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Plant Signaling & Behavior 7:9, 1084-1087; September 2012; © 2012 Landes Bioscience

1084 Plant Signaling & Behavior Volume 7 Issue 9

*Correspondence to: Igor Pottosin; Email: pottosin@ucol.mx

Submitted: 06/05/12; Revised: 06/18/12; Accepted: 06/20/12

http://dx.doi.org/10.4161/psb/ 

Polyamines and ROS Generation Under Stress:

Direct Implications for the Ion Transport

There are currently many evidence suggesting that increases of PAsunder abiotic stresses conditions are not merely collateral changes

resulting from altered cell metabolism, but in many cases forma part of protective mechanism.1-3  PAs are unique polycationicmetabolites, with a net charge of +2, +3 and +4 for putrescine,spermidine and spermine, respectively. It is not surprisingly, there-fore, that in animal cells polyamines act primarily as pore blockersin a variety of cation (including K +) channels.4,5 Such direct mech-anism in plants was demonstrated so far only for vacuolar cationFV and SV channels,6 whereas the mechanism of the PAs action(mainly, inhibition, see Figure 1) on the PM cation and K +-selectivechannels is less clear.7-9 As PAs hardly affect the K +-selective tono-plast channels,10 but suppress with a high affinity Na +-permeableFV and SV channels in this membrane, PAs increases under saltstress will tend to make the overall cation conductance of the tono-

plast more K +

-selective, assisting vacuolar Na +

 sequestration andimproving cytosolic K +/ Na + ratio at the dispense of vacuolar K +.5

Drought-induced increase in spermidine facilitates stomatal clo-sure due to selective inhibition of KIR channels, whereas KOR andPM anion channels are not affected.7 Inhibition of the PM NSCCis also believed to contribute to the salt tolerance, reducing Na +

influx and, consequently, negating membrane depolarization andNa +-induced K + efflux.11 K +  loss under salt stress is also reduced

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 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.

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

Igor Pottosin,1,* Ana-María Velarde-Buendía,1 Isaac Zepeda-Jazo,1 Oxana Dobrovinskaya1 and Sergey Shabala2

1

Centro Universitario de Investigaciones Biomédicas; Universidad de Colima; Colima, México;

2

School of Agricultural Science; University of Tasmania; Hobart, Australia

Keywords: Hydroxyl radical, polyamines, plasma membrane, ion flux, ROS-induced conductance, Ca 2+ pump and abiotic stress

 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

 with a higher PM H+ pump activity, due to a better control of themembrane potential for depolarization challenges.12 Notably, PAsstimulate the PM H+ ATPase activity 13 (Fig. 1). Yet, inhibition ofcation and K + channels by PAs is not their prime action on the iontransport 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

ROS are known to accumulate during stresses, so that oxida-tive stress tolerance is a common component of a stress response.ROS may play a dual role: as toxic by-products, eventually lead-ing to the programmed cell death or as signaling molecules.15,16  Apoplast is an important site for the ROS (O

2-, H

2O

2, OH•)

formation (Fig. 1). H2O

2  and OH•  inhibit some constitutively

expressed PM channels like KOR, KIR and NSCC17,18  andactivate another non-selective current(s), permeable for Ca 2+,ROSIC (Fig. 1). In some tissues, only OH• but not H

2O

2 induces

ROSIC.18,19 ROSIC triggers stomatal closure20 and is essential forpolarized root hair growth.19 The latter is explained by the fact

that ROSIC-related cytosolic Ca 2+

 increase in a positive feedbackmanner activates key PM ROS-producing enzyme, NADPH oxi-dase, 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 scaveng-ing of ROS by PAs22 (Fig. 1). However, oxidation of putrescine

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SHORT COMMUNICATION

by OH•: active Ca 2+  efflux, mediated by PM Ca 2+  pump andnon-selective Gd3+-sensitive passive conductance, mediating Ca 2+ influx and K +  efflux (Fig. 1). The presence of Gd3+  unmaskeda continuing pump-mediated Ca 2+  efflux. Dynamic summationof Ca 2+  efflux with passive Ca 2+  influx produces a non-monot-onous net Ca 2+  flux; at high OH•  there was net Ca 2+  influx ata steady-state. In the presence of 1 mM of PAs this influx wasreduced (putrescine), cancelled (spermidine) or converted to net

Ca 2+

 efflux (spermine). Qualitatively very similar Ca 2+

 flux behav-ior was observed also in the DEZ (Fig. 2A ). Compared with themature zone, however, OH• -induced K + efflux was much fasterand had a 3-times larger peak magnitude (Fig. Two C and D).Because OH•  induces a passive conductance, which mediatesboth K + efflux and Ca 2+ influx,18 a smaller net initial Ca 2+ efflux,observed in the DEZ as compared with the mature zone (Fig. 2Aand B) may be due to the fact that OH • -induced Ca 2+ influx islarger in DEZ (as it is for K +), so that the sum of Ca 2+ efflux (nega-tive in accord with the MIFE convention) and influx (positive)becomes less negative. Another difference was that in the mature

by DAO and higher PAs by PAO in the apoplast gives rise to theincrease of H

2O

2, which can be further converted to OH• by tran-

sient metals such as iron (mainly present in reaction centers ofcell-wall bound peroxidases23) or copper (e.g., in the reaction cen-ters of DAO) (Fig. 1). Preferential DAO expression in dicots andPAO in monocots24 may contribute to the specificity of action ofdifferent PAs. Oxidation is preceded by PAs export to the apoplastvia yet unknown mechanism (Fig. 1) and leads to induction of

ROSIC and related Ca 2+

 signal in a variety of plant responses toenvironmental stimuli18  (see references therein). Our last studyrevealed two novel joint effects of PAs and ROS (OH •) on thetransport processes across the PM (regulation routes (4) and (5)in Figure 1). First, OH• and PAs stimulated Ca 2+ efflux, sensitiveto specific Ca 2+ pump inhibitor eosine yellow. Yet PAs alone andlower OH• induced only Ca 2+ efflux, whereas at higher OH• levelsan additional, slowly developing Gd3+-sensitive Ca 2+  influx, wasobserved. This Ca 2+  influx had the same kinetics and pharma-cology as simultaneously developed OH• -induced K + efflux. Weconcluded that there are at least two flux components, activated

Figure 1. Integrative regulation of cation transport across the PM by ROS and PAs. Sequence of events, leading to the generation of H2O

2 and OH• 

in the apoplast, is depicted. H2O

2 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 Ca

2+

 (Na

+

) influx and in K 

+

 loss; (2) H2O2 from theintracellular 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 catabo lization; (4) PAs and OH• activate the PM Ca2+ pump; (5) PAs sensitize ROSIC

to OH•; this potentiation is tissue-specific.

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delimited and does not require PAs catabolization by amine oxi-dases. Most likely, PAs, which by themselves induce little or no K + flux, bind to membrane components, responsible for ROSIC andsensitize them to OH•. Similar sensitization by PAs for agonists was reported for some receptor channels in animal cells.4,25 Theapparent absence of such potentiation in DEZ (Fig. 2A ) implies

zone in the presence of PAs the OH• -induced K + efflux was sig-nificantly potentiated, whereas in DEZ no significant potentia-tion was observed (Fig. 2D). Potentiation of the OH• -induced K + efflux by PAs in the mature zone can be reproduced on isolatedprotoplasts, by patch-clamp technique in the whole cell mode(Fig. 2D, inset). This result implies that the effect is membrane

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) Ca 2+ 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 wasregistered 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 capacit ance of biological membranes (~1μF/cm2) one may calculate a conversion fac tor 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.

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between net influx and efflux in a sophisticated way depending onthe balance between (1) PAs export and catabolization; (2) forma-tion, degradation and transport of different ROS; (3) inhibitionof constitutively expressed Ca 2+-permeable NSCCs by ROS andPAs; (4) degree of ROSIC stimulation by ROS and (5) its poten-tiation by different PAs; (6) existence of feedback loops (e.g., Ca 2+ activation of the NADPH-ox); and, finally (7) ROS- and PAs-

dependent Ca 2+ pump activation.

Disclosure of Potential Conflicts of Interest 

No potential conflicts of interest were disclosed.

 Acknowledgement 

Supported by CONACyT grant CB 82913 to I.P. and ARCDiscovery grant to S.S.

that ROSIC there and in the mature zone are structurally dif-ferent. ROSIC in DEZ displays lower threshold to OH•, so thatits activation may be already saturated in the absence of PAs. Alternatively (or in addition), binding sites for PAs and/or theircoupling with a conformation that favors ROSIC, may be differ-ent in two root zones. With respect to the Ca 2+ pump activation,it may be independent from, or coupled to the H+ pump activa-

tion by PAs, which will increase driving force for H+ import (Ca 2+pump exchanges 1:1 Ca 2+  for H+). Besides, PAs cause a rapidinduction of the NO biosynthesis.26  NO in turn activates thePM H+ pump.27 The oversimplified scheme in Figure 1 does notinclude this and other possible multi-step interactions betweenROS and PAs, related to the regulation of ion transport across thePM. Yet the present model provides clues for the understanding ofa complex Ca 2+ response. As one can see, Ca 2+ flux may alternate

References

1. Kusano T, Berberich T, Tateda C, Takahashi Y.Polyamines: essential factors for growth and survival.Planta 2008; 228:367-81; PMID:18594857; http://dx.doi.org/10.1007/s00425-008-0772-7.

2. Alcázar R, Altabella T, Marco F, Bortolotti C, ReymondM, Koncz C, et al. Polyamines: molecules with regula-tory functions in plant abiotic stress tolerance. Planta2010; 231:1237-49; PMID:20221631; http://dx.doi.org/10.1007/s00425-010-1130-0.

3. Hussain SS, Ali M, Ahmad M, Siddique KHM.Polyamines: natural and engineered abiotic and bioticstress tolerance in plants. Biotechnol Adv 2011; 29:300-11; PMID:21241790; http://dx.doi.org/10.1016/j.bio-techadv.2011.01.003.

4. Williams K. Interactions of polyamines with ion chan-nels. Biochem J 1997; 325:289-97; PMID:9230104.

5. Zepeda-Jazo I, Velarde-Buendía AM, DobrovinskayaOR, Muñiz J, Pottosin II. Polyamines as regulators ofionic transport in plants. Curr Topics Plant Biol 2008;9:87-99.

6. Dobrovinskaya OR, Muñiz J, Pottosin II. Inhibitionof vacuolar ion channels by polyamines. J Membr Biol1999; 167:127-40; PMID:9916144; http://dx.doi.org/10.1007/s002329900477.

7. Liu K, Fu H, Bei Q, Luan S. Inward potassium channelin guard cells as a target for polyamine regulation ofstomatal movements. Plant Physiol 2000; 124:1315-26; PMID:11080307; http://dx.doi.org/10.1104/pp.124.3.1315.

8. Shabala S, Cuin TA, Pottosin I. Polyamines pre-vent NaCl-induced K +  efflux from pea mesophyll byblocking non-selective cation channels. FEBS Lett2007; 581:1993-9; PMID:17467698; http://dx.doi.org/10.1016/j.febslet.2007.04.032.

9. Zhao F, Song CP, He J, Zhu H. Polyamines improveK +/Na +  homeostasis in barley seedlings by regulat-ing root ion channel activities. Plant Physiol 2007;145:1061-72; PMID:17905858; http://dx.doi.org/10.1104/pp.107.105882.

10. Hamamoto S, Marui J, Matsuoka K, Higashi K,Igarashi K, Nakagawa T, et al. Characterization of atobacco TPK-type K+ channel as a novel tonoplastK+ channel using yeast tonoplasts. J Biol Chem2008; 283:1911-20; PMID:18029350; http://dx.doi.org/10.1074/jbc.M708213200.

11. Chen Z, Pottosin II, Cuin TA, Fuglsang AT, TesterM, Jha D, et al. Root plasma membrane transporterscontrolling K +/Na + homeostasis in salt-stressed barley.Plant Physiol 2007; 145:1714-25; PMID:17965172;http://dx.doi.org/10.1104/pp.107.110262.

12. Zepeda-Jazo I, Shabala S, Chen Z, Pottosin II. Na-K

transport in roots under salt stress. Plant Signal Behav2008; 3:401-3; PMID:19704579; http://dx.doi.org/10.4161/psb.3.6.5429.

13. Garufi A, Visconti S, Camoni L, Aducci P. Polyaminesas physiological regulators of 14-3-3 interaction withthe plant plasma membrane H+-ATPase. Plant CellPhysiol 2007; 48:434-40; PMID:17251201; http://dx.doi.org/10.1093/pcp/pcm010.

14. Pandolfi C, Pottosin I, Cuin T, Mancuso S, ShabalaS. Specificity of polyamine effects on NaCl-inducedion flux kinetics and salt stress amelioration in plants.Plant Cell Physiol 2010; 51:422-34; PMID:20061303;http://dx.doi.org/10.1093/pcp/pcq007.

15. Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R.Reactive oxygen species homeostasis and signallingduring drought and salinity stresses. Plant Cell Environ2010; 33:453-67; PMID:19712065; http://dx.doi.

org/10.1111/j.1365-3040.2009.02041.x.16. Mittler R, Vanderauwera S, Suzuki N, Miller G,Tognetti VB, Vandepoele K, et al. ROS signaling:the new wave? Trends Plant Sci 2011; 16:300-9;PMID:21482172; http://dx.doi.org/10.1016/j.tplants.2011.03.007.

17. Köhler B, Hills A, Blatt MR. Control of guard cell ionchannels by hydrogen peroxide and abscisic acid indi-cates their action through alternate signaling pathways.Plant Physiol 2003; 131:385-8; PMID:12586862;http://dx.doi.org/10.1104/pp.016014.

18. Zepeda-Jazo I, Velarde-Buendía AM, Enríquez-Figueroa R, Bose J, Shabala S, Muñiz-Murguía J, et al.Polyamines interact with hydroxyl radicals in activatingCa(2+) and K(+) transport across the root epidermalplasma membranes. Plant Physiol 2011; 157:2167-80; PMID:21980172; http://dx.doi.org/10.1104/pp.111.179671.

19. Foreman J, Demidchik V, Bothwell JH, Mylona P,Miedema H, Torres MA, et al. Reactive oxygen spe-cies produced by NADPH oxidase regulate plant cellgrowth. Nature 2003; 422:442-6; PMID:12660786;http://dx.doi.org/10.1038/nature01485.

20. Pei ZM, Murata Y, Benning G, Thomine S, Klüsener B, Allen GJ, et al. Calcium channels activated by hydro-gen peroxide mediate abscisic acid signalling in guardcells. Nature 2000; 406:731-4; PMID:10963598;http://dx.doi.org/10.1038/35021067.

21. Takeda S, Gapper C, Kaya H, Bell E, Kuchitsu K,

Dolan L. Local positive feedback regulation determinescell shape in root hair cells. Science 2008; 319:1241-4; PMID:18309082; http://dx.doi.org/10.1126/sci-ence.1152505.

22. Das KC, Misra HP. Hydroxyl radical scavengingand singlet oxygen quenching properties of poly-amines. Mol Cell Biochem 2004; 262:127-33;PMID:15532717; http://dx.doi.org/10.1023/B:MCBI.0000038227.91813.79.

23. Kukavica B, Mojovic M, Vuccinic Z, Maksimovi V,Takahama U, Jovanovic SV. Generation of hydroxylradical in isolated pea root cell wall, and the role ofcell wall-bound peroxidase, Mn-SOD and phenolicsin their production. Plant Cell Physiol 2009; 50:304-17; PMID:19098072; http://dx.doi.org/10.1093/pcp/pcn199.

24. Moschou PN, Paschalidis KA, Roubelakis-Angelakis

KA. Plant polyamine catabolism: The state of the art.Plant Signal Behav 2008; 3:1061-6; PMID:19513239;http://dx.doi.org/10.4161/psb.3.12.7172.

25. Ahern GP, Wang X, Miyares RL. Polyamines are potentligands for the capsaicin receptor TRPV1. J Biol Chem2006; 281:8991-5; PMID:16431906; http://dx.doi.org/10.1074/jbc.M513429200.

26. Tun NN, Santa-Catarina C, Begum T, Silveira V,Handro W, Floh EI, et al. Polyamines induce rapidbiosynthesis of nitric oxide (NO) in  Arabidopsis thali-ana   seedlings. Plant Cell Physiol 2006; 47:346-54;PMID:16415068; http://dx.doi.org/10.1093/pcp/pci252.

27. Zandonadi DB, Santos MP, Dobbss LB, OlivaresFL, Canellas LP, Binzel ML, et al. Nitric oxide medi-ates humic acids-induced root development andplasma membrane H+-ATPase activation. Planta2010; 231:1025-36; PMID:20145950; http://dx.doi.

org/10.1007/s00425-010-1106-0.