Mitochondrial sequestration of BCECF after ester loading in the giant alga Chara australis

Summary. Ratiometric fluorescent dyes are often used to monitor free ion concentrations in vivo, especially in cells that are recalcitrant to transformation with genetically encoded fluorescent markers. Although intracellular dye distributions are often found to be cytosolic, dye localisation has often not been examined in detail. We began exploring the use of BCECF (2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to monitor pH in the giant alga Chara australis and discovered that younger leaf cells could be loaded using the acetoxymethyl ester of BCECF. However, we were puzzled to find in microphotometric measurements that the fluorescence ratio appeared insensitive to manipulations affecting cytosolic pH. Confocal imaging of C. australis cells loaded with BCECF showed an accumulation of the dye in two locations: (1) on the outside of the chloroplasts in irregularly shaped stationary bodies; (2) within 1–1.5 μm structures that moved rapidly with the pericellular cytoplasmic streaming. Together with the streaming cytoplasm, these organelles were rendered stationary with 50 μM cytochalasin D. Rhodamine 123, a mitochondrionspecific dye, highlighted organelles outside of the chloroplasts, similar to those shown by BCECF in location 1. We conclude that in the cytoplasmic compartment, BCECF was sequestered within cytoplasmic mitochondria in immature and fast-growing cells and within the cortical mitochondrial system in older and slowly growing cells. Thus, BCECF-AM is unsuitable for reporting changes in cytosolic pH in C. australis but might be employed in future to study pH changes in the mitochondria. Correspondence: M. J. Beilby, Biophysics, School of Physics, University of New South Wales, Sydney 2052, Australia.     

Microbodies of the alga Chara

Chara fragilis possesses microbodies with a remarkably large size of up to 2 µm in diameter . Many of the organelles contain huge nucleoids of amorphous material or paracrystalline inclusions. After isolation of the organelles by gradient centrifugation the specific density of the microbodies was determined to be 1.25 g cm⁻³. Catalase, glycolate oxidase and hydroxypyruvate reductase as well as enzymes of the fatty acid β -oxidation pathway were demonstrated to be constituents of the microbodies in Chara indicating that they are similar to those in green leaves. The data obtained are in agreement with the view that the Charophyceae and especially the algae in the subgroup of Charales are very closely related to the land plants.     

The regulation of ammonia uptake in Chara australis

The regulation of ammonia uptake was investigated in internodalcells of the freshwater alga Chara australis. Ammonia uptakewas estimated by monitoring (i) its depletion from the bathingsolution, (ii) the uptake of radiolabelled methylamine, an analogueof ammonia, and (iii) depletion of ammonia in the unstirredlayer with the microelectrode ion-flux estimation technique(MIFE). Distribution of methylamine (¹⁴CH₃NH₃⁺) between thevacuole and cytoplasm was estimated with efflux analysis. Whencells were bathed continuously in solutions containing ammoniaor methylamine, the uptake rates of both amines decreased over12 to 48 h despite the continuing existence of a large electrochemicalgradient favouring influx of the NH⁺₄ and CH₃NH⁺₄ cations. Treatmentwith 1.0 to 10.0 mM MSX, an inhibitor of glutamine synthetase,caused the internal ammonia concentration to rise and reducedthe subsequent uptake of ammonia and methylamine by up to 70%within 2 h. These results suggest that the permease facilitatingNH⁺₄/CH₃NH⁺₄ influx is under feedback or kinetic regulationfrom either internal ammonia or an intermediate of nitrogenassimilation. Treatment with metabolic inhibitors (CCCP, azide and DCMU) andsome weak acids (DMO and butyric acid) for 30 to 60 min inhibitedmethylamine uptake, but the changes in the electrical potentialdifference across the plasma membrane could not account forthe magnitude of inhibition. The rate of cytopiasmic streaming,which is an indicator of the cellular ATP concentration in Chara,was inhibited by many of these treatments. However, under certainconditions of external pH and concentration, butyric acid couldreversibly inhibit ammonia and methylamine uptake without affectingcytoplasmic streaming, demonstrating that a decrease in cytoplasmicATP concentration was not responsible for the inhibition. Theeffect of butyric acid was rapid, causing a 60% inhibition ofuptake in 15 min. We conclude that weak acids can inhibit theNH⁺₄/CH₃NH⁺₄ permease by acidifying the cytoplasm and suggestthat this may also explain the effects of the metabolic inhibitorson ammonia and methylamine uptake. Key words: Ammonia, methylamine, uptake, regulation, Chara     

Metabolomics reveals organ-specific metabolic rearrangements during early tomato seedling development

Tomato seedlings (Solanum lycopersicum cv. MoneyMaker), grown under strictly controlled conditions, have been used to study alterations occurring in secondary metabolite biosynthetic pathways following developmental and environmental perturbations. Robustness and reproducibility of the system were confirmed using detailed statistical analyses of the metabolome. LCMS profiling was applied using whole germinated seeds as well as cotyledons, hypocotyls and roots from 3 to 9 days old seedlings to generate relative levels of 433 metabolites, of which 62 were annotated. Initial focus was given to the polyphenol pathway and several additional mass signals have been putatively annotated using high mass resolution fragmentation. Clear organ and developmental stage—specific differences were observed. Seeds accumulated saponin-like compounds; roots accumulated mainly alkaloids; cotyledons contained mainly glycosylated flavonols and; hypocotyls contained mainly anthocyanins. For each organ, the developmental changes in metabolite profiles were described by using linear mixed models. Across three independent experiments, 85 % of the metabolites showed similar developmental trends. This tomato seedling system has given us valuable additional insights into the complexity of seedling secondary metabolism. How metabolic profiles reflect an interplay between depletion of stored molecules and de novo synthesis and how the overall picture for this important crop plant contrasts to e.g. Arabidopsis are emphasised.     

Exogenous melatonin affects photosynthesis in characeae Chara australis

Melatonin was found in the fresh water characeae Chara australis. The concentrations (~4 μg/g of tissue) were similar in photosynthesizing cells, independent of their position on the plant and rhizoids (roots) without chloroplasts. Exogenous melatonin, added at 10 μM to the artificial pond water, increased quantum yield of photochemistry of photosystem II by 34%. The increased efficiency appears to be due to the amount of open reaction centers of photosystem II, rather than increased efficiency of each reaction center. More open reaction centers reflect better functionality of all photosynthetic transport chain constituents. We suggest that melatonin protection against reactive oxygen species covers not only chlorophyll, but also photosynthetic proteins in general.     

Turgor regulation in the salt-tolerant alga Chara longifolia

Chara longifolia is a salt‐tolerant Charophyte which regulates its turgor inresponse to osmotic stress. Membrane depolarization, in creased membrane conductance, and cessation of cytoplasmic streaming (due to increase in cytoplasmic Ca^{2 +} ) precede regulation in response to hypotonic stress. Measurements of these three parameters are presented here with simultaneous turgor measurements. Variability in the occurrence, rate and extent of turgor regulation in individual cells was correlated with magnitude of the stress. Hypertonic stress showed the same slow time course as was found previously, requiring several days for complete regulation. Fifty μ M nifedipine, a Ca^{2 +} channel blocker, inhibited turgor regulation. In the presence of 5 μ M nifedipine, turgor regulation was delayed. An increase in conductance preceded regulation, but membrane depolarization was less and no detectable change in cytoplasmic streaming was observed, requiring modifications to a previously presented model for turgor regulation. There was no significant difference in ⁴⁵Ca^{2 +} influx under control and stress conditions. However, the control flux was insensitive to nifedipine, whereas under stress the flux is inhibited 54% by nifedipine. We suggest that osmotic stress results in a rapid increase in a nifedipine‐sensitive Ca^{2 +} entry mechanism, followed very quickly by a decrease in the control entry mechanism.     

The Electrical Resistance of the Tonoplast of Chara australis

A critical investigation of the method usually used for measuringthe electrical resistance of the tonoplast shows that the valuethus obtained is always higher than the true value.     

Metabolomics reveals stage-specific metabolic pathways of microbial communities in two-stage anaerobic fermentation of corn-stalk

Analysis of intracellular metabolites is essential to delineate metabolic pathways of microbial communities for evaluation and optimization of anaerobic fermentation processes. The metabolomics are reported for a microbial community during two stages of anaerobic fermentation of corn stalk in a biogas digester using GC–MS. Acetonitrile/methanol/water (2:2:1, by vol) was the best extraction solvent for microbial community analysis because it yielded the largest number of peaks (>200), the highest mean summed value of identified metabolites (23) and the best reproducibility with a coefficient of variation of 30 % among four different extraction methods. Inter-stage comparison of metabolite profiles showed increased levels of sugars and sugar alcohols during methanogenesis and fatty acids during acidogenesis. Identification of stage-specific metabolic pathways using metabolomics can therefore assist in monitoring and optimization of the microbial community for increased biogas production during anaerobic fermentation.     

Effects of UV-B radiation on a charophycean alga,Chara aspera

The charophycean algal species Chara aspera was exposed for 73 days to three levels of UV-B radiation (weighted according to Caldwell's generalized plant action spectrum): 1.9 kJ m^–2 day^–1 (`no UV-B'), 6.4 KJ m<sup>–2</sup> day<sup>–1</sup> (ambient) and 10.5 kJ m<sup>–2</sup> day<sup>–1</sup> (enhanced UV-B), the latter level simulating 30% ozone reduction in The Netherlands.Charophycean algae are mainly freshwater organisms and are thought to be the algae most closely related to higher land plants. Therefore we expected that responses of charophycean algae to UV-B radiation might be more related to those observed in the higher land plants than those of other `lower' algal groups.Under elevated UV-B radiation algal length was reduced. There was no induction of UV absorbing compounds under enhanced UV-B. This might relate to a sensitive response to UV-B radiation. The charophycean algae show similar adaptations to UV-B radiation as terrestrial plants, while not having UV-screens as occur in many angiosperms. Vegetative reproduction (bulbils) increased in the presence of UV-B radiation, while generative reproduction (antheridia and oogonia) decreased.     

Metabolomics reveals reduction of metabolic oxidation in women with polycystic ovary syndrome after pioglitazone-flutamide-metformin polytherapy

Polycystic ovary syndrome (PCOS) is a variable disorder characterized by a broad spectrum of anomalies, including hyperandrogenemia, insulin resistance, dyslipidemia, body adiposity, low-grade inflammation and increased cardiovascular disease risks. Recently, a new polytherapy consisting of low-dose flutamide, metformin and pioglitazone in combination with an estro-progestagen resulted in the regulation of endocrine clinical markers in young and non-obese PCOS women. However, the metabolic processes involved in this phenotypic amelioration remain unidentified. In this work, we used NMR and MS-based untargeted metabolomics to study serum samples of young non-obese PCOS women prior to and at the end of a 30 months polytherapy receiving low-dose flutamide, metformin and pioglitazone in combination with an estro-progestagen. Our results reveal that the treatment decreased the levels of oxidized LDL particles in serum, as well as downstream metabolic oxidation products of LDL particles such as 9- and 13-HODE, azelaic acid and glutaric acid. In contrast, the radiuses of small dense LDL and large HDL particles were substantially increased after the treatment. Clinical and endocrine-metabolic markers were also monitored, showing that the level of HDL cholesterol was increased after the treatment, whereas the level of androgens and the carotid intima-media thickness were reduced. Significantly, the abundance of azelaic acid and the carotid intima-media thickness resulted in a high degree of correlation. Altogether, our results reveal that this new polytherapy markedly reverts the oxidant status of untreated PCOS women, and potentially improves the pro-atherosclerosis condition in these patients.     

Degradation of Proteins Artificially Introduced into Vacuoles of Chara australis

When an exogenous protein, bovine serum albumin, was introduced into the vacuole of a Chara australis internodal cell, it was degraded with time. This degradation proceeded only in the vacuole as far as could be observed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Degradation was inhibited by protease inhibitors such as antipain and leupeptin. Endogenous proteins introduced into the vacuole were also degraded there. Furthermore, intravacuolar cytoplasmic drops, which were often formed by cell ligation, seemed to be degraded in the vacuole. However, bovine serum albumin degradation did not proceed when mixed with isolated vacuolar sap. These results show that the vacuole in the Chara internodal cell has the capacity to degrade cellular proteins, but that cytoplasmic support is needed for this degrading activity to be maintained.     

Ion flux interaction with cytoplasmic streaming in branchlets of Chara australis

Both parts of the actin-myosin complex involved in cytoplasmic streaming could be regulated by mineral ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall and plasma membrane. The transport of K(+) and Ca(2+) along pH bands in Chara branchlet internodal cells was characterized by using the MIFE system for non-invasive microelectrode measurement of ion fluxes. Branchlets formed acidic and alkaline bands with the pH ranging from 5 to 8. Different pH patterns were observed for different sides of the branchlets. Sides with cyclosis streaming acropetally generally showed greater variation in the profiles of pH and H(+) fluxes. Although a high correlation was not found between pH bands and Ca(2+) or K(+) fluxes, there was a positive correlation between Ca(2+) and K(+) fluxes themselves for both sides of the branchlets. Application of cytochalasin D, an inhibitor of cyclosis, had no immediate effect on pH and ion fluxes, however, the time of cyclosis cessation corresponded with a dramatic change in Ca(2+) and K(+) fluxes; pH profiles and H(+) fluxes were affected within 2 h. The evidence suggests that, in Chara branchlets, pH band formation and Gd(3+)-insensitive Ca(2+) transport systems are linked to the cyclosis machinery: (i) the pH band amplitude for the acropetally streaming side was larger than that for the basipetally streaming side; (ii) cessation of cytoplasmic streaming after cytochalasin D application resulted in changed pH banding profiles and H(+), Ca(2+) and K(+) fluxes; and (iii) the application of GdCl(3) or incubation in GdCl(3) solutions did not lead to the cessation of cytoplasmic streaming, although external Ca(2+) fluxes changed.     

Partial Purification and Characterization of Aminopeptidase II from Chara australis

Aminopeptidase II, one of the two major aminopeptidases in the giant alga Chara australis, was partially purified. Its molecular weight was estimated to be about 80,000 by gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that it is composed of a single polypeptide with a molecular weight of about 85,000. Aminopeptidase II hydrolyzed alanine-2-naphthylamide more efficiently than the naphthylamides of lysine and proline, and only weakly hydrolyzed the naphthylamides of arginine, phenylalanine, valine, and leucine. The optimal pH for the hydrolysis of alanine-2-naphthylamide was near 7.0. The activity of aminopeptidase II was inhibited by the SH-reagents p-chloromercuribenzoic acid and N-ethylmaleimide and by the metal chelator 1,10-phenanthroline.     

Dependency of H+ efflux on ATP in cells of Chara australis

The internodal cell of Chara australis was made tonoplast-freeby internal perfusion with a medium containing a Ca²⁺-chelatorEGTA and the net H⁺ efflux across the plasma membrane was estimatedeither by titration of the external medium or by measuring thechange in pH in the external medium. The amount of H⁺ effluxwas high in the presence of internal ATP and low in its absence.The ATP-dependent net H⁺ efflux was about 40 nmoles/m²/sec.This amount is smaller than that estimated for the pump currenton the basis of electrical data obtained earlier (3). This discrepancyis discussed. (Received June 18, 1980; )     

pH-Sensitive Gating Kinetics of the Maxi-K Channel in the Tonoplast of Chara australis

The most frequently observed K⁺ channel in the tonoplast of Characean giant internodal cells with a large conductance (ca. 170 pS; Lühring, 1986; Laver & Walker, 1987) behaves, although inwardly rectifying, like animal maxi-K channels. This channel is accessible for patch–clamp techniques by preparation of cytoplasmic droplets, where the tonoplast forms the membrane delineating the droplet. Lowering the pH of the bathing solution, that virtually mimicks the vacuolar environment, from an almost neutral level to values below pH 7, induced a significant but reversible decrease in channel activity, whereas channel conductance remained largely unaffected. Acidification (pH 5) on both sides of the membrane decreased open probability from a maximum of 80% to less than 20%. Decreasing pH at the cytosolic side inhibited channel activity cooperatively with a slope of 2.05 and a pK _a 6.56. In addition, low pH at the vacuolar face shifted the activating voltage into a positive direction by almost 100 mV. This is the first report about an effect of extraplasmatic pH on gating of a maxi-K channel. It is suggested that the Chara maxi-K channel possesses an S4-like voltage sensor and negatively charged residues in neighboring transmembrane domains whose S4-stabilizing function may be altered by protonation. It was previously shown that gating kinetics of this channel respond to cytosolic Ca²⁺ (Laver & Walker, 1991). With regard to natural conditions, pH effects are discussed as contributing mainly to channel regulation at the vacuolar membrane face, whereas at the cytosolic side Ca²⁺ affects the channel. An attempt was made to ascribe structural mechanisms to different states of a presumptive gating reaction scheme. Received: 8 May 1998/Revised: 18 September 1998     

Some of the Electrical Characteristics of the Cell Membrane of Chara australis

Measurements of the electrical resistance and potential of thecell membranes of Chara australis have been made for over 60cells. In 17 cases the measurements extended over a period ofa few days for each cell. The most important results of theseobservations and calculations are as follows. The mean valueof the intercellular (between cells) resistance is about 1 kcm². The transcellular (vacuole to external solution) resistanceconsists of two high-resistance layers. The mean value of theelectrical resistance of the outer layer is 7 k cm². The capacityof the transcellular membrane equals about 2.5 µF cm^–2.