Tobacco leaf tissue rapidly detoxifies direct salt loads without activation of calcium and SOS signaling.

Salt stress is a major abiotic stress, responsible for declining agricultural productivity. Roots are regarded as hubs for salt detoxification, however, leaf salt concentrations may exceed those of roots. How mature leaves manage acute sodium chloride (NaCl) stress is mostly unknown. To analyze the mechanisms for NaCl redistribution in leaves, salt was infiltrated into intact tobacco leaves. It initiated pronounced osmotically-driven leaf movements. Leaf downward movement caused by hydro-passive turgor loss reached a maximum within 2 h. Salt-driven cellular water release was accompanied by a transient change in membrane depolarization but not an increase in cytosolic calcium ion (Ca2+ ) level. Nonetheless, only half an hour later, the leaves had completely regained turgor. This recovery phase was characterized by an increase in mesophyll cell plasma membrane hydrogen ion (H+ ) pumping, a salt uptake-dependent cytosolic alkalization, and a return of the apoplast osmolality to pre-stress levels. Although, transcript numbers of abscisic acid- and Salt Overly Sensitive pathway elements remained unchanged, salt adaptation depended on the vacuolar H+ /Na+ -exchanger NHX1. Altogether, tobacco leaves can detoxify sodium ions (Na+ ) rapidly even under massive salt loads, based on pre-established posttranslational settings and NHX1 cation/H+ antiport activity. Unlike roots, signaling and processing of salt stress in tobacco leaves does not depend on Ca2+ signaling.

Stalk cell polar ion transport provide for bladder-based salinity tolerance in Chenopodium quinoa.

Chenopodium quinoa uses epidermal bladder cells (EBCs) to sequester excess salt. Each EBC complex consists of a leaf epidermal cell, a stalk cell, and the bladder. Under salt stress, sodium (Na+ ), chloride (Cl- ), potassium (K+ ) and various metabolites are shuttled from the leaf lamina to the bladders. Stalk cells operate as both a selectivity filter and a flux controller. In line with the nature of a transfer cell, advanced transmission electron tomography, electrophysiology, and fluorescent tracer flux studies revealed the stalk cell's polar organization and bladder-directed solute flow. RNA sequencing and cluster analysis revealed the gene expression profiles of the stalk cells. Among the stalk cell enriched genes, ion channels and carriers as well as sugar transporters were most pronounced. Based on their electrophysiological fingerprint and thermodynamic considerations, a model for stalk cell transcellular transport was derived.

Friend or Foe? Chloride Patterning in Halophytes.

In this opinion article, we challenge the traditional view that breeding for reduced Cl- uptake would benefit plant salinity tolerance. A negative correlation between shoot Cl- concentration and plant biomass does not hold for halophytes - naturally salt tolerant species. We argue that, under physiologically relevant conditions, Cl- uptake requires plants to invest metabolic energy, and that the poor selectivity of Cl--transporting proteins may explain the reported negative correlation between Cl- accumulation and crop salinity tolerance. We propose a new paradigm: salinity tolerance could be achieved by improving the selectivity of some of the broadly selective anion-transporting proteins (e.g., for NO3->Cl-), alongside tight control of Cl- uptake, rather than targeting traits mediating its efflux from the root.

High V-PPase activity is beneficial under high salt loads, but detrimental without salinity.

The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H+ -ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and proton-pumping functions has yet to be dissected. For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging. NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt-untreated conditions, V-PPase-overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP-hyperactive cells from cell death. Furthermore, a salt-induced rise in V-PPase but not of V-ATPase pump currents was detected in nontransformed plants. The results indicate that under normal growth conditions, plants need to regulate the V-PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V-PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton-coupled Na+ sequestration.

The Role of Potassium Channels in Arabidopsis thaliana Long Distance Electrical Signalling: AKT2 Modulates Tissue Excitability While GORK Shapes Action Potentials.

Fast responses to an external threat depend on the rapid transmission of signals through a plant. Action potentials (APs) are proposed as such signals. Plant APs share similarities with their animal counterparts; they are proposed to depend on the activity of voltage-gated ion channels. Nonetheless, despite their demonstrated role in (a)biotic stress responses, the identities of the associated voltage-gated channels and transporters remain undefined in higher plants. By demonstrating the role of two potassium-selective channels in Arabidopsis thaliana in AP generation and shaping, we show that the plant AP does depend on similar Kv-like transport systems to those of the animal signal. We demonstrate that the outward-rectifying potassium-selective channel GORK limits the AP amplitude and duration, while the weakly-rectifying channel AKT2 affects membrane excitability. By computational modelling of plant APs, we reveal that the GORK activity not only determines the length of an AP but also the steepness of its rise and the maximal amplitude. Thus, outward-rectifying potassium channels contribute to both the repolarisation phase and the initial depolarisation phase of the signal. Additionally, from modelling considerations we provide indications that plant APs might be accompanied by potassium waves, which prime the excitability of the green cable.

SLAH3-type anion channel expressed in poplar secretory epithelia operates in calcium kinase CPK-autonomous manner.

Extrafloral nectaries secrete a sweet sugar cocktail that lures predator insects for protection from foraging herbivores. Apart from sugars and amino acids, the nectar contains the anions chloride and nitrate. Recent studies with Populus have identified a type of nectary covered by apical bipolar epidermal cells, reminiscent of the secretory brush border epithelium in animals. Border epithelia operate transepithelial anion transport, which is required for membrane potential and/or osmotic adjustment of the secretory cells. In search of anion transporters expressed in extrafloral nectaries, we identified PttSLAH3 (Populus tremula × Populus tremuloides SLAC1 Homologue3), an anion channel of the SLAC/SLAH family. When expressed in Xenopus oocytes, PttSLAH3 displayed the features of a voltage-dependent anion channel, permeable to both nitrate and chloride. In contrast to the Arabidopsis SLAC/SLAH family members, the poplar isoform PttSLAH3 is independent of phosphorylation activation by protein kinases. To understand the basis for the autonomous activity of the poplar SLAH3, we generated and expressed chimera between kinase-independent PttSLAH3 and kinase-dependent Arabidopsis AtSLAH3. We identified the N-terminal tail and, to a lesser extent, the C-terminal tail as responsible for PttSLAH3 kinase-(in)dependent action. This feature of PttSLAH3 may provide the secretory cell with a channel probably controlling long-term nectar secretion.

Identification of the transporter responsible for sucrose accumulation in sugar beet taproots.

Sugar beet provides around one third of the sugar consumed worldwide and serves as a significant source of bioenergy in the form of ethanol. Sucrose accounts for up to 18% of plant fresh weight in sugar beet. Most of the sucrose is concentrated in the taproot, where it accumulates in the vacuoles. Despite 30 years of intensive research, the transporter that facilitates taproot sucrose accumulation has escaped identification. Here, we combine proteomic analyses of the taproot vacuolar membrane, the tonoplast, with electrophysiological analyses to show that the transporter BvTST2.1 is responsible for vacuolar sucrose uptake in sugar beet taproots. We show that BvTST2.1 is a sucrose-specific transporter, and present evidence to suggest that it operates as a proton antiporter, coupling the import of sucrose into the vacuole to the export of protons. BvTST2.1 exhibits a high amino acid sequence similarity to members of the tonoplast monosaccharide transporter family in Arabidopsis, prompting us to rename this group of proteins 'tonoplast sugar transporters'. The identification of BvTST2.1 could help to increase sugar yields from sugar beet and other sugar-storing plants in future breeding programs.

The twins K+ and Na+ in plants.

In the earth's crust and in seawater, K(+) and Na(+) are by far the most available monovalent inorganic cations. Physico-chemically, K(+) and Na(+) are very similar, but K(+) is widely used by plants whereas Na(+) can easily reach toxic levels. Indeed, salinity is one of the major and growing threats to agricultural production. In this article, we outline the fundamental bases for the differences between Na(+) and K(+). We present the foundation of transporter selectivity and summarize findings on transporters of the HKT type, which are reported to transport Na(+) and/or Na(+) and K(+), and may play a central role in Na(+) utilization and detoxification in plants. Based on the structural differences in the hydration shells of K(+) and Na(+), and by comparison with sodium channels, we present an ad hoc mechanistic model that can account for ion permeation through HKTs.

Quantifying kinetics of net ion fluxes from plant tissues by non-invasive microelectrode measuring MIFE technique.

Non-invasive microelectrode ion flux measuring (the MIFE system) allows concurrent quantification of net fluxes of several ions with high spatial (several µm) and temporal (ca 5 s) resolution. Over the last 10 years, the MIFE system has been widely used to study various aspects of salt stress signaling and adaptation in plants. This chapter summarizes some major findings in the area such as using MIFE for deciphering the specific and non-specific components of salinity stress, resolving the role of the plasma membrane H(+)-pump in salinity responses, proving K(+) homeostasis as a key feature of salinity tolerance, and discovering the mechanisms behind the ameliorative effects of Ca(2+) and other mitigating factors (such as polyamines or compatible solutes). The full protocols for microelectrode fabrication, calibration, and use are then given, and two basic routines for measuring net K(+) and Na(+) fluxes from salinity stressed roots are described in the context of plant screening for salt stress tolerance.

The K (+) battery-regulating Arabidopsis K (+) channel AKT2 is under the control of multiple post-translational steps.

Potassium (K (+) ) is an important nutrient for plants. It serves as a cofactor of various enzymes and as the major inorganic solute maintaining plant cell turgor. In a recent study, an as yet unknown role of K (+) in plant homeostasis was shown. It was demonstrated that K (+) gradients in vascular tissues can serve as an energy source for phloem (re)loading processes and that the voltage-gated K (+) channels of the AKT2-type play a unique role in this process. The AKT2 channel can be converted by phosphorylation of specific serine residues (S210 and S329) into a non-rectifying channel that allows a rapid efflux of K (+) from the sieve element/companion cells (SE/CC) complex. The energy of this flux is used by other transporters for phloem (re)loading processes. Nonetheless, the results do indicate that post-translational modifications at S210 and S329 alone cannot explain AKT2 regulation. Here, we discuss the existence of multiple post-translational modification steps that work in concert to convert AKT2 from an inward-rectifying into a non-rectifying K (+) channel.

Competition between uptake of ammonium and potassium in barley and Arabidopsis roots: molecular mechanisms and physiological consequences.

Plants can use ammonium (NH4+) as the sole nitrogen source, but at high NH4+ concentrations in the root medium, particularly in combination with a low availability of K+, plants suffer from NH4+ toxicity. To understand the role of K+ transporters and non-selective cation channels in K+/NH4+ interactions better, growth, NH4+ and K+ accumulation and the specific fluxes of NH4+, K+, and H+ were examined in roots of barley (Hordeum vulgare L.) and Arabidopsis seedlings. Net fluxes of K+ and NH4+ were negatively correlated, as were their tissue concentrations, suggesting that there is direct competition during uptake. Pharmacological treatments with the K+ transport inhibitors tetraethyl ammonium (TEA+) and gadolinium (Gd3+) reduced NH4+ influx, and the addition of TEA+ alleviated the NH4+-induced depression of root growth in germinating Arabidopsis plants. Screening of a barley root cDNA library in a yeast mutant lacking all NH4+ and K+ uptake proteins through the deletion of MEP1-3 and TRK1 and TRK2 resulted in the cloning of the barley K+ transporter HvHKT2;1. Further analysis in yeast suggested that HvHKT2;1, AtAKT1, and AtHAK5 transported NH4+, and that K+ supplied at increasing concentrations competed with this NH4+ transport. On the other hand, uptake of K+ by AtHAK5, and to a lesser extent via HvHKT2;1 and AtAKT1, was inhibited by increasing concentrations of NH4+. Together, the results of this study show that plant K+ transporters and channels are able to transport NH4+. Unregulated NH4+ uptake via these transporters may contribute to NH4+ toxicity at low K+ levels, and may explain the alleviation of NH4+ toxicity by K+.

Ionic relations and osmotic adjustment in durum and bread wheat under saline conditions.

Wheat breeding for salinity tolerance has traditionally focussed on Na+ exclusion from the shoot, but its association with salinity tolerance remains tenuous. Accordingly, the physiological significance of shoot Na+ exclusion and maintenance of an optimal K+ : Na+ ratio was re-evaluated by studying NaCl-induced responses in 50 genotypes of bread wheat (Triticum aestivum L.) and durum wheat (Triticum turgidum L. ssp. durum) treated with 150 mM NaCl. Overall, Na+ exclusion from the shoot correlated with salinity tolerance in both species and this exclusion was more efficient in bread compared with durum wheat. Interestingly, shoot sap K+ increased significantly in nearly all durum and bread wheat genotypes. Conversely, the total shoot K+ content declined. We argue that this increase in shoot sap K+ is needed to provide efficient osmotic adjustment under saline conditions. Durum wheat was able to completely adjust shoot sap osmolality using K+, Na+ and Cl-; it had intrinsically higher levels of these solutes. In bread wheat, organic osmolytes must contribute ~13% of the total shoot osmolality. In contrast to barley (Hordeum vulgare L.), NaCl-induced K+ efflux from seedling roots did not predict salinity tolerance in wheat, implying that shoot, not root K+ retention is important in this species.

A root's ability to retain K+ correlates with salt tolerance in wheat.

Most work on wheat breeding for salt tolerance has focused mainly on excluding Na(+) from uptake and transport to the shoot. However, some recent findings have reported no apparent correlation between leaf Na(+) content and wheat salt tolerance. Thus, it appears that excluding Na(+) by itself is not always sufficient to increase plant salt tolerance and other physiological traits should also be considered. In this work, it was investigated whether a root's ability to retain K(+) may be such a trait, and whether our previous findings for barley can be extrapolated to species following a 'salt exclusion' strategy. NaCl-induced kinetics of K(+) flux from roots of two bread and two durum wheat genotypes, contrasting in their salt tolerance, were measured under laboratory conditions using non-invasive ion flux measuring (the MIFE) technique. These measurements were compared with whole-plant physiological characteristics and yield responses from plants grown under greenhouse conditions. The results show that K(+) flux from the root surface of 6-d-old wheat seedlings in response to salt treatment was highly correlated with major plant physiological characteristics and yield of greenhouse-grown plants. This emphasizes the critical role of K(+) homeostasis in plant salt tolerance and suggests that using NaCl-induced K(+) flux measurements as a physiological 'marker' for salt tolerance may benefit wheat-breeding programmes.

Compatible solutes mitigate damaging effects of salt stress by reducing the impact of stress-induced reactive oxygen species.

Under abiotic stress conditions, rapid increases in reactive oxygen species (ROS) levels occurs within plant cells. Although their role as a major signalling agent in plants is now acknowledged, elevated ROS levels can result in an impairment of membrane integrity. Similar to our previous findings on imposition of salt stress, application of the hydroxyl radical (OH(*)) to Arabidopsis roots results in a massive efflux of K(+) from epidermal cells. This is likely to cause significant damage to cell metabolism. Since K(+) loss also occurs after salt application and salt stress leads to increased cellular ROS levels, we suggest that at least some of the detrimental effects of salinity is due to damage by its resulting ROS on K(+) homeostasis. We also observed a comparative reduction in K(+) efflux by compatible solutes after both oxidative and salt stress. Thus, we propose that under saline conditions, compatible solutes mitigate the oxidative stress damage to membrane transporters. Whether this amelioration is due to free-radical scavenging or by direct protection of transporter systems, warrants further investigation.

Compatible solutes reduce ROS-induced potassium efflux in Arabidopsis roots.

Reactive oxygen species (ROS) are known to be primarily responsible for the impairment of cellular function under numerous abiotic and biotic stress conditions. In this paper, using non-invasive microelectrode ion flux measuring (MIFE) system, we show that the application of a hydroxyl radical (OH*)-generating Cu2+/ascorbate (Cu/a) mixture to Arabidopsis roots results in a massive, dose-dependent efflux of K+ from epidermal cells in the elongation zone. Pharmacological experiments suggest that both outward-rectifying K+ channels and non-selective cation channels (NSCCs) mediate such effluxes. Low (5 mM) concentrations of compatible solutes (glycine betaine, proline, mannitol, trehalose or myo-inositol) significantly reduces OH*-induced K+ efflux, similar to our previous reports for NaCl-induced K+ efflux. Importantly, a significant reduction in K+ efflux was found using osmolytes with no reported free radical scavenging activity, as well as those for which a role in free radical scavenging has been demonstrated. This indicates that compatible solutes must play other (regulatory) roles, in addition to free radical scavenging, in mitigating the damaging effects of oxidative stress.

Spectral and dose dependence of light-induced ion flux responses from maize leaves and their involvement in leaf expansion growth.

Two types of segments (intact leaf tissue and isolated mesophyll tissue respectively) were isolated from basal (still growing) and tip (non-growing) maize leaf regions. The leaf segments were exposed to different light qualities (blue or red light) and quantities, and net fluxes of K+, Ca2+ and H+ were measured non-invasively using ion-selective vibrating microelectrodes (the MIFE technique). A clear dose dependency of all ion flux responses on both red (RL) and blue (BL) light fluence rate was found. We provide evidence that light-induced K+ flux kinetics are different between growing and non-growing tissues and attribute this difference to the direct involvement of RL-induced K+ flux in turgor-driven leaf expansion growth controlled by the epidermis, as well as to the charge-balancing role of K+ in the leaf mesophyll. Generally, BL was much more efficient in stimulating K+ uptake in the growing basal region compared with RL. We also show a much stronger influence of RL on Ca2+ fluxes in the basal region compared with BL, which argues in favor of the importance of RL in Ca2+ signaling during leaf growth.

Amino acids regulate salinity-induced potassium efflux in barley root epidermis.

The amino acid content increases substantially in salt-stressed plants. The physiological relevance of this phenomenon remains largely unknown. Using the MIFE ion flux measuring technique, we studied the effects of physiologically relevant concentrations of 26 amino acids on NaCl-induced K(+) flux from barley root epidermis. We show that 21 (of 26) amino acids caused a significant mitigation of the NaCl-induced K(+) efflux, while valine and ornithine substantially enhanced the detrimental effects of salinity on K(+) homeostasis. Our results suggest that physiologically relevant concentrations of free amino acids might contribute to plant adaptive responses to salinity by regulating K(+) transport across the plasma membrane, thus enabling maintenance of an optimal K(+)/Na(+) ratio as opposed to being merely a symptom of plant damage by stress. Investigating the specific mechanisms of such amelioration remains a key issue for future studies.

Exogenously supplied compatible solutes rapidly ameliorate NaCl-induced potassium efflux from barley roots.

It has been suggested that the role of compatible solutes in plant stress responses is not limited to conventional osmotic adjustment, but also includes some other regulatory or osmoprotective functions. In this study, we hypothesized that one such function is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, a feature that may confer salt tolerance in many species, particularly in barley. This hypothesis was investigated using the non-invasive microelectrode ion flux (MIFE) measuring technique. We show that low (0.5-5 mM) concentrations of exogenously supplied proline or betaine significantly reduced NaCl-induced K+ efflux from barley roots in a dose-response manner. This effect was instantaneous, implying that large intracellular concentrations of compatible solutes are not required for an amelioratory role. Exogenously supplied betaine also significantly enhanced NaCl-induced H+ efflux, but only in pre-incubated roots, implying some alternative mechanism of regulation. Sap K+ and Na+ analysis and membrane potential measurements are also consistent with the model that one function of compatible solutes is in maintaining cytosolic K+ homeostasis by preventing NaCl-induced K+ leakage from the cell, possibly through the enhanced activity of H+-ATPase, controlling voltage-dependent outward-rectifying K+ channels and creating the electrochemical gradient necessary for secondary ion transport processes. These data provide the first direct evidence for regulation of ion fluxes across the plasma membrane by physiologically relevant low concentrations of compatible solutes.

Potassium activities in cell compartments of salt-grown barley leaves.

Triple-barrelled microelectrodes measuring K(+) activity (a(K)), pH and membrane potential were used to make quantitative measurements of vacuolar and cytosolic a(K) in epidermal and mesophyll cells of barley plants grown in nutrient solution with 0 or 200 mM added NaCl. Measurements of a(K) were assigned to the cytosol or vacuole based on the pH measured. In epidermal cells, the salt treatment decreased a(K) in the vacuole from 224 to 47 mM and in the cytosol from 68 to 15 mM. In contrast, the equivalent changes in the mesophyll were from 235 to 150 mM (vacuole) and 79 to 64 mM (cytosol). Thus mechanisms exist to ameliorate the effects of salt on a(K) in compartments of mesophyll cells, presumably to minimize any deleterious consequences for photosynthesis. Thermodynamic calculations showed that K(+) is actively transported into the vacuole of both epidermal and mesophyll cells of salinized and non- salinized plants. Comparison of the values of a(K) in K(+)-replete, non-salinized leaf cells with those previously measured in root cells of plants grown under comparable conditions indicates that cytosolic a(K) is similar in cells of both organs, but vacuolar a(K) in leaf cells is approximately twice that in roots. This suggests differences in the regulation of vacuolar a(K), but not cytosolic a(K), in leaf and root cells.