Tracey Ann Cuin

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.

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 Ca2+. At the same time, no significant difference was found between contrasting cultivars in their unidirectional 22Na+ 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.

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 Ca2+, tetraethylammonium, Ba2+, 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.

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.

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 roots 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.

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.

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.

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 plants adaptation to salinity.

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.

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.