Cell-Type-Specific H+-ATPase Activity in Root Tissues Enables K+ Retention and Mediates Acclimation of Barley (Hordeum vulgare) to Salinity Stress.

While the importance of cell type specificity in plant adaptive responses is widely accepted, only a limited number of studies have addressed this issue at the functional level. We have combined electrophysiological, imaging, and biochemical techniques to reveal the physiological mechanisms conferring higher sensitivity of apical root cells to salinity in barley (Hordeum vulgare). We show that salinity application to the root apex arrests root growth in a highly tissue- and treatment-specific manner. Although salinity-induced transient net Na+ uptake was about 4-fold higher in the root apex compared with the mature zone, mature root cells accumulated more cytosolic and vacuolar Na+, suggesting that the higher sensitivity of apical cells to salt is not related to either enhanced Na+ exclusion or sequestration inside the root. Rather, the above differential sensitivity between the two zones originates from a 10-fold difference in K+ efflux between the mature zone and the apical region (much poorer in the root apex) of the root. Major factors contributing to this poor K+ retention ability are (1) an intrinsically lower H+-ATPase activity in the root apex, (2) greater salt-induced membrane depolarization, and (3) a higher reactive oxygen species production under NaCl and a larger density of reactive oxygen species-activated cation currents in the apex. Salinity treatment increased (2- to 5-fold) the content of 10 (out of 25 detected) amino acids in the root apex but not in the mature zone and changed the organic acid and sugar contents. The causal link between the observed changes in the root metabolic profile and the regulation of transporter activity is discussed.

Hyper, a hydrogen peroxide sensor, indicates the sensitivity of the Arabidopsis root elongation zone to aluminum treatment.

Emerging evidence indicates that some reactive oxygen species (ROS), such as the superoxide anion radical and hydrogen peroxide (H2O2), are central regulators of plant responses to biotic and abiotic stresses. Thus, the cellular levels of ROS are thought to be tightly regulated by an efficient and elaborate pro- and antioxidant system that modulates the production and scavenging of ROS. Until recently, studies of ROS in plant cells have been limited to biochemical assays and the use of fluorescent probes; however, the irreversible oxidation of these fluorescent probes makes it impossible to visualize dynamic changes in ROS levels. In this work, we describe the use of Hyper, a recently developed live cell probe for H2O2 measurements in living cells, to monitor oxidative stress in Arabidopsis roots subjected to aluminum treatment. Hyper consists of a circularly permuted YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide-binding protein (OxyR), and is a H2O2-specific ratiometric, and therefore quantitative, probe that can be expressed in plant and animal cells. Now we demonstrate that H2O2 levels drop sharply in the elongation zone of roots treated with aluminum. This response could contribute to root growth arrest and provides evidence that H2O2 is involved in early Al sensing.

Kinetics of xylem loading, membrane potential maintenance, and sensitivity of K(+) -permeable channels to reactive oxygen species: physiological traits that differentiate salinity tolerance between pea and barley.

Salt sensitive (pea) and salt tolerant (barley) species were used to understand the physiological basis of differential salinity tolerance in crops. Pea plants were much more efficient in restoring otherwise depolarized membrane potential thereby effectively decreasing K(+) efflux through depolarization-activated outward rectifying potassium channels. At the same time, pea root apex was 10-fold more sensitive to physiologically relevant H2 O2 concentration and accumulated larger amounts of H2 O2 under saline conditions. This resulted in a rapid loss of cell viability in the pea root apex. Barley plants rapidly loaded Na(+) into the xylem; this increase was only transient, and xylem and leaf Na(+) concentration remained at a steady level for weeks. On the contrary, pea plants restricted xylem Na(+) loading during the first few days of treatment but failed to prevent shoot Na(+) elevation in the long term. It is concluded that superior salinity tolerance of barley plants compared with pea is conferred by at least three different mechanisms: (1) efficient control of xylem Na(+) loading; (2) efficient control of H2 O2 accumulation and reduced sensitivity of non-selective cation channels to H2 O2 in the root apex; and (3) higher energy saving efficiency, with less ATP spent to maintain membrane potential under saline conditions.

Cross-talk between reactive oxygen species and polyamines in regulation of ion transport across the plasma membrane: implications for plant adaptive responses.

Many stresses are associated with increased accumulation of reactive oxygen species (ROS) and polyamines (PAs). PAs act as ROS scavengers, but export of putrescine and/or PAs to the apoplast and their catabolization by amine oxidases gives rise to H2O2 and other ROS, including hydroxyl radicals ((•)OH). PA catabolization-based signalling in apoplast is implemented in plant development and programmed cell death and in plant responses to a variety of biotic and abiotic stresses. Central to ROS signalling is the induction of Ca(2+) influx across the plasma membrane. Different ion conductances may be activated, depending on ROS, plant species, and tissue. Both H2O2 and (•)OH can activate hyperpolarization-activated Ca(2+)-permeable channels. (•)OH is also able to activate both outward K(+) current and weakly voltage-dependent conductance (ROSIC), with a variable cation-to-anion selectivity and sensitive to a variety of cation and anion channel blockers. Unexpectedly, PAs potentiated (•)OH-induced K(+) efflux in vivo, as well as ROSIC in isolated protoplasts. This synergistic effect is restricted to the mature root zone and is more pronounced in salt-sensitive cultivars compared with salt-tolerant ones. ROS and PAs suppress the activity of some constitutively expressed K(+) and non-selective cation channels. In addition, both (•)OH and PAs activate plasma membrane Ca(2+)-ATPase and affect H(+) pumping. Overall, (•)OH and PAs may provoke a substantial remodelling of cation and anion conductance at the plasma membrane and affect Ca(2+) signalling.

Polyamines cause plasma membrane depolarization, activate Ca2+-, and modulate H+-ATPase pump activity in pea roots.

Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress.

Polyamines interact with hydroxyl radicals in activating Ca(2+) and K(+) transport across the root epidermal plasma membranes.

Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROS affect the intracellular Ca(2+) dynamics by activating a range of nonselective Ca(2+)-permeable channels in plasma membrane (PM). Using patch-clamp and noninvasive microelectrode ion flux measuring techniques, we have characterized ionic currents and net K(+) and Ca(2+) fluxes induced by hydroxyl radicals (OH(•)) in pea (Pisum sativum) roots. OH(•), but not hydrogen peroxide, activated a rapid Ca(2+) efflux and a more slowly developing net Ca(2+) influx concurrent with a net K(+) efflux. In isolated protoplasts, OH(•) evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K(+) efflux in intact roots. This current displayed a low ionic selectivity and was permeable to Ca(2+). Active OH(•)-induced Ca(2+) efflux in roots was suppressed by the PM Ca(2+) pump inhibitors eosine yellow and erythrosine B. The cation channel blockers gadolinium, nifedipine, and verapamil and the anionic channel blockers 5-nitro-2(3-phenylpropylamino)-benzoate and niflumate inhibited OH(•)-induced ionic currents in root protoplasts and K(+) efflux and Ca(2+) influx in roots. Contrary to expectations, polyamines (PAs) did not inhibit the OH(•)-induced cation fluxes. The net OH(•)-induced Ca(2+) efflux was largely prolonged in the presence of spermine, and all PAs tested (spermine, spermidine, and putrescine) accelerated and augmented the OH(•)-induced net K(+) efflux from roots. The latter effect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alter intracellular Ca(2+) homeostasis by modulating both Ca(2+) influx and efflux transport systems at the root cell PM.

Natural variation in primary root growth and K+ retention in roots of habanero pepper (Capsicum chinense) under salt stress.

In this work, we analysed the natural variation in mechanisms for protection against salt stress in pepper varieties (Capsicum chinense Jacq. cv. Rex, Chichen-Itza and Naranja and Capsicum annuum L. cv. Padron), considering primary root growth and viability of the post-stressed seedlings. NaCl-induced K+ and H+ efflux in roots was also studied by ion-selective microelectrodes under application of pharmacological agents. In these pepper varieties, the magnitude of the K+ leakage in the roots positively correlated with growth inhibition of the primary root in the presence of NaCl, with Rex variety showing a higher level of tolerance than Chichen-Itza. The K+ leakage and the activity of the H+ pump in the roots were dependent on the NaCl concentration. Pharmacological analysis indicated that the NaCl-induced K+ leakage was mediated by TEA+-sensitive KOR channels but not by NSCC channels. In addition, we present evidence for the possible participation of proline, and a Na+-insensitive HAK K+ transporter expressed in habanero pepper roots for maintaining K+ homeostasis under salt stress conditions.

Mechanisms of salt tolerance in habanero pepper plants (Capsicum chinense Jacq.): Proline accumulation, ions dynamics and sodium root-shoot partition and compartmentation.

Despite its economic relevance, little is known about salt tolerance mechanisms in pepper plants. To address this question, we compared differences in responses to NaCl in two Capsicum chinense varieties: Rex (tolerant) and Chichen-Itza (sensitive). Under salt stress (150 mM NaCl over 7 days) roots of Rex variety accumulated 50 times more compatible solutes such as proline compared to Chichen-Itza. Mineral analysis indicated that Na(+) is restricted to roots by preventing its transport to leaves. Fluorescence analysis suggested an efficient Na(+) compartmentalization in vacuole-like structures and in small intracellular compartments in roots of Rex variety. At the same time, Na(+) in Chichen-Itza plants was compartmentalized in the apoplast, suggesting substantial Na(+) extrusion. Rex variety was found to retain more K(+) in its roots under salt stress according to a mineral analysis and microelectrode ion flux estimation (MIFE). Vanadate-sensitive H(+) efflux was higher in Chichen-Itza variety plants, suggesting a higher activity of the plasma membrane H(+)-ATPase, which fuels the extrusion of Na(+), and, possibly, also the re-uptake of K(+). Our results suggest a combination of stress tolerance mechanisms, in order to alleviate the salt-induced injury. Furthermore, Na(+) extrusion to apoplast does not appear to be an efficient strategy for salt tolerance in pepper plants.

Patch-clamp protocols to study cell ionic homeostasis under saline conditions.

The patch-clamp technique was designed to measure any electrogenic transport across the whole cell and organelle (vacuolar) membranes and excised membrane patches. Here, we describe preparation of protoplasts and vacuoles, as well as patch-clamp assays, to detect the functional expression of K(+) and cation channels of plasma membrane and tonoplast, as well as plasma membrane anion channels and vacuolar and plasma membrane H(+) pumps. All of these contribute to the intracellular ionic homeostasis under saline conditions.

Synergism between polyamines and ROS in the induction of Ca ( 2+) and K (+) fluxes in roots.

Stress conditions cause increases in ROS and polyamines levels, which are not merely collateral. There is increasing evidence for the ROS participation in signaling as well as for polyamine protective roles under stress. Polyamines and ROS, respectively, inhibit cation channels and induce novel cation conductance in the plasma membrane. Our new results indicate that polyamines and OH (•) also stimulate Ca ( 2+) pumping across the root plasma membrane. Besides, polyamines potentiate the OH (•) -induced non-selective current and respective passive K (+) and Ca ( 2+) fluxes. In roots this synergism, however, is restricted to the mature zone, whereas in the distal elongation zone only the Ca ( 2+) pump activation is observed. Remodeling the plasma membrane ion conductance by OH (•) and polyamines would impact K (+) homeostasis and Ca ( 2+) signaling under stress.

Salt-sensitive and salt-tolerant barley varieties differ in the extent of potentiation of the ROS-induced K(+) efflux by polyamines.

Generation of high levels of polyamines and reactive oxygen species (ROS) is common under stress conditions. Our recent study on a salt-sensitive pea species revealed an interaction between natural polyamines and hydroxyl radicals in inducing non-selective conductance and stimulating Ca(2+)-ATPase pumps at the root plasma membrane (I. Zepeda-Jazo, A.M. Velarde-Buendía, R. Enríquez-Figueroa, B. Jayakumar, S. Shabala, J. Muñiz, I. Pottosin, Polyamines interact with hydroxyl radicals in activating Ca2+ and K+ transport across the root epidermal plasma membranes, Plant Phys. 157 (2011) 1-14). In this work, we extended that study to see if interaction between polyamines and ROS may determine the extent of genotypic variation in salinity tolerance. This work was conducted using barley genotypes contrasting in salinity tolerance. Similar to our findings in pea, application of hydroxyl radicals-generating Cu(2+)/ascorbate mixture induced transient Ca(2+) and K(+) fluxes in barley roots. Putrescine and spermine alone induced only transient Ca(2+) efflux and negligible K(+) flux. However, both putrescine and spermine strongly potentiated hydroxyl radicals-induced K(+) efflux and respective non-selective current. This synergistic effect was much more pronounced in a salt-sensitive cultivar Franklin as compared to a salt-tolerant TX9425. As retention of K(+) under salt stress is a key determinant of salinity tolerance in barley, we suggest that the alteration of cytosolic K(+) homeostasis, caused by interaction between polyamines and ROS, may have a substantial contribution to genetic variability in salt sensitivity in this species.