Synergism between polyamines and ROS in the induction of Ca²⁺ and K⁺ fluxes in roots

Abstract

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 Ca²⁺ pumping across the root plasma membrane. Besides, polyamines potentiate the OH^•-induced non-selective current and respective passive K⁺ and Ca²⁺ fluxes. In roots this synergism, however, is restricted to the mature zone, whereas in the distal elongation zone only the Ca²⁺ pump activation is observed. Remodeling the plasma membrane ion conductance by OH^• and polyamines would impact K⁺ homeostasis and Ca²⁺ signaling under stress.

Polyamines and ROS Generation Under Stress: Direct Implications for the Ion Transport

There are currently many evidence suggesting that increases of PAs under abiotic stresses conditions are not merely collateral changes resulting from altered cell metabolism, but in many cases form a part of protective mechanism.^1-3 PAs are unique polycationic metabolites, with a net charge of +2, +3 and +4 for putrescine, spermidine and spermine, respectively. It is not surprisingly, therefore, that in animal cells polyamines act primarily as pore blockers in a variety of cation (including K⁺) channels.^4,5 Such direct mechanism in plants was demonstrated so far only for vacuolar cation FV and SV channels,⁶ whereas the mechanism of the PAs action (mainly, inhibition, see Figure 1) on the PM cation and K⁺-selective channels is less clear.^7-9 As PAs hardly affect the K⁺-selective tonoplast channels,¹⁰ but suppress with a high affinity Na⁺-permeable FV and SV channels in this membrane, PAs increases under salt stress will tend to make the overall cation conductance of the tonoplast more K⁺-selective, assisting vacuolar Na⁺ sequestration and improving cytosolic K⁺/ Na⁺ ratio at the dispense of vacuolar K⁺.⁵ Drought-induced increase in spermidine facilitates stomatal closure due to selective inhibition of KIR channels, whereas KOR and PM anion channels are not affected.⁷ Inhibition of the PM NSCC is also believed to contribute to the salt tolerance, reducing Na⁺ influx and, consequently, negating membrane depolarization and Na⁺-induced K⁺ efflux.¹¹ K⁺ loss under salt stress is also reduced with a higher PM H⁺ pump activity, due to a better control of the membrane potential for depolarization challenges.¹² Notably, PAs stimulate the PM H⁺ ATPase activity¹³ (Fig. 1). Yet, inhibition of cation and K⁺ channels by PAs is not their prime action on the ion transport across the PM. Depending on growth conditions, root zone and PAs species, the effect of PAs on the Na⁺-induced K⁺ efflux may be either inhibitory or stimulatory.¹⁴

Figure 1. Integrative regulation of cation transport across the PM by ROS and PAs. Sequence of events, leading to the generation of H₂O₂ and OH^• in the apoplast, is depicted. H₂O₂ is formed in reactions, catalyzed by SOD and amine oxidases, and is converted to OH^• by iron or copper via Fenton reaction. PAs, if not available in the external medium, need to be exported to the apoplast from the cytosol. (1) PAs and ROS inhibit constitutively expressed cation channels, while PAs also activate the H⁺ pump. These actions will lead to a decrease in Ca²⁺ (Na⁺) influx and in K⁺ loss; (2) H₂O₂ from the intracellular side (in some tissues, also from extracellular one) and external OH^• activate ROSIC, facilitating K⁺ efflux and Ca²⁺ (Na⁺) uptake; (3) dual role of PAs: ROS can act as a scavenger or as a source of H₂O_2, due to their catabolization; (4) PAs and OH^• activate the PM Ca²⁺ pump; (5) PAs sensitize ROSIC to OH^•; this potentiation is tissue-specific.

ROS are known to accumulate during stresses, so that oxidative stress tolerance is a common component of a stress response. ROS may play a dual role: as toxic by-products, eventually leading to the programmed cell death or as signaling molecules.^15,16 Apoplast is an important site for the ROS (O₂^-, H₂O₂, OH^•) formation (Fig. 1). H₂O₂ and OH^• inhibit some constitutively expressed PM channels like KOR, KIR and NSCC^17,18 and activate another non-selective current(s), permeable for Ca²⁺, ROSIC (Fig. 1). In some tissues, only OH^• but not H₂O₂ induces ROSIC.^18,19 ROSIC triggers stomatal closure²⁰ and is essential for polarized root hair growth.¹⁹ The latter is explained by the fact that ROSIC-related cytosolic Ca²⁺ increase in a positive feedback manner activates key PM ROS-producing enzyme, NADPH oxidase, localized in the tips of growing root hairs.²¹

Polyamines and ROS: Cross Talks Revealed

The simplest way for PAs and ROS interaction is a direct scavenging of ROS by PAs²² (Fig. 1). However, oxidation of putrescine by DAO and higher PAs by PAO in the apoplast gives rise to the increase of H₂O₂, which can be further converted to OH^• by transient metals such as iron (mainly present in reaction centers of cell-wall bound peroxidases²³) or copper (e.g., in the reaction centers of DAO) (Fig. 1). Preferential DAO expression in dicots and PAO in monocots²⁴ may contribute to the specificity of action of different PAs. Oxidation is preceded by PAs export to the apoplast via yet unknown mechanism (Fig. 1) and leads to induction of ROSIC and related Ca²⁺ signal in a variety of plant responses to environmental stimuli¹⁸ (see references therein). Our last study revealed two novel joint effects of PAs and ROS (OH^•) on the transport processes across the PM (regulation routes (4) and (5) in Figure 1). First, OH^• and PAs stimulated Ca²⁺ efflux, sensitive to specific Ca²⁺ pump inhibitor eosine yellow. Yet PAs alone and lower OH^• induced only Ca²⁺ efflux, whereas at higher OH^• levels an additional, slowly developing Gd³⁺-sensitive Ca²⁺ influx, was observed. This Ca²⁺ influx had the same kinetics and pharmacology as simultaneously developed OH^• -induced K⁺ efflux. We concluded that there are at least two flux components, activated by OH^•: active Ca²⁺ efflux, mediated by PM Ca²⁺ pump and non-selective Gd³⁺-sensitive passive conductance, mediating Ca²⁺ influx and K⁺ efflux (Fig. 1). The presence of Gd³⁺ unmasked a continuing pump-mediated Ca²⁺ efflux. Dynamic summation of Ca²⁺ efflux with passive Ca²⁺ influx produces a non-monotonous net Ca²⁺ flux; at high OH^• there was net Ca²⁺ influx at a steady-state. In the presence of 1 mM of PAs this influx was reduced (putrescine), cancelled (spermidine) or converted to net Ca²⁺ efflux (spermine). Qualitatively very similar Ca²⁺ flux behavior was observed also in the DEZ (Fig. 2A). Compared with the mature zone, however, OH^• -induced K⁺ efflux was much faster and had a 3-times larger peak magnitude (Fig. Two C and D). Because OH^• induces a passive conductance, which mediates both K⁺ efflux and Ca²⁺ influx,¹⁸ a smaller net initial Ca²⁺ efflux, observed in the DEZ as compared with the mature zone (Fig. 2A and B) may be due to the fact that OH^• -induced Ca²⁺ influx is larger in DEZ (as it is for K⁺), so that the sum of Ca²⁺ efflux (negative in accord with the MIFE convention) and influx (positive) becomes less negative. Another difference was that in the mature zone in the presence of PAs the OH^• -induced K⁺ efflux was significantly potentiated, whereas in DEZ no significant potentiation was observed (Fig. 2D). Potentiation of the OH^• -induced K⁺ efflux by PAs in the mature zone can be reproduced on isolated protoplasts, by patch-clamp technique in the whole cell mode (Fig. 2D, inset). This result implies that the effect is membrane delimited and does not require PAs catabolization by amine oxidases. Most likely, PAs, which by themselves induce little or no K⁺ flux, bind to membrane components, responsible for ROSIC and sensitize them to OH^•. Similar sensitization by PAs for agonists was reported for some receptor channels in animal cells.^4,25 The apparent absence of such potentiation in DEZ (Fig. 2A) implies that ROSIC there and in the mature zone are structurally different. ROSIC in DEZ displays lower threshold to OH^•, so that its activation may be already saturated in the absence of PAs. Alternatively (or in addition), binding sites for PAs and/or their coupling with a conformation that favors ROSIC, may be different in two root zones. With respect to the Ca²⁺ pump activation, it may be independent from, or coupled to the H⁺ pump activation by PAs, which will increase driving force for H⁺ import (Ca²⁺ pump exchanges 1:1 Ca²⁺ for H⁺). Besides, PAs cause a rapid induction of the NO biosynthesis.²⁶ NO in turn activates the PM H⁺ pump.²⁷ The oversimplified scheme in Figure 1 does not include this and other possible multi-step interactions between ROS and PAs, related to the regulation of ion transport across the PM. Yet the present model provides clues for the understanding of a complex Ca²⁺ response. As one can see, Ca²⁺ flux may alternate between net influx and efflux in a sophisticated way depending on the balance between (1) PAs export and catabolization; (2) formation, degradation and transport of different ROS; (3) inhibition of constitutively expressed Ca²⁺-permeable NSCCs by ROS and PAs; (4) degree of ROSIC stimulation by ROS and (5) its potentiation by different PAs; (6) existence of feedback loops (e.g., Ca²⁺ activation of the NADPH-ox); and, finally (7) ROS- and PAs- dependent Ca²⁺ pump activation.

Figure 2. Comparison of OH^•-induced Ca²⁺ and K⁺ fluxes in the pea root DEZ and mature zone and of their modulation by PAs. Ca²⁺ (A) and K⁺ (C) responses to OH^• in the DEZ. OH^• are generated by application of 1 mM CuCl₂/Na-ascorbate (Cu/A) mixture as indicated by arrows. To reveal the impact of PAs, 1 mM of spermine, spermidine or putrescine were added to the experimental chamber 10 min before the Cu/A application. K⁺ and Ca²⁺ fluxes were measured simultaneously by MIFE technique as described previously.¹⁸ Negative values correspond to ion efflux. Comparison of peak (hollow bars) and steady-state (30 min after the start of treatment; filled bars) Ca²⁺ flux responses in pea DEZ and mature root zones to the treatment with OH^•; alone or in a combination with 1 mM of a PA. Comparison of K⁺ flux responses (peak values) in DEZ and mature root zone (D). Six to seven roots were tested for each condition; data are presented as mean ± SE. The inset shows the density of a steady-state (30 min after the start of treatment) ionic current induced by OH^• alone or in a combination with 1 mM of putrescine or spermine. Currents were evaluated by means of patch-clamp technique in the whole cell mode, applied to single epidermal protoplasts, isolated from pea mature root zone.¹⁸ Briefly, current-voltage relation of the OH^• -induced current was approximated by a linear fit, and a mean current increment in a response to a 60 mV depolarization (such depolarization was registered in intact pea roots in response to 1 mM Cu/A, Pottosin and Shabala, unpublished) was estimated and divided by the whole cell capacitance (in pF). Using specific capacitance of biological membranes (~1 μF/cm²) one may calculate a conversion factor 1 pA/ pF ~100 nmol m⁻² s⁻¹ to compare K⁺ flux (by MIFE) and OH^• -induced current, mainly carried by K⁺ (by patch-clamp). Six to seven protoplasts were assayed for each of these treatments.

Glossary

Abbreviations:

DEZ

distal elongation zone

KOR

outward rectifying K⁺ channel

KIR

inward rectifying K⁺ channel

NADPH-ox

NADPH oxidase

NO

nitric oxide

NSCC

non-selective cation channels

OH^•

hydroxyl radical

PAs

polyamines

PAO

polyamine oxidase

POD

peroxidase

DAO

diamine oxidase

PM

plasma membrane

ROSIC

ROS-induced conductance

ROS

reactive oxygen species

SOD

superoxide dismutase