Calcium transport across plant membranes: mechanisms and functions.

Contents Summary 49 I. Introduction 49 II. Physiological and structural characteristics of plant Ca2+ -permeable ion channels 50 III. Ca2+ extrusion systems 61 IV. Concluding remarks 64 Acknowledgements 64 References 64 SUMMARY: Calcium is an essential structural, metabolic and signalling element. The physiological functions of Ca2+ are enabled by its orchestrated transport across cell membranes, mediated by Ca2+ -permeable ion channels, Ca2+ -ATPases and Ca2+ /H+ exchangers. Bioinformatics analysis has not determined any Ca2+ -selective filters in plant ion channels, but electrophysiological tests do reveal Ca2+ conductances in plant membranes. The biophysical characteristics of plant Ca2+ conductances have been studied in detail and were recently complemented by molecular genetic approaches. Plant Ca2+ conductances are mediated by several families of ion channels, including cyclic nucleotide-gated channels (CNGCs), ionotropic glutamate receptors, two-pore channel 1 (TPC1), annexins and several types of mechanosensitive channels. Key Ca2+ -mediated reactions (e.g. sensing of temperature, gravity, touch and hormones, and cell elongation and guard cell closure) have now been associated with the activities of specific subunits from these families. Structural studies have demonstrated a unique selectivity filter in TPC1, which is passable for hydrated divalent cations. The hypothesis of a ROS-Ca2+ hub is discussed, linking Ca2+ transport to ROS generation. CNGC inactivation by cytosolic Ca2+ , leading to the termination of Ca2+ signals, is now mechanistically explained. The structure-function relationships of Ca2+ -ATPases and Ca2+ /H+ exchangers, and their regulation and physiological roles are analysed.

Mechanisms of thaxtomin A-induced root toxicity revealed by a thaxtomin A sensitive Arabidopsis mutant (ucu2-2/gi-2).

KEY MESSAGE: The Arabidopsis mutant ( ucu2 - 2/gi - 2 ) is thaxtomin A, isoxaben and NPA-sensitive indicated by root growth and ion flux responses providing new insights into these compounds mode of action and interactions. Thaxtomin A (TA) is a cellulose biosynthetic inhibitor (CBI) that promotes plant cell hypertrophy and cell death. Electrophysiological analysis of steady-state K(+) and Ca(2+) fluxes in Arabidopsis thaliana roots pretreated with TA for 24 h indicated a disturbance in the regulation of ion movement across the plant cell membrane. The observed inability to control solute movement, recorded in rapidly growing meristematic and elongation root zones, may partly explain typical root toxicity responses to TA treatment. Of note, the TA-sensitive mutant (ucu2-2/gi-2) was more susceptible with K(+) and Ca(2+) fluxes altered between 1.3 and eightfold compared to the wild-type control where fluxes altered between 1.2 and threefold. Root growth inhibition assays showed that the ucu2-2/gi-2 mutant had an increased sensitivity to the auxin 2,4-D, but not IAA or NAA; it also had increased sensitivity to the auxin efflux transport inhibitor, 1-naphthylphthalamic acid (NPA), but not 2,3,5- Triiodobenzoic acid (TIBA), when compared to the WT. The NPA sensitivity data were supported by electrophysiological analysis of H(+) fluxes in the mature (but not elongation) root zone. Increased sensitivity to the CBI, isoxaben (IXB), but not dichlobenil was recorded. Increased sensitivity to both TA and IXB corresponded with higher levels of accumulation of these toxins in the root tissue, compared to the WT. Further root growth inhibition assays showed no altered sensitivity of ucu2-2/gi-2 to two other plant pathogen toxins, alternariol and fusaric acid. Identification of a TA-sensitive Arabidopsis mutant provides further insight into how this CBI toxin interacts with plant cells.

Nax loci affect SOS1-like Na+/H+ exchanger expression and activity in wheat.

Salinity stress tolerance in durum wheat is strongly associated with a plant's ability to control Na(+) delivery to the shoot. Two loci, termed Nax1 and Nax2, were recently identified as being critical for this process and the sodium transporters HKT1;4 and HKT1;5 were identified as the respective candidate genes. These transporters retrieve Na(+) from the xylem, thus limiting the rates of Na(+) transport from the root to the shoot. In this work, we show that the Nax loci also affect activity and expression levels of the SOS1-like Na(+)/H(+) exchanger in both root cortical and stelar tissues. Net Na(+) efflux measured in isolated steles from salt-treated plants, using the non-invasive ion flux measuring MIFE technique, decreased in the sequence: Tamaroi (parental line)>Nax1=Nax2>Nax1:Nax2 lines. This efflux was sensitive to amiloride (a known inhibitor of the Na(+)/H(+) exchanger) and was mirrored by net H(+) flux changes. TdSOS1 relative transcript levels were 6-10-fold lower in Nax lines compared with Tamaroi. Thus, it appears that Nax loci confer two highly complementary mechanisms, both of which contribute towards reducing the xylem Na(+) content. One enhances the retrieval of Na(+) back into the root stele via HKT1;4 or HKT1;5, whilst the other reduces the rate of Na(+) loading into the xylem via SOS1. It is suggested that such duality plays an important adaptive role with greater versatility for responding to a changing environment and controlling Na(+) delivery to the shoot.

Receptor kinase-mediated control of primary active proton pumping at the plasma membrane.

Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2 and PSY1R observed might provide a general paradigm for regulation of plasma membrane proton transport by receptor kinases.

Enhanced resistance to the cellulose biosynthetic inhibitors, thaxtomin A and isoxaben in Arabidopsis thaliana mutants, also provides specific co-resistance to the auxin transport inhibitor, 1-NPA.

BACKGROUND: Thaxtomin A (TA) is a phytotoxin produced by plant pathogenic Streptomyces spp. responsible for potato common scab. TA inhibits cellulose biosynthesis in expanding plant tissues and is essential for disease induction. Auxin treatment of various plant tissues has been repeatedly demonstrated to inhibit TA toxicity and to reduce common scab. This work utilises Arabidopsis thaliana mutants with resistance to cellulose biosynthesis inhibitors (CBIs) to investigate the interaction between TA, other CBIs and auxins. RESULTS: Three CBI resistant A. thaliana mutants; txr1-1 (tolerance to TA), ixr1-1 (tolerance to isoxaben - IXB) and KOR1 (cellulose deficiency), showed no altered root growth response to treatment with natural or synthetic auxins, nor with the auxin efflux transport inhibitor 2,3,5-Triiodobenzoic acid (TIBA). However, all mutants had significantly enhanced tolerance to 1-napthylphthalamic acid (NPA), another auxin efflux transport inhibitor, which blocks polar auxin transport at a site distinct from TIBA. NPA tolerance of txr1-1 and ixr1-1 was further supported by electrophysiological analysis of net H+ fluxes in the mature, but not elongation zone of roots. All three mutants showed increased tolerance to IXB, but only txr1-1 showed tolerance to TA. No mutant showed enhanced tolerance to a third CBI, dichlobenil (DCB). CONCLUSIONS: We have demonstrated that plant tolerance to TA and IXB, as well as cell wall synthesis modifications in roots, have resulted in specific co-resistance to NPA but not TIBA. This suggests that CBI resistance has an impact on polar auxin efflux transport processes associated with the NPA binding protein. We also show that NPA inhibitory response in roots occurs in the mature root zone but not the elongation zone. Responses of mutants to CBIs indicate a similar, but not identical mode of action of TA and IXB, in contrast to DCB.

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells.

Plant cell growth and stress signaling require Ca²âº influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca²âº-permeable conductance that permits Ca²âº influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²âº-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca²âº- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²âº in response to OH•. An OH•-activated Ca²âº conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²âº-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²âº in plants.

Endomembrane Ca2+-ATPases play a significant role in virus-induced adaptation to oxidative stress.

Although the role of Ca2+ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the role of active Ca2+ efflux systems in this process. In our recent paper, we reported Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana and showed the critical role of plasma membrane Ca2+/H+ exchangers in this process. The current study continues this research. Using biochemical and electrophysiological approaches, we reveal that both endomembrane P2A and P2B Ca2+-ATPases play significant roles in adaptive responses to oxidative stress by removing excessive Ca2+ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca2+ efflux systems in acquired tolerance to oxidative stress and open up prospects for practical applications in agriculture, after in-depth comprehension of the fundamental mechanisms involved in common responses to environmental factors at the genomic, cellular and organismal levels.

Assessing the role of root plasma membrane and tonoplast Na+/H+ exchangers in salinity tolerance in wheat: in planta quantification methods.

This work investigates the role of cytosolic Na+ exclusion in roots as a means of salinity tolerance in wheat, and offers in planta methods for the functional assessment of major transporters contributing to this trait. An electrophysiological protocol was developed to quantify the activity of plasma membrane Na+ efflux systems in roots, using the microelectrode ion flux estimation (MIFE) technique. We show that active efflux of Na+ from wheat root epidermal cells is mediated by a SOS1-like homolog, energized by the plasma membrane H+-ATPase. SOS1-like efflux activity was highest in Kharchia 65, a salt-tolerant bread wheat cultivar. Kharchia 65 also had an enhanced ability to sequester large quantities of Na+ into the vacuoles of root cells, as revealed by confocal microscopy using Sodium Green. These findings were consistent with the highest level of expression of both SOS1 and NHX1 transcripts in plant roots in this variety. In the sensitive wheat varieties, a greater proportion of Na+ was located in the root cell cytosol. Overall, our findings suggest a critical role of cytosolic Na+ exclusion for salinity tolerance in wheat and offer convenient protocols to quantify the contribution of the major transporters conferring this trait, to screen plants for salinity tolerance.

Plasma membrane Ca²+ transporters mediate virus-induced acquired resistance to oxidative stress.

This paper reports the phenomenon of acquired cross-tolerance to oxidative stress in plants and investigates the activity of specific Ca²+ transport systems mediating this phenomenon. Nicotiana benthamiana plants were infected with Potato virus X (PVX) and exposed to oxidative [either ultraviolet (UV-C) or H2O2] stress. Plant adaptive responses were assessed by the combined application of a range of electrophysiological (non-invasive microelectrode ion flux measurements), biochemical (Ca²+- and H+-ATPase activity), imaging (fluorescence lifetime imaging measurements of changes in intracellular Ca²+ concentrations), pharmacological and cytological transmission electrone microscopy techniques. Virus-infected plants had a better ability to control UV-induced elevations in cytosolic-free Ca²+ and prevent structural and functional damage of chloroplasts. Taken together, our results suggest a high degree of crosstalk between UV and pathogen-induced oxidative stresses, and highlight the crucial role of Ca²+ efflux systems in acquired resistance to oxidative stress in plants.

Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death.

Reactive oxygen species (ROS) are central to plant stress response, signalling, development and a multitude of other processes. In this study, the plasma-membrane hydroxyl radical (HR)-activated K(+) channel responsible for K(+) efflux from root cells during stress accompanied by ROS generation is characterised. The channel showed 16-pS unitary conductance and was sensitive to Ca(2+), tetraethylammonium, Ba(2+), Cs(+) and free-radical scavengers. The channel was not found in the gork1-1 mutant, which lacks a major plasma-membrane outwardly rectifying K(+) channel. In intact Arabidopsis roots, both HRs and stress induced a dramatic K(+) efflux that was much smaller in gork1-1 plants. Tests with electron paramagnetic resonance spectroscopy showed that NaCl can stimulate HR generation in roots and this might lead to K(+)-channel activation. In animals, activation of K(+)-efflux channels by HRs can trigger programmed cell death (PCD). PCD symptoms in Arabidopsis roots developed much more slowly in gork1-1 and wild-type plants treated with K(+)-channel blockers or HR scavengers. Therefore, similar to animal counterparts, plant HR-activated K(+) channels are also involved in PCD. Overall, this study provides new insight into the regulation of plant cation transport by ROS and demonstrates possible physiological properties of plant HR-activated K(+) channels.

Xylem ionic relations and salinity tolerance in barley.

Control of ion loading into the xylem has been repeatedly named as a crucial factor determining plant salt tolerance. In this study we further investigate this issue by applying a range of biophysical [the microelectrode ion flux measurement (MIFE) technique for non-invasive ion flux measurements, the patch clamp technique, membrane potential measurements] and physiological (xylem sap and tissue nutrient analysis, photosynthetic characteristics, stomatal conductance) techniques to barley varieties contrasting in their salt tolerance. We report that restricting Na(+) loading into the xylem is not essential for conferring salinity tolerance in barley, with tolerant varieties showing xylem Na(+) concentrations at least as high as those of sensitive ones. At the same time, tolerant genotypes are capable of maintaining higher xylem K(+)/Na(+) ratios and efficiently sequester the accumulated Na(+) in leaves. The former is achieved by more efficient loading of K(+) into the xylem. We argue that the observed increases in xylem K(+) and Na(+) concentrations in tolerant genotypes are required for efficient osmotic adjustment, needed to support leaf expansion growth. We also provide evidence that K(+)-permeable voltage-sensitive channels are involved in xylem loading and operate in a feedback manner to maintain a constant K(+)/Na(+) ratio in the xylem sap.

Electrical signalling and cytokinins mediate effects of light and root cutting on ion uptake in intact plants.

Nutrient acquisition in the mature root zone is under systemic control by the shoot and the root tip. In maize, exposure of the shoot to light induces short-term (within 1-2 min) effects on net K+ and H+ transport at the root surface. H+ efflux decreased (from -18 to -12 nmol m(-2) s(-1)) and K+ uptake (approximately 2 nmol m(-2) s(-1)) reverted to efflux (approximately -3 nmol m(-2) s(-1)). Xylem probing revealed that the trans-root (electrical) potential drop between xylem vessels and an external electrode responded within seconds to a stepwise increase in light intensity; xylem pressure started to decrease after a approximately 3 min delay, favouring electrical as opposed to hydraulic signalling. Cutting of maize and barley roots at the base reduced H+ efflux and stopped K+ influx in low-salt medium; xylem pressure rapidly increased to atmospheric levels. With 100 mm NaCl added to the bath, the pressure jump upon cutting was more dramatic, but fluxes remained unaffected, providing further evidence against hydraulic regulation of ion uptake. Following excision of the apical part of barley roots, influx changed to large efflux (-50 nmol m(-2) s(-1)). Kinetin (2-4 microM), a synthetic cytokinin, reversed this effect. Regulation of ion transport by root-tip-synthesized cytokinins is discussed.

Potassium transport and plant salt tolerance.

Salinity is a major abiotic stress affecting approximately 7% of the world's total land area resulting in billion dollar losses in crop production around the globe. Recent progress in molecular genetics and plant electrophysiology suggests that the ability of a plant to maintain a high cytosolic K+/Na+ ratio appears to be critical to plant salt tolerance. So far, the major efforts of plant breeders have been aimed at improving this ratio by minimizing Na+ uptake and transport to shoot. In this paper, we discuss an alternative approach, reviewing the molecular and ionic mechanisms contributing to potassium homeostasis in salinized plant tissues and discussing prospects for breeding for salt tolerance by targeting this trait. Major K+ transporters and their functional expression under saline conditions are reviewed and the multiple modes of their control are evaluated, including ameliorative effects of compatible solutes, polyamines and supplemental calcium. Subsequently, the genetic aspects of inheritance of K+ transport 'markers' are discussed in the general context of salt tolerance as a polygenic trait. The molecular identity of 'salt tolerance' genes is analysed, and prospects for future research and breeding are examined.

Root plasma membrane transporters controlling K+/Na+ homeostasis in salt-stressed barley.

Plant salinity tolerance is a polygenic trait with contributions from genetic, developmental, and physiological interactions, in addition to interactions between the plant and its environment. In this study, we show that in salt-tolerant genotypes of barley (Hordeum vulgare), multiple mechanisms are well combined to withstand saline conditions. These mechanisms include: (1) better control of membrane voltage so retaining a more negative membrane potential; (2) intrinsically higher H(+) pump activity; (3) better ability of root cells to pump Na(+) from the cytosol to the external medium; and (4) higher sensitivity to supplemental Ca(2+). At the same time, no significant difference was found between contrasting cultivars in their unidirectional (22)Na(+) influx or in the density and voltage dependence of depolarization-activated outward-rectifying K(+) channels. Overall, our results are consistent with the idea of the cytosolic K(+)-to-Na(+) ratio being a key determinant of plant salinity tolerance, and suggest multiple pathways of controlling that important feature in salt-tolerant plants.

Expression of animal CED-9 anti-apoptotic gene in tobacco modifies plasma membrane ion fluxes in response to salinity and oxidative stress.

Apoptosis, one form of programmed cell death (PCD), plays an important role in mediating plant adaptive responses to the environment. Recent studies suggest that expression of animal anti-apoptotic genes in transgenic plants may significantly improve a plant's ability to tolerate a variety of biotic and abiotic stresses. The underlying cellular mechanisms of this process remain unexplored. In this study, we investigated specific ion flux "signatures" in Nicotiana benthamiana plants transiently expressing CED-9 anti-apoptotic gene and undergoing salt- and oxidative stresses. Using a range of electrophysiological techniques, we show that expression of CED-9 increased plant salt and oxidative stress tolerance by altering K(+) and H(+) flux patterns across the plasma membrane. Our data shows that PVX/CED-9 plants are capable of preventing stress-induced K(+) efflux from mesophyll cells, so maintaining intracellular K(+) homeostasis. We attribute these effects to the ability of CED-9 to control at least two types of K(+)-permeable channels; outward-rectifying depolarization-activating K(+) channels (KOR) and non-selective cation channels (NSCC). A possible scenario linking CED-9 expression and ionic relations in plant cell is suggested. To the best of our knowledge, this study is the first to link "ion flux signatures" and mechanisms involved in regulation of PCD in plants.

Polyamines prevent NaCl-induced K+ efflux from pea mesophyll by blocking non-selective cation channels.

Despite numerous reports implicating polyamines in plant salinity responses, the specific ionic mechanisms of polyamine-mediated adaptation to salt-stress remain elusive. In this work, we show that micromolar concentrations of polyamines are efficient in preventing NaCl-induced K(+) efflux from the leaf mesophyll, and that this effect can be attributed to the inhibition of non-selective cation channels in mesophyll. The inhibition by externally applied polyamines developed slowly over time, suggesting a cytosolic mode of action. Overall, we suggest that elevated levels of cellular polyamine may modulate the activity of plasma membrane ion channels, improving ionic relations and assisting in a plant's adaptation to salinity.

Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein.

Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM H+ -ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM H+ -ATPase AHA2 at a novel site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between the PM H+ -ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the calcium binding protein SCaBP1 and that high external pH can trigger an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM H+ -ATPase regulation.

Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance.

The accumulation of compatible solutes is often regarded as a basic strategy for the protection and survival of plants under abiotic stress conditions, including both salinity and oxidative stress. In this work, a possible causal link between the ability of contrasting barley genotypes to accumulate/synthesize compatible solutes and their salinity stress tolerance was investigated. The impact of H(2)O(2) (one of the components of salt stress) on K(+) flux (a measure of stress 'severity') and the mitigating effects of glycine betaine and proline on NaCl-induced K(+) efflux were found to be significantly higher in salt-sensitive barley genotypes. At the same time, a 2-fold higher accumulation of leaf and root proline and leaf glycine betaine was found in salt-sensitive cultivars. The total amino acid content was also less affected by salinity in salt-tolerant cultivars. In these, potassium was found to be the main contributor to cytoplasmic osmolality, while in salt-sensitive genotypes, glycine betaine and proline contributed substantially to cell osmolality, compensating for reduced cytosolic K(+). Significant negative correlations (r= -0.89 and -0.94) were observed between Na(+)-induced K(+) efflux (an indicator of salt tolerance) and leaf glycine betaine and proline. These results indicate that hyperaccumulation of known major compatible solutes in barley does not appear to play a major role in salt-tolerance, but rather, may be a symptom of salt-susceptibility.

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels.

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.

Salinity-induced ion flux patterns from the excised roots of Arabidopsis sos mutants.

The SOS signal-transduction pathway is known to be important for ion homeostasis and salt tolerance in plants. However, there is a lack of in planta electrophysiological data about how the changes in signalling and ion transport activity are integrated at the cellular and tissue level. In this study, using the non-invasive ion flux MIFE technique, we compared net K+, H+ and Na+ fluxes from elongation and mature root zones of Arabidopsis wild type Columbia and sos mutants. Our results can be summarised as follows: (1) SOS mutations affect the function of the entire root, not just the root apex; (2) SOS signalling pathway is highly branched; (3) Na+ effects on SOS1 may by-pass the SOS2/SOS3 complex in the root apex; (4) SOS mutation affects H+ transport even in the absence of salt stress; (5) SOS1 mutation affects intracellular K+ homeostasis with a plasma membrane depolarisation-activated outward-rectifying K+ channel being a likely target; (6) H+ pump also may be a target of SOS signalling. We provide an improved model of SOS signalling and discuss physiological mechanisms underlying salt stress perception and signalling in plants. Our work shows that in planta studies are essential for understanding the functional genomics of plant salt tolerance.