Proton Extrusion Research Articles

A Novel Calcium-Activated Potassium Channel Controls Membrane Potential and Intracellular pH in Trypanosoma cruzi.

Trypanosoma cruzi develops in environments where nutrient availability, osmolarity, ionic concentrations, and pH undergo significant changes. The ability to adapt and respond to such conditions determines the survival and successful transmission of T. cruzi. Ion channels play fundamental roles in controlling physiological parameters that ensure cell homeostasis by rapidly triggering compensatory mechanisms. Combining molecular, cellular and electrophysiological approaches we have identified and characterized the expression and function of a novel calcium-activated potassium channel (TcCAKC). This channel resides in the plasma membrane of all 3 life stages of T. cruzi and shares structural features with other potassium channels. We expressed TcCAKC in Xenopus laevis oocytes and established its biophysical properties by two-electrode voltage clamp. Oocytes expressing TcCAKC showed a significant increase in inward currents after addition of calcium ionophore ionomycin or thapsigargin. These responses were abolished by EGTA suggesting that TcCAKC activation is dependent of extracellular calcium. This activation causes an increase in current and a negative shift in reversal potential that is blocked by barium. As predicted, a single point mutation in the selectivity filter (Y313A) completely abolished the activity of the channels, confirming its potassium selective nature. We have generated knockout parasites deleting one or both alleles of TcCAKC. These parasite strains showed impaired growth, decreased production of trypomastigotes and slower intracellular replication, pointing to an important role of TcCAKC in regulating infectivity. To understand the cellular mechanisms underlying these phenotypic defects, we used fluorescent probes to evaluate intracellular membrane potential, pH, and intracellular calcium. Epimastigotes lacking the channel had significantly lower cytosolic calcium, hyperpolarization, changes in intracellular pH, and increased rate of proton extrusion. These results are in agreement with previous reports indicating that, in trypanosomatids, membrane potential and intracellular pH maintenance are linked. Our work shows TcCAKC is a novel potassium channel that contributes to homeostatic regulation of important physiological processes in T. cruzi and provides new avenues to explore the potential of ion channels as targets for drug development against protozoan parasites.

Characterization of natural genetic variation identifies multiple genes involved in salt tolerance in maize

Progressive decline in irrigation water is forcing farmers to use brackish water which increases soil salinity and exposes the crop plants to salinity. Maize, one of the most important crops, is sensitive to salinity. Salt tolerance is a complex trait controlled by a number of physiological and biochemical processes. Scant information is available on the genetic architecture of salt tolerance in maize. We evaluated 399 inbred lines for six early vigor shoot and root traits upon exposure of 18-day seedlings to salinity (ECiw = 16 dS m-1) stress. Contrasting response of shoot and root growth to salinity indicated a meticulous reprogramming of resource partitioning by the plants to cope with the stress. The genomic analysis identified 57 single nucleotide polymorphisms (SNP) associated with early vigor traits. Candidate genes systematically associated with each SNP include both previously known and novel genes. Important candidates include a late embryogenesis protein, a divalent ion symporter, a proton extrusion protein, an RNA-binding protein, a casein kinase 1, and an AP2/EREBP transcription factor. The late embryogenesis protein is associated with both shoot and root length, indicating a coordinated change in resource allocation upon salt stress. Identification of a casein kinase 1 indicates an important role for Ser/Thr kinases in salt tolerance. Validation of eight candidates based on expression in a salt-tolerant and a salt-sensitive inbred line supported their role in salt tolerance. The candidate genes identified in this investigation provide a foundation for dissecting genetic and molecular regulation of salt tolerance in maize and related grasses.

The role of N form supply for PGPM‐host plant interactions in maize

AbstractThe form of nitrogen (N) supply has a significant impact on rhizosphere chemistry and root growth responses of higher plants. The respective effects are also employed as management options to improve nutrient acquisition and to minimize nutrient losses in cropping systems. However, surprisingly little is known concerning the interactions with rhizosphere biota. In this study, we investigated the effects of selected bacterial and fungal inoculants with proven plant growth‐promoting and phosphate (P)‐solubilizing potential (plant growth‐promoting microorganisms, PGPM) in maize with nitrate or stabilized ammonium supply, on soils with limited P availability and sparingly soluble rock phosphate (Rock‐P) applied as P fertilizer. The combination of the bacterial inoculants Pseudomonas sp. DSMZ 13134 (Proradix) and Bacillus amyloliquefaciens FZB42 with ammonium sulphate fertilization, stabilized with the nitrification inhibitor 3,4‐dimethylpyrazole‐phosphate (DMPP), resulted in a superior shoot biomass production (79–111%) and shoot P accumulation (109–235%) as compared with nitrate supply. This effect could be partially attributed to (1) ammonium‐induced rhizosphere acidification via increased root extrusion of protons, (2) promotion of root hair elongation, and (3) increased shoot concentrations of hormonal growth regulators (indole‐3‐acetic acid, zeatin, gibberellic acid). The effects, induced by the microbial inoculants were mainly related to increased root length development (43–44%), associated with a 60% increase in auxin production potential. No inoculant effects were detected on root hair elongation or on chemical modifications of the rhizosphere involved in P solubilisation, such as rhizosphere acidification, release of carboxylates or secretory phosphohydrolases. However, the ammonium‐induced stimulation of root hair elongation increased preferential sites for root colonization by the selected inoculants, which may explain the increase in rhizosphere abundance of PGPMs, exemplarily recorded for the fungal inoculant Trichoderma harzianum OMG16 (210%). The presented data suggest a network of positive interactions between stabilized ammonium fertilization and plant growth‐promoting functions of various bacterial and fungal PGPM inoculants. This offers perspectives to increase the efficiency and the reproducibility of PGPM‐assisted fertilization strategies.

Soil carbonate drives local adaptation in Arabidopsis thaliana.

High soil carbonate limits crop performance especially in semiarid or arid climates. To understand how plants adapt to such soils, we explored natural variation in tolerance to soil carbonate in small local populations (demes) of Arabidopsis thaliana growing on soils differing in carbonate content. Reciprocal field-based transplants on soils with elevated carbonate (+C) and without carbonate (-C) over several years revealed that demes native to (+C) soils showed higher fitness than those native to (-C) soils when both were grown together on carbonate-rich soil. This supports the role of soil carbonate as a driving factor for local adaptation. Analyses of contrasting demes revealed key mechanisms associated with these fitness differences. Under controlled conditions, plants from the tolerant deme A1(+C) native to (+C) soil were more resistant to both elevated carbonate and iron deficiency than plants from the sensitive T6(-C) deme native to (-C) soil. Resistance of A1(+C) to elevated carbonate was associated with higher root extrusion of both protons and coumarin-type phenolics. Tolerant A1(+C) also had better Ca-exclusion than sensitive T6(-C) . We conclude that Arabidopsis demes are locally adapted in their native habitat to soils with moderately elevated carbonate. This adaptation is associated with both enhanced iron acquisition and calcium exclusion.

Novel hybrid action of GABA mediates inhibitory feedback in the mammalian retina.

The stream of visual information sent from photoreceptors to second-order bipolar cells is intercepted by laterally interacting horizontal cells that generate feedback to optimize and improve the efficiency of signal transmission. The mechanisms underlying the regulation of graded photoreceptor synaptic output in this nonspiking network have remained elusive. Here, we analyze with patch clamp recording the novel mechanisms by which horizontal cells control pH in the synaptic cleft to modulate photoreceptor neurotransmitter release. First, we show that mammalian horizontal cells respond to their own GABA release and that the results of this autaptic action affect cone voltage-gated Ca2+ channel (CaV channel) gating through changes in pH. As a proof-of-principle, we demonstrate that chemogenetic manipulation of horizontal cells with exogenous anion channel expression mimics GABA-mediated cone CaV channel inhibition. Activation of these GABA receptor anion channels can depolarize horizontal cells and increase cleft acidity via Na+/H+ exchanger (NHE) proton extrusion, which results in inhibition of cone CaV channels. This action is effectively counteracted when horizontal cells are sufficiently hyperpolarized by increased GABA receptor (GABAR)-mediated HCO3− efflux, alkalinizing the cleft and disinhibiting cone CaV channels. This demonstrates how hybrid actions of GABA operate in parallel to effect voltage-dependent pH changes, a novel mechanism for regulating synaptic output.

ZnT2 is an electroneutral proton-coupled vesicular antiporter displaying an apparent stoichiometry of two protons per zinc ion.

Zinc is a vital trace element crucial for the proper function of some 3,000 cellular proteins. Specifically, zinc is essential for key physiological processes including nucleic acid metabolism, regulation of gene expression, signal transduction, cell division, immune- and nervous system functions, wound healing, and apoptosis. Consequently, impairment of zinc homeostasis disrupts key cellular functions resulting in various human pathologies. Mammalian zinc transport proceeds via two transporter families ZnT and ZIP. However, the detailed mechanism of action of ZnT2, which is responsible for vesicular zinc accumulation and zinc secretion into breast milk during lactation, is currently unknown. Moreover, although the putative coupling of zinc transport to the proton gradient in acidic vesicles has been suggested, it has not been conclusively established. Herein we modeled the mechanism of action of ZnT2 and demonstrated both computationally and experimentally, using functional zinc transport assays, that ZnT2 is indeed a proton-coupled zinc antiporter. Bafilomycin A1, a specific inhibitor of vacuolar-type proton ATPase (V-ATPase) which alkalizes acidic vesicles, abolished ZnT2-dependent zinc transport into intracellular vesicles. Moreover, using LysoTracker Red and Lyso-pHluorin, we further showed that upon transient ZnT2 overexpression in intracellular vesicles and addition of exogenous zinc, the vesicular pH underwent alkalization, presumably due to a proton-zinc antiport; this phenomenon was reversed in the presence of TPEN, a specific zinc chelator. Finally, based on computational energy calculations, we propose that ZnT2 functions as an antiporter with a stoichiometry of 2H+/Zn2+ ion. Hence, ZnT2 is a proton motive force-driven, electroneutral vesicular zinc exchanger, concentrating zinc in acidic vesicles on the expense of proton extrusion to the cytoplasm.

Temperature dependence of mitoflash biogenesis in cardiac mitochondria

Mitochondrial flashes (mitoflashes) represent fundamental biochemical and biophysical dynamics of the organelle, involving sudden depolarization of mitochondrial membrane potential (ΔΨm), bursting production of reactive oxygen species (ROS), and accelerated extrusion of matrix protons. Here we investigated temperature dependence of mitoflash biogenesis as well as ΔΨm oscillations, a subset of which overlapping with mitoflashes, in both cardiac myocytes and isolated respiring cardiac mitochondria. Unexpectedly, we found that mitoflash biogenesis was essentially temperature-independent in intact cardiac myocytes, evidenced by the constancy of frequency as well as amplitude and rise speed over 5 °C–40 °C. Moderate temperature dependence was found in single mitochondria charged by respiratory substrates, where mitoflash frequency was decreased over 5 °C–20 °C with Q10 of 0.74 for Complex I substrates and 0.83 for Complex II substrate. In contrast, ΔΨm oscillation frequency displayed a negative temperature dependence at 5 °C–20 °C with Q10 of 0.82 in intact cells, but a positive temperature dependence at 25 °C - 40 °C with Q10 of 1.62 in isolated mitochondria charged with either Complex I or Complex II substrates. Moreover, the recovery speed of individual mitoflashes exhibited mild temperature dependence (Q10 = 1.14–1.22). These results suggest a temperature compensation of mitoflash frequency at both the mitochondrial and extra-organelle levels, and underscore that mitoflashes and ΔΨm oscillations are related but distinctly different mitochondrial functional dynamics.

Effect of Ionization State on Voltage-Sensor Structure in Resting State of the Hv1 Channel.

Voltage-gated proton-selective channels (Hv1) mediate proton extrusion during intracellular acidification. Hv1 is gated by the proton electrochemical gradient. Intracellular ionizable residues in Hv1 have been proposed to serve as proton-binding sites for pH-dependent gating, but detailed descriptions remain unclear. Here, molecular dynamics (MD) simulations were performed to investigate the effect of ionization states of charged residues on the X-ray structure of Hv1. Modification of the protonation state of acidic residues affected the resting conformation of Hv1 by disrupting salt bridges between S4 and the other segments. Upon protonation, conformational changes enabled the displacement of the S4 arginines toward the extracellular side and increased the mobility of hydrophobic residues at the gate. The aqueous crevice was considerably wider with increased hydration in the pore. Solvation free energies of the pore residues were low at the extra- and intracellular entrances, whereas the narrowest region exhibited the energy barrier for water translocation. Our MD data showed that water molecules in the upper and lower pore oriented differently. In neutral pH, the pore water oriented its dipole pointing away from the voltage-sensing domain center, whereas the opposite direction of the water dipole was observed in acidic pH.

The Effect of Naphthazarin on the Growth, Electrogenicity, Oxidative Stress, and Microtubule Array in Z. mays Coleoptile Cells Treated With IAA.

Naphthazarin (5,8-dihydroxy-1,4-naphthoquinone, DHNQ) is a naturally occurring 1,4-naphthoquinone derivative. In this study, we focused on elucidating the toxic effect of this secondary metabolite on the growth of plant cells. The dose–response curves that were obtained for the effects of DHNQ on endogenous and IAA-induced growth in maize coleoptile segments differ in shape; in the first case, it is linear, while in the presence of auxin it is bell-shaped with the maximum at 1 μM. It was found that DHNQ at almost all concentrations studied, when added to the incubation medium inhibited endogenous growth (excluding naphthazarin at 0.001 μM) as well as growth in the presence of IAA. Simultaneous measurements of the growth and external medium pH of coleoptile segments indicated that DHNQ diminished or eliminated proton extrusion at all of the concentrations that were used. Interestingly, the oxidative stress in maize coleoptile cells, which was measured as hydrogen peroxide (H2O2) production, catalase activity, redox activity and malondialdehyde (MDA) content, increased at the lower concentrations of DHNQ (<1 μM), thus suggesting a specific character of its action. It was also found that naphthazarin at concentration higher than 0.1 μM caused the depolarization of the membrane potential (Em). An analysis of the organization and anisotropy of the cortical microtubules showed that naphthazarin at all of the concentrations that were studied changed the IAA-induced transverse microtubule reorientation to an oblique reorientation. Our results indicate that naphthazarin diminished the growth of maize coleoptile cells by a broad spectrum of its toxic effects, thereby suggesting that naphthazarin might be a hypothetical component of new bioherbicides and biopesticides.

Proteomic analysis of Rhizobium freirei PRF 81T reveals the key role of central metabolic pathways in acid tolerance

Soil acidity strongly affects microbial diversity and represents a constraint to legume production. Rhizobium freirei is a common bean symbiont recognized by its tolerance to environmental stresses, including low pH. The protein expression profiles of R. freirei PRF 81T grown in pH 6.8 and 4.8 were analyzed to clarify the mechanisms responsible for acid tolerance in this species. Bacteria were grown up to exponential phase in tryptone-yeast (TY) medium at pH 6.8 and 4.8. Whole-cell protein extracts were separated by two-dimensional electrophoresis (2-DE), and spots that showed statistical difference in their relative volumes (%vol) between the treatments were selected and excised for identification by MALDI-TOF mass spectrometry. Data showed that protein synthesis was increased at pH 4.8, which consequently raises energy demand. Differential expression of membrane-associated proteins suggested an increased proton extrusion and decreased influx, while central metabolism seemed to be through inducing acid-consuming catabolic pathways and preventing fatty acid biosynthesis. Consequently, the respiratory chain was stimulated along with the production of reactive oxygen species (ROS). The broad range of metabolic pathways modulated by acidified pH endorses the adaptive response to environmental stresses as a multigenic character in R. freirei PRF 81. Moreover, our data emphasize the key role of central metabolism in acid stress tolerance.

Bioconjugation strategy for cell surface labelling with gold nanostructures designed for highly localized pH measurement

Regulation of intracellular pH is critically important for many cellular functions. The quantification of proton extrusion in different types of cells and physiological conditions is pivotal to fully elucidate the mechanisms of pH homeostasis. Here we show the use of gold nanoparticles (AuNP) to create a high spatial resolution sensor for measuring extracellular pH in proximity of the cell membrane. We test the sensor on HepG2 liver cancer cells and MKN28 gastric cancer cells before and after inhibition of Na+/H+ exchanger. The gold surface conjugation strategy is conceived with a twofold purpose: i) to anchor the AuNP to the membrane proteins and ii) to quantify the local pH from AuNP using surface enhanced Raman spectroscopy (SERS). The nanometer size of the cell membrane anchored sensor and the use of SERS enable us to visualize highly localized variation of pH induced by H+ extrusion, which is particularly upregulated in cancer cells.

Proton Sensor GPR68 Is Essential to Maintain Myeloid Malignancies

Despite the improvement of chemotherapy and targeted therapy, drug resistance still remains a challenge for long term disease free survival in aggressive leukemia patients. Recently, enhanced glycolysis is observed in acute myeloid leukemia (AML), and in association with poor clinical outcomes and chemoresistance. The byproducts of glycolysis include lactate and protons (H+), which contribute to intracellular acidosis. The extrusion of protons further results in extracellular acidosis. A group of G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A), have been demonstrated to respond to extracellular acidosis, resulting in activation of downstream signaling pathways that regulate pleotropic cellular processes. However, it remains unclear whether these proton-sensing GPCRs contribute to the etiology of AML.Here, we performed genomic examination of leukemia (via cBioPortal). Among 660 leukemia patients, only one patient exhibited deletion of GPR132. Other than this single case, we found no genetic mutations or cytogenetic abnormalities pertaining to proton-sensing GPCRs. Examination of transcripts of these proton-sensing GPCRs revealed that GPR68 was upregulated in both pediatric and adult AML. AML patients with higher levels of GPR68 were associated with shorter overall survival. To understand the function of GPR68 in AML, we knocked down GPR68 in AML cell lines with shRNA targeting GPR68 (shGPR68). GPR68 knockdown markedly induced apoptosis, and reduced colony formation and proliferation in AML cells. This result indicates that myeloid malignancies acquire a dependency on GPR68 function.In response to extracellular H+ or overexpression, GPR68 activates Ca2+ pathway. To determine the molecular mechanism by which GPR68 overexpression supports leukemia cell growth and survival, we examined the intracellular Ca2+ levels (i.e. [Ca2+]i) in primary AML samples. Compared with CD34+ normal hematopoietic cells, all primary AML specimens tested exhibited increased [Ca2+]i, consistent with GPR68 overexpression in AML cells. Meanwhile, shGPR68 reduced [Ca2+]i in all AML cell lines tested, indicating that overexpressed GPR68 activates the Ca2+ pathway in AML. Given that enhanced glycolysis leads to extracellular acidosis, we tested whether glycolysis-mediated local acidosis could also explain enhanced GPR68 activation in AML. Indeed, inhibition of glycolysis by 2-deoxyglucose (2-DG) reduced [Ca2+]i in most of the AML cell lines tested, indicating that glycolysis is likely responsible for enhanced GPR68 activation in AML as well. Next, we attempted to identify the Ca2+-dependent molecular mechanism that mediates the prosurvival effects due to GPR68 activation. We screened a series of pharmacological inhibitors for their efficacy in reducing cell growth and inducing apoptosis. Among the inhibitors tested, only a calcineurin (CaN) inhibitor, Cyclosporine, dramatically reduced cell growth and induced apoptosis in AML cells. This finding raises the possibility that GPR68 promotes AML cell survival through activating the Gq/11/Ca2+/CaN pathway.In summary, we find that the myeloid malignancies acquire a dependency on GPR68 signaling pathway, and inhibition of GPR68 might provide a novel therapeutic strategy for AML, especially in those developing chemoresistance. DisclosuresNo relevant conflicts of interest to declare.

Lactobacillus vini: mechanistic response to stress by medium acidification.

This work describes the response of Lactobacillusvini, a bacterium found as a contaminant in winemaking and fuel ethanol fermentation processes, to acid stress caused by inorganic or weak organic acids. First, we observed for the first time that bacterial cells become resistant to lysis by lysozyme when submitted to acidic stress. Then, the predicted intracellular acidification can be reversed by the presence of arginine, histidine and glutamine. However, these molecules were not able to reverse the effect of resistance to lysis, indicating the independence of these mechanisms. In general, a reduction in the expression of the main genes involved in the synthesis and deposition of material in the cell wall was observed, whereas the genes involved in the reabsorption of this structure showed increased expression. These data suggested that L. vini responds to the acidification of the medium through early entry into the stationary phase, firing two signals for cell wall remodelling and maintenance of intracellular pHin a coordinated way, most probably by alkalization and the proton extrusion process. If this picture is conserved among lactobacilli, it may not only have an impact on research associated with fermentation processes, but also on that associated with probiotic improvement.

Monovalent cation transporters at the plasma membrane in yeasts.

Maintenance of proper intracellular concentrations of monovalent cations, mainly sodium and potassium, is a requirement for survival of any cell. In the budding yeast Saccharomyces cerevisiae, monovalent cation homeostasis is determined by the active extrusion of protons through the Pma1 H+ -ATPase (reviewed in another chapter of this issue), the influx and efflux of these cations through the plasma membrane transporters (reviewed in this chapter), and the sequestration of toxic cations into the vacuoles. Here, we will describe the structure, function, and regulation of the plasma membrane transporters Trk1, Trk2, Tok1, Nha1, and Ena1, which play a key role in maintaining physiological intracellular concentrations of Na+ , K+ , and H+ , both under normal growth conditions and in response to stress.

Overcoming transporter-mediated multidrug resistance in cancer: failures and achievements of the last decades.

Multidrug resistance (MDR) is a complex phenomenon caused by numerous reasons in cancer chemotherapy. It is related to the abnormal tumor metabolism, precisely increased glycolysis and lactic acid production, extracellular acidification, and drug efflux caused by transport proteins. There are few strategies to increase drug delivery into cancer cells. One of them is the inhibition of carbonic anhydrases or certain proton transporters that increase extracellular acidity by proton extrusion from the cells. This prevents weakly basic chemotherapeutic drugs from ionization and increases their penetration through the cancer cell membrane. Another approach is the inhibition of MDR proteins that pump the anticancer agents into the extracellular milieu and decrease their intracellular concentration. Physical methods, such as ultrasound-mediated sonoporation, are being developed, as well. To increase the efficacy of sonoporation, various microbubbles are used. Ultrasound causes microbubble cavitation, i.e., periodical pulsation of the microbubble, and destruction which results in formation of temporary pores in the cellular membrane and increased permeabilization to drug molecules. This review summarizes the main approaches to reverse MDR related to the drug penetration along with its applications in preclinical and clinical studies.

The effect of ocean acidification on tropical coral calcification: Insights from calcification fluid DIC chemistry

Ocean acidification typically reduces calcification in tropical marine corals but the mechanism for this process is not understood. We use skeletal boron geochemistry (B/Ca and δ11B) to reconstruct the calcification fluid DIC of corals cultured over both high and low seawater pCO2 (180, 400 and 750 μatm). We observe strong positive correlations between calcification fluid pH and concentrations of the DIC species potentially implicated in aragonite precipitation (be they CO32−, HCO3− or HCO3− + CO32−). Similarly, with the exception of one outlier, the fluid concentrations of precipitating DIC species are strongly positively correlated with coral calcification rate. Corals cultured at high seawater pCO2 usually have low calcification fluid pH and low concentrations of precipitating DIC, suggesting that a reduction in DIC substrate at the calcification site is responsible for decreased calcification. The outlier coral maintained high pHCF and DICCF at high seawater pCO2 but exhibited a reduced calcification rate indicating that the coral has a limited energy budget to support proton extrusion from the calcification fluid and meet other calcification demands. We find no evidence that increasing seawater pCO2 enhances diffusion of CO2 into the calcification site. Instead the overlying [CO2] available to diffuse into the calcification site appears broadly comparable between seawater pCO2 treatments, implying that metabolic activity (respiration and photosynthesis) generates a similar [CO2] in the vicinity of the calcification site regardless of seawater pCO2.

BR-INSENSITIVE1 regulates hydrotropic response by interacting with plasma membrane H+-ATPases in Arabidopsis

ABSTRACTArabidopsis roots sense the moisture gradient in soils and grow toward an area with higher water potential – a process called hydrotropism. Our previous study has shown that the apoplastic proton extrusion in root tips is influenced by brassinosteroids (BRs) receptor BR-INSENSITIVE1 (BRI1) and is crucial for hydrotropic response in Arabidopsis. Here we show that BRI1 interacts directly not only with Arabidopsis plasma membrane H+-ATPase 2 (AHA2) but also with Arabidopsis plasma membrane H+-ATPase 7 (AHA7). Therefore, BRI1 may affect hydrotropic response via regulating the activities of AHA2 and AHA7.

Single and combined effects of Cd and Pb on the growth, medium pH, membrane potential and metal contents in maize (Zea mays L.) coleoptile segments

The mechanisms of the toxic effects of Cd and Pb on plant cell growth are still poorly understood. In particular, little is known about their interactive effects, which usually occur in the environment. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were performed with maize (Zea mays) coleoptile segments, which is a classical model system for studies of plant cell elongation growth. Cadmium and lead, which were added at 0.1 mM, reduced the endogenous and IAA-induced elongation growth of maize coleoptile cells. When both metals were added together or in sequence, their effect on IAA-induced growth was more toxic. The medium pH changes, which were measured simultaneously with growth, indicated that while Pb stopped IAA-induced proton extrusion, Cd only partially diminished it. Although Cd was generally more accumulated than Pb in the maize coleoptile segments, when IAA was added together with Pb, it significantly increased the accumulation of the metal. The short-term electrophysiological experiments showed that the addition of Cd caused the depolarisation of the membrane potential (Em), whereas Pb caused membrane hyperpolarisation. In the long-term electrophysiological experiments, it was found that the Cd-induced Em changes are complex. In conclusion, these results suggest that the effects of Cd and Pb as well as their combination on the elongation growth of maize coleoptile cells and the accumulation of the metals result, among others, from different ionic mechanisms by which each metal change the membrane potential of the cells.

Dissecting the Acid Stress Response of Rhizobium tropici CIAT 899.

Rhizobium tropici CIAT899 is a nodule-forming α-proteobacterium displaying intrinsic resistance to several abiotic stress conditions such as low pH and high temperatures, which are common in tropical environments. It is a good competitor for Phaseolus vulgaris (common bean) nodule occupancy at low pH values, however little is known about the genetic and physiological basis of the tolerance to acidic conditions. To identify genes in R. tropici involved in pH stress response we combined two different approaches: (1) A Tn5 mutant library of R. tropici CIAT899 was screened and 26 acid-sensitive mutants were identified. For 17 of these mutants, the transposon insertion sites could be identified. (2) We also studied the transcriptomes of cells grown under different pH conditions using RNA-Seq. RNA was extracted from cells grown for several generations in minimal medium at 6.8 or 4.5 (adapted cells). In addition, we acid-shocked cells pre-grown at pH 6.8 for 45 min at pH 4.5. Of the 6,289 protein-coding genes annotated in the genome of R. tropici CIAT 899, 383 were differentially expressed under acidic conditions (pH 4.5) vs. control condition (pH 6.8). Three hundred and fifty one genes were induced and 32 genes were repressed; only 11 genes were induced upon acid shock. The acid stress response of R. tropici CIAT899 is versatile: we found genes encoding response regulators and membrane transporters, enzymes involved in amino acid and carbohydrate metabolism and proton extrusion, in addition to several hypothetical genes. Our findings enhance our understanding of the core genes that are important during the acid stress response in R. tropici.

Tomato plants ectopically expressing Arabidopsis GRF9 show enhanced resistance to phosphate deficiency and improved fruit production in the field

Agronomic performance of transgenic tomato overexpressing functional genes has rarely been investigated in the field. In an attempt to improve low-phosphate (P) stress tolerance of tomato (Solanum lycopersicum) plants and promote tomato fruit production in the field, an expression vector containing cDNA to an Arabidopsis 14-3-3 protein, General Regulatory Factor 9 (GRF9), driven by a cauliflower mosaic virus 35S promoter, was transferred into tomato plants. Transgenic expression of GRF9 was ascertained by quantitative real-time PCR analysis. The degree of low-P tolerance in transgenic plants was found to be significantly greater than that in wild-type plants, and reflected in improved root development and enhanced P content under hydroponic conditions. For transgenic tomato, roots had higher P uptake, as evidenced by tissue P content and relative expression of the genes LePT1 and LePT2 in both normal and low-P hydroponic solutions. GRF9 overexpressors had greatly enhanced proton extrusion from roots and heightened activity of the plasma-membrane H+-ATPase (PM H+-ATPase) in roots under low-P hydroponic conditions. Thus, in addition to enhanced root development, higher expression of genes coding for phosphate transporters and improved capacity for acidification in the rhizosphere emerged as key mechanisms underpinning improved P acquisition in transgenic tomato plants in soil. Subsequent field trials measuring tomato fruit production at two P levels, indicated that GRF9 can indeed improve total tomato production and may play a role in early fruit maturity. Our results suggest that the heterologous Arabidopsis GRF9 gene can confer resistance to P deficiency in transgenic tomato plants and promote fruit production.