System Potentials, a Novel Electrical Long-Distance Apoplastic Signal in Plants, Induced by Wounding 1

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System Potentials, a Novel Electrical Long-Distance

Apoplastic Signal in Plants, Induced by Wounding

Matthias R. Zimmermann, Heiko Maischak, Axel Mitho¨fer, Wilhelm Boland, and Hubert H. Felle*

Botanisches Institut I, Justus-Liebig-Universita¨t, D–35390 Giessen, Germany (M.R.Z., H.H.F.); and

Max-Planck-Institut fu¨r Chemische O

¨ kologie, D–07745 Jena, Germany (H.M., A.M., W.B.)

Systemic signaling was investigated in both a dicot (Vicia faba) and a monocot (Hordeum vulgare) plant. Stimuli were applied to

one leaf (S-leaf), and apoplastic responses were monitored on a distant leaf (target; T-leaf) with microelectrodes positioned in

substomatal cavities of open stomata. Leaves that had been injured by cutting and to which a variety of cations were

subsequently added caused voltage transients at the T-leaf, which are neither action potentials nor variation potentials: with

respect to the cell interior, the initial polarity of these voltage transients is hyperpolarizing; they do not obey the all-or-none

rule but depend on both the concentration and the type of substance added and propagate at 5 to 10 cm min

. This response is

thought to be due to the stimulation of the plasma membrane H

-ATPase, a notion supported by the action of fusicoccin, which

also causes such voltage transients to appear on the T-leaf, whereas orthovanadate prevents their propagation. Moreover,

apoplastic ion ﬂux analysis reveals that, in contrast to action or variation potentials, all of the investigated ion movements (Ca

,

K

, H

, and Cl

) occur after the voltage change begins. We suggest that these wound-induced “system potentials” represent a

new type of electrical long-distance signaling in higher plants.

Understanding systemic signaling in plants has long

been recognized as a major scientiﬁc challenge. In

principle, the systemic signaling induced by wound-

ing and/or pathogen or herbivore attack may be

realized by either chemical or electrical signals. Chem-

ical signals have been shown to be involved in long-

distance signaling, propagating likely from organ to

organ either through the vascular system or as vola-

tiles that are released into the atmosphere, carrying the

message not only to organs within a plant but possibly

to neighboring plants as well (Heil and Silva Bueno,

2007; Heil and Ton, 2008; Howe and Jander, 2008;

Mitho¨fer et al., 2009). Other studies suggest that upon

wounding, electrical signals may travel through

phloem and/or xylem elements (Davies, 1987; Rhodes

et al., 1996). Interestingly, such electrical signals have

also been shown to affect systemic leaves, for example,

by regulating genes (Graham et al., 1986; Wildon et al.,

1992; Stankovic´ and Davies, 1997; Herde et al., 1998).

Among other genes, proteinase inhibitor (Pin) and

calmodulin mRNA have been up-regulated in tomato

(Solanum lycopersicum) upon wounding and the appli-

cation of heat stimuli (Stankovic´ and Davies, 1997).

Plants that elicited no electrical signal did not accu-

mulate Pin mRNA (Stankovic´ and Davies, 1997). In

particular, the induction of Pin genes is striking be-

cause these proteinase inhibitors are induced upon

insect herbivory as a defense reaction (Green and

Ryan, 1972). Proteinase inhibitors either harm the

attackers or simply prevent insects from feeding

(Koiwa et al., 1997). Although, in principle, cellular

reactions in plants have also been demonstrated to

follow the release of electrical signals induced by heat,

chilling, or electric voltage, to what extent such signals

carry speciﬁc information in nonspecialized plants or

organs is disputed.

In plants, a variety of electrical phenomena have

been described and have to be considered as signal-

transducing events. Whereas local voltage transients,

due to system resistance, will vanish after a distance of

a few millimeters and hence have no relevance for

systemic signal transfer, action potentials (APs) and

so-called variation potentials (VPs; for review, see

Davies, 2006) may carry information over long dis-

tances from organ to organ. As demonstrated recently

(Felle and Zimmermann, 2007), even if the channel

activation was insufﬁcient to trigger an AP, subthresh-

old depolarizations may have propagated along the

stimulated leaf without proceeding to neighboring

leaves, either because not enough channels were acti-

vated or because no signal-conducting connection

existed between these leaves. Although such voltage

changes suffer a decrement, information can be carried

much farther than through simple voltage transients.

APs and VPs are well documented in the literature

(Davies, 2006). As electrical signals are fast, they may

act as forerunners of slower traveling chemical signals,

which might be located throughout the plant. For

This work was supported by the Deutsche Forschungsgemein-

schaft (grant nos. Fe 213/15–1 and Fe 213/15–2) and the Max Planck

Society.

* Corresponding author; e-mail hubert.felle@bio.uni-giessen.de.

The author responsible for distribution of materials integral to the

ﬁndings presented in this article in accordance with the policy

described in the Instructions to Authors (www.plantphysiol.org) is:

Hubert H. Felle (hubert.felle@bio.uni-giessen.de).

www.plantphysiol.org/cgi/doi/10.1104/pp.108.133884

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Ó 2009 American Society of Plant Biologists

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instance, such signals might be released from injuries

caused by herbivorous insects. Still, single APs, as “all-

or-none” phenomena, likely do not contain much

information regarding the kind of threat or stress

that caused them; they may serve as general stress

signals, however, which cause responses at the level of

gene expression, primarily transcription but also

translation (Wildon et al., 1992; Stankovic´ and Davies,

1997). Additional and speciﬁc information regarding

the nature of the threat may come from chemical

signals that are either transported within phloem and/

or xylem elements or transferred through volatile

substances (De Bruxelles and Roberts, 2001; Heil and

Silva Bueno, 2007; Mitho¨fer et al., 2009).

So far, APs and VPs have been thought to be the only

kind of electrical long-distance signals. In this study,

we demonstrate a novel type of electrical signal that

propagates systemically (i.e. from leaf to leaf), varies

with intensity as well as with the nature of the original

stimulus, and, therefore, is no AP. Since the initial

direction of the signal is hyperpolarizing (with respect

to the cell interior), it does not qualify for a VP in the

classical sense (Stahlberg and Cosgrove, 1997). Thus,

we propose the existence of a new electrical signal

type, called “system potential” (SP). It operates by

stimulating the plasma membrane H

-ATPase, which

may hold and transport information systemically

within the whole plant or at least in parts of the plant.

A detailed ion ﬂux analysis is given. We demonstrate

that this signal can be triggered by substances that are

added to a leaf injured by cutting and transmitted

systemically in Hordeum vulgare and Vicia faba as well

as in a variety of other plants.

RESULTS

Release of SPs

Electrical signals were set off after mechanical injury

to a leaf (cut) and the subsequent addition of inorganic

cations (Ca

, K

, Mg

, or Na

) or Glu to the site of

injury. In accordance with the setup shown in Figure 1,

the signals had to propagate ﬁrst in the basipetal

direction (S-leaf) and then in the acropetal direction

through the stem to the T-leaf (20–25 cm in H. vulgare,

20–40 cm in V. faba) to be monitored with apoplasti-

cally positioned microprobes (Felle and Zimmermann,

2007). Without mechanically injuring the leaves, sig-

nals were either not released or weak. Cutting alone

caused an immediate fast transient voltage jump of a

few millivolts on the T-leaf but had no obvious signif-

icant effect thereafter. As shown in Figure 2, after

wounding and stimulation with Glu, we found two

clearly different voltage responses in the T-leaf apo-

plast: APs with the usual polarity and/or voltage

transients in the inverse direction. A similar chain of

events occurs due to other stimuli (e.g. inorganic ions).

As demonstrated recently (Felle and Zimmermann,

2007), inorganic cations (Ca

, K

, Na

, and Mg

)

applied to the cut leaf may or may not trigger an AP

that propagates systemically from leaf to leaf. When it

does not, a voltage transient like the one shown in

Figure 2B appears at the T-leaf. This response, called

SP, is both substrate and concentration dependent (Fig.

3). Of the ions tested, Ca

proved to be the most

effective. Although SPs could be recorded with Ca

concentrations at 1 m

(kinetics not shown), mostly

higher concentrations were used to obtain the best

responses possible. Typically, the SPs propagated at 5

to 10 cm min

. Since an SP had to propagate ﬁrst

basipetally and then acropetally to reach the systemic

leaf, a direct effect of the applied ion (e.g. through

long-distance transport) can be excluded. Moreover,

the responses to the different cations (Fig. 3A) clearly

indicate that the anion (Cl

) is not involved. Mannitol

(50 m

), a nondepolarizing agent, had no effect (data

not shown). Apart from H. vulgare and V. faba, SPs

were also recorded on Nicotiana tabacum, Phaseolus

lunatus, and Zea mays (data not shown).

Extracellular Versus Intracellular Responses

To prove that apoplastic and intracellular voltage

changes correspond, control experiments were per-

formed in which the intracellular and extracellular

responses to Ca

were measured simultaneously (Fig.

4). The observation that the extracellular voltage

change considerably exceeds the change in membrane

potential is based on the fact that the electrical resis-

tances of apoplast and symplast differ.

The Effects of H

Pump Stimulation or Inhibition

Fusicoccin (FC) is a well-known fungal toxin that stim-

ulates H

extrusion and hyperpolarizes the plasma

membrane of plants (Marre`, 1979). When added to the

S-leaf, after due time 1

M

FC causes a hyperpolarizing

Figure 1. Basic setup to measure apoplastic voltage and ion activities

with microelectrodes positioned in substomatal cavities of open sto-

mata of H. vulgare or V. faba. Stimuli were applied to one leaf (S-leaf),

while the responses were measured in a distant target leaf (T-leaf). The

tip of the T-leaf was submerged in AAF (5 m

KCl and 0.1 m

CaCl

, pH

5), and the solution was put to earth with a blunt reference electrode

ﬁlled with 0.5

KCl.

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response at the T-leaf similar to the one detected with

the cations (Fig. 5B). An SP-initiating Ca

treatment

applied to the same leaf after an FC treatment no

longer had any effect (kinetics not shown). Orthova-

nadate, a P-type ATPase inhibitor, generally prevented

the propagation of SPs (Fig. 5, B and C). After

orthovanadate was injected into the apoplast of the

target leaf in H. vulgare (Fig. 5A) and the solution had

been absorbed by the affected tissue, tests were carried

out. Electrodes were positioned roughly 2 cm before

and 2 cm behind the orthovanadate-treated area.

Noninvasive control “light/dark” tests proved the

full responsiveness of the tissue at both electrodes.

Figure 5B shows the FC response at electrode 1 and the

missing response behind the orthovanadate-treated

area at electrode 2. SPs generated by Ca

were like-

wise stopped from propagating after orthovanadate

treatment at the S-leaf (data not shown). Inﬁltrating

the leaf with artiﬁcial apoplastic ﬂuid (AAF; 2 m

KCl

and 0.1 m

CaCl

, pH 5) used as a control did not stop

the propagation of the signal but did weaken it (con-

trol AAF). Adding orthovanadate to the wounded

region before cation treatment prevented the release of

an SP (kinetics not shown) but, after due propagation

time, massively hyperpolarized the apoplastic poten-

tial (depolarized the membrane potential) on the T-leaf

about 30 cm away from the stimulus site (Fig. 5C). To

test whether the orthovanadate could have been trans-

ported during the elapsed time from the site of stim-

ulation to the receiving electrode, 25 m

KCl was

added to the wounded region and a K

selective

microelectrode was placed at the neighboring leaf to

pick up K

shifts. As shown in Figure 5C, in the ﬁrst 30

min, no increase of apoplastic [K

] was recorded.

Ion Movements

As recently demonstrated (Felle and Zimmermann,

2007), release and propagation of APs are causally

linked to ion movements, due to the selective activa-

tion of channels. Since SPs are essentially hyperpolar-

izing events, most likely generated and transmitted by

+

pump activation, it was of interest whether this

would show up in the ion movements as well. Figure 6

shows the ion movements that occur during their

pertinent voltage changes (SPs); these movements

have been aligned to point out the temporal sequence

of the ion movements. Apoplastic Ca

, K

, and H

ac-

tivities decreased; only Cl

increased. All of the ion

movements were transient, and none of the ion move-

ments occurred before the voltage changes.

DISCUSSION

In H. vulgare and V. faba as well as in other plants

such as N. tabacum, P. lunatus, and Z. mays (data not

shown), we demonstrate a new kind of electrical long-

distance signal that propagates systemically, the SP. As

shown in Figures 2 and 3, in combination with me-

chanical wounding, SP signals are triggered by inor-

ganic cations such as Ca

, K

, Na

, and Mg

organic compounds such as Glu. Obviously, SPs are

neither VPs nor APs. In Table I, the most basic char-

acteristics of the three kinds of signals are compared:

unlike the primary polarity in VPs and APs, the

Figure 3. Systemic apoplastic voltage transients (SPs), generated by different inorganic cations, added to a cut-wounded leaf

(S-leaf) and monitored on the T-leaf. A, Response of H. vulgare to 100 m

, Mg

, K

, or Ca

. B and C, Responses of H.

vulgare and V. faba, respectively, to different Ca

concentrations, as indicated. For all panels, one experimental set each of three

equivalent tests is represented.

Figure 2. Typical systemic apoplastic voltage signals (AP and SP)

measured successively on one leaf. A, Following a cut injury, 10 m

Glu was applied to one leaf of H. vulgare and an AP was measured on a

distant leaf (distance in this recording of 33 cm) with a blunt micro-

electrode positioned in the substomatal cavity of an open stoma. B,

After the stimulus site was rinsed with AAF, a second Glu application

was made that did not trigger an AP but a hyperpolarization, an SP.

Representative examples of ﬁve equivalent measurements each are

shown. E(apo), Apoplastic voltage.

Electrical Long-Distance Signals

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primary polarity of SPs is reversed; moreover, SPs do

not obey all-or-none conditions and are not caused by

a hydraulic pressure surge or activation of ion chan-

nels. As the amplitude of the SPs is modiﬁed by the

concentration as well as by the nature of a substance,

the level of information potentially transferred by SPs

should be higher than that transferred by a single AP,

which cannot be modulated in amplitude.

SPs Are Due to a Stimulation of the H

-ATPase

What is the nature of these SPs and how are they

generated? In a recent paper, we demonstrated that in

H. vulgare, APs were triggered by a variety of sub-

stances like inorganic ions, Glu, et cetera (Felle and

Zimmermann, 2007). The release of a systemic AP was

shown to be critical: when the stimulus was too weak

to activate enough channels or the system resistance

around the nodi was too high, APs were either not

released at all or did not propagate to the T-leaf.

However, even weak stimuli are sufﬁcient to generate

an SP, providing that a substantial depolarization

occurs. To explain this, one has to recall that the two

main functions of a plasma membrane H

-ATPase are

to generate a transmembrane electrochemical H

gra-

dient and to maintain a constant H

turnover, both of

which are necessary for transmembrane transport

processes. The undisturbed membrane establishes a

dynamic equilibrium (i.e. a stable membrane poten-

tial), transmembrane ion gradients, and ion ﬂuxes. As

soon as this equilibrium is disturbed, for example, by a

depolarization of any kind, the H

-ATPase will re-

Figure 4. Simultaneous intracellular and extracellular (apoplastic) recordings of SPs triggered by Ca

on distant leaves of H.

vulgare or V. faba. Intracellular measurements were carried out on mesophyll cells while the apoplastic voltage electrode was

positioned in a neighboring stoma. Kinetics representative of four equivalent tests each are shown. Em, Membrane potential;

E(apo), apoplastic voltage.

Figure 5. Responses to FC and orthovanadate. A, Principle of testing the effect of 1 m

orthovanadate on the FC effect. Part of the

T-leaf (shaded area) was pressure injected with the vanadate (dissolved in AAF). Capillary forces made the solution spread

through the apoplast. Electrode 1 picked up the FC signal before the vanadate zone, and electrode 2 picked up the signal behind

it. In a control experiment, the leaf was injected with AAF only and the FC effect was measured at electrode 2. B, FC (1

) added

to a cut-injured leaf of H. vulgare caused a hyperpolarizing voltage transient on the T-leaf (electrode 1). Orthovanadate (1 m

)

inhibited the signal propagation to electrode 2, while AAF left it almost unaffected. C, Voltage response to 1 m

orthovanadate

added to an S-leaf injured by cutting and measured at the T-leaf. In the inset, to test for a possible mass ﬂow from the S-leaf to the

T-leaf, 25 m

KCl was added to the cut wound at the S-leaf and K

activity was measured at the T-leaf. Representative kinetics of

at least three equivalent experiments each are shown. The distance of stimulus to electrodes on the T-leaf was about 25 cm for

electrode 1 and about 30 cm for electrode 2. E(apo), Apoplastic voltage.

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spond with increased activity in order to restore the

resting state of the membrane potential. This effect, a

(partial or total) repolarization, is quite common and

follows the addition of cations or cotransported sub-

strates to the external phase of the plasma membrane.

Such a scenario apparently happens in the wounded

leaf region after cations are added. Clearly, the initial

depolarization will suffer a decrement and vanish

within a short distance (millimeters) from the origin,

while the H

-ATPase stimulation sustains yields of

hyperpolarization (or apoplastic depolarization); this

hyperpolarization obviously propagates and can be

picked up intracellularly as well as within the apoplast

of the target leaf. On the other hand, a primary

hyperpolarization following pump stimulation by FC

does not have to be transformed but propagates as it is.

Actually, SPs may be equivalent to the late recovery

section of an AP (Fig. 2), which has been demonstrated

to depend on pump activity (Felle and Zimmermann,

2007). Therefore, we suggest that the phenomena

shown here reﬂect nothing but hyperpolarization

caused by a temporary stimulation of the plasma

membrane H

-ATPase(s). The observation that an

FC-induced hyperpolarization actually propagates

from one leaf to another strongly supports our hy-

pothesis, which is backed up by the following obser-

vations: (1) after FC treatment, other agents no longer

trigger SPs; (2) orthovanadate, a potent inhibitor of

P-type H

-ATPases, prevents the propagation of SPs;

and (3) the depolarization induced at the site of

orthovanadate addition is carried systemically from

one leaf to another, indicating that an inhibition of the

pump is also carried systemically. The effects shown

here cannot be explained by mass transport, which

would be far more time consuming, and the observa-

tion that an increase in K

(used as transport test

substance) at the stimulus site is not carried to the

measuring site on the T-leaf supports this notion (Fig.

5C). Evidently, SPs are self-propagating systemic

events, which, unlike APs, do not need the mediation

of channel activation. Because of the low area density

for channels (1–3 per

mm), the release of an AP

requires a channel to indirectly communicate through

a substantial voltage change of a certain (probably

critical) membrane area and the subsequent activation

of voltage-gated channels or ligand activation (e.g.

Glu). In contrast, propagation by pumps is conceivable

by direct protein-protein interactions and activation

transfer by molecular contact, because of their much

higher area density (approximately 1,000 per

mm).

Moreover, in particular for the H

-ATPase in plant

plasma membranes, oligomerization has been demon-

strated upon activation that was mediated by 14-3-3

proteins (Ottmann et al., 2007).

The Chain of Events

Whereas with APs a Ca

inﬂux clearly precedes the

rapid voltage response and anion efﬂux is responsible

Figure 6. Apoplastic ion movements recorded as pX values on a V. faba

leaf (T-leaf) during SPs [E(apo)], released after injuries by cutting the

S-leaf and the subsequent addition of 50 m

Ca

2+

. SPs recorded together

with the ion movements are denoted as pCa, pK, pCl, and pH. SPs were

aligned (dashed line) to accentuate the temporal sequence of the ion

movements. Arrows denote the moment of stimulation. Representative

examples of at least three equivalent measurements each are shown.

Table I. Comparison of basic characteristics of APs, VPs, and SPs

Characteristic

APs

VPs

SPs

Induction

Voltage threshold

Rapid turgor increase

Plasma membrane

depolarization

Propagation

Self-propagating

Non-self-propagating

Self-propagating

Rate

20–400 cm min

10 s to several minutes

5–10 cm min

Mechanism

Activation of ion channels

(Ca

, Cl

, K

)

Inactivation of the H

pump

Activation of the H

pump

Ion movements and

triggers Cl

efﬂux and

Causalities unclear

Ion movements follow voltage

Direction

Depolarization

Hyperpolarization

Duration of initial

voltage change

,20 s

10 s to several minutes

8–12 min

Signal

All or none

Graded signals of variable size

Signals depend on stimulus

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for the typical AP “breakthrough,” the ion ﬂuxes

accompanying the SP occur not prior to the voltage

response but after its onset (Fig. 6), indicating that

these ion movements are a result of the SP rather than

its cause. This interpretation is also strongly supported

by the observation that the direction of K

, Ca

, and Cl

ﬂuxes clearly follows the voltage changes (i.e. changes

in driving force). The development of apoplastic

pH appears to be the only characteristic that APs,

VPs, and SPs have in common, namely an alkaliniza-

tion. It has been demonstrated that apoplastic

pH increase is a typical stress response to drought

(Wilkinson, 1999), salt stress, low temperature, and

fungal attack (Felle et al., 2005), and oxygen shortage

(Felle, 2006). Despite the apparently logical assump-

tion that pump activation should acidify the apoplastic

pH and alkalize the cytoplasm, there is ample evi-

dence in the literature that pump activity and pH

(changes) are not necessarily related. The observation

that the apoplastic pH increases in the recovery phase

of APs, when the pump actively repolarizes or even

hyperpolarizes the plasma membrane (Felle and

Zimmermann, 2007), clearly shows that pump activity

and apoplastic pH or changes thereof are not related.

Apart from being buffered by weak acids/bases or

being part of biochemical reactions that produce/

consume H

, pH is a general mediator and regulator

of membrane transport that also involves other ions

than H

. Acid base chemistry (Stewart, 1983) pre-

dicts that a pH change within a given compartment

is not dependent on a transmembrane H

displace-

ment but on a change of the strong ion ratio. FC, for

example, which undoubtedly stimulates the plasma

membrane H

pump and H

extrusion, may lead to

external alkalinization (Ullrich et al., 1991) and may

acidify the cytoplasm (Bertl and Felle, 1985), or it

may hardly affect the pH of either side of the mem-

brane (Ullrich and Novacky, 1990). Here, we observe

that an insigniﬁcant initial pH decrease is followed

by a substantial transient pH increase while the

voltage recovers. The apoplastic alkalinization ob-

served here may have several causes. (1) Due to the

known poor selectivity of anion channels (Hedrich

et al., 1994; Schmidt and Schroeder, 1994), the efﬂux

of Cl

2

is accompanied by the efﬂux of a variety of

organic acids; as soon as these enter the acidic

apoplast, protons are bound and cause a pH in-

crease. (2) Due to the hyperpolarization, the driving

force for H

cotransport is increased, which could

drain the apoplast of H

to some extent. (3) Cell walls

contain high concentrations of uronic acids with pK

values similar to that of polygalacturonic acid.

Thus, either cations of the apoplast are reversibly

retained as free hydrated ions or they become im-

mobilized. An activity decrease of cations (Fig. 6)

means more free negative charges that can be occu-

pied by H

, which will increase the apoplastic pH, as

demonstrated (Felle, 1998). Thus, the somewhat un-

expected pH response would not contradict our

pump hypothesis.

The Novelty of SPs

Hyperpolarization during long-distance signaling

has been reported before. (1) Eschrich et al. (1988)

investigated the transmission of electric signals in

sieve tubes of zucchini (Cucurbita pepo) and observed

AP-like depolarizations induced by the addition of 100

Suc at the petiole, which was picked up after 10 to

40 s as hyperpolarization at the fruit (40 cm away).

Although no deﬁnite interpretation was given, it is

possible that the phenomenon is similar to what we

are describing here. On the other hand, the observation

that a hyperpolarization at the petiole turned into a

depolarization at the fruit would make this interpre-

tation unlikely. (2) Fromm and Eschrich (1993) showed

that 2 m

MgSO

added to the roots of willow (Salix

viminalis) causes an immediate rapid and transient

hyperpolarization, which is transmitted without dec-

rement at 5 cm s

to the leaf mesophyll. Since the

velocity of these transmissions, their duration, and

their lack of decrement were very different from what

we found, we suggest that these AP-like phenomena

have nothing in common with the SPs. (3) Fromm et al.

(1997), testing the effects of phytohormones on the

endogenous current in willow roots, observed an

abscisic acid-induced hyperpolarization that was

transferred from the root to the tip. Lautner et al.

(2005) describe chilling-induced hyperpolarizations in

poplar (Populus species) leaves, which propagated

basipetally. In both reports, the authors presented

evidence that hyperpolarizations were caused by K

channel activation; this is not comparable to the SPs,

where K

ﬂuxes were shown to be the result and not

the cause of the voltage changes (Fig. 6).

Again, this study is not about long-distance signal-

ing in highly specialized plants or organs; it is directed

toward the ordinary plant that encounters stresses and

hazards to which it must respond quickly and appro-

priately. In such plants, APs and VPs have so far been

considered the only relevant electrical long-distance

signals, a view that ought to be reconsidered. In Table

I, the most essential characteristics of APs, VPs, and

SPs are compared with each other, showing that SPs

do not have much in common with APs or VPs.

Whereas due to their all-or-none characteristics, APs

do not carry much information with respect to the

nature or intensity of the triggering stimulus, SPs are

modulated in amplitude as well as in their interde-

pendent ion ﬂuxes, from which the plant or the af-

fected organ may be able to gain information about the

nature and intensity of the threat or injury. Thus, the

SPs described here are a basic kind of self-propagating

signal that may occur regularly when the membrane

potential is shifted considerably from its set value

through the combination of an injury and a chemical

stimulus, the result of which is challenged by H

pump activity changes. This way, the systemic organ

encounters a broad spectrum of information regarding

the kind of disturbance suffered some distance away.

The information transferred lies not only in the voltage

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change but also in the changes in ion activities on both

sides of the membrane: pH changes will alter enzyme

activities and gene activation, K

changes will cause

water ﬂow and cell turgor, and Ca

inﬂux will in-

crease cytosolic free Ca

and modulate signal chains.

Whether SPs are in fact a main electric signal trans-

mission following, for instance, herbivore attack in

order to initiate systemic defense or priming is the

focus of our ongoing research. Preliminary data sug-

gest that this likely is the case.

MATERIALS AND METHODS

Plant Material

Plants of Vicia faba and Hordeum vulgare ‘Ingrid’ (Deutsche Saatveredelung)

were grown from seed in a plastic pot under a 12-h/12-h light/dark regime at

°C to 25°C in a greenhouse. Intact 40- to 50-cm-long V. faba or three- to four-

leafed H. vulgare plants were used throughout the experiments.

Recording Apoplastic Voltage

Whole V. faba plants or H. vulgare plants were mounted on a cuvette inside

a Faraday cage. As described earlier (Felle et al., 2000; Felle and Zimmermann,

2007), the target leaves were tightly ﬁxed on a Plexiglas plate with a double

adhesive tape to prevent movement during the measurements. The leaves

were illuminated with a cold light lamp (Leica; KL1500; Wetzlar) to induce

stomatal opening. Under optical control (microscope) using a 20

3 long-

distance objective, two or three blunt electrodes (tip diameter about 5

mm;

ﬁlled with 0.5

KCl/agar) were positioned at an angle of 40

° to 50° in

substomatal cavities of neighboring open stomata. The earth electrode (ﬁlled

with 0.5

KCl) was placed at the cut tip of this leaf, submerged in a solution

comprising 10 m

KCl, 1 m

CaCl

, and 1 m

(MES + Tris), and mixed to pH

5. Electrodes were connected with a high-impedance ampliﬁer (World Preci-

sion Instruments; FD223); kinetics were recorded by a pen chart recorder

(Linseis; L2200). As soon as the tips of the electrodes came into contact with

the apoplastic ﬂuid, the electrical circuit was closed. The voltages given

depend on the distance and the apoplastic network resistance between the

voltage and the earth electrodes. Membrane potential measurements were

carried out with sharp tips (0.5

mm) by inserting them into a mesophyll cell.

To test nonbiotic systemic responses, a leaf (S-leaf) was cut with scissors

and the test solution (stimulus) was immediately applied; the response to this

treatment was monitored with two or three electrodes on a different leaf

(T-leaf; Fig. 1). The solutions tested were KCl, CaCl

, MgCl

, NaCl, Glu, FC, and

orthovanadate at concentrations given in the ﬁgures or respective legends.

Ion-Selective Microelectrodes

Ion-selective microelectrodes were fabricated and used as described before

(Felle et al., 2000; Felle and Zimmermann, 2007). Brieﬂy, all microprobes were

fabricated from 1.5-mm (o.d.) borosilicate glass tubing (Hilgenberg). Capil-

laries were pulled on a two-stage puller (List-L/P-3P-A) to tips of 2 to 5

mm,

which were heat polished over a platinum wire to prevent injury to cells.

These capillaries were ﬁlled with 0.5

KCl to be used as apoplastic voltage

electrodes. To prepare ion-selective electrodes, capillaries were heated to

200

°C in an oven for 1 h and silanized internally by repeatedly dipping the

blunt end of the hot capillaries into a 0.02% tributylsilane/chloroform solu-

tion. The capillaries were kept at 200

°C for approximately 1 h before the

silanization procedure was repeated. Silanized capillaries (cooled to room

temperature) were backﬁlled with the respective sensor mixture (H

, 95297;

; 21196; K

, 60398; Cl

, 24899; Fluka Chemical) and topped up with the

reference solution (i.e. K

and Cl

electrodes, 100 m

KCl; Ca

electrode,

1 m

M

CaCl

; H

electrode, 0.5

KCl buffered to pH 6 with MES + Tris). All

electrodes were stored for several days before use. Double-barreled electrodes

were fabricated from double-barreled tubing (Hilgenberg). After being pulled

to about 5-

mm tips, barrels were ﬁlled with the respective sensor as described

above. Ready-to-use electrodes were connected through a suitable electrode

holder (World Precision Instruments) with a high-impedance (10

V) ampli-

ﬁer (WPI-FD223; World Precision Instruments). Kinetics were recorded on a

chart (Linseis; L2200). Signals coming from the ion-selective electrodes consist

of both apoplastic voltage and ion-speciﬁc voltage, generated at the electrode

tips. To obtain the net signal, traces were subtracted from each other through

the differential ampliﬁer.

Pressure Injection

A glass pipette (tip diameter, 5–10

mm) was used to inject orthovanadate

into the T-leaf apoplast; the pipette was positioned in the substomatal cavity of

open stomata. After applying pressure of approximately 50 psi through a

pneumatic picopump (World Precision Instruments), the injected ﬂuid spon-

taneously dispersed within the leaf apoplast. To inﬁltrate the entire width of

the leaf, this procedure had to be repeated in different stomata three or four

times.

Conventions

With an intracellular recording, a depolarization of the plasma membrane

occurs when the cell interior becomes less negative, whereas for an apoplastic

recording, the reverse argument holds true. To avoid confusion, throughout

this article we follow the convention and call an apoplastic hyperpolarization

a depolarization. Since apoplastic voltage can be inﬂuenced by a variety of

processes and, therefore, unlike a membrane potential is not clearly deﬁned,

we give no absolute values, just the polarity (

6) together with relative voltage.

Received December 8, 2008; accepted December 22, 2008; published January 7,

2009.

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