Journal of Experimental Botany, Vol. 49, No. 319, pp. 239–247, February 1998
Effects of salicylic acid on sugar and amino acid uptake
Andrée Bourbouloux1,3, Philippe Raymond2 and Serge Delrot1
1 Laboratoire de Physiologie et Biochimie Végétales, ERS CNRS 6099, Bâtiment Botanique,
40 Avenue du Recteur Pineau, F-86022 Poitiers Cédex, France
2 INRA, Station de Physiologie Végétale, F-33883 Villenave d’Ornon Cédex, France
Received 30 April 1997; Accepted 19 September 1997
Abstract
Ageing of sugar beet (Beta vulgaris L.) leaf discs
induces a large stimulation of sugar and amino acid
uptake. This may result from an hormonal imbalance.
The effects of various hormones added during the
ageing period were studied on sugar and amino acid
uptake. Salicylic acid (SA) inhibited both valine and
sucrose uptake in a concentration-dependent manner
from 10 mM to 200 mM. This inhibition was specific for
SA: various mono- and di-substituted analogues of
benzoic acid did not significantly affect uptake, except
for 2,4-dichlorobenzoic acid. SA inhibited the active
component of uptake, and the inhibition was reversible
upon its removal. SA did not affect the amounts of
transcripts encoding the sucrose transporter and the
amounts of the sucrose transporter protein. SA
decreased medium acidification due to the activity of
the plasma membrane H+-ATPase, without affecting
the amounts of ATPase transcripts and ATPase protein.
SA significantly decreased the energy charge of the
tissues during ageing. It is suggested that SA may
affect the uptake of sugars and amino acids by indirect
inhibition of the plasma membrane H+-ATPase energizing this process.
Key words: Ageing, Beta vulgaris, energy charge, membrane transport, nutrient uptake.
Introduction
Flotation of excised leaf tissues for a few hours on a
simple medium (ageing) induces important changes in
metabolic activities, including a stimulation of the uptake
of organic compounds such as hexoses, sucrose and amino
acids (Sakr et al., 1993). Uptake rates of these nutrients
depends on the activity of their respective H+-cotransporters and on the plasmalemma ATPase which energizes
this transport. Stimulation of sucrose uptake by ageing
results from an increased activity of the plasma membrane
H+-ATPase energizing the sucrose transporter, whereas
stimulation of hexose and valine uptake involves both an
increased activity of the ATPase and of the corresponding
hexose and amino acid transporter (Noubhani et al.,
1996; Sakr et al., 1997).
The effects induced by ageing may result from excision
of leaf tissues, removal of the lower epidermis and cutting
of discs which provokes wounding responses in the tissues
and may affect the hormonal balance. Volatile methyl
jasmonate can be released from plant tissues, especially
if wounded (Albrecht et al., 1993); the levels of amino1-cyclopropane carboxylic acid synthase transcripts level
increase largely between 1 h and 4 h after wounding in
sliced tomato fruit (Li et al., 1992), and it is well known
that auxins and cytokinins are involved in the formation
of wound-induced callus. Therefore, the increase of nutrient uptake which develops with ageing may be affected
by hormones. This hypothesis was tested in sugar beet
leaf discs using several phytohormones or hormone-like
substances.
The effects of phytohormones on nutrient transport
have been studied largely at the physiological level with
whole plants or isolated organs. These data led to the
general conclusion that auxin, gibberellin and cytokinin
would be able to promote assimilate transport (Morris,
1996), whereas abscisic acid may be stimulatory (Clifford
et al., 1986) or inhibitory (Estruch et al., 1991). In this
3 To whom correspondence should be addressed. Fax: +33 549 45 41 86. E-mail: [email protected]
Abbreviations: 2,4-Cl BA, 2,4-dichlorobenzoic acid; 2,4-OH BA, 2,4-dihydroxybenzoic acid; 2,6-Cl BA, 2,6-dichlorobenzoic acid; p-OH BA,
4-hydroxybenzoic acid; 2,6-OH BA, 2,6-dihydroxybenzoic acid; CCCP, carbonyl-cyanide m-chlorophenylhydrazone; DEPC, diethyl pyrocarbonate; m-OH
BA, 3-hydroxybenzoic acid; PD, transmembrane potential difference; PMV, plasma membrane vesicles; SA, salicylic acid (o-hydroxybenzoic acid); SSC,
sodium (chloride) sodium citrate buffer; TCA, trichloracetic acid.
© Oxford University Press 1998
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Bourbouloux et al.
system, it turned out that SA induced the most dramatic
effects. SA belongs to a group of plant phenolics widely
distributed in plants. Early studies have described its
flower-inducing effects related to inhibition of ethylene
synthesis (Leslie and Romani, 1986), and its involvement
in heat production in plants ( Raskin et al., 1989). SA is
now considered as an hormone-like substance and is
recognized as a key molecule involved in local and
systemic responses to viral infection (Ryan and Farmer,
1992). To our knowledge, only a few studies in the
literature have been devoted to the effect of SA (or its
derivatives) on the uptake properties of plant tissues, and
they dealt only with ion uptake. These facts and recent
results led us to investigate in more detail the mode of
action of this compound, which has never been studied
for its effects on assimilate transport.
Materials and methods
Plant material
Sugar beet (Beta vulgaris L.) mature leaves were obtained from
plants grown in conditions previously described (Lemoine et
al., 1991). Discs (6 mm in diameter) were excised from leaves
after peeling off the lower epidermis and floated in the dark on
a medium (basal medium) containing 300 mM mannitol,
0.5 mM CaCl , 0.25 mM MgCl and 20 mM MES (pH 5.0).
2
2
Upon flotation for a few hours on this medium, plant tissues
undergo the ageing phenomenon which induces, within 12 h, a
marked stimulation of sugar and amino acid uptake (Sakr et
al., 1993).
Uptake assays
Leaf discs were floated in the light for 30 min at 25 °C on basal
medium added with 1 mM -[3,4(n)-3H ] valine (11.1
MBq mmol−1, final activity) and 1 mM U-[14C ] sucrose (7.4
MBq mmol−1, final activity). The leaf discs were rinsed 3×3 min
on fresh basal medium to remove apoplastic label and their
radioactivity was measured by liquid scintillation counting.
Depending of the experiments, the leaf discs were pretreated
for various times during the ageing period with phytohormones
and hormone-like substances. The compounds tested included
auxin (indole-3-acetic acid, IAA), abscisic acid (ABA), gibberellin A (GA ), jasmonic acid (JA), and mainly SA, as detailed
3
3
in the Results.
Electrophysiological measurements
The PD of mesophyll cells was measured with standard
electrophysiological techniques. Leaf tissues without lower
epidermis were stuck (peeled side up) to the bottom of a Petri
dish with an inert paste ( Terostat). The tissues were rinsed with
3 ml of the medium previously described (pH 5.0) and incubated
for 30 min with the same buffered medium before PD measurements. A glass micropipette (tip diameter <1 mm; tip resistance
from 5–30 MV) was inserted into a mesophyll cell. The output
signal between this electrode and a reference (filled with 3 M
KCl in 1% agar) was recorded with the equipment previously
described (Mounoury et al., 1984). SA or analogues were
dissolved in the buffered medium and added to the fresh tissues
during the recording to determine the initial impact of SA on
the membrane.
pH measurements
Mature leaf tissues (1 g) without lower epidermis were incubated
on 20 ml of an aerated medium containing 300 mM mannitol,
0.5 mM CaCl and 0.25 mM MgCl (initial pH 6.1). In some
2
2
experiments, the variations of the pH of the incubation medium
were recorded continuously. After a lag phase of about 90 min,
the pH of the medium decreased for several hours due to the
activity of the proton-pumping plasma membrane ATPase
(Noubhani et al., 1996). pH measurements were run with fresh
tissues treated or not with SA or analogues during the recording.
In this case, the benzoic compounds were added to the
incubating medium at the beginning of the rapid acidification
phase. In other experiments, tissues were treated (or not,
control ) with SA or its analogues just before the start of the
pH recordings.
In some experiments, the buffering capacity of the medium
and leaf tissues was assessed by measuring the pH shift induced
by addition of 150 ml 0.01 N HCl.
ELISA tests and Western blot analysis
Purified PMV were obtained by phase partitioning of microsomal fractions (Noubhani et al., 1996) from three different
sets of plant material: fresh, aged (12 h) and SA-treated leaf
discs (100 mM, 12 h). ELISA tests were run on the purified
PMV to estimate the amounts of the sucrose transporter and
of the ATPase as already described (Li et al., 1992). Antisera
directed against the Arabidopsis thaliana sucrose transporters
AtSUC1 or AtSUC2 (Stadler and Sauer, 1996) and the
Arabidopsis thaliana plasma membrane H+-ATPase (antibody
721 directed against the central cytoplasmic domain of the
isoform 3; Pardo and Serrano, 1989) were used as the primary
antibody. Anti-AtSUC1 and anti-AtSUC2 sera were diluted at
1/100 and anti-ATPase serum at 1/500. Horseradish peroxidaseconjugated goat antirabbit antibody was used at 1/2000 as the
secondary antibody.
Western blots were performed after separation of the plasma
membrane proteins by electrophoresis on a 10–22% SDS-PAGE
gel and transfer on 0.45 mm nitrocellulose membrane (Noubhani
et al., 1996). The efficiency of transfer was checked by the
Rouge Ponceau method. The antibodies were used in the same
conditions as for ELISA tests.
Northern experiments
Total RNA was extracted from aged and SA-treated leaf discs.
Leaf discs (600–800 mg FW ) were frozen in liquid nitrogen
and ground in a cold mortar with 1.8 ml of grinding medium
(25 mM TRIS-HCl pH 8.0, 25 mM EDTA, 75 mM NaCl, 1%
SDS, and 1 M b-mercaptoethanol ). The aqueous extract was
mixed twice with an equal volume of phenol/chloroform/
isoamyl alcohol (25/24/1, by vol.) to remove proteins and rinsed
with chloroform/isoamyl alcohol (24/1, v/v). Ten M LiCl was
added in the RNA-containing aqueous phase to obtain a 2 M
LiCl mixture which was kept at 4 °C during the night. After
centrifugation (10 000 g, 15 min), the pellet was rinsed with 2 M
LiCl and resuspended in DEPC-treated water. RNA was
precipitated at −20 °C for 20 min after the addition of absolute
ethanol/300 mM Na acetate (3/1, v/v). The precipitated RNA
was pelleted at 10 000 g (20 min) and washed twice with 1 ml
of cold 70% ethanol to discard contaminating polysaccharides
and remaining salts. After extraction, the RNA pellet was
resuspended in DEPC-treated water and stored at −70 °C.
RNA denaturation and Northern blots were run essentially
as described by Noubhani et al. (1996). RNA samples were
fractionated by a denaturating electrophoresis on agarose gels
containing 2.2 M formaldehyde, using a MOPS buffer (100 mM
Salicylic acid and membrane transport
MOPS pH 7.0, 40 mM Na acetate, 5 mM EDTA). They were
transferred on 0.45 mm nylon membranes by capillarity and
fixed by heating at 80 °C for 2 h. Prehybridization was run for
4 h in a medium containing 0.25 M Na phosphate buffer
(pH 7.2), 6.6% SDS, 1 mM EDTA (pH 8.0) with 1% (w/w) BSA.
Northern blot analyses were run using the sugar beet sucrose
transporter probe BvSut1 (accession number X 83850) and the
Arabidopsis plasmalemma H+-ATPase probes pma2 and pma4
(Boutry et al., 1989). The probes were labelled with [32P]-dCTP
to a specific activity of about 5–8 108 dpm mg−1 by the random
priming technique with a DNA labelling kit (Amersham France,
Les Ulis). Hybridization was run in the same medium added
with the labelled probe for 16 h at room temperature for
ATPases and at 65 °C for sucrose transporter probes. The
membranes were then washed in SSC (3 M NaCl and 0.3 M
Na citrate pH 7.0 for 1×SSC ) media (from 2× to 0.1×SSC )
and 0.1% SDS at 42 °C for ATPases and at 65 °C for sucrose
transporter probes.
Quantification of adenine nucleotides
Leaf discs were isolated as previously described, divided into
two sets and floated on a simple medium in the presence or
absence of 100 mM SA. Samples were harvested every 2 h during
the 12 h ageing period, immediately frozen in liquid N and
2
kept at −80 °C. The extraction of adenine nucleotides was
derived from Saglio and Pradet (1980). Four frozen discs were
dropped into 2 ml of 0.6 M TCA in diethylether at −20 °C and
homogenized with a Polytron apparatus; 2.5 ml of aqueous
0.6 M TCA were added and homogenization was repeated. The
homogenizer was then rinsed with 2.5 ml of aqueous TCA and
the extracts were pooled together. TCA was removed by three
extractions with 40 ml of diethylether. Remaining traces of
ether were evacuated by air bubbling and the pH of the extracts
was raised to 5.0 with diluted NaOH. The nucleotides were
assayed by a bioluminescence method using a Pico ATP
biophotometer. ADP and AMP were converted to ATP
according to Saglio and Pradet (1980). Internal standardization
was used in all ATP determinations.
Statistical analysis
For comparison among treatments, an analysis of variance was
used. Student’s t-test was used in cases of equal variances to
compare two mean values from samples with a normal
distribution. For each treatment, means followed by different
letters are significantly different at P≤0.05 or otherwise stated.
241
that these hormones only prevented partially the effects
of ageing. The strongest inhibition was observed with SA
on both valine and sucrose uptake. Therefore, further
experiments were run with SA.
Effect of SA applied for the ageing period on the nutrient
uptake
A concentration range study showed that a SA concentration of 10 mM applied during the ageing period was
needed to induce a slight but significant decrease in
sucrose (P≤0.02, Fig. 1) and valine (data not shown)
uptake. Yet, the inhibition was more marked with
100 mM SA.
Whatever the SA concentration, addition of CCCP, a
protonophore which uncouples oxidative phosphorylation
in mitochondria and collapses the transplasmalemma pH
gradient, strongly inhibited sucrose uptake to a constant
level (Fig. 1) that was below the uptake in fresh tissues.
These results indicate that SA affects the active phase of
total sucrose uptake. Parallel experiments run with valine
in these conditions led to the same conclusion (data
not shown).
Specificity of the SA effect
Analogues of SA (mono- and di-substituted benzoic acids)
were tested on valine and sucrose uptake in comparison
with SA ( Table 1). Contrary to SA (which is o-OH BA),
m-OH and p-OH BA did not affect valine and sucrose
uptake. Both 2,4-OH BA and 2,4-Cl BA (chlorinated
derivative dissolved in DMSO) significantly inhibited
valine (P≤0.001 in both cases) and sucrose (P≤0.02 and
0.001, respectively) uptake. However, inhibition by
2,4-OH BA was only marginal, whereas inhibition by
2,4-Cl BA was similar to SA inhibition.
Results
Effect of different phytohormones on the nutrient uptake
The effects of IAA, ABA, GA , JA or SA on ageing3
induced increase of solute uptake were tested. The hormones or hormone-like compounds were added at a
concentration of 100 mM during the 12 h ageing period,
but they were omitted during the 30 min uptake period
that followed ageing. As already described (Sakr et al.,
1993), ageing induced a marked stimulation of sucrose
and valine uptake compared to fresh tissues. Three compounds (ABA, JA and SA) significantly inhibited the
ageing-induced valine uptake (P≤0.05) (data not shown).
Even after hormonal treatment, uptake in aged tissues
was always higher than in fresh tissues, which indicates
Fig. 1. Concentration-dependence of SA-induced inhibition on sucrose
uptake by aged sugar beet leaf discs. SA was added at the indicated
concentration during the ageing period. CCCP (10 mM ) was present
during the uptake period as indicated. Each point is the mean of 30
independent measurements (±SE of the mean). For each experimental
series (i.e. sucrose or sucrose+CCCP), different letters indicate that
values are statistically different at P≤0.05.
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Bourbouloux et al.
Table 1. Comparative effects of SA and related compounds on
sucrose and valine uptake
All compounds were present at 100 mM during the ageing period, 2,4-Cl
BA and 2,6-Cl BA were dissolved in 0.3% DMSO (final concentration
in the medium). A corresponding control was run. Values represent the
mean of 20 independent measurements±SE. For each nutrient, different
letters indicate that values are statistically different at P≤0.05.
Treatment
Leaf tissues were floated with or without SA for 2, 3 or 6 h at the
beginning or at the end of the ageing period. At the end of this period,
the tissues were rinsed with initial medium and kept in the dark before
the incubation with labelled solutes. Valine and sucrose uptake was
measured at the end of the ageing period. Values represent the mean of
20 independent measurements±SE. For each nutrient, different letters
indicate that values are statistically different at P≤0.05.
Nutrient uptake (nmol cm−2)
Valine
Control
SA
m-OH BA
p-OH BA
2,6-OH BA
2,4-OH BA
2,6-Cl BA
2,4-Cl BA
Control DMSO
Table 2. SA sensitivity of leaf tissues during a 12 h ageing period
33.4±1.0
5.5±0.3
32.7±0.3
33.7±0.2
34.1±0.7
29.5±0.3
30.8±0.2
5.9±0.4
26.4±0.5
Sucrose
a
b
a
a, c
a, c
d
e
b
f
12.9±1.2 g
3.1±0.2 h
12.5±0.7 g, i
12.9±0.3 g, i, j
12.1±0.6 g, i, j, k
9.6±0.5 l
11.8±0.2 g, i, k
3.7±0.2 m
11.2±0.2 g, i, k
Optimizing the effects of SA on uptake
In non-treated tissues, maximal uptake was reached
before 12 h of ageing (Sakr et al., 1993). A series of
experiments were run to optimize the inhibitory effects of
SA on ageing-induced increase in sugar and amino acid
uptake (data not shown). Tissues were treated with SA
concentrations ranging from 1–100 mM during 12, 24 and
36 h, before the uptake period (30 min). In untreated
samples, increased duration of ageing did not result in
any further stimulation of uptake compared to tissues
aged for 12 h. SA-induced inhibition of valine and sucrose
uptake was concentration-dependent from 10–100 mM.
That effect was not time-dependent (from 12–36 h of
treatment), maximal inhibition being reached after a 12 h
treatment. Valine uptake was slightly more sensitive to
SA than sucrose uptake: after a 12 h treatment with
100 mM SA, inhibition of valine uptake reached
76.0±2.0% compared to 62.1±4.0% for sucrose.
Since maximal inhibition was reached after 12 h of
ageing, further experiments were run to determine whether
the presence of SA was necessary throughout the ageing
period. Leaf tissues were incubated for short times (2, 3
or 6 h) with SA before the uptake assay ( Table 2). A 2 h
treatment with SA was sufficient to observe a significant
(P≤0.05) inhibition of uptake, although increasing the
duration of treatment resulted in a stronger inhibition.
Treatments equal in duration were slightly more efficient
when they were applied just before the incubation period
( Table 2), which suggests that the SA effect is reversible.
Reversibility of SA effect
The reversibility of SA-induced inhibition was studied to
test whether this inhibition was due to irreversible cell
damage. After a 12 h treatment with SA (100 or 200 mM ),
the tissues were rinsed and kept on the fresh initial
medium during 12 or 24 h before the uptake assay.
Time of SA-treatment
(h)
Control without SA
0–2
10–12
0–3
9–12
0–6
6–12
Control with SA
Nutrient uptake (nmol cm−2)
Valine
Sucrose
38.0±1.2 a
27.1±0.8 b
26.3±0.9 b, c
24.4±0.9 c
18.6±0.6 d
16.2±1.2 d, e
13.9±0.6 e
8.3±0.7 f
14.7±0.5 g
11.6±0.5 h
10.2±0.5 h, i
11.8±0.3 h
10.2±0.6 h, i, j
10.0±0.5 i, j, k
9.0±0.6 i, j, k
5.7±0.4 l
Control consisted in treatment with SA during 12 h. The
results indicate that a good recovery in the uptake capacity of the tissues occurred within 12 h after SA removal,
at least for the tissues treated with 100 mM SA (Fig. 2).
Much less recovery was observed for tissues treated with
200 mM SA, suggesting that the compound was toxic at
this concentration.
Effects of SA and analogues on the acidifying activity of the
tissues
Because the uptake of sugars and amino acids is mediated
by proton cotransporters, SA inhibition may result both
from direct and indirect effects on the permeability of the
membrane to protons and/or on the activity of the protonpumping ATPase and transporters. To clarify the mode
Fig. 2. Uptake recovery from SA-induced inhibition. Leaf discs were
floated on a medium with or without SA (100 or 200 mM ) for 12 h.
One set was immediately fed with labelled nutrients for 30 min; other
sets were kept floating on a simple medium for an additional 12 or 24 h
before the uptake assay. For each nutrient, the columns represent the
uptake successively in control (without SA treatment), after a 100 mM
SA-treatment for 12 h, after a 100 mM SA-treatment for 12 h and
recovery in a simple medium for 12 or 24 h, after a 200 mM SA-treatment
for 12 h, and after a 200 mM SA-treatment for 12 h and recovery in a
simple medium for 12 or 24 h. Each point is the mean of 30 independent
measurements (±SE of the mean). For each nutrient, different letters
indicate that values are statistically different at P≤0.05.
Salicylic acid and membrane transport
of action of SA, its effect was first investigated on the
acidifying activity of the tissues, which is due to the
plasma membrane H+-ATPase.
Fresh sugar beet leaf tissues acidified the incubating
medium to an average value of 4.7 within 3–4 h
(Noubhani et al., 1996). Addition of SA to fresh tissues
after the start of the acidification (i.e. about 1.5 h after
the beginning of incubation) inhibited this process in a
concentration-dependent manner ( Fig. 3A). Addition of
50 mM SA, which had only a marginal effect on uptake
( Fig. 1), only significantly delayed the acidification of the
medium for 2.5 h while 100 mM SA which markedly
hindered uptake (Fig. 1), completely inhibited the H+
excretion for at least 4 h ( Fig. 3A). A concentration of
10 mM, which only slightly but significantly affected the
uptake ( Fig. 1), was only slightly effective on the ATPase
activity between 50 and 140 min after the addition of SA.
Control experiments showed that addition of SA did not
change the buffering capacity of the medium and tissues
(data not shown). Evaluations of H+-excretion by backtitration measurements with a NaOH solution gave similar results and led to identical conclusions.
Addition of the SA analogue p-OH BA only delayed
the acidification for about 1 h; the dihydroxyle analogue
2,4-OH BA significantly (P≤0.05) decreased the rate of
H+-excretion over longer periods than did p-OH BA
( Fig. 3B). These data are in good agreement with the
effects of these compounds on the uptake of sucrose and
valine ( Table 1). Addition of 2,4-Cl BA after the start of
the incubation of the tissues resulted in immediate, large
and uncontrolled pH shifts. For this reason, the compound was added before addition of the tissues, at an
initial pH of 6.1. For comparison, SA was assayed in a
similar way. As shown on Fig. 3C, the rate of excretion
of H+ of untreated tissues increased sharply and regularly
between 150 and 300 min after the beginning of the
recording before decreasing to very small or even slightly
negative values till the end of the experimentation. The
initial increase in the apparent H+-excretion was largely
or totally (0.05≤P≤0.001 between 160 and 310 min after
the beginning of the recording) inhibited by SA and
2,4-Cl BA (two compounds hindering the uptake of
nutrients by the tissues). After about 6 h after the beginning of the recording, the rates of H+-excretion were not
significantly different between all series.
Electrophysiological measurements
The resting PD recorded in fresh control tissues ranged
between −127 and −159 mV (−139.6±1.4 mV; n=45).
Addition of SA (10–200 mM, final concentration) did not
significantly alter the PD, at least within 45 min after the
addition, although the cells responded immediately to
10 mM CCCP by a large depolarization and to 10 mM
fusicoccin which induced a strong hyperpolarization (data
not shown).
243
Fig. 3. Effects of SA and analogues on the acidifying activity of sugar
beet leaf tissues. (A) Apparent cumulative H+-excretion. SA (10 to
100 mM, final concentration) was added to the tissues (arrow) a few
minutes after the beginning of the natural acidification of the incubation
medium by the tissues. Each point is the mean of n experiments (±SE
of the mean). Control: n=11; 100 mM SA: n=6; 50 mM SA and 10 mM
SA: n=5. (B) Same expression and experimental procedure as in (A).
Hydroxylated-BA derivatives were used at 100 mM (final concentration).
Each point is the mean of n experiments (±SE of the mean). Control:
n=11; p-OH BA and 2,4-OH BA: n=5. (C ) Effect of SA and 2,4-Cl
BA on the rate of apparent H+-excretion. Tissues were added to the
medium containing or not SA or 2,4-Cl BA (100 mM ). Each point is
the mean of n experiments (± SE of the mean). Control: n=11; SA:
n=8; 2,4-Cl BA: n=10. All the series of data were submitted to an
ANOVA test and then to a t-test. Significant differences between mean
values are noticed in the Results.
Attempts were also made to measure the PD in mesophylls cells of tissues aged for 12 h in presence or in
absence of SA. However, impalement of the cells of
SA-treated tissues turned out to be impossible, presumably due to a change in cell wall composition and rigidity.
244
Bourbouloux et al.
SA effect on the level of ATPase and sucrose transporter
transcripts
The amounts of total RNA recovered from aged (control )
and SA-treated tissues (at 10, 50 and 100 mM ) were
408±41, 455±29, 455±26, and 434±13 mg g−1 FW,
respectively (means of 6 extractions±SE ). Therefore,
treatment with SA did not affect within 12 h the amount
of RNA extracted from the tissues.
The pma2 ATPase probe and the BvSut1 sucrose
transporter probe reacted in aged or SA-treated tissues
with a single band at 3.5 kb (Fig. 4A) and 1.5 kb
( Fig. 4B), respectively, which is in good agreement with
the expected molecular size of transcripts hybridizing with
these probes. The pma4 probe gave the same results as
pma2 (data not shown). SA did not significantly affect
the levels of the ATPase and sucrose transporter transcripts ( Fig. 4).
SA effects on the amounts of PM H+-ATPase and sucrose
transporter
Western blotting of plasma membrane proteins with antibody 721 directed against the central part of the plasma
membrane H+-ATPase yielded a net major band at
97 kDa in samples from fresh, aged and SA-treated tissues
( Fig. 5A). Although equal amounts of proteins had been
loaded, the immunoreaction was stronger in aged tissues
compared to control (fresh tissues), as previously
described by Noubhani et al. (1996). SA did not significantly affect the intensity of the band observed after ageing
( Fig. 5A). Quantifications of the amounts of the H+ATPase and of the sucrose transporter with ELISA tests
both showed similar results ( Table 3).
Western analysis of sugar beet plasma membrane proteins with antibody AtSUC1 gave a single band at 42 kDa
in samples from fresh, aged and SA-treated tissues
( Fig. 5B). The intensity of the bands appeared similar in
PMV from fresh and aged tissues and SA did not affect
this intensity ( Fig. 5B). However, ELISA measurements
showed that ageing induced a significant rise in the
amount of the protein while SA-treatment did not affect
it ( Table 3). AtSUC2 antiserum did not immunoreact
Table 3. ELISA estimation of the amount of plasma membrane
proteins reacting with the anti-ATPase and the anti-AtSUC1
serum in PMV fractions from fresh, aged and SA-treated tissues
during the ageing period
Values represent the mean of triplicate measurements (±SE ) from six
(ATPase) and four (sucrose transporter) independent experiments. For
each protein, different letters indicate that values are statistically
different at P≤0.05.
Tissue treatment
Fig. 4. Northern analysis of RNA extracted from leaf tissues aged in
the absence (C ) or the presence of 100 mM SA (SA). Twenty mg of
total RNA were deposited in each lane. They were hybridized with the
pma2 (A) or BvSut1 (B) probe. Position of the molecular mass markers
is shown on the left. Similar patterns were obtained with RNA extracts
from 4 (pma2) and 5 (BvSut1) independent experiments.
Fresh
Aged
SA-treated
Absorbance
ATPase
Sucrose transporter
0.68±0.04 a
1.29±0.03 b
1.25±0.03 b
0.24±0.02 c
0.35±0.01 d
0.34±0.01 d
Fig. 5. Western analysis of plasma membrane proteins from fresh (f ), aged (a) or SA-treated leaves (SA) with anti-H+-ATPase serum (A) and
with anti-sucrose transporter serum (B). (A) Ten mg of proteins were deposited in each lane and treated with the anti-plasmalemma H+-ATPase
antibody 721. Similar results were obtained when 5 to 25 mg of proteins were deposited. (B) Seventy-five mg of proteins were deposited in each lane
and the blots were treated with the anti-sucrose transporter AtSUC1 serum. Position of the molecular mass markers is shown on the left. The
experiments were repeated three times with independent samples and similar results.
Salicylic acid and membrane transport
with any band in PMV from sugar beet leaves (data
not shown).
Changes in content of adenine nucleotides during ageing
The ATP content of untreated tissues increased about
25% during the first 2 h of the ageing period, was steady
for the following 6 h and then clearly decreased ( Fig. 6A).
ADP levels followed a similar pattern, whereas AMP was
very small throughout this period. Compared to control
tissues, treatment with SA lowered the ATP and ADP
contents, with ATP being relatively more affected than
ADP. As a result, the ATP/ADP ratio was constant
between 2 h and 12 h incubation in untreated aged tissues,
but decreased continuously and significantly (P≤0.05)
during the same period in SA-treated samples (Fig. 6B).
SA also prevented the initial rise in ATP/ADP ratio which
normally occurred during the first 2 h of ageing. The
energy charge of the cells, which is a good indicator of
their metabolic activity was fairly constant throughout
the 12 h ageing period in untreated tissues (Fig. 6B). In
the presence of SA, the energy charge was lower than in
control samples, and it decreased between 6 h and 12 h.
Fig. 6. Metabolic activity of sugar beet leaf discs during the ageing
period. (A) Adenine nucleotides content was measured on tissues
incubated for various times on a buffered medium in the presence or in
the absence of 100 mM SA. (B) Left axis: ATP/ADP ratio in the same
leaf discs as in (A). Right axis: Energy charge in the same leaf discs as
in (A). In (A) and (B), each point is the mean of three experiments in
triplicate (±SE of the mean).
245
Discussion
Wounding of leaf tissues by excision, removal of the
lower epidermis and cutting of discs may affect the
hormonal balance of the tissues (see Introduction).
Therefore, the increase of nutrient uptake which develops
with ageing may be controlled by hormones. In this
system, most of the tested phytohormones did not significantly change uptake of sucrose and valine either in
fresh or in aged tissues. However, JA inhibited valine
uptake and SA was the only compound preventing the
stimulation of both sucrose and valine uptake which
normally develops during ageing.
Although hormonal control of assimilate transport has
been studied and reported for IAA, cytokinins, gibberellins and ABA (see references cited in Introduction), the
effects of SA on uptake of sugars and amino acids have
never been tested. To our knowledge, SA was only shown
to inhibit K+ (Harper and Balke, 1981; Schulz et al.,
1993) and phosphate (Glass, 1975) uptake in cereal roots.
Treatment of the beet leaf tissues with 100 mM SA for
a few hours prevents the stimulation of sucrose and valine
uptake normally induced by ageing ( Table 2). These
conditions of treatment are less drastic than those used
in most studies where concentrations ranging from
0.5–10 mM, and treatment durations up to 7 d have been
used (Harper and Balke, 1981; Li N. et al., 1992; Schulz
et al., 1993; Mur et al., 1996). After a 3 h treatment of
Nicotiana plumbaginifolia leaves with only 50 mM SA,
Willekens et al. (1994) observed a strong induction of
acid chitinases transcripts, but in this case, SA was
infiltrated in the leaves. In the present experiments, simple
flotation of the tissues on a 10 mM SA solution was also
efficient, but the use of 100 mM SA resulted in a more
consistent inhibition of uptake (Fig. 1).
Several results indicate that the inhibition observed is
largely reversible upon removal of SA ( Table 2; Fig. 2).
This recovery may be due to the possible metabolism of
the molecule (breakdown or conjugation followed by
compartmentation) and/or the development of a repair
process.
Among many SA-derivatives, only aspirin (Chen and
Klessig, 1991; Kim et al., 1992) and chlorinated derivatives ( Kauss et al., 1994) were as effective as SA in
inducing systemic acquired resistance; in particular, neither hydroxyderivatives (except 2,3- and 2,4-hydroxybenzoic acids which showed some effect) ( Kim et al.,
1992) nor catechol, the breakdown product of SA
( Friedrich et al., 1995), presented any effect. The same
type of specificity was observed in the present experiments
which showed that SA, 2,4-Cl BA and to a lesser extent
2,4-OH BA were the only tested derivatives hindering
both valine and sucrose transport ( Table 1). Therefore,
it was concluded that the interaction between SA and
nutrient transport is specific.
246
Bourbouloux et al.
Further experiments were run to determine how SA
may affect the uptake of sugars and amino acids. This
inhibition might have been due to a decrease in the
activity of the transporters, of the H+-ATPase or to a
general effect on membrane permeability. These data
show that SA affected neither the amount of transcripts
encoding the sucrose transporter (Fig. 4B) nor the
amount of this protein ( Fig. 5B; Table 3). Since the
activity of the H+/solute cotransporters depends on the
proton motive force across the plasma membrane, any
modification induced by SA of either transmembrane
DpH or Dy or both could interfere with uptake. SA did
not apparently affect the cation permeability of the plasma
membrane (electrophysiological data), showing that it did
not behave as a protonophore in the short term. In
contrast, SA (and 2,4-Cl BA, which also inhibited sugar
and amino acid uptake) decreased the acidifying activity
of the tissues (Fig. 3A), without affecting either the level
of ATPase transcripts ( Fig. 4A) or the amount of plasma
membrane ATPase (Fig. 5A).
The decrease in the acidifying activity of the tissues
may be due to the decrease of the ATP and of the energy
charge levels that were measured (Fig. 6). The energy
charge of the cell is considered as a good indicator of
metabolic activity. The intracellular ATP concentration
falls for about 1 h after excision of corn roots (Gronewald
and Hanson, 1982), but recovers with continuous washing. The data in Fig. 6A and B may reflect the same
phenomenon. Indeed, the initial increase observed in
control sets may correspond to the recovery phase following an initial ATP loss, because about 1 h was needed to
prepare the samples. The energy charge measured in
control tissues was about 0.82, which corresponds to a
classical value in wheat leaves (Pradet and Raymond,
1983), and was maintained during the whole ageing
period. After SA treatment, the decrease in the energy
charge was about 0.15 unit ( Fig. 6). High concentrations
of SA have been shown to uncouple phosphorylation in
mitochondria (Macri et al., 1986). In addition, SA, which
provokes a well-known temperature rise during flowering
of thermogenic species, induced a similar rise in the leaves
of tobacco, a non-thermogenic species ( Van Der Straeten
et al., 1995). SA stimulated both total respiration and
the alternative pathway, with the latter being more stimulated than the cytochrome pathway. During the transfer
of electrons from NADH to O via the alternative path2
way, only one-third as much ATP is formed as during
transfer via the cytochrome pathway, suggesting a break
effect in the phosphorylation-coupled electron transfer
chain. In the present experiments, the energy charge of
SA-treated tissues stabilized at a level below the control
during the first 6 h of treatment with SA (Fig. 6B). This
suggests that the energy-providing processes of the cell
were certainly affected (although in a reversible way,
Table 2) during the first 6 h of ageing. These effects were
small in comparison with the drastic fall due to anoxia
for example (0.7 unit) (Saglio et al., 1980). However, a
small decrease in the energy charge may affect the physiology of the cell, as previously shown for germinating oil
seeds (Al-Ani et al., 1985).
In maize root tips placed under anoxia the energy
charge was lowered ( Xia et al., 1995) and the proton
efflux resumed at a slower rate ( Xia and Roberts, 1996).
These facts agree with the idea that the H+-ATPase of
the plasmalemma becomes ATP-limited: the possible regulation of this ATPase as a function of ATP avaibility has
been discussed previously (Saint-Ges et al., 1991).
Likewise, in the present experiments, SA decreases both
the energy charge and the acidifying activity of the tissue,
which may result in inhibition of proton-coupled uptake
of sucrose and valine. However, a more direct effect of
SA on the activity of the transporters cannot be ruled out.
The fact that the PD was hyperpolarized by fusicoccin
after SA treatment is not inconsistent with the conclusion
that SA inhibits ATPase activity through an effect on the
ATP levels. Indeed, fusicoccin (which does not act directly
on the ATPase) induces both an increase in V
and a
max
decrease in the apparent K for ATP (Johansson et al.,
m
1993). Either of these effects alone would allow an
increased activity, even under non-saturating ATP.
These SA effects should be considered in many experiments described sofar, where long durations of SA treatment have been used on intact plants or excised leaf
tissues. They may also be related to the fact that gene
expression, including that involved in systemic acquired
resistance, depends on the sugar status of the cell (Herbers
et al., 1996).
Acknowledgements
This work was supported by the Centre National de la
Recherche Scientifique (ERS 6099). We are grateful to Professor
M Boutry ( University of Louvain-La-Neuve, Belgium) for the
gift of the pma2 and pma4 probes, to Dr J Riesmeier (Max
Planck Institute, Golm, Germany) for gift of BvSUT1 probe,
and to Professor N Sauer ( University of Erlangen, Germany)
for gift of AtSUC1 and AtSUC2 antisera. We thank Monique
Gaudillère for help in adenine nucleotides measurements. Partial
financial support was also provided by the Conseil Régional
Poitou-Charentes.
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