Ecology 92, 218 (2011* Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.

The central vacuole is the largest Ca2+ store in a mature plant cell. Ca2+ release from this store contributes to Ca2+-mediated intracellular signalling in a variety of physiological responses. However, the routes for vacuolar Ca2+ release are not well characterized. To date, at least two voltage-dependent and two ligand-gated Ca2+-permeable channels have been reported in plant vacuoles. However, the so-called VVCa (vacuolar voltage-gated Ca2+) channel most probably is not a separate channel but is identical to another voltage-dependent channel-the so-called SV (slow vacuolar) channel. Studies in the last few years have added a new dimension to our knowledge of SV channel-mediated ion transport and the mechanisms of its regulation by multiple natural factors. Recently, the SV channel was identified as the product of the TPC1 gene in Arabidopsis. In contrast, the TPC1 channel from other species was thought to be localized in the plasma membrane. A re-evaluation of this work under the assumption that the TPC1 channel is generally a vacuolar channel provides interesting insights into the physiological function of the TPC1/SV channel. Considerably less is known about vacuolar Ca2+ channels that are supposed to be activated by inositol 1,4,5-trisphosphate or cADP ribose. The major problems are controversial reports about functional characteristics, and a remarkable lack of homologues of animal ligand-gated Ca2+ channels in higher plants. To help understand Ca2+-mediated intracellular signalling in plant cells, a critical update of existing experimental evidence for vacuolar Ca2+ channels is presented.

In order to test the hypothesis that slowly activating vacuolar (SV) channels mediate Ca2+‐induced Ca2+ release the voltage‐ and Ca2+‐dependence of these K+ and Ca2+‐ permeable channels were studied in a quantitative manner. The patch‐clamp technique was applied to barley (Hordeum vulgare L.) mesophyll vacuoles in the whole vacuole and vacuolar‐free patch configuration. Under symmetrical ionic conditions the current‐voltage relationship of the open SV channel was characterized by a pronounced inward rectification. The single channel current amplitude was not affected by changes in cytosolic Ca2+ whereas an increase in vacuolar Ca2+ decreased the unitary current in a voltage‐dependent manner. The SV channel open‐probability increased with positive potentials and elevated cytosolic Ca2+, but not with elevated cytosolic Mg2+. An increase of cytosolic Ca2+ shifted the half‐activation potential to more negative voltages, whereas an increase of vacuolar Ca2+ shifted the half‐activation potential to more positive voltages. At physiological vacuolar Ca2+ activities (50 μM to 2 mM) changes in cytosolic Ca2+ (5 μM to 2 mM) revealed an exponential dependence of the SV channel open‐probability on the electrochemical potential gradient for Ca2+ (ΔμCa). At the Ca2+ equilibrium potential (ΔμCa = 0) the open‐probability was as low as 0.4%. Higher open‐probabilities required net Ca2+ motive forces which would drive Ca2+ influx into the vacuole. Under conditions favouring Ca2+ release from the vacuole, however, the open‐probability further decreased. Based on quantitative analysis, it was concluded that the SV channel is not suited for Ca2+‐induced Ca2+ release from the vacuole.

Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca 2+ binding sites have been characterized in cytosolic proteins. However, little is known about Ca 2+ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca 2+ . However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca 2+ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca 2+ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.

In contrast to vertical gene transfer from parent to offspring, horizontal (or lateral) gene transfer moves genetic information between different species. Bacteria and archaea often adapt through horizontal gene transfer. Recent analyses indicate that eukaryotic genomes, too, have acquired numerous genes via horizontal transfer from prokaryotes and other lineages. Based on this we raise the hypothesis that horizontally acquired genes may have contributed more to adaptive evolution of eukaryotes than previously assumed. Current candidate sets of horizontally acquired eukaryotic genes may just be the tip of an iceberg. We have recently shown that adaptation of the thermoacidophilic red alga Galdieria sulphuraria to its hot, acid, toxic-metal laden, volcanic environment was facilitated by the acquisition of numerous genes from extremophile bacteria and archaea. Other recently published examples of horizontal acquisitions involved in adaptation include ice-binding proteins in marine algae, enzymes for carotenoid biosynthesis in aphids, and genes involved in fungal metabolism. Editor's suggested further reading in BioEssays Jumping the fine LINE between species: Horizontal transfer of transposable elements in animals catalyses genome evolution Abstract.

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca(2+)-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channel's closed conformations. Vacuolar Ca2+ and Mg2+ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a 'permeable blocker'. Vacuolar Ca(2+)-with a much higher affinity than Mg(2+)-slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca(2+)-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channel's closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca2+ at the vacuolar side. The specificity for Ca2+ compared to Mg2+ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca2+ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.

SummaryIn contrast to the vacuolar ion channels which are gated open by an increase of cytosolic Ca 2+ the vacuolar ion currents at resting cytosolic Ca2+are poorly explored. Therefore, this study was performed to investigate the properties of the so-called fast-activating vacuolar (FV) current which dominates the electrical characteristics of the tonoplast at physiological free Ca 2+ concentrations. Patch-clamp measurements were performed on whole barley (Hordeum vulgare) mesophyll vacuoles and on excised tonoplast patches. Single ion channels were identified, which, based on their selectivity, activation kinetics, Ca 2+-and voltage-dependence, carry the whole-vacuole FV current. Reversal potential determinations indicated a K ÷ over CI-permeability ratio of about 30. Both inward and outward whole-vacuole currents as well as the activity of single FV channels were inhibited by an increase of cytosolic Ca 2 +, with a Kd ~ 6 IxM. At physiological vacuolar Ca 2+ activities, the FV channel is an outward-rectifying potassium channel. The FV channel was activated in less than a few milliseconds both by negative and positive potential steps, having a minimal activity that is 40 mV negative of the K + equilibrium potential. It is proposed that transport of K + through this cation channel controls the electrical potential difference across the tonoplast.

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