Download Induction of wound response gene expression in tomato leaves by

Planta (2001) 212: 431±435
Induction of wound response gene expression in tomato leaves
by ionophores
Andreas Schaller, David Frasson
Institute of Plant Sciences, ETH-ZuÈrich, UniversitaÈtstrasse 2, 8092 ZuÈrich, Switzerland
Received: 16 May 2000 / Accepted: 12 August 2000
Abstract. Three ionophores were used to investigate a
potential role of the plasma-membrane (PM) potential in
the regulation of systemic wound-response gene expression in tomato (Lycopersicon esculentum Mill.) plants.
Valinomycin, nigericin, and gramicidin, which a€ect the
PM potential by dissipating H+ and K+ gradients,
respectively, induced the rapid accumulation of woundresponse gene transcripts. Transcript induction by gramicidin was kinetically, qualitatively and quantitatively
similar to systemin-induced transcript accumulation. On
a molar basis, gramicidin and nigericin, which a€ect
gradients of both H+ and K+, were more e€ective than
the K+-selective valinomycin. Hyperpolarization of the
PM by fusicoccin, on the other hand, repressed woundresponse gene expression and, at the same time, induced
salicylic acid (SA) accumulation and the expression of
pathogenesis-related proteins. We show here that the
inhibition of the wound response after fusicoccin treatment is not mediated by elevated concentrations of SA
but is likely a direct e€ect of PM hyperpolarization. The
data indicate a role for the PM potential in the di€erential regulation of wound and pathogen defense responses.
Key words: Ionophore ± Lycopersicon (wound
response) ± Plasma-membrane potential ± Systemin ±
Wound response
Introduction
In tomato plants, herbivory and mechanical wounding
trigger the accumulation of systemic wound-response
proteins (SWRPs) in the wounded as well as in systemic
leaves (Ryan and Pearce 1998; Ryan 2000). The induction
Abbreviations: AIP ˆ 2-aminoindan-2-phosphonic acid; FC ˆ
fusicoccin; PM ˆ plasma membrane; SA ˆ salicylic acid; SWRPs ˆ
systemic wound response proteins
Correspondence to: A. Schaller;
E-mail: [email protected]; Fax: +41-1-6321084
of defense-gene expression is mediated by the 18-aminoacid peptide systemin (Pearce et al. 1991). In response to
wounding, systemin is thought to be released into the
apoplast, where it interacts with a plasma membrane
(PM) receptor (Meindl et al. 1998; Scheer and Ryan
1999) thereby triggering an intracellular signaling cascade resulting in SWRP-gene activation. Intracellular
signaling events include the activation of a myelin basic
protein protein kinase (Stratmann and Ryan 1997), the
activation of phospholipase A2 (NarvaÂez-VaÂsquez et al.
1999), and the release of linolenic acid from membrane
lipids (Conconi et al. 1996). Linolenic acid is the
substrate of oxylipin biosynthesis via the octadecanoid
pathway resulting in the production of jasmonic acid.
Together with ethylene (O'Donnell et al. 1996), jasmonic
acid induces the expression of defense genes. While this
sequence of events has been established and well
documented by C.A. Ryan and his group (for review
see Ryan 2000), the initial stages of systemin signaling,
i.e. the stimulus-response coupling at the level of the PM,
are still poorly understood.
Evidence is accumulating, however, that changes in
the ionic ¯uxes across the PM are among the earliest
cellular responses triggered by systemin (reviewed by
Schaller 1999). In cell-suspension cultures of Lycopersicon peruvianum, systemin causes H+in¯ux/K+e‚ux
resulting in a dose-dependent, transient alkalinization of
the growth medium (Felix and Boller 1995; Schaller
1998). Alkalinization of the extracellular space and
depolarization of the PM potential in response to
systemin has also been observed in tomato mesophyll
cells (Moyen and Johannes 1996). Furthermore, systemin
was shown to cause a rapid increase in the cytosolic Ca2+
concentration due to both the in¯ux of extracellular Ca2+
as well as the release of Ca2+ from intracellular stores
(Moyen et al. 1998). Chelation of extracellular Ca2+ and
the inhibition of PM Ca2+ channels was shown to
prevent systemin-induced alkalinization of the apoplast,
indicating that for this response to external systemin the
in¯ux of Ca2+ is required (Schaller and Oecking 1999).
Interestingly, very similar observations have been
made for oligogalacturonides, a second class of extra-
432
cellular signaling molecules that induce the expression of
SWRP genes (Bishop et al. 1984). Oligogalacturonides
cause an in¯ux of Ca2+, PM depolarization, and
extracellular alkalinization in tobacco cell cultures
(Mathieu et al. 1991). In tomato leaf cells, a depolarization of the PM in response to oligogalacturonides was
observed and the inhibition of the PM H+-ATPase has
been implicated in this response (Thain et al. 1995).
The PM H+-ATPase builds up and maintains a
proton gradient across the PM at the expense of ATP
(reviewed by Palmgren 1991). Inhibition of the H+ATPase resulting in alkalinization of the apoplast and
PM depolarization caused the induction of SWRP-gene
expression (Schaller and Oecking 1999). Activation of
the pump by the fungal toxin fusicoccin (FC), on the
other hand, results in a hyperpolarization of the PM and
extracellular acidi®cation (Marre 1979). Interestingly,
proton pump activation by FC suppressed the systemininduced membrane depolarization/alkalinization response as well as the wound-, systemin-, and oligogalacturonide-induced expression of SWRP genes
(Doherty and Bowles 1990; Schaller and Oecking
1999). These ®ndings indicate membrane depolarization/extracellular alkalinization (or the underlying ion
¯uxes) as early steps in the signaling pathway for
defense-gene expression. In this study, evidence is
presented for these ion ¯uxes being both necessary and
sucient for the induction of SWRP-gene expression.
Materials and methods
A. Schaller et al.: Wound response gene expression in tomato leaves
Nigericin, on the other hand, is of the carboxylic type and
facilitates the exchange of H+ and K+ ions across the
PM driven by their respective chemical gradients.
Gramicidin is a channel-forming peptide exhibiting
selectivity for monovalent cations and, hence, e€ects
the dissipation of H+ and K+ gradients (GoÂmez-Puyou
and GoÂmez-Lojero 1977). All three ionophores caused a
dose-dependent accumulation of SWRP transcripts in
leaves of tomato plants (Fig. 1). On a molar basis,
nigericin was found to be at least 10 times as active as
valinomycin, causing a substantial induction of SWRP
transcripts at a concentration as low as 10 nM. At 1 lM,
both ionophores induced SWRP transcripts to levels
approaching those observed after treatment with saturating concentrations of systemin (5 nM). Gramicidin
also induced SWRP-gene expression. The response
appeared to be biphasic: SWRP transcripts were induced
above control levels by gramicidin at 30 nM. Concentrations of 100 and 300 nM were less e€ective, while
strong induction was observed at >1 lM gramicidin.
The biphasic appearance of the gramicidin response,
while observed reproducibly in two independent experiments, remains unexplained. The ion-conducting gramicidin channel is a transmembrane dimer and channel
opening is controlled by the monomer-dimer equilibrium. Two distinct dimer conformations are known to exist
di€ering in their ion-conducting properties (Wallace
2000, and references therein) and may be the reason for
the complex dose-dependence of the gramicidin response.
The induction kinetics of SWRP transcripts were
rapid for all three ionophores (Fig. 2). An increase in
Growth and treatment of plants
Tomato (Lycopersicon esculentum cv. Castlemart II) plants were
grown as described previously (Schaller et al. 2000). Experimental
plants were 12±14 d old and had two fully developed leaves. For
bioassays, compounds to be tested were supplied to tomato
seedlings cut at the base of their stem via the transpiration stream
and the expression of SWRP genes was analyzed on RNA gel blots
as described previously (Schaller and Oecking 1999; Schaller et al.
2000). Systemin and 2-aminoindan-2-phosphonic acid (AIP) were
added from aqueous stock solutions at the concentrations indicated
in the text and ®gure legends. Fusicoccin (Sigma), valinomycin,
nigericin and gramicidin A (all from Fluka, Buchs, Switzerland)
were added from 1 mM stock solutions in ethanol or dimethyl
sulfoxide, respectively. The concentration of the solvent never
exceeded 1% during the feeds and, at this concentration, did not
have any e€ect on defense-gene expression in tomato leaves.
Assay of salicylic acid (SA) concentration
Leaf SA concentrations were determined as described by Raskin
et al. (1989) with minor modi®cations (Malamy et al. 1992;
Schaller and Oecking 1999).
Results and discussion
Three mechanistically distinct ionophores (valinomycin,
nigericin, and gramicidin) were tested for their e€ects on
defense-gene expression. Valinomycin is a neutral
ionophore dissipating the electrochemical K+ gradient.
Fig. 1. Dose-dependence of SWRP-transcript induction by ionophores. Tomato seedlings with two expanded leaves were used for
feeding experiments. They were cut at the base of their stems and
supplied through the transpiration stream for 1 h with bu€ered
solutions (10 mM phosphate bu€er pH 6.0) of systemin (sys, 5 nM),
or valinomycin, nigericin or gramicidin at the concentrations indicated
(lM). After 8 h, leaf tissue was harvested and RNA was extracted.
Five micrograms of total RNA was analyzed by RNA gel blotting
and probed for transcripts of three typical SWRPs, i.e. proteinase
inhibitor II (PI-II), threonine deaminase (TD), and leucine aminopeptidase (LAP). Duplicate gels were stained with ethidium bromide
as a control of RNA loading. The representative result of one of two
independent experiments is shown
A. Schaller et al.: Wound response gene expression in tomato leaves
Fig. 2. Kinetics of SWRP-transcript induction by ionophores.
Tomato plants were treated (cf. Fig. 1) with valinomycin (2 lM),
nigericin (2 lM) or gramicidin (1 lM) for 1 h and then transferred to
water. At the time points indicated (h), leaf tissue was harvested and
RNA was extracted. Seven micrograms of total RNA was analyzed
by RNA gel blotting and probed for transcripts of proteinase
inhibitors I and II (PI-I, PI-II), cathepsin D inhibitor (CDI), leucine
aminopeptidase (LAP), and threonine deaminase (TD), and a
duplicate gel was stained with ethidium bromide as a control of
RNA loading. The experiment was repeated twice with similar results
transcript abundance was observed as early as 1 h after
treatment. For valinomycin (2 lM) and nigericin
(2 lM), the transcript levels of most of the SWRPs
analyzed peaked between 4 and 6 h after treatment of
plants. In the case of gramicidin (1 lM) treatment,
SWRP transcripts continued to accumulate throughout
the 8-h experiment (Fig. 2). Therefore, the time course
as well as the extent of gramicidin-induced transcript
accumulation resembles the induction of defense-gene
expression by systemin (Ryan 2000). This is the ®rst
demonstration of SWRP-gene activation by ionophores.
We did not analyze the e€ects of valinomycin, nigericin,
and gramicidin on the PM potential in tomato leaf cells.
Considering the well-known e€ects of these ionophores
on ion permeability, however, the data suggest that
changes in ion permeability leading to membrane
depolarization and extracellular alkalinization may be
sucient for the induction of defense-gene expression.
This interpretation is consistent with the previously
observed accumulation of SWRP transcripts in leaves of
tomato plants after treatment with inhibitors of the PM
H+-ATPase (Schaller and Oecking 1999). It is not yet
clear, however, whether the signal is carried by any one
of the ion ¯uxes in particular, by the extracellular
alkalinization, or, alternatively, by cytosolic acidi®cation (Roos et al. 1998). Furthermore, it remains to be
established how the inducing ion ¯uxes relate to
the systemic transmission of the wound stimulus. On
the one hand, membrane depolarization/extracellular
433
alkalinization is induced by systemin (Felix and Boller
1995; Moyen and Johannes 1996; Schaller and Oecking
1999), which is a candidate molecule for a systemically
transmitted chemical signal (Pearce et al. 1991; Ryan
2000). On the other hand, depolarization of the PM has
also been implicated in electrical and hydraulic mechanisms of systemic wound signal transduction (Wildon
et al. 1992; Malone 1996; Vian et al. 1996; Stankovic
and Davies 1997; Herde et al. 1998). It seems likely that
chemical, electrical, and hydraulic mechanisms of signaling are not exclusive but rather interact.
Hyperpolarization of the PM by FC-mediated activation of the PM H+-ATPase has previously been
shown to suppress both systemin-induced alkalinization
of the apoplast and the induction of SWRP-gene
expression suggesting that depolarization/alkalinization
is not only sucient but also necessary for the activation
of defense genes (Schaller and Oecking 1999). However,
FC had additional e€ects in that it induced the expression of pathogenesis-related (PR) genes as well as the
accumulation of SA in leaves of tomato plants (Fig. 3;
Roberts and Bowles 1999; Schaller and Oecking 1999;
Schaller et al. 2000). Salicylic acid (SA) is known to
exert an inhibitory e€ect on the induction of SWRPgene expression (Doherty et al. 1988; PenÄa-CorteÂs et al.
1993; Doares et al. 1995). Hence, the suppression of
SWRP-gene induction by FC may be an indirect e€ect
caused by elevated levels of SA, rather than a direct
consequence of PM hyperpolarization. To di€erentiate
between the two alternatives we attempted to suppress
FC-induced SA accumulation and investigated whether
or not the inhibitory e€ect of FC on SWRP-transcript
induction persists in the absence of SA accumulation.
In SA biosynthesis, the conversion of phenylalanine
to cinnamic acid by phenylalanine ammonia-lyase
(PAL) is an essential step (Coquoz et al. 1998). A
speci®c in-vivo inhibitor of PAL activity, 2-aminoindane-2-phosphonic acid (AIP), has been described (ZonÂ
and Amrhein 1992) and has been used to assess the role
of SA in pathogen defense responses (Mauch-Mani and
Slusarenko 1996) and in the FC-mediated induction of
PR gene expression (Schaller et al. 2000). Neither AIP,
nor FC when supplied individually had an e€ect on
SWRP-gene expression in tomato leaves (data not
shown). In FC-treated tomato plants, however, AIP
e€ectively inhibited the accumulation of SA (Fig. 3;
Schaller et al. 2000) but did not alleviate the suppression
of SWRP transcripts (Fig. 3). Hence, notwithstanding
the known inhibitory e€ect of SA on SWRP-gene
induction (Doherty et al. 1988; PenÄa-CorteÂs et al.
1993; Doares et al. 1995), the repression of SWRP-gene
expression after FC treatment is not mediated by SA. It
rather seems to be a direct consequence of FC-mediated
PM hyperpolarization. The data may provide an explanation for the early observation of an inhibitory e€ect of
auxins on SWRP-gene activation. In detached potato
leaves, 2,4-dichlorophenoxy acetic acid (2,4-D) inhibited
the accumulation of chymotrypsin inhibitor I, i.e. an
SWRP (Ryan 1968). Furthermore, naphthalene acetic
acid inhibited the expression of the chloramphenicol
acetyl transferase reporter gene under regulation of the
434
A. Schaller et al.: Wound response gene expression in tomato leaves
in terms of their hyperpolarizing activity, which counteracts systemin-mediated depolarization of the PM.
Conclusions
We have shown the ecient dose- and time-dependent
induction of SWRP-transcript accumulation by the
ionophores valinomycin, nigericin, and the channelforming peptide gramicidin. Furthermore, we showed
that the inhibitory e€ect of FC on SWRP-transcript
accumulation persists under conditions of inhibited SA
biosynthesis, indicating that it is the membrane-hyperpolarizing activity of FC that inhibits SWRP-transcript
induction. The data provide further support for a role of
the PM potential and the PM H+-ATPase as its major
electrogenic pump in the di€erential regulation of the
wound and pathogen defense responses.
We thank Dr. N. Amrhein for critical reading of the manuscript
and for support. This work was supported by grants of the Swiss
National Science Foundation to A.S.
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Fig. 3. The e€ect of AIP on the FC-mediated induction of SA and
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supplied through the transpiration stream with bu€ered solutions
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