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.

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.

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.

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.

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.

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.