Control of basal jasmonate signalling and defence through modulation of intracellular cation flux capacity.

Unknown mechanisms tightly regulate the basal activity of the wound-inducible defence mediator jasmonate (JA) in undamaged tissues. However, the Arabidopsis fatty acid oxygenation upregulated2 (fou2) mutant in vacuolar two-pore channel 1 (TPC1D454N ) displays high JA pathway activity in undamaged leaves. This mutant was used to explore mechanisms controlling basal JA pathway regulation. fou2 was re-mutated to generate novel 'ouf' suppressor mutants. Patch-clamping was used to examine TPC1 cation channel characteristics in the ouf suppressor mutants and in fou2. Calcium (Ca2+ ) imaging was used to study the effects fou2 on cytosolic Ca2+ concentrations. Six intragenic ouf suppressors with near wild-type (WT) JA pathway activity were recovered and one mutant, ouf8, affected the channel pore. At low luminal calcium concentrations, ouf8 had little detectable effect on fou2. However, increased vacuolar Ca2+ concentrations caused channel occlusion, selectively blocking K+ fluxes towards the cytoplasm. Cytosolic Ca2+ concentrations in unwounded fou2 were found to be lower than in the unwounded WT, but they increased in a similar manner in both genotypes following wounding. Basal JA pathway activity can be controlled solely by manipulating endomembrane cation flux capacities. We suggest that changes in endomembrane potential affect JA pathway activity.

Gene transfers shaped the evolution of de novo NAD+ biosynthesis in eukaryotes.

NAD(+) is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD(+) biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD(+) biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD(+) biosynthesis in eukaryotes was shaped by numerous gene transfers.

Horizontal gene acquisitions by eukaryotes as drivers of adaptive evolution.

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.

Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote.

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.

Calcium Signals from the Vacuole.

The vacuole is by far the largest intracellular Ca(2+) store in most plant cells. Here, the current knowledge about the molecular mechanisms of vacuolar Ca(2+) release and Ca(2+) uptake is summarized, and how different vacuolar Ca(2+) channels and Ca(2+) pumps may contribute to Ca(2+) signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca(2+) transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca(2+) channels that could elicit cytosolic [Ca(2+)] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca(2+) release, but the molecular mechanism of this Ca(2+) release still awaits elucidation. Some vacuolar Ca(2+) channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca(2+) signaling still has to be demonstrated. Ca(2+) pumps in addition to establishing long-term Ca(2+) homeostasis can shape cytosolic [Ca(2+)] transients by limiting their amplitude and duration, and may thus affect Ca(2+) signaling.

Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis.

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.

A novel calcium binding site in the slow vacuolar cation channel TPC1 senses luminal calcium levels.

Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca²âº binding sites have been characterized in cytosolic proteins. However, little is known about Ca²âº binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca²âº. 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 Ca²âº binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca²âº binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.

pH regulation in an acidophilic green alga - a quantitative analysis.

Short-term cytosolic pH regulation has three components: H(+) binding by buffering groups; H(+) transport out of the cytosol; and H(+) transport into the vacuole. To understand the large differences plants show in their tolerance to acidic environments, these three components were quantified in the acidophilic unicellular green alga Eremosphaera viridis. Intracellular pH was recorded using ion-selective microelectrodes, whereas constant doses of weak acid were applied over different time intervals. A mathematical model was developed that describes the recorded cytosolic pH changes. Recordings of cytosolic K(+) and Na(+) activities, and application of anion channel inhibitors, revealed which ion fluxes electrically compensate H(+) transport. The cytosolic buffer capacity was beta = 30 mM. Acidification resulted in a substantial and constant H(+) efflux that was probably driven by the plasmalemma H(+)-ATPase, and a proportional pH regulation caused by H(+) pumped into the vacuole. Under severe cytosolic acidification (> or = 1 pH) more than 50% of the ATP synthesized was used for H(+) pumping. While H(+) influx into the vacuole was compensated by cation release, H(+) efflux out of the cell was compensated by anion efflux. The data presented here give a complete and quantitative picture of the ion fluxes during acid loading in an acidophilic green plant cell.

Vacuolar ion channels in the liverwort Conocephalum conicum.

As a liverwort Conocephalum conicum belongs to the oldest terrestrial plants1 and is phylogenetically located between green algae and higher plants. Recent patch-clamp recordings on Conocephalum vacuoles2,3 demonstrate ion channels very similar to higher plants and clearly different from vacuolar ion channels described in green algae. Here we summarize the features of a vacuolar cation channel and a vacuolar anion channel that both are common in terrestrial plants but are not detected in green algae, and we speculate about the molecular identity of these channels in the liverwort Conocephalum.

Characteristics of anion channels in the tonoplast of the liverwort Conocephalum conicum.

Isolated vacuoles of the liverwort Conocephalum conicum thallus cells were investigated using the patch-clamp technique. At high cytosolic Ca(2+) activities, slowly activating currents were evoked by positive potentials. The currents were conducted by the SV (slow-vacuolar) channel. When isolation of vacuoles was carried out at high Mg(2+) and low Ca(2+) concentration and the same proportion of the cations was kept in the bath, currents were recorded at negative potentials. Once activated, these currents persisted even after replacing Mg(2+) with K(+) in the bath. Sr(2+) and Ba(2+) were also effective activators of the currents. With a Cl(-) gradient, 10 mM in the bath and 100 mM in the lumen, currents were significantly reduced and the current-voltage characteristics shifted towards the reversal potential of Cl(-), indicating Cl(-) selectivity. Currents almost vanished after substituting Cl(-) with gluconate. They were strongly reduced by anion channel inhibitors 4,4'-diisothicyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mM), anthracene-9-carboxylic acid (A9C; 2 mM) and ethacrinic acid (0.5 mM). Single-channel recordings revealed a 32 pS channel activating at negative voltages. It is concluded that the currents at negative potentials are carried by anion channels suitable for conducting anions from the cytosol to the vacuole. The anion channels were weakly calcium dependent, remaining active at physiological calcium concentration. The channels were almost equally permeable to Cl(-), NO3(-) and SO4(2-), and much less permeable to malate(2-). Anion channels did not respond to ATP addition. cAMP (10 microM) had a weak effect on anion channels. Protein kinase A (0.4 U) added to the medium caused no significant effect on anion channels.

Mechanism of luminal Ca2+ and Mg2+ action on the vacuolar slowly activating channels.

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.

KCO1 is a component of the slow-vacuolar (SV) ion channel.

The Arabidopsis double pore K+ channel KCO1 was fused to green fluorescent protein and expressed in tobacco protoplasts. Microscopic analysis revealed a bright green fluorescence at the vacuolar membrane. RT-PCR experiments showed that KCO1 is expressed in the mesophyll. Vacuoles from Arabidopsis wild-type and kco1 knockout plants were isolated for patch-clamp analyses. Currents mediated by slow-activating vacuolar (SV) channels of mesophyll cell vacuoles were significantly smaller in kco1 plants compared to the wild-type. This shows that KCO1 is involved in the formation of SV channels.