes in cytosolic [Ca²⁺] and [H⁺] were detected by use of the fluorescent dyes acetoxy methyl ester of calcium‐binding benzofuran and acetoxy methyl ester of 2′, 7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6) carboxyfluorescein, respectively, and for vacuolar pH, fluorescent 6‐carboxyfluorescein and confocal microscopy were used. Addition of NaCl induced a higher increase in [Ca²⁺]_cyt in the salt‐tolerant cv. Pokkali than in the salt‐sensitive cv. BRRI Dhan29. From inhibitor studies, we conclude that the internal stores appear to be the major source for [Ca²⁺]_cyt increase in Pokkali, although the apoplast is more important in BRRI Dhan29. The [Ca²⁺]_cyt measurements in rice also suggest that Na⁺ should be sensed inside the cytosol, before any increase in [Ca²⁺]_cyt occurs. Moreover, our results with individual mesophyll protoplasts suggest that ionic stress causes an increase in [Ca²⁺]_cyt and that osmotic stress sharply decreases [Ca²⁺]_cyt in rice. The [pH]_cyt was differently shifted in the two rice cultivars in response to salt stress and may be coupled to different activities of the H⁺‐ATPases. The changes in vacuolar pH were correlated with the expressional analysis of rice vacuolar H⁺‐ATPase in these two rice cultivars.     

Acidocalcisomes as calcium- and polyphosphate-storage compartments during embryogenesis of the insect Rhodnius prolixus Stahl

Background

The yolk of insect eggs is a cellular domain specialized in the storage of reserve components for embryo development. The reserve macromolecules are stored in different organelles and their interactions with the embryo cells are mostly unknown. Acidocalcisomes are lysosome-related organelles characterized by their acidic nature, high electron density and large content of polyphosphate bound to several cations. In this work, we report the presence of acidocalcisome-like organelles in eggs of the insect vector Rhodnius prolixus.

Methodology/Principal findings

Characterization of the elemental composition of electron-dense vesicles by electron probe X-ray microanalysis revealed a composition similar to that previously described for acidocalcisomes. Following subcellular fractionation experiments, fractions enriched in acidocalcisomes were obtained and characterized. Immunofluorescence showed that polyphosphate polymers and the vacuolar proton translocating pyrophosphatase (V-H⁺-PPase, considered as a marker for acidocalcisomes) are found in the same vesicles and that these organelles are mainly localized in the egg cortex. Polyphosphate quantification showed that acidocalcisomes contain a significant amount of polyphosphate detected at day-0 eggs. Elemental analyses of the egg fractions showed that 24.5±0.65% of the egg calcium are also stored in such organelles. During embryogenesis, incubation of acidocalcisomes with acridine orange showed that these organelles are acidified at day-3 (coinciding with the period of yolk mobilization) and polyphosphate quantification showed that the levels of polyphosphate tend to decrease during early embryogenesis, being approximately 30% lower at day-3 compared to day-0 eggs.

Conclusions

We found that acidocalcisomes are present in the eggs and are the main storage compartments of polyphosphate and calcium in the egg yolk. As such components have been shown to be involved in a series of dynamic events that may control embryo growth, results reveal the potential involvement of a novel organelle in the storage and mobilization of inorganic elements to the embryo cells.     

The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

H+ Fluxes at Plasmalemma Level: In Vivo Evidence for a Significant Contribution of the Ca2+-ATPase and for the Involvement of its Activity in the Abscisic Acid-Induced Changes in Egeria Densa Leaves

Abstract: The features of Ca²⁺ fluxes, the importance of the Ca²⁺ pump‐mediated H⁺/Ca²⁺ exchanges at plasmalemma level, and the possible involvement of Ca²⁺‐ATPase activity in ABA‐induced changes of H⁺ fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca²⁺ flux was evident, a conspicuous Ca²⁺ influx (about 1.1 ìmol g^?1 FW h^?1) occurred. The concomitant efflux of Ca²⁺ was markedly reduced by treatment with 5 íM eosin Y (EY), a specific inhibitor of the Ca²⁺‐ATPase, that completely blocked the transport of Ca²⁺ after the first 20 ‐ 30 min. The decrease in Ca²⁺ efflux induced by EY was associated with a significant increase in net H⁺ extrusion (?ÄH⁺) and a small but significant cytoplasmic alkalinization. The shift of external [Ca²⁺] from 0.3 to 0.2 mM (reducing Ca²⁺ uptake by about 30 %) and the hindrance of Ca²⁺ influx by La³⁺ were accompanied by progressively higher ?ÄH⁺ increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca²⁺ influx by an nH⁺/Ca²⁺ exchange. The ABA‐induced decreases in ?ÄH⁺ and pH_cyt were accompanied by a significant increase in Ca²⁺ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H⁺ fluxes are due to activation of the plasmalemma Ca²⁺‐ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca²⁺ pump in removing from the cytoplasm the Ca²⁺ taken up, and the importance of the contribution of Ca²⁺ pump‐mediated H⁺/Ca²⁺ fluxes in bringing about global changes of H⁺ fluxes at plasmalemma level.     

A vacuolar H-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress

Background

Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.).

Methods

The vacuolar H⁺ pumps, the H⁺-ATPase and H⁺-PP_iase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops.

Results

In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H⁺ transport, which was in turn at least 3-folds faster than that mediated by the H⁺-PP_iase. Its PP_i hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PP_i-dependent H⁺-gradient. This effect was antagonized by plant rehydration as the H⁺-PP_iase activity was highly stimulated, reassuming a coupled PP_i-driven H⁺ pumping. Maize tonoplasts exhibited 2–4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PP_i-dependent H⁺ pumping in response to stress relief, resulting in an enhanced H⁺-pumps coupling efficiency.

Conclusion

These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H⁺-pumps, which differ between weeds and crops upon water deficit.

General significance

The evidences highlight an unusual modulation of the H⁺-PP_iase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.