FEBS Letters 581 (2007) 1993–1999

Polyamines prevent NaCl-induced K+ efflux from pea mesophyllby blocking non-selective cation channels

Sergey Shabalaa,*, Tracey A. Cuina, Igor Pottosinb

a School of Agricultural Science, University of Tasmania, Hobart, Tas 7001, Australiab Biomedical Center, University of Colima, 28045 Colima, Mexico

Received 23 March 2007; revised 12 April 2007; accepted 13 April 2007

Available online 24 April 2007

Edited by Julian Schroeder

Abstract Despite numerous reports implicating polyamines inplant salinity responses, the specific ionic mechanisms of poly-amine-mediated adaptation to salt-stress remain elusive. In thiswork, we show that micromolar concentrations of polyaminesare efficient in preventing NaCl-induced K+ efflux from the leafmesophyll, and that this effect can be attributed to the inhibitionof non-selective cation channels in mesophyll. The inhibition byexternally applied polyamines developed slowly over time, sug-gesting a cytosolic mode of action. Overall, we suggest that ele-vated levels of cellular polyamine may modulate the activity ofplasma membrane ion channels, improving ionic relations andassisting in a plant’s adaptation to salinity.� 2007 Published by Elsevier B.V. on behalf of the Federation ofEuropean Biochemical Societies.

Keywords: Potassium; Salinity; Membrane; Putrescine;Spermine; Ion transport

1. Introduction

Polyamines (putrescine, spermidine, and spermine) are plant

growth regulators, critical for a number of developmental pro-

cesses, including cell division, somatic embryogenesis, root

growth, floral initiation, and flower and fruit development

[1,2]. Despite some significant variations between species, the

reported estimated levels of free concentrations of polyamines

(PA) in plant cells are as follows: Spm4+, from a few, up to tens

of micromolar; Spd3+, tens to hundreds of micromolar; and

Put2+, up to several millimolar [1–4]. In addition to their role

in plant development, PA may also play an important role in

plant stress responses. Several fold increases in the level of free

PA in plant cells in response to a variety of abiotic stresses,

including salinity, have been reported [1,5,6]. Furthermore,

since the early 1990s, it has been demonstrated that salt-resis-

tant plant varieties contain higher polyamine levels under

stress conditions [7,8] and more recent work has shown that

the application of exogenous PA can improve the adaptation

of plants to salt-stress [9,10]. Overexpression of genes for

ADC or ODC, two key enzymes involved in putrescine biosyn-

Abbreviations: KOR, potassium outward-rectifying channel; NSCC,non-selective cation channel; Vr, reversal potential

*Corresponding author. Fax: +61 3 6226 2642.E-mail address: Sergey.Shabala@utas.edu.au (S. Shabala).

0014-5793/$32.00 � 2007 Published by Elsevier B.V. on behalf of the Feder

doi:10.1016/j.febslet.2007.04.032

thesis, improves plant salt-tolerance in tobacco [11] and oat

[12]. In addition, increased salt-tolerance was reported in

plants over-expressing SAMDC (S-adenosylmethionine decar-

boxylase; [13]) and SPDS (spermidine synthase; [14]), while

loss-of-function Arabidopsis mutants deficient in polyamine

biosynthesis, showed a higher sensitivity to salt-stress [15,16].

At the same time, salt-induced injury in the adc2-1 mutant

was partly reverted by putrescine addition [17]. All this points

towards the importance of PA in adaptive mechanisms against

salt-stress. However, the specific ionic mechanisms of PA-med-

iated adaptation to salinity remain elusive.

One plausible hypothesis is that elevated polyamine levels in

the cell cytosol modulate the activity of plasma membrane ion

channels in plant tissues, thus improving ionic relations and

assisting plants in their adaptation to salinity. Some evidence

supporting this hypothesis comes from the animal literature.

Polyamines are organic polycations, protonated at a physio-

logical pH, which can potentially interact with a variety of

cellular targets including nucleic acids and proteins. In recent

years, specific interactions of polyamines with a number of dif-

ferent types of ion channels, including KIR inward-rectifying

K+ channels in animal tissues, have been reported [18–21]. In

plant systems, cytosolic polyamines have been shown to be effi-

cient inhibitors of both slow (SV) and fast (FV) vacuolar cat-

ion channels [22,23] as well as potent blockers of inward K+

currents across the plasma membrane of guard cells [5]. How-

ever, none of these channels are considered to be major players

in mediating plant adaptive responses to salinity [24,25].

One of the ‘‘hallmark’’ cellular responses to salt-stress is a

massive K+ efflux, observed in both root [26–30] and leaf

[27,31–33] tissues. Such K+ efflux is initiated within seconds

of the acute NaCl stress and may last for several hours

[28,29], reducing the intracellular K+pool [34,35], and signifi-

cantly impairing cell metabolism. Consistent with the key role

of K+ homeostasis in salt-tolerance mechanisms [24], a reduc-

tion of K+ efflux correlates with increased salt-tolerance

[28,29,34]. Electrophysiological and pharmacological studies

have suggested that NaCl-induced K+ efflux across the plasma

membrane is mediated by at least two transport systems,

namely outward-rectifying K+ permeable channels (KOR)

and non-selective cation (NSCC) channels [27,36]. It has also

been shown that various substances known to ameliorate the

detrimental effects of salinity on plants (such as supplementary

Ca2+ or compatible solutes), are efficient in preventing NaCl-

induced K+ efflux [26,30,31,36,37]. Is it possible that polyam-

ines play the same role?

The above question was addressed by a comprehensive

electrophysiological study of the effects of polyamine on

ation of European Biochemical Societies.

1994 S. Shabala et al. / FEBS Letters 581 (2007) 1993–1999

NaCl-induced K+ fluxes and currents from pea leaf mesophyll

tissues (using the non-invasive ion flux measuring (the MIFE)

technique) and protoplasts isolated from these tissues (using

the patch-clamp technique). We report the presence of NSCC

in pea mesophyll tissue and show that polyamines are efficient

in preventing K+ efflux from mesophyll cells, most likely by

blocking these NSCC. Both MIFE and patch-clamp experi-

ments show a clear time-dependency of the polyamine effect

on K+ transport across the cell membrane, suggesting a cyto-

solic mode of action.

mol

m-2

s-1

100 NaCla

-200

-100

0

100

2. Materials and methods

2.1. Plant materialSeeds of the Argenteum mutant of Pisum sativum L. were grown in a

standard potting mix [38] in a glasshouse (16 h/8 h light/darkness) for20–30 days. Fully unfolded, but still growing leaves were used forexperiments.

2.2. Potassium flux measurementsThe leaf epidermis was gently removed, and mesophyll segments of

about 5 · 7 mm were cut and left floating in a shallow Petri dish filledwith nutrient solution (0.1 mM CaCl2 + 0.2 mM KCl) for severalhours, essentially as described elsewhere [32,38]. Various concentra-tions of polyamines (as chloride salts) were added at various times dur-ing pre-incubation to certain samples. One hour prior to measurement,mesophyll segments were immobilized in a Perspex holder, then placedinto a measuring chamber filled with the appropriate solution. Netfluxes of K+ were measured using the non-invasive ion-selective vibrat-ing microelectrode (the MIFE) technique (University of Tasmania,Hobart), generally as described by in our previous publications[33,36,39], and calculated from the measured differences in electro-chemical potential for each ion between these two positions as de-scribed earlier [39,40].

Time, min

Net

K+

flux,

n

0

50

100

150

200

250

300

350

400

Control 10 min 2h 4h

Pea

k ef

flux,

nm

olm

-2s-1

Pre-incubation time

b

-600

-500

-400

-300

0 10 20 30 40 50

Control

Put2+

Spm4+

Fig. 1. (a) Transient K+ fluxes measured from pea mesophyll tissue inresponse to 100 mM NaCl treatment from control samples and fromleaf segments pre-incubated in 1 mM of either Put2+ or Spm4+ for 2–3 h. Results for Spd3+ were in between those for Spm4+ and Put2+ (notshown). Means ± S.E. (n = 6–8 leaf segments isolated from 4 to 5different leaves for each treatment). (b) The peak magnitude of NaCl-induced K+ efflux from pea mesophyll segments pre-incubated in1 mM Put2+ for various amounts of time before adding 50 mM NaCl.Mean ± S.E. (n = 4–7). The sign convention is ‘‘efflux negative’’ for allMIFE measurements.

2.3. Protoplast isolationProtoplasts from leaf mesophyll cells were isolated from plasmoly-

sed leaves using a procedure adapted from [27]. Briefly, the adaxial leafepidermis was removed with forceps. Peeled leaves were floated(peeled-side down) on enzyme solution (2% (w/v) Cellulase OnozukaRS [Yakult Honsha, Tokyo, Japan], 1.2% (w/v) cellulysin [CalBio-chem, Nottingham, UK], 0.1% (w/v) pectolyase Y-23 [Yakult Honsha,Tokyo, Japan], 0.1% (w/v) bovine serum albumin [Sigma], 10 mMKCl, 10 mM CaCl2, 2 mM MgCl2, 2 mM MES, pH 5.7 adjusted withTris base, 760 mOsm adjusted with DD-sorbitol) at 28–30 �C and 90 rpmrotation (an orbital shaker). After 12–15 min incubation, leaves werewashed in a ‘‘wash’’ solution (as above, minus enzymes) for 3–5 minunder the above conditions. Protoplasts were then released by gentlyshaking the plasmolysed leaf tissue in 1.5 mL of ‘‘release’’ solution(1 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.6, 380 mOsm adjustedwith DD-sorbitol) directly in the measuring chamber. About 10 mL ofthe ‘‘sealing’’ solution (as above, but osmolality adjusted to480 mOsm) was then pumped through the chamber to remove cell deb-ris and all unattached protoplasts.

2.4. Patch-clamp electrophysiologyPea mesophyll protoplasts of 20–25 lm diameter were patch-

clamped in the whole-cell mode. GX resistance seals were obtainedin the ‘‘sealing’’ solution (1 mM CaCl2, 5 mM KCl, 2 mM MES, pH5.6, 480 mOsM adjusted with DD-sorbitol). Basic ‘‘pipette solution’’(PS) contained (in mM): 100 KCl, 2 MgCl2, 0.8 CaCl2 (100 nM freeCa2+), 2 EGTA, 10 HEPES, pH 7.2 adjusted with Tris base. Osmolal-ity of the PS was 540 mOsm. In some experiments, 50 mM NaCl wasused in the bath solution instead of 5 mM KCl. Polyamines were addedas a 50 mM stock made in the appropriate solution, with both pH andosmolality adjusted to the required level.

Measurements were made using an Axopatch 200 patch-clampamplifier (Axon Instruments, USA) in the conventional whole-cell con-figuration as described in our previous publications [27,41]. Membranepotentials were clamped at �80 mV throughout experiments, and volt-

age pulses applied in steps (�160 to + 100 mV range, with 20 mVincrements). Typical access resistance was 18–23 MX, and whole cellcapacitance; 15–20 pF.

2.5. Membrane potential measurementsConventional KCl-filled Ag/AgCl microelectrodes [36] with tip

diameter �0.5 lm were used. Measurements were taken from at leastfive individual leaf segments for each treatment, and recorded for1.5–2 min after the initial cell penetration.

3. Results

Leaf pre-incubation in 1 mM concentrations of polyamines

significantly (P < 0.001) prevented NaCl-induced K+ efflux

from pea mesophyll (Fig. 1a), with a 85–90% inhibition ob-

served for Spm4+ and Spd3+, and 90–95% inhibition for

Put2+ (measured for steady-state conditions, 30 min after NaCl

treatment). When analyzed for the first 5 min of stress, the effi-

ciency of polyamine block followed the sequence: Put2+ >

Spd3+ > Spm4+. The effect of polyamines was time-dependent,

with increasing duration of pre-treatment resulting in a stron-

ger inhibition of the NaCl-induced K+ leak (illustrated for

Put2+ in Fig. 1b). A clear dose-dependency of polyamine effect

S. Shabala et al. / FEBS Letters 581 (2007) 1993–1999 1995

on NaCl-induced K+ efflux was found (Fig. 2), with Km values

being 40 and 50 lM for Put2+ and Spm4+, respectively (the

dose-dependence of Spd3+ was not studied).

Membrane potential measurements suggested that cell pre-

treatment with polyamines reduced the magnitude of NaCl-in-

duced depolarization of the membrane potential (Fig. 3). In

general, Spm4+ was more efficient than Put2+ in preventing

membrane depolarization. The efficiency of the polyamine ef-

fect also increased with increasing concentrations of polyam-

ines (Fig. 3).

Further characterization of the effects of polyamines on the

electrophysiological characteristics of mesophyll cells was con-

ducted using the patch-clamp technique. Several types of ion

channels were observed in pea mesophyll in the whole-cell

Polyamine concentration, μM

Net

K+

flux,

nm

ol m

-2s-1

-100

-90

-80

-70

-60

-50

-40

-30

-20

-10

00 200 400 600 800 1000

Put

SpmPut + NaCl

Spm + NaCl

Fig. 2. Dose-dependence of polyamine effects on K+ flux from peamesophyll. Leaf segments were pre-incubated in an appropriateconcentration of either Put2+ or Spm4+ in the presence or absence ofsalt-stress (50 mM). Net K+ fluxes were measured 1 h after salttreatment when a steady-state level was achieved. Mean ± S.E.(n = 24). At least five individual leaf segments (collected from differentleaves) were measured for each treatment, with 4–5 spots on each.

50NaCl 50NaCl 50NaCl 50NaCl

Mem

bran

e po

tent

ial,

mV

-130

-120

-110

-100

-90

-80

-70

-60

Control 50NaCl 1Put2+ 1Spm4+5 5Put2+ 5Spm4+

Fig. 3. Membrane potential of pea mesophyll cells at various NaCl/polyamine ratios. Cells were pre-incubated in an appropriate concen-tration of either Put2+ or Spm4+ for 1 h prior to NaCl treatment.Membrane potentials were measured 30–40 min after salinity treat-ment, after net K+ fluxes stabilized (see Fig. 1a). Mean ± S.E. (n = 20).

configuration; the most abundant of which (observed in

>80% of protoplasts) were the instantaneously activated cur-

rents (Fig. 4a). The reversal potential (Vr) for all currents

was about �60 mV under standard conditions (5 mM K+ in

the bath; 100 mM K+ in the pipette) (Fig. 4e). This is close

to the EK (�72 mV), implying their high cationic selectivity.

However, as the instantaneously activated currents also medi-

ated Na+ fluxes (Fig. 4b), as well as Ca2+ fluxes (data not

shown); we term the respective channels as NSCC (for non-

selective cation channels). These channels were our focus for

further investigation.

Both Put2+ and Spm4+ (1 mM concentration) were effective

in blocking �70% of NSCC inward currents (as illustrated

for Put2+ in Fig. 4c). Also reduced was the outward current

(�30% reduction 3 min after Spm4+ application, significant

at P < 0.05; �20% reduction 3 min after Put2+ application,

+60 to +100 mV range). The above blocking effect of polyam-

ines on NSCC currents showed a clear time-dependency (illus-

trated for Put2+ in Fig. 4f). The characteristic time of this

process was 4–5 min as estimated from the single exponential

fit of the normalized channel-mediated current at �160 mV

(Fig. 4e). It should be mentioned that in each experiment,

polyamines were added only after the stabilization of the cur-

rent amplitude in the control (7–10 min after obtaining the

whole-cell configuration), after the absence of run-downs was

confirmed.

Adding 50 mM NaCl to the bath solution dramatically (2.5-

fold, significant at P < 0.01) increased the magnitude of inward

NSCC currents, without any substantial effect on the chord

conductance of the outward current (Fig. 4b and e). There

was also an obvious shift in Vr values (from �60 mV in

5 mM KCl bath, to �20 mV in 5 mM KCl + 50 mM NaCl

bath), suggesting that NSCC was indeed conducting Na+.

Adding 1 mM Put2+ significantly (P < 0.05) inhibited the in-

ward (Na+) current (Fig. 4c). The protoplast washout with

50 mM NaCl solution showed only partial reversibility of

polyamine effects on NSCC currents (Fig. 4d).

Another type of the channel observed in some patches was

KOR channels (Fig. 5). Accordingly, we undertook a study

on the effect of polyamines on KOR currents in whole-cell

patch-clamp experiments. This was not a trivial task, as

KOR channels were present only in �10% of successful

patches, and the overall success rate of seal formation on

pea mesophyll was rather low (�5%) in our experiments.

Nonetheless, we were able to analyze PA effects on two sam-

ples with KOR activity (Fig. 5). In both cases, no effect of

PA on the time-dependent K+ outward current was found

(illustrated for 1 mM Spm4+ in Fig. 5), although instantaneous

inward currents (through NSCC) were substantially sup-

pressed.

4. Discussion

Elevated Na+ levels in the leaf apoplast cause a substantial

K+ efflux from mesophyll cells [27,31–33]. With K+ being an

activator of a large number of enzymes, the consequences of

such a K+ loss to cell metabolism are rather drastic.

Reports on the effect of polyamines on plasma membrane

K+ transport date back two decades [42]. In this work, we

show that micromolar concentrations of externally applied

polyamines (Km = 40–50 lM; Fig. 2) substantially reduced

0

20

40

60

80

100

120

0 5 10 15

60 mV

-160 mV

a

b

c

d

50 Na bath

control

+ 1 mM Put2+

washout

200 pA

1 s

V, mV

I, pA

-180 -120 -60 0 60

K bath

Na bath

Put2+

200

100

- 200

- 100

e

Time, min

Nor

mal

ized

cur

rent

, %

fy = 72 e-0.19x + 28

Fig. 4. Non-selective cation channels (NSCC) in pea mesophyll cells and the effect of polyamines on their currents. (a)–(d) Typical examples ofrecordings of NSCC currents. (a) Control (5 mM KCl + 1 mM CaCl2). (b) �50 mM NaCl in the bath; (c) �50 mM NaCl + 1 mM Put2+ (after15 min); (d) Washout (>30 min in 50 mM NaCl). (e) I–V relationships for instantaneous currents (mean ± S.E.; n = 4–7). (f) Normalized protoplastcurrents as a function of time for Put2+ effects. Mean ± S.E. (n = 4). Solid line is a best fit to monoexponential function with an offset.

Fig. 5. Outward-rectifying K+ currents (mediated by KOR channel) in pea mesophyll cells are not blocked by exogenously applied polyamines. Atypical example of KOR recording from a small outside-out vesicle (�0.4 pF) isolated from pea protoplast is shown. From a holding potential of�80 mV voltage steps from �160 mV up to + 100 mV were applied in 20 mV increments. Adding 1 mM Spm4+ to the bath did not substantiallyinhibit outward K+ current, while instantaneous inward currents (mediated by NSCC) was strongly suppressed. The bath contained (in mM): 1CaCl2, 5 KCl, 2 MES-KOH (pH 5.6), and pipette solution: 100 KCl, 2 MgCl2, 0.1 lM free Ca2+, 10 HEPES–Tris (pH 7.2).

1996 S. Shabala et al. / FEBS Letters 581 (2007) 1993–1999

the magnitude of the NaCl-induced K+ efflux from pea meso-

phyll tissue (Figs. 1 and 2).

The inhibitory effect of externally applied polyamines on

both K+ efflux (Fig. 1b) and on NSCC-mediated currents

(Fig. 4f) developed slowly over time, thus ruling out a direct

inhibition of membrane cation conductance from outside the

cell. More likely, polyamines are transported across the plasma

membrane [43,44] and exert their inhibitory effects from the

cytosolic side. Therefore, the potency of polyamine block will

depend on how fast a threshold concentration is built up with-

in the cytosol. Interestingly, in situ effects of exogenous poly-

amines (measured in MIFE experiments) are much slower

then those observed in patch-clamp measurements, suggesting

that removing the cell wall may cause severe perturbation to

S. Shabala et al. / FEBS Letters 581 (2007) 1993–1999 1997

cell metabolism (and, possibly, transport properties). More

likely, however, is that in MIFE experiments, a substantial

amount of applied polyamines is strongly bound to cell walls.

It has been reported that polyamines adsorb selectively on

plant cell walls according to their valency (up to 70% of the to-

tal wall CEC for Put2+; up to 90% for Spd3+ and Spm4+ [45]).

Thus, it is reasonable to assume that it should take far longer

for the same amount of polyamine to be accumulated in the

cytosol of intact cells (compared with isolated protoplasts) to

reach the same threshold concentration. Furthermore, stron-

ger Spm4+ binding to the cell wall [45] may be an explanation

of why Put2+ was more efficient in preventing NaCl-induced

K+ efflux during the first moments of acute salt-stress

(Fig. 1a). The only report of effect of polyamines on plasma

membrane ion channels in plant cells so far is from a study

of K+ inward-rectifying (KIR) channels in Vicia guard cell

[5]. Polyamines in that case indirectly inhibited KIR current

from the cytosolic side. Similar to our study, no large specific-

ity of polyamine effect was observed: at 1 mM concentration

Put2+, Spd3+ and Spm4+ inhibited 40%, 60%, and 55% of

KIR-mediated currents, respectively [5]. In contrast to our

data for NSCC, up to 15 min incubation with externally ap-

plied polyamines did not affect the KIR current amplitude.

One possible explanation is that different cell types, such as

guard and mesophyll cells, possess different capacities for poly-

amine transport across the plasma membrane.

To the best of our knowledge, no direct information about

the free cytosolic polyamine levels is available in the literature.

However, experiments on protoplasts with radiolabelled [14C]-

polyamines suggested that only 42% of Put2+ and 28% Spd3+

were compartmentalized in the vacuole, with less then 2% of

free polyamines found in either the chloroplasts or mitochon-

dria [46]. Thus, it might be reasonable to assume that a substan-

tial portion of free polyamines, not bound to the cell wall, may

reside in the cytosol under our conditions. Keeping in mind the

Km values for Put2+ and Spm4+ effect on NaCl-induced K+ ef-

flux being 40–50 lM, even resting PA levels may be sufficient to

(at least partially) block K+ efflux from the cell. The efficiency

of this block will increase if the total polyamine pool is in-

creased under stress conditions. Polyamine levels increase in re-

sponse to a variety of abiotic stresses, showing a stress-specific

pattern. When it comes to salt-stress, most researchers are

unanimous in their reports of a significant elevation in Spd3+

or Spm4+. Data for Put2+ is rather controversial, with both ele-

vation [17,47–49] and decline [50–52] reported. The impact of

salt-stress on free Put2+ content also appears to be tissue- and

cultivar-specific [48,51]. However, polyamine control of

NaCl-induced K+ efflux showed little specificity, with Put2+

being as efficient as Spd3+ or Spm4+ (Figs. 1 and 2). Therefore,

it is the size of the total pool of free polyamines, rather than

specific polyamine content, that is important to control intra-

cellular K+ homeostasis, at least in pea mesophyll.

Polyamines are ideally suited as physiological channel block-

ers. While being efficient channel blockers, most inorganic

polycations (e.g. Al3+; Gd3+; La3+) are, at the same time,

highly toxic to cell metabolism, so cannot be accumulated in

the cytosol at the concentrations needed for ‘safe’’ control of

intracellular homeostasis. Polyamines are the only organic

polycations present in sufficient quantities to perform the role

of channel blockage, without compromising cell metabolism.

The simplest explanation for the observed reduction of

NaCl-induced K+ efflux by polyamines is a blockage of

KOR channels. However, in the case of pea mesophyll cells,

KOR currents were insensitive to polyamines (Fig. 5). This

observation is consistent with reports on polyamine effects

on the KOR currents in guard cells [5]. Moreover, KOR chan-

nels were expressed in less than 10% of successfully patched

protoplasts in our experiments. Thus, the involvement of some

other K+-permeable channel should be considered.

It is generally agreed that NaCl-induced depolarization of

the membrane potential is caused by excessive Na+ entry into

the cytosol. NSCC are often cited as a major route for this Na+

uptake, at least in root tissue [53,54]. Evidence for leaves is

scarce, although Na+-permeable cation channels were reported

earlier for Arabidopsis mesophyll cells [27], and Elzenga and

Van Volkenburgh [56] have reported TEA-insensitive cation

channels in the plasma membrane of pea epidermal cells that

poorly discriminate between potassium, sodium and lithium.

In this work, non-selective, K+- and Na+-permeable cation

channels was reported in pea mesophyll protoplasts (Fig. 3).

Although its detailed characterization is outside the scope of

this paper, the general features of this channel (current rectifi-

cation, instantaneous activation, selectivity) are very similar to

previously reported NSCC in plant root [53], guard [55] or leaf

epidermal [56] cells. Importantly, both inward- and outward-

currents were effectively blocked by polyamines (illustrated

for Put2+ in Fig. 4 and Spm4+ in Fig. 5).

Taken together with MIFE flux measurements and mem-

brane potential determinations, these results suggest the follow-

ing scenario. Under saline conditions, Na+ enters the cytosol

through NSCC, inducing plasma membrane depolarization

(Fig. 3). This results in increased K+ efflux (Fig. 1) through

depolarization-activated outward-rectifying KOR channels

[27] and NSCC channels. Both Put2+ and Spm4+ block NSCC

(from the cytosolic side, in a time-dependent manner), reducing

the magnitude of NaCl-induced plasma membrane depolariza-

tion and, consequently the depolarization-induced K+ efflux

through KOR. In addition to this mechanism, polyamines

may directly block outward K+ currents through NSCC (Figs.

3 and 4) or activate the plasma membrane H+-ATPase [4], so

restoring the membrane potential. One way or another, poly-

amine regulation of membrane transport activity would enable

the maintenance of an optimal cytosolic K+/Na+ ratio for cell

metabolism. This scenario may explain both the reported

observations of reduced Na+ accumulation in leaves [10] and

higher K+/Na+ ratios [9] in the shoot of polyamine-treated

plants grown in the presence of NaCl.

A direct confirmation for this model could come from mea-

surements of polyamine effects on net Na+ flux from salinized

mesophyll tissue. Unfortunately, for the moment, these mea-

surements are completely out of question due to the poor selec-

tivity of all available Na+ LIX (see [28] for details). The acute

NaCl stress causes significant K+ and Ca2+ efflux (due to plas-

ma membrane depolarization and resulting from Na+/Ca2+

Donnan exchange in the cell wall, respectively). This efflux is

measured by the Na+ LIX (sensitive to both K+ and Ca2+;

[28]) and interpreted as an apparent Na+ efflux, thus com-

pletely masking any Na+ uptake.

In summary, our results suggest that the observed stress-in-

duced elevation in the level of endogenous polyamines under

saline conditions, may represent an important adaptive mech-

anism; reducing the uptake of Na+ and leakage of K+ from

mesophyll cells, thus assisting plants in their adaptation to

salinity.

1998 S. Shabala et al. / FEBS Letters 581 (2007) 1993–1999

Acknowledgements: This work was supported by ARC Discovery andUniv. Tasmania IRGS grants to S. Shabala.

References

[1] Evans, P.T. and Malmberg, R.L. (1989) Do polyamines have rolesin plant development? Annu. Rev. Plant. Physiol. Plant Mol. Biol.40, 235–269.

[2] Galston, A.W. and Kaur-Sawhney, R. (1990) Polyamines in plantphysiology. Plant Physiol. 94, 406–410.

[3] Flores, H.E. and Galston, A.W. (1982) Analysis of polyamines inhigher-plants by high-performance liquid-chromatography. PlantPhysiol. 69, 701–706.

[4] Reggiani, R., Zaina, S. and Bertani, A. (1992) PlasmalemmaATPase in rice coleoptiles – stimulation by putrescine andpolyamines. Phytochemistry 31, 417–419.

[5] Liu, K., Fu, H.H., Bei, Q.X. and Luan, S. (2000) Inwardpotassium channel in guard cells as a target for polyamineregulation of stomatal movements. Plant Physiol. 124, 1315–1325.

[6] Alcazar, R., Marco, F., Cuevas, J.C., Patron, M., Ferrando, A.,Carrasco, P., Tiburcio, A. and Altabella, T. (2006) Involvement ofpolyamines in plant response to abiotic stress. Biotechnol. Lett.28, 1867–1876.

[7] Basu, R. and Ghosh, B. (1991) Polyamines in various rice (Oryzasativa) genotypes with respect to sodium-chloride salinity. Physiol.Plant. 82, 575–581.

[8] Erdei, L., Trivedi, S., Takeda, K. and Matsumoto, H. (1990)Effects of osmotic and salt stresses on the accumulation ofpolyamines in leaf segments from wheat-varieties differing in saltand drought tolerance. J. Plant Physiol. 137, 165–168.

[9] Lakra, N., Mishra, S.N., Singh, D.B. and Tomar, P.C. (2006)Exogenous putrescine effect on cation concentration in leaf ofBrassica juncea seedlings subjected to Cd and Pb along withsalinity stress. J. Environ. Biol. 27, 263–269.

[10] Ndayiragije, A. and Lutts, S. (2006) Exogenous putrescinereduces sodium and chloride accumulation in NaCl treated calliof the salt-sensitive rice cultivar I Kong Pao. Plant GrowthRegulat. 48, 51–63.

[11] Kumira, R. and Rajam, M.V. (2002) Alteration in polyaminetitres during Agrobacterium-mediated transformation of indicarice with ornithine decarboxylase gene affects plant regenerationpotential. Plant Sci. 162, 769–777.

[12] Roy, M. and Wu, R. (2001) Arginine decarboxylase transgeneexpression and analysis of environmental stress tolerance intransgenic rice. Plant Sci. 160, 869–875.

[13] Wi, S.J., Kim, W.T. and Park, K.Y. (2006) Overexpression ofcarnation s-adenosylmethionine decarboxylase gene generates abroad-spectrum tolerance to abiotic stresses in transgenic tobaccoplants. Plant Cell Rep. 25, 1111–1121.

[14] Kasukabe, Y., He, L.X., Nada, K., Misawa, S., Ihara, I. andTachibana, S. (2004) Overexpression of spermidine synthaseenhances tolerance to multiple environmental stresses and up-regulates the expression of various stress regulated genes intransgenic Arabidopsis thaliana. Plant Cell Physiol. 45, 712–722.

[15] Kasinathan, V. and Wingler, A. (2004) Effect of reduced argininedecarboxylase activity on salt tolerance and on polyamineformation during salt stress in Arabidopsis thaliana. Physiol.Plant. 121, 101–107.

[16] Yamaguchi, K., Takahashi, Y., Berberich, T., Imai, A., Miyazaki,A., Takahashi, T., Michael, A. and Kusano, T. (2006) Thepolyamine spermine protects against high salt stress in Arabidop-sis thaliana. FEBS Lett. 580, 6783–6788.

[17] Urano, K., Yoshiba, Y., Nanjo, T., Ito, T., Yamaguchi-Shino-zaki, K. and Shinozaki, K. (2004) Arabidopsis stress-induciblegene for arginine decarboxylase AtADC2 is required for accu-mulation of putrescine in salt tolerance. Biochem. Biophys. Res.Commun. 313, 369–375.

[18] Lopatin, A.N., Makhina, E.N. and Nichols, C.G. (1994) Potas-sium channel block by cytoplasmic polyamines as the mechanismof intrinsic rectification. Nature 372, 366–369.

[19] Lu, Z. and Ding, L. (1999) Blockade of a retinal cGMP-gatedchannel by polyamines. J. Gen. Physiol. 113, 35–43.

[20] Bowie, D., Lange, G.D. and Mayer, M.L. (1998) Activity-dependent modulation of glutamate receptors by polyamines. J.Neurosci. 18, 8175–8185.

[21] Gomez, M. and Hellstrand, P. (1999) Endogenous polyaminesmodulate Ca2+ channel activity in guinea-pig intestinal smoothmuscle. Pflugers Archiv. 438, 445–451.

[22] Bruggemann, L.I., Pottosin, I.I. and Schonknecht, G. (1998)Cytoplasmic polyamines block the fast-activating vacuolar cationchannel. Plant J. 16, 101–105.

[23] Dobrovinskaya, O.R., Muniz, J. and Pottosin, I.I. (1999) Inhibi-tion of vacuolar ion channels by polyamines. J. Membr. Biol. 167,127–140.

[24] Maathuis, F.J.M. and Amtmann, A. (1999) K+ nutrition and Na+

toxicity: The basis of cellular K+/Na+ ratios. Ann. Bot. 84, 123–133.

[25] Tester, M. and Davenport, R. (2003) Na+ tolerance and Na+

transport in higher plants. Ann. Bot. 91, 503–527.[26] Shabala, S., Shabala, L. and Van Volkenburgh, E. (2003) Effect of

calcium on root development and root ion fluxes in salinisedbarley seedlings. Funct. Plant Biol. 30, 507–514.

[27] Shabala, S., Demidchik, V., Shabala, L., Cuin, T.A., Smith, S.J.,Miller, A.J., Davies, J.M. and Newman, I.A. (2006) ExtracellularCa2+ ameliorates NaCl-induced K+ loss from Arabidopsis rootand leaf cells by controlling plasma membrane K+-permeablechannels. Plant Physiol. 141, 1653–1665.

[28] Chen, Z., Newman, I., Zhou, M., Mendham, N., Zhang, G. andShabala, S. (2005) Screening plants for salt tolerance by measur-ing K+ flux: a case study for barley. Plant Cell Environ. 28, 1230–1246.

[29] Chen, Z.H., Zhou, M.X., Newman, I.A., Zhang, G.P. andShabala, S. (2007) Potassium and sodium relations in salinisedbarley tissues as a basis of differential salt tolerance. Funct. PlantBiol. 34, 150–162.

[30] Cuin, T.A. and Shabala, S. (2005) Exogenously supplied com-patible solutes rapidly ameliorate NaCl-induced potassium effluxfrom barley roots. Plant Cell Physiol. 46, 1924–1933.

[31] Shabala, S. (2000) Ionic and osmotic components of salt stressspecifically modulate net ion fluxes from bean leaf mesophyll.Plant Cell Environ. 23, 825–837.

[32] Shabala, S., Babourina, O. and Newman, I. (2000) Ion-specificmechanisms of osmoregulation in bean mesophyll cells. J. Exp.Bot. 51, 1243–1253.

[33] Shabala, S., Shabala, L., Van Volkenburgh, E. and Newman, I.(2005) Effect of divalent cations on ion fluxes and leaf photo-chemistry in salinized barley leaves. J. Exp. Bot. 56, 1369–1378.

[34] Carden, D.E., Walker, D.J., Flowers, T.J. and Miller, A.J. (2003)Single-cell measurements of the contributions of cytosolic Na+

and K+ to salt tolerance. Plant Physiol. 131, 676–683.[35] Cuin, T.A., Miller, A.J., Laurie, S.A. and Leigh, R.A. (2003)

Potassium activities in cell compartments of salt-grown barleyleaves. J. Exp. Bot. 54, 657–661.

[36] Shabala, L., Cuin, T.A., Newman, I. and Shabala, S. (2005)Salinity-induced ion flux patterns from the excised roots ofArabidopsis sos mutants. Planta 222, 1041–1050.

[37] Cuin, T.A. and Shabala, S. (2007) Amino acids regulate salinity-induced potassium efflux in barley root epidermis. Planta 225,753–761.

[38] Shabala, S. and Hariadi, Y. (2005) Effects of magnesiumavailability on the activity of plasma membrane ion transportersand light-induced responses from broad bean leaf mesophyll.Planta 221, 56–65.

[39] Shabala, S.N., Newman, I.A. and Morris, J. (1997) Oscillations inH+ and Ca2+ ion fluxes around the elongation region of cornroots and effects of external pH. Plant Physiol. 113, 111–118.

[40] Newman, I.A. (2001) Ion transport in roots: measurements offluxes using ion-selective microelectrodes to characterize trans-porter function. Plant Cell Environ. 24, 1–14.

[41] Demidchik, V., Shabala, S.N., Coutts, K.B., Tester, M.A. andDavies, J.M. (2003) Free oxygen radicals regulate plasma mem-brane Ca2+ and K+-permeable channels in plant root cells. J. CellSci. 116, 81–88.

[42] de Agazio, M., Giardina, M.C. and Grego, S. (1988) Effect ofexogenous putrescine, spermidine, and spermine on K+ uptakeand H+ extrusion through plasma membrane in maize rootsegments. Plant Physiol. 87, 176–178.

S. Shabala et al. / FEBS Letters 581 (2007) 1993–1999 1999

[43] Antognoni, F., Casali, P., Pistocchi, R. and Bagni, N. (1994)Kinetics and calcium-specificity of polyamine uptake in carrotprotoplasts. Amino Acids 6, 301–309.

[44] Giromini, L., Paina, A., Cerana, R. and Colombo, R. (1994)Putrescine channels in the plasma membrane of Arabidopsisthaliana. Plant Physiol. 105, 921–992.

[45] Messiaen, J. and van Cutsem, P. (1999) Polyamines and pectines.II. Modulation of pectic-signal transduction. Planta 208, 247–256.

[46] Cohen, S.S. (1998) A Guide to the Polyamines, University Press,Oxford, 624p.

[47] Legocka, J. and Kluk, A. (2005) Effect of salt and osmotic stresson changes in polyamine content and arginine decarboxylaseactivity in Lupinus luteus seedlings. J. Plant Physiol. 162, 662–668.

[48] Bagni, N., Ruiz-Carrasco, K., Franceschetti, M., Fornale, S.,Fornasiero, R.B. and Tassoni, A. (2006) Polyamine metabolismand biosynthetic gene expression in Arabidopsis thaliana undersalt stress. Plant Physiol. Biochem. 44, 776–786.

[49] Liu, J.-H., Nada, K., Honda, C., Kitashiba, H., Wen, X.-P.,Pang, X.-M. and Moriguchi, T. (2006) Polyamine biosynthesis ofapple callus under salt stress: importance of the arginine decar-boxylase pathway in stress response. J. Exp. Bot. 57, 2589–2599.

[50] Lin, C.C. and Kao, C.H. (2002) NaCl-induced changes inputrescine content and diamine oxidase activity in roots of riceseedlings. Biol. Plant 45, 633–636.

[51] Maiale, S., Sanchez, D.H., Guirado, A., Vidal, A. and Ruiz, O.A.(2004) Spermine accumulation under salt stress. J. Plant Physiol.161, 35–42.

[52] Zapata, P.J., Serrano, M., Pretel, A.T., Amoros, A. and Botella,M.A. (2004) Polyamines and ethylene changes during germinationof different plant species under salinity. Plant Sci. 167, 781–788.

[53] Spalding, E.P., Slayman, C.L., Goldsmith, M.H.M., Gradmann,D. and Bertl, A. (1992) Ion channels in Arabidopsis plasmamembrane – transport characteristics and involvement in light-induced voltage changes. Plant Physiol. 99, 96–102.

[54] Demidchik, V. and Tester, M. (2002) Sodium fluxes throughnonselective cation channels in the plasma membrane of protop-lasts from Arabidopsis roots. Plant Physiol. 128, 379–387.

[55] Wolf, T., Guinot, D.R., Hedrich, R., Dietrich, P. and Marten, I.(2005) Nucleotides and Mg2+ ions differentially regulate K+

channels and non-selective cation channels present in cellsforming the stomatal complex. Plant Cell Physiol. 46, 1682–1689.

[56] Elzenga, J.T.M. and Van Volkenburgh, E. (1994) Characteriza-tion of ion channels in the plasma membrane of epidermal cells ofexpanding pea (Pisum sativum Arg) leaves. J. Membr. Biol. 137,227–235.