Electrochemical potential enables dormant spores to integrate environmental signals.

The dormant state of bacterial spores is generally thought to be devoid of biological activity. We show that despite continued dormancy, spores can integrate environmental signals over time through a preexisting electrochemical potential. Specifically, we studied thousands of individual Bacillus subtilis spores that remain dormant when exposed to transient nutrient pulses. Guided by a mathematical model of bacterial electrophysiology, we modulated the decision to exit dormancy by genetically and chemically targeting potassium ion flux. We confirmed that short nutrient pulses result in step-like changes in the electrochemical potential of persistent spores. During dormancy, spores thus gradually release their stored electrochemical potential to integrate extracellular information over time. These findings reveal a decision-making mechanism that operates in physiologically inactive cells.

Collaboration between NDH and KEA3 Allows Maximally Efficient Photosynthesis after a Long Dark Adaptation.

In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around PSI (CET). K+ Efflux Antiporter3 (KEA3) is a putative thylakoid H+/K+ antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis (Arabidopsis thaliana). The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m-2 s-1 after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent down-regulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m-2 s-1, the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent down-regulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO2 fixation was delayed after overnight dark adaptation.

Modulation of K+ translocation by AKT1 and AtHAK5 in Arabidopsis plants.

Root cells take up K+ from the soil solution, and a fraction of the absorbed K+ is translocated to the shoot after being loaded into xylem vessels. K+ uptake and translocation are spatially separated processes. K+ uptake occurs in the cortex and epidermis whereas K+ translocation starts at the stele. Both uptake and translocation processes are expected to be linked, but the connection between them is not well characterized. Here, we studied K+ uptake and translocation using Rb+ as a tracer in wild-type Arabidopsis thaliana and in T-DNA insertion mutants in the K+ uptake or translocation systems. The relative amount of translocated Rb+ to the shoot was positively correlated with net Rb+ uptake rates, and the akt1 athak5 T-DNA mutant plants were more efficient in their allocation of Rb+ to shoots. Moreover, a mutation of SKOR and a reduced plant transpiration prevented the full upregulation of AtHAK5 gene expression and Rb+ uptake in K+ -starved plants. Lastly, Rb+ was found to be retrieved from root xylem vessels, with AKT1 playing a significant role in K+ -sufficient plants. Overall, our results suggest that K+ uptake and translocation are tightly coordinated via signals that regulate the expression of K+ transport systems.

Each of the chloroplast potassium efflux antiporters affects photosynthesis and growth of fully developed Arabidopsis rosettes under short-day photoperiod.

In Arabidopsis thaliana, the chloroplast harbors three potassium efflux antiporters (KEAs), namely KEA1 and KEA2 in the inner envelope and KEA3 in the thylakoid membrane. They may play redundant physiological roles as in our previous analyses of young developing Arabidopsis rosettes under long-day photoperiod (16 h light per day), chloroplast kea single mutants resembled the wild-type plants, whereas kea1kea2 and kea1kea2kea3 mutants were impaired in chloroplast development and photosynthesis resulting in stunted growth. Here, we aimed to study whether chloroplast KEAs play redundant roles in chloroplast function of older Arabidopsis plants with fully developed rosettes grown under short-day photoperiod (8 h light per day). Under these conditions, we found defects in photosynthesis and growth in the chloroplast kea single mutants, and most dramatic defects in the kea1kea2 double mutant. The mechanism behind these defects in the single mutants involves reduction in the electron transport rate (kea1 and kea3), and stomata conductance (kea1, kea2 and kea3), which in turn affect CO2 fixation rates. The kea1kea2 mutant, in addition to these alterations, displayed reduced levels of photosynthetic machinery. Taken together, our data suggest that, in addition to the previously reported roles in chloroplast development in young rosettes, each chloroplast KEA affects photosynthesis and growth of Arabidopsis fully developed rosettes.

Plastidial transporters KEA1, -2, and -3 are essential for chloroplast osmoregulation, integrity, and pH regulation in Arabidopsis.

Multiple K(+) transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K(+) transporters is limited. Potassium transport and a well-balanced K(+) homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K(+) transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higher-order loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na(+)/H(+) antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis.

Regulation of potassium transport in plants under hostile conditions: implications for abiotic and biotic stress tolerance.

Intracellular potassium homeostasis is a prerequisite for the optimal operation of plant metabolic machinery and plant's overall performance. It is controlled by K(+) uptake, efflux and intracellular and long-distance relocation, mediated by a large number of K(+) -selective and non-selective channels and transporters located at both plasma and vacuolar membranes. All abiotic and biotic stresses result in a significant disturbance to intracellular potassium homeostasis. In this work, we discuss molecular mechanisms and messengers mediating potassium transport and homeostasis focusing on four major environmental stresses: salinity, drought, flooding and biotic factors. We argue that cytosolic K(+) content may be considered as one of the 'master switches' enabling plant transition from the normal metabolism to 'hibernated state' during first hours after the stress exposure and then to a recovery phase. We show that all these stresses trigger substantial disturbance to K(+) homeostasis and provoke a feedback control on K(+) channels and transporters expression and post-translational regulation of their activity, optimizing K(+) absorption and usage, and, at the extreme end, assisting the programmed cell death. We discuss specific modes of regulation of the activity of K(+) channels and transporters by membrane voltage, intracellular Ca(2+) , reactive oxygen species, polyamines, phytohormones and gasotransmitters, and link this regulation with plant-adaptive responses to hostile environments.

A mathematical model of action potential in cells of vascular plants.

A mathematical model of action potential (AP) in vascular plants cells has been worked out. The model takes into account actions of plasmalemma ion transport systems (K(+), Cl(-) and Ca(2+) channels; H(+)- and Ca(2+)-ATPases; 2H(+)/Cl(-) symporter; and H(+)/K(+) antiporter), changes of ion concentrations in the cell and in the extracellular space, cytoplasmic and apoplastic buffer capacities and the temperature dependence of active transport systems. The model of AP simulates a stationary level of the membrane potential and ion concentrations, generation of AP induced by electrical stimulation and gradual cooling and the impact of external Ca(2+) for AP development. The model supports a hypothesis about participation of H(+)-ATPase in AP generation.

Pathophysiology of mitochondrial volume homeostasis: potassium transport and permeability transition.

Regulation of mitochondrial volume is a key issue in cellular pathophysiology. Mitochondrial volume and shape changes can occur following regulated fission-fusion events, which are modulated by a complex network of cytosolic and mitochondrial proteins; and through regulation of ion transport across the inner membrane. In this review we will cover mitochondrial volume homeostasis that depends on (i) monovalent cation transport across the inner membrane, a regulated process that couples electrophoretic K(+) influx on K(+) channels to K(+) extrusion through the K(+)-H(+) exchanger; (ii) the permeability transition, a loss of inner membrane permeability that may be instrumental in triggering cell death. Specific emphasis will be placed on molecular advances on the nature of the transport protein(s) involved, and/or on diseases that depend on mitochondrial volume dysregulation.

Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance.

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S. cerevisiae cells in the exponential growth phase. Altogether, the contributions of six K+ transporters to the maintenance of a stable membrane potential were tested. As confirmed by the observed hyperpolarization of trk1 trk2 deletion strains, the diS-C3(3) assay is a suitable method for comparative studies of the membrane potential of yeast strains differing in the presence/absence of one or more cation transporters. We have shown that the presence of the Tok1 channel strongly influences membrane potential: deletion of the TOK1 gene results in significant plasma membrane depolarization, whereas strains overexpressing the TOK1 gene are hyperpolarized. We have also proved that plasma membrane potential is not the only parameter determining the hygromycin B sensitivity of yeast cells, and that the role of intracellular transporters in protecting against its toxic effects must also be considered.

A Na+:H+ antiporter and a molybdate transporter are essential for arsenite oxidation in Agrobacterium tumefaciens.

Transposon Tn5-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an Agrobacterium tumefaciens soil isolate, strain 5A. In one mutant, the transposon interrupted modB, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in mrpB, which is part of a seven-gene operon encoding an Mrp-type Na+:H+ antiporter complex. Complementation experiments with mod and mrp operons PCR cloned from the genome-sequenced A. tumefaciens strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of A. tumefaciens. As expected, the mrp mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in A. tumefaciens is involved in Na+ circulation across the membrane. Gene expression studies (lacZ reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the mrp operon in response to As(III) exposure, whereas expression of the mod operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.

Do physiological roles foster persistence of drug/multidrug-efflux transporters? A case study.

Drug and multidrug resistance have greatly compromised the compounds that were once the mainstays of antibiotic therapy. This resistance often persists despite reductions in the use of antibiotics, indicating that the proteins encoded by antibiotic-resistance genes have alternative physiological roles that can foster such persistence in the absence of selective pressure by antibiotics. The recent observations that Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both confer alkali tolerance offer a striking case study in support of this hypothesis.

A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana.

Electroneutral monovalent cation/proton antiport across the chloroplast envelope has been shown previously to have an important regulatory effect on stromal pH and thereby on photosynthetic carbon reduction. Here we report that an Arabidopsis nuclear gene, AtCHX23, encodes a putative Na(+)(K(+))/H(+) exchanger and functions in the adjustment of pH in the cytosol and possibly in maintaining a high pH level in the chloroplast stroma. The AtCHX23 protein is localized in the chloroplast envelope. Plastids from chx23 mutants had straight thylakoids but lacked grana lamellae. chx23 mutant leaves were pale yellow and had a much reduced chlorophyll content. The chlorophyll content of chx23 was increased by growing in medium at low (4.0) pH and decreased by growing at high (7.0) pH. The cytosolic pH in the leaves of the mutant was significantly higher than that in the wild type. chx23 mutants displayed a high sensitivity to NaCl. Together, these data indicate that CHX23 is a probable chloroplast Na(+)(K(+))/H(+) exchanger important for pH homeostasis and chloroplast development and function.