AtSTP3, a green leaf-specific, low affinity monosaccharide-H+ symporter of Arabidopsis thaliana

This paper describes the expression analyses of the AtSTP3 gene of Arabidopsis thaliana, the functional characterization of the encoded protein as a new monosaccharide transporter, and introduces the AtSTP gene family. The kinetic properties of the AtSTP3 protein (for sugar transport protein 3) were studied in a hexose transport deficient mutant of Schizosaccharomyces pombe. AtSTP3 represents a new monosaccharide transporter that is composed of 514 amino acids and has a calculated molecular mass of 55·9 kDa. Kinetic analyses in yeast showed that AtSTP3 is a low affinity, energy‐dependent H ⁺ symporter with a K_m for D ‐glucose of 2 m M . RNase protection analyses revealed that AtSTP3 is expressed in leaves and floral tissue of Arabidopsis. This expression pattern of the AtSTP3 gene was confirmed in AtSTP3 promoter‐ β ‐glucuronidase (GUS) plants showing AtSTP3‐driven GUS activity in green leaves, such as cotelydons, rosette and stalk leaves and sepals. Wounding caused an induction of GUS activity in the transgenic plants and an increase of AtSTP3 mRNA levels in Arabidopsis wild‐type plants. Polymerase chain reaction analyses with degenerate primers identified additional new AtSTP genes and revealed that AtSTP3 is the member of a large family of at least 14 homologous genes coding for putative monosaccharide‐H ⁺ symporters (AtSTPs).     

Characterization of csi52, a Cs+ resistant mutant of Arabidopsis thaliana altered in K+ transport

Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Although details on the structure and function of these transporters are beginning to emerge many prominent questions remain concerning how these proteins function in planta. Such questions can be addressed through the use of well-defined transport mutants. Csi52, a caesium-insensitive mutant of Arabidopsis thaliana which is defective in potassium transport, is further characterized here using conventional electrophysiology, patch-clamp and radiometric approaches to identify the nature of the potassium transport lesion. Rb⁺ uptake experiments reveal a reduced uptake in csi52 in both the high- and low-affinity uptake range. Patch-clamp analysis indicates that the activity of the predominant inward rectifying channel observed in wild-type cells is extremely low in root protoplasts isolated from csi52, whereas outward rectifying channel activity is comparable between wild-type and mutant. Rb⁺ uptake studies show that in both wild-type and csi52 the high-affinity uptake pathway is considerably less sensitive to Cs⁺ than the low-affinity pathway with K_1/2 values for Cs⁺ of around 1.3 and 0.2 mM, respectively. Furthermore, K⁺ starvation leads to a larger relative increase in high-affinity K⁺ uptake in the mutant than the wild-type. The results demonstrate the Cs⁺ sensitivity of each individual uptake pathway is comparable in wild-type and csi52 but the high-affinity pathway is less Cs⁺ sensitive (in both wild-type and csi52). Therefore, the larger shift toward high-affinity uptake in the mutant compared with the wild-type under K⁺-starvation conditions will endow the mutant with a higher degree of overall Cs⁺ resistance. The data supply evidence for the hypothesis that the csi52 mutation lies within a gene that regulates the activity of several potassium transport systems and coordinates their relative contribution to overall root K⁺ uptake.     

Effects of amiodarone on K+, internal pH and Ca2+ homeostasis in Saccharomyces cerevisiae

In this study, amiodarone, at very low concentrations, produced a clear efflux of K⁺. Increasing concentrations also produced an influx of protons, resulting in an increase of the external pH and a decrease of the internal pH. The K⁺ efflux resulted in an increased plasma membrane potential difference, responsible for the entrance of Ca²⁺ and H⁺, the efflux of anions and the subsequent changes resulting from the increased cytoplasmic Ca²⁺ concentration, as well as the decreased internal pH. The Δ tok1 and Δ nha1 mutations resulted in a smaller effect of amiodarone, and Δ trk1 and Δ trk2 showed a higher increase of the plasma membrane potential. Higher concentrations of amiodarone also produced full inhibition of respiration, insensitive to uncouplers and a partial inhibition of fermentation. This phenomenon appears to be common to a large series of cationic molecules that can produce the efflux of K⁺, through the reduction of the negative surface charge of the cell membrane, and the concentration of this cation directly available to the monovalent cation carriers, and/or producing a disorganization of the membrane and altering the functioning of the carriers, probably not only in yeast.     

Comparative analysis of leaf-type ferredoxin-NADP+ oxidoreductase isoforms in Arabidopsis thaliana

Physiological roles of the two distinct chloroplast-targeted ferredoxin-NADP⁺ oxidoreductase (FNR) isoforms in Arabidopsis thaliana were studied using T-DNA insertion line fnr1 and RNAi line fnr2 . In fnr2 FNR1 was present both as a thylakoid membrane-bound form and as a soluble protein, whereas in fnr1 the FNR2 protein existed solely in soluble form in the stroma. The fnr2 plants resembled fnr1 in having downregulated photosynthetic properties, expressed as low chlorophyll content, low accumulation of photosynthetic thylakoid proteins and reduced carbon fixation rate when compared with wild type (WT). Under standard growth conditions the level of F₀'rise' and the amplitude of the thermoluminescence afterglow (AG) band, shown to correlate with cyclic electron transfer (CET), were reduced in both fnr mutants. In contrast, when plants were grown under low temperatures, both fnr mutants showed an enhanced rate of CET when compared with the WT. These data exclude the possibility that distinct FNR isoforms feed electrons to specific CET pathways. Nevertheless, the fnr2 mutants had a distinct phenotype upon growth at low temperature. The fnr2 plants grown at low temperature were more tolerant against methyl viologen (MV)-induced cell death than fnr1 and WT. The unique tolerance of fnr2 plants grown at low temperature to oxidative stress correlated with an increased level of reduced ascorbate and reactive oxygen species (ROS) scavenging enzymes, as well as with a scarcity in the accumulation of thylakoid membrane protein complexes, as compared with fnr1 and WT. These results emphasize a critical role for FNR2 in the redistribution of electrons to various reducing pathways, upon conditions that modify the photosynthetic capacity of the plant.     

Cd2+ uptake, Cd2+ binding and loss of cell K+ by a Cd-sensitive and a Cd-resistant strain of Saccharomyces cerevisiae

Abstract Uptake of Cd²⁺ into Cd-resistant cells was approximately four times lower than in Cd-sensitive cells of Saccharomyces cerevisiae . Binding of Cd²⁺ to the yeast cells increased during incubation of the cells in the presence of Cd²⁺. The increase in the binding was much higher for wild-type cells than for Cd-resistant cells. This increased binding is ascribed to permeabilization of part of the cells. There is no single relation between the relative rate of K⁺ efflux and the cellular Cd content as has been found previously for wild-type cells. The rates of K⁺ efflux were much less than those found for the wild-type cells. Only with short incubation periods of the cells with Cd²⁺ was the same dependence found between the efflux of K⁺ and the cellular Cd content for both types of cell. The discrepancies found after extended incubation of the cells with Cd²⁺ are ascribed to the fact that Cd-provoked K⁺ release proceeds via an all-or-nothing process and that K⁺ released from permeabilized cells can be reaccumulated in still intact cells. The latter proceeds more efficiently in Cd-resistant cells than in wild-type cells.     

The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+ antiporter activities of oat and tobacco tonoplast vesicles

Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca²⁺/H⁺ and Cd²⁺/H⁺ antiporter activities of tonoplast vesicles from non‐transformed, wild‐type plants. Solubilization and reconstitution of oat‐seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6‐fold loss of protein, about a 6‐fold enhancement of Cd²⁺/H⁺ antiport specific activity (at 10 µM Cd²⁺), and almost complete loss of Ca²⁺/H⁺ antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd²⁺ concentration‐dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca²⁺ concentration‐dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and non‐linear. Cd²⁺ pre‐treatment of oat vesicles reduced verapamil inhibition of Cd²⁺/H⁺ activity and verapamil binding to vesicles, while Ca²⁺ pre‐treatment was much less protective of Ca^2+/H⁺ activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd²⁺/H⁺ and Ca²⁺/H⁺. Our observation that solubilization and reconstitution have similar effects on both Cd²⁺/H⁺ and Ca²⁺/H⁺ activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.     

Early changes of Cl− efflux and H+ extrusion induced by osmotic stress in Arabidopsis thaliana cells

In various plant materials changes in turgor pressure, following hyper- or hypo-osmotic stress, were associated with the activation or inactivation of the plasma membrane H⁺-ATPase, respectively. To see if the turgor changes might indirectly influence H⁺-ATPase activity by regulating ion fluxes through plasma membrane, we investigated, in cultured cells of Arabidopsis thaliana (L.) Heynh., the early effects of hyper- and hypo-osmotic stress on Cl^? fluxes in comparison, in the case of hyper-osmotic treatment, with its effect on net H⁺ extrusion. The results obtained showed that hyper-osmotic stress (200 mM mannitol) quickly reduced Cl^? efflux (?70%) from cells preloaded with ³⁶C1^? for 18 h. This inhibiting effect was independent of the simultaneous mannitol-induced stimulation of Cl^? influx and rapidly reversible after removal of the hyper-osmotic treatment. The inhibition of Cl^? efflux was associated with a stimulation of net H⁺ extrusion, and these two effects showed the same dependence on the external mannitol concentration. Fusicoccin (FC, 20 µM), which stimulated H⁺ extrusion to about the same extent as 200 mM mannitol, did not affect Cl^? efflux. When cells preloaded with ³⁶C1^? for 18 h in the presence of mannitol (from 25 up to 200 mM) were eluted in a mannitol-free medium an early and strong increase in Cl^? efflux was found. The increase of Cl- efflux was already detectable for a small hypo-osmotic jump (25 mM), and was reduced (?50%) by the anion channel inhibitor A9C (300 µM). These results lead to exclude a direct causal relationship mediated by E_m changes between the effects of osmoticum on Cl^? efflux and net H⁺ extrusion, and favour the view that the changes in turgor pressure induced by hyper/hypo-osmotic stress may respectively induce an early inactivation/activation of stretch-sensitive anion channels.     

Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H⁺-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae , even after deletion of the enzyme's carboxy terminus. Functional chimerical H⁺-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H⁺-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H⁺-ATPase. The reverse Ala-598 →Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H⁺-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K _m values for MgATP and decreased V _max compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H⁺-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.     

Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress

Na⁺ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PP_i-dependent H⁺ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na⁺/H⁺ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na⁺ was mainly accumulated in the roots. Under a more severe salt treatment, Na⁺ was equally distributed in shoots and roots. In contrast to what was observed with Na⁺, all the salt treatments reduced the shoot K⁺ content. Dissipation by Na⁺ of the H⁺ gradient generated by the tonoplast H⁺-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na⁺/H⁺ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na⁺/H⁺ exchange activity, which displayed saturation kinetics for Na⁺ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na⁺/H⁺ antiports. No Na⁺/H⁺ exchange was detected in vesicles from control roots. The activity was specific for Na⁺. since K⁺ added to the assay medium slightly dissipated H⁺ gradients and displayed non-saturating kinetics for all salt treatments. Apparent K_m for Na⁺/H⁺ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, V_max remaining unchanged. The results suggest that the existence of a specific Na⁺/H⁺ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity.     

Critical role of divalent cations and Na+ efflux in Arabidopsis thaliana salt tolerance

Detrimental effects of salinity on plants are known to be partially alleviated by external Ca²⁺. Previous work demonstrated that the Arabidopsis SOS3 locus encodes a Ca²⁺‐binding protein with similarities to CnB, the regulatory subunit of protein phosphatase 2B (calcineurin). In this study, we further characterized the role of SOS3 in salt tolerance. We found that reduced root elongation of sos3 mutants in the presence of high concentrations of either NaCl or LiCl is specifically rescued by Ca²⁺ and not Mg²⁺, whereas root growth is rescued by both Ca²⁺ and Mg²⁺ in the presence of high concentrations of KCl. Phenocopies of sos3 mutants were obtained in wild‐type plants by the application of calmodulin and calcineurin inhibitors. These data provide further evidence that SOS3 is a calcineurin‐like protein and that calmodulin plays an important role in the signalling pathways involved in plant salt tolerance. The origin of the elevated Na : K ratio in sos3 mutants was investigated by comparing Na⁺ efflux and influx in both mutant and wild type. No difference in Na⁺ influx was recorded between wild type and sos3; however, sos3 plants showed a markedly lower Na⁺ efflux, a property that would contribute to the salt‐oversensitive phenotype of sos3 plants.     

Li+-regulated 1-aminocyclopropane-1-carboxylate synthase gene expression in Arabidopsis thaliana

In Arabidopsis thaliana, 1-aminocyclopropane-1-carboxylate synthase (ACS) is encoded by a multigene family consisting of at least five members whose expression is induced by hormones, developmental signals, and protein synthesis inhibition. Li⁺, known to interfere with the phosphoinositide (PI) second messenger system by inhibiting the activity of inositol-phosphate phosphatases, is one of the strongest inducers of ACC synthase activity in plants. Treatment of etiolated Arabidopsis seedlings with LiCl results in a rapid induction of the ACS5 gene. Also, LiCl represses the cycloheximide (CHX)-induced accumulation of the ACS2 mRNA. The effects of Li⁺ on the expression of ACS5 and ACS2 are specific, dose-dependent, and can be reversed by Ca²⁺ and mimicked by the protein kinase inhibitor K-252a. The results suggest that the regulation of some ACS genes by various inducers may involve protein kinase activity, which in turn may be controlled through an inositol 1,4,5-triphosphate (IP₃)-mediated Ca²⁺ mobilization. Since plants contain no Li⁺, the cation appears to unmask pre-existing biochemical capacity that may be utilized by various unknown transducers during plant growth and development.     

Na+/H+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the salt-sensitive Plantago media

Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na⁺ ions. A salt-sensitive ( Plantago media L.) and a salt-tolerant ( P. maritima L.) species were compared with respect to Na⁺/H⁺ antiport activities at the tonoplast. After exposure of the plants to 50 m M NaCl for 6 days isolated tonoplast vesicles of P. maritima showed Na⁺/H⁺ antiport activity with saturation kinetics and a K_m of 2.4 m M Na⁺, NaCl-grown P. media and the control plants of both species showed no antiport activity. Selectivity of the antiport system for Na⁺ was high and was determined by adding different chloride salts after formation of a Δ pH in the vesicles. Specific tonoplast ATPase activities were similar in the two species and did not alter after exposure to NaCl stress.     

Cellular localization of the Ca2+ binding TCH3 protein of Arabidopsis

TCH3 is an Arabidopsis t ou ch ( TCH ) gene isolated as a result of its strong and rapid upregulation in response to mechanical stimuli, such as touch and wind. TCH3 encodes an unusual calcium ion-binding protein that is closely related to calmodulin but has the potential to bind six calcium ions. Here it is shown that TCH3 shows a restricted pattern of accumulation during Arabidopsis vegetative development. These data provide insight into the endogenous signals that may regulate TCH3 expression and the sites of TCH3 action. TCH3 is abundant in the shoot apical meristem, vascular tissue, the root columella and pericycle cells that give rise to lateral roots. In addition, TCH3 accumulation in cells of developing shoots and roots closely correlates with the process of cellular expansion. Following wind stimulation, TCH3 becomes more abundant in specific regions including the branchpoints of leaf primordia and stipules, pith parenchyma, and the vascular tissue. The consequences of TCH3 upregulation by wind are therefore spatially restricted and TCH3 may function at these sites to modify cell or tissue characteristics following mechanical stimulation. Because TCH3 accumulates specifically in cells and tissues that are thought to be under the influence of auxin, auxin levels may regulate TCH3 expression during development. TCH3 is upregulated in response to low levels of exogenous indole-3-acetic acid (IAA), but not by inactive auxin-related compounds. These results suggest that TCH3 protein may play roles in mediating physiological responses to auxin and mechanical environmental stimuli.     

Sodium transport measured in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits

Right-side-out plasma membrane vesicles were isolated from wheat roots using an aqueous polymer two-phase system. The purity and orientation of the vesicles were confirmed by marker enzyme analysis. Membrane potential (Ψ)-dependent ²²Na⁺ influx and sodium/proton (Na⁺/ H⁺) antiport-mediated efflux across the plasma membrane were studied using these vesicles. Membrane potentials were imposed on the vesicles using either K⁺ gradients in the presence of valinomycin or H⁺ gradients. The ΔΨ was quantified by the uptake of the lipophilic cation tetraphenylphosphonium. Uptake of Na⁺ into the vesicles was stimulated by a negative ΔΨ and had a K_m for extrav-esicular Na⁺ of 34.8 ± 5.9 mol m³. The ΔΨ-dependent uptake of Na⁺ was similar in vesicles from roots of hexaploid (cv. Troy) and tetraploid (cv. Langdon) wheat differing in a K⁺/Na⁺ discrimination trait, and was also unaffected by growth in 50 mol m^?3 NaCl. Inhibition of ΔΨ-dependent Na⁺ uptake by Ca²⁺ was greater in the hexaploid than in the tetraploid. Sodium/proton antiport was measured as Na⁺-dependent, amiloride-inhibited pH gradient formation in the vesicles. Acidification of the vesicle interior was measured by the uptake of ¹⁴C-methylamine. The Na⁺/H⁺ antiport had a K_m, for intravesicular Na⁺ of between 13 and 19 mol m^?3. In the hexaploid, Na⁺/H⁺ antiport activity was greater when roots were grown in the presence of 50 mol m^?3NaCl, and was also greater than the activity in salt-grown tetraploid wheat roots. Antiport activity was not increased in a Langdon 4D chromosome substitution line which carries a trait for K⁺/Na⁺ discrimination. It is concluded that neither of the transport processes measured is responsible for the Na⁺/K⁺ discrimination trait located on the 4D chromosome of wheat.     

Actual escape area and lateral escape from the xylem of the alkali ions Na+, K+, Rb+ and Cs+ in tomato

Approximation of the total escape area of the xylem in an inbred line of tomato (Ly-copersicon escutentum Mill. cv. Tiny Tim) with help of the frequency distribution of xylem vessel radii provides the possibility to calculate realistic escape constant values from uptake experiments of several elements into tomato stem segments. Comparison of the lateral escape rates of ²⁴Na⁺, ⁴²K⁺, ⁸⁶Rb⁺ and ¹³⁴Cs⁺ indicate that Na⁺ escape is rate-limited by its uptake into a rather constant number of surrounding cells, regardless of changes in the total escape area of the xylem vessels. The escape of K⁺, Rb⁺ and Cs⁺ seems to be proportional to the surface area of the xylem vessels and their escape is apparently controlled by their transport across the cell walls of the transport channels. The calculated small values for the escape rate constants (apparent permeability of the xylem cell walls, ca 2–3 · 10−⁹ m s−⁷) are probably due to the presence of lignin in the xylem cell walls, the discrimination between ions as a result of differing affinities and selectivities and the presence of other solutes in the applied solution.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Na+/H+ antiporter activity of the SOS1 gene: lifetime imaging analysis and electrophysiological studies on Arabidopsis seedlings

Based on sequence analysis, the salt overly sensitive (SOS1) gene has been suggested to function as a Na⁺/H⁺ antiporter located at the plasma membrane of plant cells, being expressed mostly in the meristem zone of the root and in the parenchyma cells surrounding the vascular tissue of the stem. In this study, we compared net H⁺ and Ca²⁺ fluxes and intracellular pH and [Ca²⁺]_cyt in the root meristem zone of Arabidopsis wild‐type (WT) and sos mutants before and after salt stress. In addition, we studied the effect of pretreatment with amiloride (an inhibitor of Na⁺/H⁺ antiporters) on net ion fluxes, intracellular pH and intracellular Ca²⁺ activity ([Ca²⁺]_cyt) in WT plants and sos1 mutants before and after salt stress. Net ion fluxes were measured using microelectrode ion flux estimation (MIFE) and intracellular pH and [Ca²⁺]_cyt using fluorescence lifetime imaging microscopy (FLIM) techniques. During the first 15 min after NaCl application, sos1 mutants showed net H⁺ efflux and intracellular alkalinization in the meristem zone, whereas sos2 and sos3 mutants and WT showed net H⁺ influx and slight intracellular acidification in the meristem zone. Treatment with amiloride led to intracellular acidification and lower net H⁺ flux in WT plants and to a decrease in intracellular Ca²⁺ in WT and sos1 plants. WT plants pretreated with amiloride did not show positive net H⁺ flux and intracellular acidification. After NaCl application, internal pH shifted to higher values in WT and sos1 plants. However, absolute values of H⁺ fluxes were higher and internal pH values were lower in WT plants pretreated with amiloride compared with sos1 mutants. Therefore, the SOS1 transporter is involved in H⁺ influx into the meristem zone of Arabidopsis roots, or it may function as a Na⁺/H⁺ antiporter. Amiloride affects SOS1 and other Na⁺/H⁺ antiporters in plant cells because of its ability to decrease the H⁺ gradient across the plasma membrane.     

Calmodulin/Ca2+-ATPase interaction at the Arabidopsis thaliana plasma membrane is dependent on calmodulin isoform showing isoform-specific Ca2+ dependencies

Arabidopsis thaliana plasma membrane (PM) Ca²⁺-ATPase is a type IIB P-type ATPase, which binds calmodulin (CaM) to an autoinhibitory N-terminal domain. Here, we took advantage of the fact that PM isolated from cultured cells mainly contains At -ACA8, the first cloned A. thaliana PM Ca²⁺-ATPase, to analyse its interaction with CaM in detail. Analysis of the ability of different peptides designed from At -ACA8 N-terminus to compete with the native protein for binding of bovine brain CaM (bbCaM) showed that peptide ⁴¹I-T⁶³ had the same affinity of the native protein [apparent dissociation constant (KD) at 10 µ M free Ca²⁺ about 25 n M ], thus localizing At -ACA8 CaM-binding site within this sequence. The interaction of At -ACA8 N-terminus with bbCaM, as determined by surface plasmon resonance, was rapid, and slowly but was fully reversible. Analysis of Ca²⁺-ATPase activation as a function of the concentration of different isoforms of A. thaliana CaM showed that Ca²⁺-ATPase is activated to similar extent by bbCaM and by different isoforms of homologous CaM. However, the affinity for the divergent A. thaliana isoform CaM8 was lower than that for canonical CaM isoforms such as A. thaliana CaM2, CaM4 and CaM6 or bbCaM. The apparent KD for CaM isoforms of the native enzyme increased with the decrease of free Ca²⁺ concentration, suggesting that enzyme conformation is affected by Ca²⁺. Binding of CaM isoforms to At -ACA8 N-terminus was affected differently by free Ca²⁺ concentration, suggesting that plant CaMs may have different affinities for Ca²⁺.