Polyamines Interact With Hydroxyl Radicals

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Polyamines Interact with Hydroxyl Radicals inActivating Ca 2+ and K + Transport across the RootEpidermal Plasma Membranes 1[W]

Isaac Zepeda-Jazo 2,3 , Ana Marıá Velarde-Buendı á2 , René Enrı́ quez-Figueroa, Jayakumar Bose,Sergey Shabala, Jesu ´ s Mun ˜ iz-Murguı á, and Igor I. Pottosin*

Centro Universitario de Investigaciones Biome ´dicas, Universidad de Colima, 28045 Colima, Mexico (I.Z.-J.,A.M.V.-B., R.E.-F., J.M.-M., I.I.P.); and School of Agricultural Science, University of Tasmania, Hobart,Tasmania 7001, Australia (J.B., S.S.)

Reactive oxygen species (ROS) are integral components of the plant adaptive responses to environment. Importantly, ROSaffect 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 ux measuring techniques, we have characterized ionic currentsand net K + and Ca 2+ uxes induced by hydroxyl radicals (OH

d

) in pea (Pisum sativum) roots. OHd

, but not hydrogen peroxide,activated a rapid Ca 2+ efux and a more slowly developing net Ca 2+ inux concurrent with a net K + efux. In isolated pro-toplasts, OH

d

evoked a nonselective current, with a time course and a steady-state magnitude similar to those for a K + efux inintact roots. This current displayed a low ionic selectivity and was permeable to Ca 2+. Active OH

d

-induced Ca 2+ efux in roots wassuppressed 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 niumate inhibited OH

d

-inducedionic currents in root protoplasts and K + efux and Ca 2+ inux in roots. Contrary to expectations, polyamines (PAs) did not inhibitthe OH

d

-induced cation uxes. The net OHd

-induced Ca 2+ efux was largely prolonged in the presence of spermine, and all PAstested (spermine, spermidine, and putrescine) accelerated and augmented the OH

d

-induced net K + efux from roots. The lattereffect was also observed in patch-clamp experiments on root protoplasts. We conclude that PAs interact with ROS to alterintracellular Ca 2+ homeostasis by modulating both Ca 2+ inux and efux transport systems at the root cell PM.

Increased reactive oxygen species (ROS) productionis a common denominator of plant adaptive responsesto a large number of abiotic and biotic stresses (Mittler,2002). ROS are produced by cell wall-associated perox-idases, apoplastic diamine and polyamine oxidases,plasma membrane(PM)NADPH oxidase, and oxidasesand peroxidases in mitochondria, chloroplasts, andperoxisomes (Mahalingam and Fedoroff, 2003). A combination of increasing ROS production and limitedenergy resources to replenish the antioxidant activityresults in ROS accumulation (Taylor et al., 2004). Themost abundant types of ROS in plants are hydrogenperoxide (H 2O2) and the two free oxygen radicals,namely superoxide radical (

d

O22 ) and hydroxyl radical

(OHd

), the latter being the most reactive with biomol-ecules and structures. The lifetime of the OH

d

is only102 9 s, which implies that it acts within 1 nm from thepoint of its formation and does not cross the membrane(Mori and Schroeder, 2004). Generation of the OH

d

inroots, via the activity of intrinsic peroxidases, is aprerequisite for cell wall loosening and normal rootgrowth (Schopfer et al., 2002; Liszkay et al., 2004). Theextent of ROS accumulation is ultimately a determinantof whether ROS production is part of a signal mecha-nism (at low levels) or a harmful event (at high levels)for plants (Foyer and Noctor, 2005; Miller et al., 2010).Thus, stress-specic modulation of ROS productionand scavenging is crucial. Up-regulation of ROS-

responsive transcripts under osmotic stress was con-ned almost exclusively to shoots, whereas duringsalinity, these changes were observed almost exclu-sively in roots (Davletova et al., 2005a, 2005b; Milleret al., 2010).

Ion channels have long been considered as potentialROS targets. In the PM, H 2O2 activates the hyper-polarization-activated nonselective Ca 2+-permeablechannels (Pei et al., 2000; Demidchik et al., 2007) andinhibits outward- and inward-rectifying potassiumchannels (Kö hler et al., 2003). The induction of Ca 2+inux by H 2O2 in guard cells mediates elicitor- orabscisic acid-induced stomatal closure (Lee et al.,1999; Pei et al., 2000; Schroeder et al., 2001). OH

d

has

1 This work was supported by Consejo Nacional de Ciencia yTecnologıá (grant no. CB 82913 to I.I.P. and fellowships to I.Z.-J. andA.M.V.-B.), University of Tasmania Visiting Fellowship to I.I.P., andthe Australian Research Council (grant no. DP1094663 to S.S.).

2 These authors contributed equally to the article.3 Present address: Instituto de Biotecnologı á, Universidad Nacio-

nal Autó noma de Me´xico, Cuernavaca, Morelos 62210, Mexico.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

ndings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Igor I. Pottosin ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.111.179671

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activated a nonselective Ca 2+-permeable current in themature zone of Arabidopsis ( Arabidopsis thaliana)roots, whereas external H 2O2 does not evoke anycurrent in this tissue (Demidchik et al., 2003, 2007).The OH

d

-induced current was implicated in elonga-tion via Ca 2+ signaling (Foreman et al., 2003). Also, in

Arabidopsis roots, OH

d

was shown to activate theconstitutively expressed outward-rectifying K + cur-rent, as part of a programmed cell death scenario(Demidchik et al., 2010). Therefore, it appears thatdifferent ROS may have rather different spectra of physiological activities and differ in their effects onthe PM ion channels.

The accumulation of the polyamines (PAs) putrescine(Put 2+), spermidine (Spd 3+), and spermine (Spm 4+) isanother common component of stress responses of plants and correlates with plant stress resistance(Bouchereau et al., 1999; Walters, 2003). The up- ordown-regulation of genes involved in the biosynthesisand degradation of PAs was reported to modulate plantsensitivity to drought, salt, osmotic, oxidative, and coldstresses (Kusano et al., 2007a, 2007b; Rhee et al., 2007;Alcázar et al., 2010; Takahashi and Kakehi, 2010). Understress conditions, PAs can act either directly, as chemicalchaperones for DNA and other macromolecules, orindirectly, as positive regulators of stress responsegenes (Rhee et al., 2007). PAs normally reduce themembrane leakage induced by abiotic stresses in plants.In addition to unspecic increase of the membrane“rigidness,” due to the immobilization of negativelycharged phospholipids, more specic effects could bein work, such as activation of the antioxidant enzymes by PAs (Groppa and Benavides, 2008; Gill and Tuteja,

2010). In plants, PAs may play a dual role, as free radicalscavengers and antioxidant enzyme activators but alsoas a source of H 2O2 (Takahashi and Kakehi, 2010). If thelatter mechanism prevails, instead of preventing it, PAscould increase the oxidative damage (Mohapatra et al.,2009, and refs. therein).

Among immediate molecular targets for PAs, ionchannels and receptors are receiving growing atten-tion. In animal cells, PAs block a variety of K + andother cation-selective channels (Drouin and Hermann,1994; Ficker et al., 1994; Lopatin et al., 1994; Bä hringet al., 1997; Lu and Ding, 1999). In plants, PAs inhibitPM Shaker-type K + channels in guard, cortical, epi-

dermal, and xylem parenchyma cells (Liu et al., 2000;Zhao et al., 2007), nonselective cation channels inmesophyll and root PM (Shabala et al., 2007; Zhaoet al., 2007), as well as nonselective cation fast andslow vacuolar channels (Bru ¨ ggemann et al., 1998;Dobrovinskaya et al., 1999a, 1999b). PAs are the onlyorganic polycations that are present in sufcient quan-tities under stress to play the role of channels blockerswithout compromising cell metabolism (Alca ´zar et al.,2010). At the same time, PAs could act as cofactors inthe activation of PM H + pumps (Reggiani et al., 1992;Liu et al., 2005b; Garu et al., 2007).

If the blockage of PM K + and nonselective cationchannels were the dominant effect of PAs, it would

assist the retention of intracellular K + and the reduc-tion of Na + inux under salt stress, thus amelioratingits detrimental effects on plant ionic homeostasis(Zepeda-Jazo et al., 2008). However, recent results byPandol et al. (2010) suggested that PAs, depending ongrowth conditions or particular root zone, could either

suppress the salt-induced K+

efux or stimulate it.Such stimulation may take place via cross talk betweenPAs and ROS, while the latter are produced in theapoplast via PA catabolization by amino oxidases, asalready mentioned. Indeed, it was shown recently thatPAs may be actively exported to the apoplast and beoxidized there, generating ROS; the latter will activateCa2+ entry across the PM. Such a pathway was shownto mediate abscisic acid-induced stomatal closure inVicia fabaguard cells (An et al., 2008), the production of volatile terpenoids in lima bean ( Phaseolus lunatus )leaves (Ozawa et al., 2009), and to control pollen tubegrowth (Wu et al., 2010). Importantly, salt stress alsoprovokes the exodus of PAs into the apoplast, with itsfurther oxidation and ROS production. Depending onthe conditions, this may result in either a toleranceresponse or lead to programmed cell death (Moschouet al., 2008a). A coproduction of PAs and ROS and theirinterplay under stresses, therefore, may inuence PMion conductance in different ways, affecting in partic-ular Ca 2+ signaling.

In this work, we have studied ROS-induced Ca 2+and K + uxes and currents in pea ( Pisum sativum ) rootsand tested the effects of PAs on their kinetics. Ourresults suggest a novel mechanism, where PAs act ascofactors in the ROS induction of PM Ca 2+-permeablenonselective current and as inducers of the active Ca 2+

efux across the PM.

RESULTS

Kinetics and Pharmacology of the OHd

-Induced K + andCa 2+ Fluxes in Pea Roots

OHd

was generated by the application of copperascorbate (Cu/A; Biaglow et al., 1997; Halliwell andGutteridge, 1999). Briey, being reduced by ascorbate,copper catalyzes a sequence of one-electron reduc-tion steps from molecular oxygen to

d

O22 , from

d

O22 to

H 2O2, and nally, from H 2O2 to OHd

. The last step is

analogous to the classical Fenton reaction, where re-duced Fe 2+ is normally used instead of Cu + (Biaglowet al., 1997).

Net Ca 2+ and K + uxes induced by 1 m M Cu/Awere measured by the noninvasive microelectrodeion ux measuring (MIFE) technique from pea rootepidermis (for details, see “Materials and Methods”).The concentration of 1 m M Cu/A was selected here tomake our results comparable with previously pub-lished studies on roots (Demidchik et al., 2003, 2010;Cuin and Shabala, 2007). This treatment provoked along-lasting (more than 40-min) net K + efux frompea roots (Fig. 1A), while the Ca 2+ ux showed a morecomplex kinetics, undergoing a switch from a rapidly

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developed net Ca 2+ efux immediately after Cu/Aapplication to a net Ca 2+ inux 10 to 15 min later (Fig.1B). The application of ascorbate (1 m M) alone did nothave any signicant ( P , 0.05) effect on net ion uxes,whereas 1 m M Cu 2+ evoked a relatively small K + andCa 2+ efux (Fig. 1, C and D). As Cu 2+ alone acts as a

superoxide radical scavenger (Schopfer et al., 2002),the observed ux stimulation may be related to OHd

production, due to the reduction of Cu 2+ added byintrinsic apoplastic ascorbate. Ca 2+ and K + ux re-sponses were observed also at lower Cu/A concen-trations (see below).

The identity of ion ux components evoked by 1 m MCu/A was subjected to a pharmacological analysis.Pretreatment with the nonspecic cation channel blocker gadolinium (Gd 3+; 0.1 mM) strongly dimin-ished the development of the OH

d

-induced K + efuxand abolished Ca 2+ inux, favoring net Ca 2+ efux(Fig. 1A). Eosine yellow (EY; 0.5 m M), a specic inhibitor of the PM Ca 2+ pump (Romani et al., 2004;Beffagna et al., 2005), almost completely suppressedthe OH

d

-induced Ca 2+ efux (Fig. 1B) without a sig-nicant effect on the OH

d

-induced K + efux (Fig. 1A).Pretreatment with another uorescein derivative,erythrosine B (0.5 m M), caused a decrease of the OH

d

-induced Ca 2+ efux by 77% ( n = 10) without signicanteffects on Ca 2+ inux or K + efux ( P , 0.05). Thus, atleast two transport systems appear to mediate theobserved Ca 2+ uxes. One of them is an active Ca 2+efux system. This system is rapidly activated by OH

d

and is sensitive to EY (Fig. 1B), suggesting the Ca 2+-ATPase as a possible candidate (White and Broadley,2003). The second (slower) transport component ap-

pears to be passive, it is sensitive to Gd3+

, and it couldmediate both Ca 2+ inux and K + efux (Fig. 1, A and B).

Slower (steady-state) Ca 2+ and K + ux componentswere subjected to a further analysis, where pharmaco-logical agents were added directly to the experimentalchamber after 30 min of OH

d

treatment. Figure 2 showsthat the application of 0.1 m M Gd3+, the Ca 2+ channel blockers nifedipine and verapamil, which also block

nonselective cation channels activated by OH

d

(Demid-chik et al., 2003; Demidchik and Maathuis, 2007), and5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB)and niumic acid, which block several anionic chan-nels in plants (Roberts, 2006), all cause signicant ( P ,0.05) inhibition of K + and Ca 2+ uxes. The substantialinhibition of Ca 2+ inux by Gd 3+, nifedipine, andniumate unmasked a continuing Ca 2+ efux (Fig.2B). Therefore, both OH

d

-induced Ca 2+ efux and inuxappeared to occur at steady state, but at high (1 m M)Cu/A concentration, the inux dominates over theefux, resulting in a net Ca 2+ inux.

PAs Potentiate the OH

d

-Induced Ion Fluxes in Pea RootsIn pea roots, the concentration of free PAs ranged

from submillimolar (Spm 4+ and Spd 3+) to low milli-molar (up to 4 m M for Put 2+; Shen and Galston, 1985).Therefore, we have tested the effects of Put 2+, Spd 3+, orSpm 4+ at 1 mM concentration on the OH

d

-induced K +and Ca 2+ uxes. Similar concentrations, justied bynatural PA contents, were tested previously againstPM channels in different plant tissues (Liu et al., 2000;Shabala et al., 2007; Zhao et al., 2007). Each of threePAs added in combination with 1 m M Cu/A aug-mented and accelerated the OH

d

-induced K + efux(Fig. 3). Simultaneous application of Cu/A with either

1 mM Put2+

or Spd3+

suppressed the Ca2+

inux andslightly diminished the peak Ca 2+ efux. At the same

Figure 1. Effects of Gd3+ and EY on the OHd

-induced K+ and Ca 2+ uxes in pea roots. OH

d

radicals were generated by mixing 1 m M CuCl2with 1 mM sodium ascorbate in the bath at thetimes indicated by arrows. A, K+ uxes werestrongly suppressed in the presence of Gd 3+ (0.1mM; triangles) but were insensitive to EY (0.5 m M;squares). B, Gd3+ at 0.1 mM suppressed the OH

d

-induced Ca 2+ inux, whereas the presence of 0.5m M EY in the bath abolished the Ca2+ efux. The

sign convention “efux negative” applies to allMIFE measurements. Data are means 6 SE; n = 6,4, and 4 individual roots for control conditions(1 mM Cu/A only), Gd3+, and EY treatments,respectively. C and D, Control experiments,where 1 m M CuCl2 (Cu; white triangles) or sodiumascorbate (A; black circles) was applied sepa-rately. Data are means 6 SE; n = 3 or 4 individualroots, respectively.

Polyamines Potentiate OHd

-Induced Ion Fluxes

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time, in the presence of Spm 4+, the OHd

-induced Ca 2+efux was greatly prolonged (Fig. 4). The effects of Put 2+ or Spm 4+ alone and on the background of var-iable concentrations of Cu/A are described below.

Physiological Concentrations of Redox-Active TransientMetals Catalyze the Activation of K + and Ca 2+ Fluxes

The apoplastic space normally contains submillimo-lar concentrations of ascorbate (Pignocchi and Foyer,2003; Kukavica et al., 2009). In legume nodules, copperconcentration is 10 to 50 m M, whereas the concentrationof iron, another redox-active metal, is between 0.1 and0.7 mM (Becana and Klucas, 1992). Similar contents of

catalytically active copper and iron, equivalent toapproximately 30 m M and 0.3 m M, were found in pearoot cell walls (Kukavica et al., 2009). The productionof OH

d

by the Cu/A mixture depends only weaklyon the ascorbate concentration, within a range of 0.1 to4.0 mM, but strongly on the copper concentration(Biaglow et al., 1997). In light of the above, we havetested the effects of different copper concentrations onthe induction of K + and Ca 2+ uxes using a xed (0.1mM) concentration of ascorbate. Results shown inFigure 5A indicate that at physiological concentrations(0.01–0.1 mM) of Cu 2+, the Cu/A mixture evokeda relatively small K + efux (10–20 nmol m 2 2 s2 1);this, however, was strongly (up to 10-fold) potentiated

in the presence of Spm 4+ or Put 2+. Spm4+ alone didnot provoke a signicant K + efux (3 6 5 nmol m 2 2 s2 1),whereas Put 2+ induced a K + efux of 41 6 5 nmolm 2 2 s2 1. However, even in the latter case, the K + efuxproduced by a combination of 0.01 to 0.1 m M Cu2+ (+0.1mM ascorbate) and 1 m M Put2+ was twice as high

compared with the sum of uxes produced by Cu/Aand Put 2+ separately. The synergistic effect of PAs withROS, albeit less pronounced in relative terms, is con-served up to the highest (1 m M) Cu/A concentrationtested.

At the same time, Cu/A-induced Ca 2+ efux reachedits maximum level already at the copper concentrationof 0.1 mM (Fig. 5B). This implies a lower threshold forthe activation of the PM Ca 2+ efux system as comparedwith the induction of passive cation permeability, me-diating both K + efux and Ca 2+ inux. However, theaddition of Put 2+ or Spm 4+ alone also provoked Ca 2+

Figure 2. Pharmacology of the steady-state OHd

-induced K+ and Ca 2+

uxes in pea roots. Drugs (0.1 m M Gd3+, nifedipine, verapamil, NPPB,or niumic acid) were introduced to the bath 30 min after the inductionof the ion uxes by 1 mM Cu/A. A, Net K+ uxes. B, Net Ca2+ uxes. Ionuxes were averaged over 5-min periods immediately before (as acontrol) and after drug addition. Fluxes are plotted as means 6 SE, withthe number of individual roots for each treatment indicated in paren-theses.

Figure 3. Natural PAs potentiate OHd

-induced K+ efux in pea roots.Roots were treated either by 1 m M Cu/A alone (circles) or in acombination with 1 m M Put2+ (A), Spd3+ (B), or Spm4+ (C). Data aremeans 6 SE; n = 6 to 7 individual roots assayed for each treatment.

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transient efux, very similar in peak magnitude andduration to the maximal one produced by Cu/A.Steady-state Ca 2+ inux was observed only at thehighest (1 m M) Cu2+ concentration; it was greatlydiminished in the presence of Put 2+ and converted toa net efux in the presence of Spm 4+ (Fig. 5C).

As compared with copper, iron is a 10 times moreabundant transient metal ion in pea roots, and it alsohas a higher midpoint redox potential, so that even inthe absence of ascorbate it would be approximatelyhalf reduced and capable, therefore, of convertingH2O2 to OH

d

via the Fenton reaction (Becana andKlucas, 1992; Biaglow et al., 1997; Kukavica et al.,2009). Figure 6 shows that 0.5 m M Fe2+ provoked a sub-stantial Ca 2+ efux and less pronounced K + efux.These could be induced by Fe 2+-catalyzed conver-sion of the intrinsic H 2O2 to the OH

d

. H 2O2 alone doesnot provoke any signicant K + or Ca 2+ ux, but oncesupplemented with 0.5 m M Fe2+, it caused a massive K +efux and Ca 2+ inux.

Patch-Clamp Characterization of the OHd

-Induced IonCurrents in Pea Root Protoplasts

Most (approximately 80%) protoplasts preparedfrom the pea root mature zone displayed only small(less than 20 pA) and unspecic leak currents whenassayed in the whole-cell mode. In a few cases, proto-plasts expressed either outward-rectifying K +-selectiveor weakly voltage-dependent nonselective cation cur-rents, classied on the basis of their reversal potentials,approximately 2 70 and approximately 2 20 mV, re-spectively, as compared with E K = 2 72 mV and E Cl =+57 mV. These currents were rapidly abolished by theapplication of 1 m M Cu/A (Fig. 7). After a delay of afew minutes, a new current started to develop. Nor-

Figure 5. Effects of Put2+ and Spm4+ on the OHd

-induced K+ and Ca2+

uxes in pea roots as a function of Cu 2+ concentration. K + (A) and Ca2+

(B and C) uxes are shown with 1, 0.1, and 0.01 m M CuCl2 (+0.1 mMsodium ascorbate) only (white circles) or in a combination with 1 m MSpm4+ (black squares) or Put2+ (white triangles). Ca2+ uxes weremeasured as the average response in the rst 5 min after the applicationof treatment (peak values in B) or 30 min after the application of treatment (steady state in C). Arrows indicate the condition of zero Cu/Aadded (1 m M Spm4+ or Put2+ only). Data are means 6 SE; n = 4 to7 individual roots for each treatment.

Figure 4. Effects of PAs on the OHd

-induced Ca 2+ uxes in pea roots.Fluxes were evoked either by the addition of 1 m M Cu/A alone (circles)or in the presence of 1 m M Put2+ (A), Spd3+ (B), or Spm4+ (C). Data aremeans 6 SE; n = 6 to 7 for each treatment, except Spm 4+ (n = 14).


-Induced Ion Fluxes





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mally, this current became apparent at about 10 minafter the application of Cu/A and reached a steady-

state level after 30 to 40 min of incubation (Fig. 8A).The addition of 1 m M Put 2+ or Spm 4+ alone did notevoke any current (data not shown). When 1 m M Put 2+or Spm 4+ was added simultaneously with 1 m M Cu/A,

the current that developed in a response to ROS grewup faster and reached a larger amplitude as comparedwith the application of 1 m M Cu/A alone (Fig. 8B;Supplemental Fig. S1). PAs (Put 2+ and Spm 4+) greatlyreduced the delay in the development of OH

d

-inducedcurrent and augmented two or four times, respectively,

the maximal current magnitude (Fig. 8C).To check whether the OHd

-induced current conductsCa2+, we increased the bath CaCl 2 concentration to 20m M. If the current conducts cations (Ca 2+ in this case) better than anions (Cl 2 ), one should expect an increaseof the inward current due to the increased Ca 2+ inux,without or with a little increase of the outward one. Onthe contrary, if the current is carried mainly by anions,an increased inux of Cl 2 needs to be reected by anincrease of the outward current. As can be seen fromthe data presented in Figure 9, A and B, it was theinward current that increased by up to 100% at 2 160mV, and this could only be explained by the fact thatOH

d

-induced current mediates a substantial calciuminux. However, the OH

d

-induced current could alsoconduct other cations, even as large as tetraethylam-monium (TEA +; Fig. 9B). It should be noted that theincrease of the external salt (Ca 2+ or TEA+ plus Cl 2 )concentration had little effect on the reversal potentialof the OH

d

-induced current, which was close to zero inall cases. This behavior is consistent with a weakpreference between cations and anions for their en-trance into the pore from either membrane side, indi-cating a small difference in their relative permeability.Yet, cations appear to be conducted easier across themembrane (i.e. they display a higher absolute perme-ability), reected by a larger increment of the respec-

tive current, inward versus outward, upon theincrease of salt concentration in the bath (for the basesof selective permeability, see Hille, 2001). To verify thehypothesis of a dual cation and Cl 2 , permeability of

Figure 6. H2O 2 alone is unable to induce K+ and Ca 2+ uxes in pea

roots. K+ (A) and Ca2+ (B) uxes were recorded after the application of 5 mM H2O2 (white squares), 0.5 m M FeSO4 (white circles), or 5 mMH2O 2 after 0.5 mM FeSO4 (black circles). Data are means 6 SE; n = 5 to6 individual roots for each treatment.

Figure 7. OHd

inhibits ionic currents constitutively expressed in pea root protoplasts. After the achievement and stabilization of whole-cell conguration, a control record of currents as a response to a standard voltage steps protocol (depicted at the top) wasundertaken. Original records from the two distinct protoplasts are presented. A, Protoplast expressing outward-rectifyingpotassium current. B, Another protoplast expressing a nonselective cation current. Bottom traces show currents recorded in thesame protoplast as above 3 minafter the applicationof 1 m M Cu/A into the bath. Bath contained 5 m M and pipette contained 100mM KCl; for detailed solution compositions, see “Materials and Methods.” Dashed lines indicate zero current levels.

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2003, 2010). To verify this relation, we have analyzedthe kinetics and pharmacology of OH

d

-induced Ca 2+and K + uxes in pea roots, applying a noninvasiveMIFE technique. The results presented in Figures

1 and 2 suggest that there are at least two kineticcomponents of the OHd

-induced uxes: (1) a relativelyslow K + efux paralleled by approximately 20-foldsmaller Ca 2+ inux; and (2) a rapidly developed Ca 2+efux. This tentative component separation was fur-ther conrmed by a pharmacological analysis, as theactive Ca 2+ efux was sensitive to the Ca 2+ pump-specic inhibitors EY (Fig. 1B) and erythrosine B.Fluorescein derivatives EY and erythrosine B alsoinhibit the PM H + pump, but at 1,000-fold higherconcentrations (De Michelis et al., 1993). On the otherhand, both supposedly passive processes, K + efuxand Ca 2+ inux, were blocked by Gd 3+, nifedipine,

verapamil, niumate, or NPPB (Fig. 2). We suggested,therefore, that both K + efux and Ca 2+ inux aremediated by a nonselective passive conductance. Sev-eral lines of evidence are consistent with this sugges-tion. First, the kinetics of the OH

d

-induced currentmeasured in the whole-cell mode in patch experi-ments on individual root protoplasts was similar tothat for K + efux and Ca 2+ inux in MIFE experimentson intact roots. Second, the steady-state magnitudeof the outward (mainly carried by K +) OH

d

-inducedcurrent, 12 pA pF 2 1 at +80 mV, is equivalent to 1,200nmol m 2 2 s2 1, fairly comparable to the OH

d

-inducedK+ efux reported by the MIFE technique (Fig. 1).Third, a 10- fold increase of external Ca 2+ provoked an

increase of the inward current by 50% to 100% (be-tween 2 120 and 2 160 mV; Fig. 9B). This implies that,initially, the Ca 2+ fraction was about 5% to 10% of thetotal current, which is comparable to the relation between K + and Ca 2+ ux magnitudes in MIFE exper-iments (Fig. 1). Such a relation is to be expected for a

nonselective channel, whose relative conductance fordifferent ions is determined by the relation of theirconcentrations in experimental media, in this case,more than 1 order of magnitude higher for K + ascompared with Ca 2+. Finally, the inhibition pattern of the OH

d

-induced current in patch-clamp experiments(Fig. 10) was qualitatively similar to that obtained byMIFE for the K + efux in intact roots (Fig. 2A), withGd 3+ being the most potent blocker. One may note thatpositively charged compounds, Gd 3+ and verapamil,were more potent in MIFE experiments on intact rootsthan in patch-clamp experiments on isolated proto-plasts. A similar quantitative difference was reported before by Demidchik and coworkers (2003). This dif-ference may be expected due to a lower ionic strengthin MIFE experiments, which tends to increase thenegative surface potential and the local concentrationof cations; surface potential also may be reduced dueto the removal of cell walls upon protoplast isolation.

In studies on plant cells, no selective blockers weredeveloped against any type of ion channel. Moreover,some blockers that were previously considered to berelatively selective (e.g. dihydropyridines such as ni-fedipine or phenylalkylamines such as verapamil,which block voltage-dependent Ca 2+-selective chan-nels in animal cells) have different targets in plantcells. These block several types of nonselective Ca 2+-

permeable channels and even some outward-rectifyingK+ channels (Demidchik and Maathuis, 2007). There-fore, one may not rely on a single blocker and needs totest a variety of broad-spectrum inhibitors.

The effects of TEA +, Gd3+, and verapamil on theOH

d

-induced current reported here were qualitativelysimilar to those reported for the Arabidopsis rootmature epidermis (Demidchik et al., 2003), althoughwith somewhat lower afnity. An apparent lack of cation/anion selectivity of the OH

d

-induced currentforced us to test additionally some anionic pore blockers; these were proved to be equally efcient(Figs. 2 and 10). Yet, a stilbene derivative, 4,4-diiso-

thiocyanostilbene-2,2-disulfonate, irreversibly modi-fying and inhibiting some anion channels andtransporters, was inefcient in our case. No signicantinhibition of OH

d

-induced current was found for eitherruthenium red (a known inhibitor of Ca 2+ uniporterand a variety of Ca 2+ and Ca 2+-permeable channels) oramiloride (an inhibitor of cation transporters and somenonselective cation channels).

A poor ion selectivity of the OHd

-induced currentand its sensitivity to both cation and anion channelinhibitors (Figs. 9 and 10) seems surprising at rstglance. Yet, there are multiple reports on plant PMchannels with a low cation-to-anion selectivity (forreview, see Demidchik et al., 2002; for experimental

Figure 10. Pharmacology of the OHd

-induced ion currents in pea rootprotoplasts. Relative ion currents in the presence of Gd 3+ (0.1 mM),nifedipine (0.1 m M), verapamil (0.1 mM), quinine (0.5 mM), NPPB (0.1mM), or niumate (0.1 mM) in the bath are shown. Negative (black) andpositive (white) bars (means 6 SE; n = 3–8 protoplasts for eachtreatment) correspond to currents measured at 2 160 mV and +80mV, respectively; control-specic currents ( n = 25) at these potentialswere 2 29 6 1 and 12 6 1 pA pF2 1, respectively.

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evidence, see Zepeda-Jazo et al., 2008). When it comesto OH

d

-induced currents in the root PM, it should benoted that they were never rigorously tested for cationover anion selectivity, as these currents were onlyassayed under pseudosymmetric ionic conditions, inthe absence of steep salt gradients across the mem-

brane. Under these ionic conditions, reversal potentialvalues were close to zero, which was a compromise between equilibrium potential values for Cl 2 , mono-valent, and divalent cations (Foreman et al., 2003). Thisobservation might imply a signicant anion permeability of the OH

d

-induced currents, not only a weakdiscrimination between different cations. Indeed, un-der the ionic conditions of the experiment presented inFigure 9C, the reversal potential of the whole-cellcurrent was substantially different from zero, butagain, it was in the middle between equilibrium po-tentials for K + and Cl 2 . Besides, substitution of the 90%of intracellular Cl 2 with a large nonpermeable anion(HEPES) unraveled a contribution of the Cl 2 inux tothe inward current, which dramatically decreasedwithout a signicant change of the outward one. Onthe contrary, the H 2O2-activated Ca

2+-permeable chan-nels in guard cells and the root elongation zonedisplay a somewhat better selectivity among cations(e.g. lacking permeability for TEA +) and a clear pref-erence of cation over Cl 2 (Pei et al., 2000; Demidchiket al., 2007). As shown previously (Demidchik et al.,2007) and in this paper (Fig. 6), H 2O2-activated chan-nels are not present in the mature root zone, at leastin the plant species studied. Thus, two different ROSspecies, OH

d

and H 2O2, activate distinct Ca2+-permeable

channels in the root PM.

Physiological Concentrations of Copper and Iron CanCatalyze OH

d

Production, Which Induces a SubstantialK+ Leak

The ion uxes and currents discussed so far wereinduced by high (1 m M) Cu/A concentration. Al-though even higher Cu/A concentrations were usedto demonstrate the role of OH

d

in cell wall looseningand stretching during the elongation process (Schopfer,2001) and equivalent concentrations were applied forthe activation of Ca 2+-permeable channels in roots

(Demidchik et al., 2003; Foreman et al., 2003), theeffects of lower Cu/A concentrations need to be stud-ied to reveal the thresholds for OH

d

-activated currents.Demidchik and coworkers (2010), using long-livingOH

d

-specic spin-trap 5,5-dimethyl-1-pyrroline- N -oxide, have shown that production of OH

d

by 1 mMCu/A in pure solution without plants was approxi-mately equivalent to that produced by intact Arabi-dopsis roots subjected to 100 m M NaCl. However, inthe presence of living roots naturally producing H 2O2,the generation of OH

d

induced by 1 m M Cu/A in-creased severalfold and was equivalent to 3- or 5-foldof its production upon the application of 250 or 100 m MNaCl, respectively. Extreme (several hundred micro-

moles) and lethal OHd

production occurs in illumi-nated photosynthetic tissues treated with herbicides(paraquat) or upon the inhibition of ascorbate perox-idase (Babbs et al., 1989; Burkhard and Heber, 1996).However, even a much lower physiological (10 m M)concentration of copper reduced by ascorbate is able to

generate 2 m mol of OH

d

within 10 min (Biaglow et al.,1997). In biological systems, not only total OHd

gener-ation but also the location of the production site withrespect to the target molecules and scavenging mecha-nisms existing within this locality is important. Ourdata imply that at up to 0.1 m M Cu/A, only small K +efux resulted from the mature zone of pea roots(Fig. 5A). However, this efux was strongly (up to1 order of magnitude) potentiated by PAs, reaching100 to 200 nmol m 2 2 s2 1 (see discussion of the mecha-nism of PA action below). On the other hand, iron(another redox-active transient metal capable of generating OH

d

via the Fenton reaction) is present atpea cell walls at 10-fold higher concentrations thancopper, mainly as a part of peroxidase reaction centers(Becana and Klucas, 1992; Kukavica et al., 2009).Moreover, on an equimolar iron basis, the peroxidaseiron is a 1 to 2 orders of magnitude better Fentonreaction catalyst as compared with the inorganic ironor Fe-EDTA (Chen and Schopfer, 1999). In our exper-iments, inorganic iron alone at 0.5 m M concentrationprovoked a relatively large (up to 100 nmol m 2 2 s2 1) K+efux, which grew severalfold upon the application of millimolar H 2O2 (Fig. 6A). For a comparison, K

+ efuxfrom the root mature zone, induced by 50 to 100 m MNaCl, was (in nmol m 2 2 s2 1) 100 to 200 for Arabidop-sis, 100 for maize ( Zea mays; Pandol et al., 2010), 100

to 400 for barley ( Hordeum vulgare) varieties differentin their salt tolerance (Chen et al., 2007), and about 50on average for different wheat ( Triticum aestivum)cultivars (Cuin et al., 2008). Therefore, physiologicalconcentrations of copper (supplemented by PAs) andiron are capable of catalyzing OH

d

production suf-cient to induce quite a signicant K + leak from pearoots persisting tens of minutes.

PAs Potentiate the OHd

-Induced PMPassive Conductance

The kinetics of the OH

d

-induced cation currents andK+ uxes across the PM were further modulated byPAs. PAs are present at high, submillimolar to milli-molar, concentrations in pea roots. Spm 4+ and Spd 3+are more abundant near root apices, whereas Put 2+is almost equally distributed between the root baseand apex (Shen and Galston, 1985). When it comes tothe known effects of PAs on passive membrane con-ductance, only the inhibitory effects, either direct orindirect, on K +-selective and nonselective cation chan-nels were documented for plants so far (Bru ¨ ggemannet al., 1998; Dobrovinskaya et al., 1999a, 1999b; Liuet al., 2000; Shabala et al., 2007; Zhao et al., 2007). Thus, benecial roles of PAs during stresses were discussed


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in this context. As an example, the PA contribution tosalt stress tolerance in plants was explained on the basis of their inhibition of Na + inux, reducing theconsequent membrane depolarization and K + efux(Shabala et al., 2007; Zhao et al., 2007; Zepeda-Jazoet al., 2008). However, our recent study (Pandol et al.,

2010) showed that PAs, in particularly Spm4+

, depend-ing on the growing condition and root zone, canreduce NaCl-induced K + efux, cause no change, oreven stimulate it. Clearly, for explanations of theeffects of externally applied PAs in vivo, one needsto consider not only the effects of PAs per se but also of PA metabolization associated with ROS generation aswell as their roles in ROS scavenging as early effects.In particular, all natural PAs are powerful OH

d

scav-engers, at concentrations above 1.5 m M eliminatingvirtually all OH

d

produced by a mixture of 0.2 m MH2O2 with 0.04 m M iron (Das and Misra, 2004). On theother hand, oxidation of Spm 4+ and Spd 3+ by poly-amine oxidase, and Put 2+ by diamine oxidase, leads tothe formation of H 2O2 (Moschou et al., 2008b), whichcould be further converted to OH

d

. External applica-tion of PAs to roots mimics their export to apoplast,which occurred in response to different environmen-tal cues, and is associated with a stimulation of Ca 2+inux by ROS, generated as a result of PA oxidation(An et al., 2008; Moschou et al., 2008a; Ozawa et al.,2009; Wu et al., 2010). In addition to the Ca 2+ inux,overall PM leakage could increase, also leading to K +loss. Thus, catabolization of PAs and ROS genera-tion may outweigh their roles as ROS scavengers,activators of the antioxidant enzymes, and micro-somal NADPH oxidase inhibitors (Papadakis and

Roubelakis-Angelakis, 2005; Mohapatra et al., 2009;Takahashi and Kakehi, 2010). Increase of ROS pro-duction due to PA catabolization may indeed takeplace in intact pea roots. Indirect evidence for this isthat Put 2+ alone provoked a much higher K + efux ascompared with Spm 4+ (Fig. 5A), which may reect amuch higher apoplast expression of diamine oxidaseas compared with polyamine oxidase, in the apoplastof dicots, particularly pea (Moschou et al., 2008b). Thecontribution of such a mechanism should be ruled out,however, in the case of isolated protoplasts, becauseapoplastic amine oxidases are washed out upon theprotoplast isolation procedure (Kaur-Sawhney et al.,

1981). Besides, we have not observed any membranecurrent stimulation by treatment with PAs alone. Still,PAs may act as OH

d

scavengers, but this could onlypreclude or handicap the development of OH

d

-induced ionic currents. Therefore, exogenous PAscould stimulate the induction of ionic currents inprotoplasts by OH

d

only if PAs are acting as cofactorsin this process. To the best of our knowledge, thispossibility has not been considered in the literature sofar. However, it was reported that PAs may stabilizethe binding of other cofactors essential for the ionchannel activity, such as PIP 2 for Kir and plant ShakerK+ channels (Liu et al., 2005a; Xie et al., 2005). Thus, theprecise biophysical mechanisms of this synergism

between PAs and ROS interaction warrant a separateinvestigation.

PAs and OHd

Activate Ca 2+ Pumping in Intact Roots

Another intriguing nding of this study is the acti-

vation of active Ca2+

efux across the root epidermis by OHd

and PAs (Figs. 1B, 4, and 5). This ux compo-nent has a substantially lower threshold for its activa-tion by OH

d

as compared with the K + efux (Fig. 5,A and B). Stimulation of the active EY-sensitive Ca 2+efux across the PM by ROS was reported in a singlestudy on an Arabidopsis cell culture (Romani et al.,2004). It was shown that ROS generated by oligoga-lacturonide treatment assisted Ca 2+-ATPase activation by calmodulin. However, in that paper, ROS inducednet Ca 2+ inux rst, and net Ca 2+ efux was measuredonly in 10 to 20 min; this is exactly the opposite ordercompared with the OH

d

-induced Ca 2+ ux kinetics inour work (Figs. 1B and 4). On the other hand, there is alarge body of data on animal PM and sarcoplasmicreticulum Ca 2+ pumps showing their inhibition byROS, which may originate from protein cross-linking,lipid peroxidation, and concurrent inhibition by oxi-dized forms of the calmodulin (for review, see Waring,2005). These effects develop slowly (hours), whichmay preclude their observation under the conditionsof our study.

At the same time, Put 2+ and Spm 4+ evoked Ca 2+efux that was very similar to that induced by OH

d

.One possibility is that this efux was actually caused by ROS, generated during PA catabolization. Alterna-tively/additionally, PAs in principle can activate PM

Ca2+

-ATPase in one of the ways they affect anotherP-type pump, H +-ATPase. These involve the PA-induced promotion of the interaction of 14-3-3 proteinswith the autoinhibitory domain at the H +-ATPase Cterminus (Garu et al., 2007) and the activation of the H +pump via a nitric oxide-dependent pathway (Tun et al.,2006; Arasimowicz-Jelonek et al., 2009; Zandonadi et al.,2010). Finally, if PAs activate the H + pump, they couldcause the stimulation of Ca 2+ pumping across the PMvia a coupled mechanism, as PM Ca 2+-ATPase exportstwo Ca 2+ ions in the exchange for the two imported H +ions (Beffagna et al., 2005).

Implications of PA and OHd

Effects on IntracellularCa 2+ Homeostasis

Irrespective of the precise mechanism of ROS andPA early effects on Ca 2+ uxes across the root epider-mis PM, some consequences for Ca 2+ homeostasisand signaling could be drawn from our experimen-tal data. Moderate OH

d

production results in a tran-sient increase of Ca 2+ pumping, outweighing theOH

d

-induced passive Ca 2+ inux (Fig. 5, B and C).The resultant depletion of the intracellular Ca 2+ poolshould reduce the Ca 2+-dependent PM NADPH oxi-dase activity in a feedback manner (Takeda et al.,2008), thus diminishing ROS generation. Membrane-

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bound NADPH oxidase is an electrogenic enzyme thattransports an electron from the interior to the exteriorof the cell upon oxygen reduction, and it is stimulated by membrane hyperpolarization. The latter may begenerated by an increased PM pump activity. As anexample, activation of the H +-ATPase by fusicoccin

results in higher OH

d

production (Liszkay et al., 2004).However, activation of the nonselective ion conduc-tance by OH

d

(Fig. 9) would tend to depolarize themembrane, although opposed by enhanced H + andCa2+ pumping as a response to the depolarization. ThePM H + pump is also activated by hyperosmotic stressor salt. This increases cytosolic pH, which leads to alower synthesis of NADPH, thus decreasing its avail-ability for NADPH oxidase (Beffagna et al., 2005).And, as mentioned in a previous section, the H + pumpmay be activated by PAs. As Ca 2+-ATPase operates as a1:1 Ca2+:H+ exchanger, it might be of secondary rele-vance which enzyme, H +- or Ca2+-ATPase, is theprimary target for OH

d

/ PA, while the activity of both pumps will be mutually coupled via the H +circuit. Thus, most likely, under conditions of moder-ate OH

d

production, the joint activity of H + and Ca 2+pumps would tend to diminish ROS production bythe PM NADPH oxidase. Oscillations of intracellularH+ and Ca 2+, in parallel with oscillations of ROSproduction levels, may also be expected under theseconditions.

At strong oxidative stress, here experimentally pro-voked by 1 m M Cu/A, in the absence of PAs, net Ca 2+inux was observed at steady state (after 30 minof incubation; Figs. 1B and 5C). However, on the background of PAs (Figs. 4 and 5C), it is either largely

diminished (Put2+

) or reverted to an efux (Spm4+

).The direction of the net Ca 2+ ux at any time is dened by the sign of the algebraic sum of Ca 2+ efux andinux, which are generated by distinct PM transportsystems. As can be seen from Figures 3 and 8C, atlonger incubation times, Put 2+ stimulates the OH

d

-induced passive K + efux (and, presumably, also theassociated Ca 2+ inux) more than Spm 4+ does. At thesame time, active Ca 2+ efux observed in the presenceof Put 2+ and Spm 4+ is approximately equal (Fig. 5B).Thus, at longer incubation times, one may expect arelatively higher net Ca 2+ efux in the presence of Spm 4+ as compared with Put 2+, which is indeed the

case (Fig. 4). Thus, the ratio between different PAs isimportant to dene the direction of a net Ca 2+ uxacross the PM. In the pea root mature zone, Put 2+ isaccumulated at a higher concentration than the sumof Spd 3+ and Spm 4+ concentrations (Shen and Galston,1985), although in the apoplast of pea and other dicots,the activity of diamine oxidase dominates over theactivity of polyamine oxidase (Moschou et al., 2008b).PA concentration patterns are known to be not onlyspecies, tissue, and age specic but also stress specic(Alcá zar et al., 2010). As an example, salt stress shiftsthe distribution in favor of higher PAs, Spm 4+ andSpd 3+, at the expense of Put 2+, even though the totalPA concentration changes are not very dramatic. Yet,

plants opposing this tendency (i.e. maintaining ahigher Put 2+/Spm 4+ ratio) turn out to be more salttolerant and, importantly, to better accumulate cat-ions, including Na +, as required for the osmoticadjustment (Zapata et al., 2008). Among other expla-nations, this might be related to the fact that Put 2+

could support a longer lasting potentiation of the OH

d

-induced passive conductance (Fig. 3A) and could also,in contrast to Spm 4+ and Spd 3+, allow some net Ca 2+inux at steady state (Fig. 4).

Summarizing, our study demonstrates that lowerOH

d

concentrations induce Ca 2+ pumping, while higherOH

d

levels activate also a passive Ca 2+ uptake across theroot PM. The overall direction of the net Ca 2+ ux isfurther modulated by natural PAs in a species-specicmanner. Thus, stress-induced changes in OH

d

produc-tion and in the levels of different PAs may be translatedinto intracellular Ca 2+ changes.

MATERIALS AND METHODS

Plant Material

Pea (Pisum sativum ‘Greenfeast’) seeds were surface sterilized (full-strengthcommercial bleach for 30 min) and thoroughly rinsed with distilled water.Seeds were germinated in a dark growth cabinet at +24 C in two layers of wetpaper in petri dishes for 2 to 3 d. Uniformly germinated seedlings wereselected and transferred to a bubbled hydroponic culture unit comprising a3-L plastic container over which seedlings were suspended on a plastic grid sothat their roots were almost completely immersed in the growth solution (0.5mM KCl and 0.1 m M CaCl 2). Aeration wasprovidedby oneaquariumair pumpvia exible plastic tubing. Seedlings were grown under constant (+24 C)conditions in a lighted growth cabinet until 5 d old. Roots of 8 to 10 cm longwere used for current and ux measurements.

Measurements of Ion Fluxes

Net K + and Ca 2+ uxes were measured using the noninvasive MIFEtechnique (University of Tasmania innovation). The principles of the MIFEmeasurements, microelectrode fabrication, and calibration are available inprevious publications (Shabala et al., 2001, 2007), and the theory of the MIFEmeasurements is available elsewhere (Newman, 2001; Shabala et al., 2006a).During experiments, pea seedlings were placed in 30-mL measuring cham- bers. Their roots were immobilized in a horizontal position as describedelsewhere (Cuin and Shabala, 2005) and preincubated in a new solutioncontaining 0.5 m M KCl, 0.1 mM CaCl2, 5 mM MES, and 2 m M Tris base, pH 6.0,for 1 h. Normally, two ion-selective microelectrodes, one for K + and anotherfor Ca 2+, were used in the same experiment. During measurements, electrodeswere moved between positions M1 and M2, 50 and 150 m m from the rootsurface, respectively, in the mature zone (approximately 15–20 mm from thetip). OH

d

was generated by the addition of CuCl 2 (0.01–1 mM) plus sodium

ascorbate (0.1–1 m M) into the chamber. PAs (Spm 4+, Spd 3+, and Put 2+) wereadded simultaneously with Cu/A mixture, or individually. Net K + and Ca 2+

uxes were measured for over 40 min after the treatment. Different blockerswere added 10 min before the Cu/A treatment (EY, erythrosine B, Gd 3+) or 30min later (Gd 3+, nifedipine, NPPB, niumate) as indicated.

Isolation of Pea Protoplasts from the Root Mature Zone

The isolation of root protoplasts was developed based on the previouslydescribed protocols used for mesophyll protoplasts (Demidchik and Tester,2002; Shabala et al., 2006b) using the same modications as described by Chenet al. (2007). Exodermal cylinders of root segments of pea seedlings, aftermechanical separation of steles, were placed into 3 mL of the enzyme solutioncontaining 2% (w/v) cellulase (Yakult Honsha), 1.2% (w/v) cellulysin(Calbiochem), 0.1% (w/v) pectolyase, 0.1% (w/v) bovine serum albumin, 10mM KCl, 10 mM CaCl2, and 2 m M MgCl 2, pH 5.7, adjusted with 2 m M MES, and


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osmolality was adjusted to 750 mosmol with sorbitol. After 40 min of incubation in the enzyme solution (in the dark at 30 C; agitated on a 90-rpmrotary shaker), root segments were transferred to the so-called wash solution(as above, minus enzymes) and thoroughly washed for another 2 min.Segments were then transferred into the measuring chamber lled with asolution containing 5 m M KCl, 2 mM CaCl2, 0.5 mM MgCl 2, and 2 m M MES-KOH, pH 5.7, with osmolality of 650 mosmol adjusted by D-sorbitol. By gentlyshaking, protoplasts were released into the measuring chamber used for

patch-clamp experiments (see next section). Once the protoplasts were re-leased, we washed them with EDTA-bath solution (the same as release-bathsolution plus 5 m M EDTA) to clean the cell membrane surface and thenwashed them again with release-bath solution alone. The origin of releasedprotoplasts was veried by size distribution, as within a mature root zone theaverage cortical cell is about twice as large in diameter as an epidermal one(Rost et al., 1988). The mean diameter for protoplasts used in patch-clampexperiments was 11.5 m m, which coincided with a position of a peakcorresponding to a fraction of the smallest protoplasts in our preparation(mean diameter of 11.2 m m, close to the value reported for pea protoplastsfrom the root epidermis); due to some overlapping of peaks corresponding todifferent protoplast fractions (Supplemental Fig. S2), one may not excludesome contribution of cortical protoplasts into those studied by patch clamp,with a probability of more than 60% that the patched protoplasts originatedfrom the epidermis.

Patch-Clamp ExperimentsMeasurements on individual pea root protoplasts were made using an

Axopatch 200 patch-clamp amplier (Axon Instruments) in the conventionalwhole-cell conguration as described by Shabala et al. (2006b). The basicpipette solution contained (in m M) 100 KCl,3 MgCl 2, 0.8 CaCl2, 2 K2EGTA, and5 HEPES-KOH, pH 7.4, osmolality of 650 mosmol adjusted with D-sorbitol.Membrane potentials were clamped at 2 100 mV throughout the experiments,and voltage pulses were applied in 20-mV steps, from 2 140 or 2 160 mV to+40 or +80 mV. A typical access resistance was between 12 and 30MV , compensated by 60% to 70% using the Axopatch 200 compensationcircuit, and the whole-cell capacitance was in the range between 2 and 6 pF. Tostimulate the production of OH

d

, 1 mM CuCl 2 and sodium ascorbate weremixed directly in the experimental chamber. PAs (Spm 4+ or Put 2+) were addeddirectly into the bath solution to give a nal concentration of 1 m M. Inpharmacological assays, the inhibitors Gd 3+ (0.1 mM), nifedipine (0.1 m M),

verapamil (0.1 m M), quinine (0.5 m M), amiloride (1 m M), ruthenium red (0.1mM), and NPPB and niumate (both 0.1 m M) were added directly into the bath. In selectivity measurements, external solutions with 20 m M CaCl2 orTEA-Cl were applied by bath perfusion. Anion (Cl 2 ) permeability wasveried by substitution of 100 m M Cl2 in the patch pipette with 100 m MK-HEPES (100 mM KOH plus 244 m M HEPES, pH 7.4). In the latter case, thenecessary correction for liquid junction potential between the pipette and the bath was made using the JPCalc program by P.H. Barry (University of NewSouth Wales). All chemicals were of analytical grade, purchased from Sigma.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Development of the OHd

-induced current isaccelerated in the presence of spermine.

Supplemental Figure S2. Smallest, mostly of epidermal origin, protoplastsare selected for patch-clamp assays.

Received May 6, 2011; accepted October 3, 2011; published October 6, 2011.

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