Plant Science 167 (2004) 913–923
Sugar-induced tolerance to atrazine in Arabidopsis seedlings:
interacting effects of atrazine and soluble sugars on
psbA mRNA and D1 protein levels
Cécile Sulmon, Gwenola Gouesbet, Ivan Couée∗ , Abdelhak El Amrani
Centre National de la Recherche Scientifique, Université de Rennes 1, UMR 6553 ECOBIO, Campus de Beaulieu,
bâtiment 14A, F-35042 Rennes, Cedex, France
Received 19 February 2004; received in revised form 7 May 2004; accepted 20 May 2004
Available online 19 June 2004
Abstract
In order to assess the potential environmental impact of atrazine on wild terrestrial plants, the physiological determinants for atrazine
sensitivity were investigated in the model plant Arabidopsis thaliana. Atrazine treatment arrested development of Arabidopsis at the stage
of heterotrophy–phototrophy transition, with cotyledon bleaching and seedling death. However, sucrose and, to a markedly lesser extent,
glucose were found to confer to Arabidopsis seedlings a high level of tolerance to atrazine, with maintenance of chlorophylls, carotenoids,
and D1 protein, and protection of photosystem II. Moreover, atrazine in the presence of sucrose was found to have a paradoxically positive
effect on seedling development. The effects of different carbon substrates and the analysis of Arabidopsis sugar-response mutants showed that
sucrose-induced atrazine tolerance was not due to carbon compensation of photosynthesis and probably relied on hexokinase-independent
signalling pathways. Enhancement of seedling growth, and of chlorophyll and carotenoid accumulation, by sucrose occurred both in the
absence and presence of atrazine. In contrast, whereas sucrose treatment in the absence of atrazine decreased psbA mRNA and D1 protein
levels, the combined effects of sucrose and atrazine resulted in increased levels of psbA mRNA and of D1 protein levels, which may thus
significantly contribute to the induction of atrazine tolerance.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Arabidopsis; D1 protein; Herbicide; Photosystem II; Soluble carbohydrates; Tolerance
1. Introduction
Atrazine (2-chloro-4-ethylamino-6-isopropylamine-1,3,5triazine) is an electron transport inhibitor, which binds to
the D1 protein of photosystem II (PSII) reaction centre, thus
blocking electron transfer to the plastoquinone pool [1,2].
Inhibition of PSII electron transport prevents conversion of
absorbed light energy into electrochemical energy and results in production of triplet chlorophyll and singlet oxygen,
which induce oxidative stress and final bleaching [1,2].
Studies of the impact of atrazine contaminations in the
environment have generally focused on non-target aquatic
Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea;
FW, fresh weight; HXK, hexokinase; MS, Murashige and Skoog; PSII,
photosystem II; sis, sugar insensitive; Ws, Wassilewskija
∗ Corresponding author. Tel.: +33 2 2323 5123; fax: +33 2 2323 5026.
E-mail address: [email protected] (I. Couée).
organisms [3]. In contrast, relatively little is known on the
potential impact of contaminating atrazine on wild terrestrial
plant communities, although they possess D1, the protein
target of atrazine. Emergence of atrazine-resistant weeds in
the last decades has shown that plants could develop mechanisms of atrazine tolerance, generally resulting from genetic
mutations of the psbA gene, which encodes the D1 protein,
the sole target of atrazine. Changes of D1 protein sequence
involving modified conformation of herbicide binding site
can thus confer atrazine tolerance [4].
Tolerance can also derive from existence of detoxification
pathways. For example, in sorgho, increase of glutathioneS-transferase activity confers atrazine tolerance [5]. In Setaria faberi, a maize weed, atrazine resistance results from
detoxification metabolism with increased monooxygenation
and glutathione-related reactions [6]. However, most works
on atrazine tolerance in weeds have been carried out with
high herbicide concentrations corresponding to treatment
0168-9452/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2004.05.036
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C. Sulmon et al. / Plant Science 167 (2004) 913–923
of agricultural land and under conditions where atrazine is
applied to leaves [7,8], therefore, near its biochemical target, the chloroplast-localised D1 protein. In contrast, in the
context of soil pollution and impact on wild terrestrial plant
species, contaminating concentrations of herbicide may be
lower, and absorption occurs through the root system prior
to distribution in the whole plant and interaction with the
D1 target. Risk assessment therefore requires better understanding of the genetic and physiological determinants of
plant sensitivity or tolerance to atrazine.
The present study was carried out on the cruciferous
species Arabidopsis thaliana, which is not only a model for
plant physiology and genetics, but also a wild non-humanselected species, with a broad spectrum of habitats and a
close relative to numerous other wild cruciferous species
[9]. Plants grown under axenic conditions were submitted
to a wide range of atrazine concentrations – from 5 nM to
40 ␮M – which correspond to situations of diffuse environmental pollution and agricultural treatments [10,11]. In the
course of setting up ecologically-relevant ecotoxicological
tests, carbon nutritional status was found to have a strong
impact on the sensitivity to atrazine, with sucrose treatment
inducing high tolerance level to atrazine. The present work
shows that the effects of exogenous sugars on the sensitivity to atrazine cannot be ascribed to metabolic carbon compensation of photosynthesis inhibition, and that exogenous
sucrose in the presence of atrazine had unexpected positive
effects on psbA mRNA and D1 protein expression.
2. Methods
2.1. Plant material and growth conditions
Seeds of Arabidopsis thaliana (ecotype Wassilewskija
(Ws)) were surface-sterilized for 5–10 min in 50% bayrochlore/50% ethanol, rinsed twice in absolute ethanol and
dried overnight. Surface-sterilized seeds were plated on
square Petri dishes for germination, and growth was carried
out under axenic conditions. Growth medium consisted of
0.8% (w/v) agar in 1× Murashige and Skoog (MS) basal
salt mix (Sigma, St. Louis, MO, USA) adjusted to pH 5.7.
Petri dishes were sealed with Parafilm and placed in a cold
chamber at 4 ◦ C during 48 h in order to break dormancy
and homogenize germination. Petri dishes were then transferred to a controlled growth chamber at 22 ◦ C under a 16-h
light period regime at 4500 lux. Sugars, glucose analogues,
mannitol, glycerol, sodium malate, 3-(3,4-dichlorophenyl)1,1-dimethylurea (DCMU), and atrazine (Pestanal grade,
Riedel-deHaën) were purchased from Sigma (St. Louis, MO,
USA). Sucrose, glucose, and mannitol were directly added
at various concentrations during preparation of agar-MS
media and autoclaved. Stock solutions of glucose analogues
(3-O-methylglucose and 2-deoxyglucose) and atrazine
(30 mg L−1 ) were separately dissolved in 1× MS basal salt
mix and sterilised by microfiltration through 0.2 ␮m cellu-
lose acetate filters (Polylabo, Strasbourg, France). Glucose
analogues and atrazine were axenically added to melted
agar-MS medium prior to pouring into Petri dishes. Seed
germination and seedling growth took place directly in the
media under study, unless otherwise specified.
2.2. Growth parameters
Growth parameters were measured after 15 days of cultivation. Length of primary root was measured on 11–33
seedlings grown on vertical plates. Fresh weight measurements were carried out on 5 replicas of about 2–10 pooled
seedlings each. Results were given as the mean (±S.E.M.)
of these determinations.
2.3. Chloroplast pigments and photosynthesis parameters
Pigments were extracted by pounding aerial parts of
seedlings in 80% acetone, and absorbance of the resulting extracts was measured at three wavelengths: 663 nm,
646 nm, 470 nm. Levels in these extracts, expressed as
␮g mL−1 of chlorophylls and total carotenoids (xanthophylls and carotenes), were determined from the equations
given by Lichtenthaler and Wellburn [12]. Measurements
were done on 5 replicas of 2–10 pooled seedlings each.
Results were given as the mean (±S.E.M.) of these five
determinations. Chlorophyll fluorescence and maximum
PSII efficiency (Fv /Fm ) were measured with a PAM-210
chlorophyll fluorometer system (Heinz Walz, Effeltrich,
Germany). After dark adaptation for at least 15 min, minimum fluorescence (F0 ) was determined under weak red
light. Maximum fluorescence of dark adapted leaf (Fm )
was measured under a subsequent saturating pulse of red
light, and variable fluorescence (Fv = Fm − F0 ) was determined. Measurements were carried out on at least ten
plants. Oxygen evolution was measured on whole plantlets
with a Clark-type electrode unit (Hansatech, King’s Lynn,
UK). The rate of photosynthetic oxygen evolution was estimated by subtracting the rate of dark respiration from the
oxygen evolution rate in the light [13].
2.4. Analysis of atrazine
Atrazine was extracted from ground lyophilised samples by agitation (400 rpm) for 4 h in methanol/water
(4:1, v/v). After centrifugation, supernatants were filtered
through 0.45 ␮m filters and the solvent was evaporated at
40 ◦ C. Sample volumes were then completed to 200 mL
with water. Solid-phase extraction cartridges (Oasis HLB,
Waters, Milford, MA, USA) were activated with 5 mL of
methanol followed by 5 mL of water. The 200-mL samples
were applied to the activated column, and after washing
of the columns with water, atrazine was eluted with 2 mL
of dichloromethane. The solvent was evaporated to dryness, and the solide residue was dissolved in 500 ␮L of
methanol/water (1:1, v/v). Atrazine levels in these samples
C. Sulmon et al. / Plant Science 167 (2004) 913–923
were then determined by HPLC analysis on a C18 Waters (5 ␮m, 25 cm × 4.6 mm) column with detection and
quantification by diode array.
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3. Results
3.1. Effects of exogenous glucose and sucrose on seedling
sensitivity to atrazine
2.5. RNA isolation and northern blot analysis
Total RNA from Arabidopsis seedlings was extracted
using the RNAgents® Total RNA Isolation System kit
(Promega, Madison, USA). Equal amounts of RNA (0.2 ␮g)
were mixed with sample buffer containing ethidium bromide, separated by electrophoresis through 1% (w/v)
agarose gel containing 2% (v/v) formaldehyde and MOPS
1×, and were capillary-blotted with 20× SSC onto ZetaProbe® GT genomic Tested Blotting membranes (Bio–Rad,
Hercules, USA). RNA was fixed to the membrane by UVcrosslinking. A 950-bp PCR-amplified fragment of the Arabidopsis psbA gene (Genbank accession number X79898)
was used to generate a digoxigenin (DIG)-labelled DNA
probe by random primed labelling with digoxigenin-11dUTP using DIG High Prime DNA labelling® and Detection Starter Kit II (Roche Diagnostics, Mannheim, Germany). Hybridized probes were immunodetected with an
alkaline phosphatase-conjugated anti-digoxigenin antibody
and visualized with chemiluminescence substrate CSPD
(Roche) following the supplier’s instructions (Roche Diagnostics, Germany). Prehybridization (2 h) and hybridization
(overnight) were performed in hybridization buffer at 50 ◦ C.
After hybridization, membranes were washed for 5 min
twice with 2× SSC, 0.1% SDS at room temperature, and
15 min twice with 0.1× SSC, 0.1% SDS at 50 ◦ C. Autoradiography was carried out with the Lumi-film chemiluminescent Detection Film (Boehringer Mannheim, Indianapolis,
USA).
2.6. Extraction of proteins and immunoblot analysis
Membrane-associated proteins were isolated from
seedlings according to the method of Pilgrim et al. [14].
Proteins were separated on SDS–PAGE, transferred to nitrocellulose membrane, and detected with antibodies raised
against the D1 protein. Immunoreactions were detected
by enhanced chemiluminescence (Pierce, SuperSignalTM ,
Rockford, USA) according to the instructions of the manufacturer. Anti-D1 antibody was a kind gift from Dr. Bruce
A. Diner (Central Research and Development Department,
E. I. Du Pont de Nemours & Company, USA). This antibody was raised against the C-terminal region of the mature
D1 polypeptide.
2.7. Statistical analysis
Statistical analysis was conducted with the Minitab package. Curve adjustment was carried out by the least-square
methods. Pairwise comparison of means used the ANOVA
and the Mann–Whitney statistical tests. Values of significance (P) are given in brackets.
The effects of atrazine on seedling development became
significant in the concentration range of 100 nM–250 nM,
where plants showed reduced growth of primary root
and aerial organs (Fig. 1A and B). Fig. 1(C) shows that
chlorophyll level started to decrease significantly in the
atrazine concentration range of 50–100 nM (P = 0.0161).
Concentration-dependent inhibition of root growth could
be significantly fitted to a hyperbolic curve (R2 = 0.93)
giving an I50 of 120 nM for atrazine inhibition. At 500 nM
atrazine, seedlings were bleached and their development
was arrested after cotyledon expansion.
In the presence of 80 mM sucrose or glucose, no effect on
seedling development was observed at atrazine concentrations lower than 500 nM. At higher atrazine concentrations,
in the range 1–40 ␮M (Fig. 1D), a slight reduction of root
growth and of leaf greening could then be observed. However, these effects remained weak in comparison to those
observed in the absence of exogenous sugars (Fig. 1A).
Although sugar-treated seedlings presented apparent loss
of chlorophylls in cotyledons and first true leaves, they
maintained development of root and aerial organs. Fig. 1E
shows primary root length of Arabidopsis seedlings grown
on 80 mM sucrose or glucose at varying atrazine concentrations from 1 to 40 ␮M. In the presence of 80 mM exogenous
sugars, 1–10 ␮M atrazine levels gave a slight inhibitory effect on primary root growth development (P < 10−4 ). In
contrast, higher concentrations of atrazine paradoxically resulted in enhanced root growth (P = 0.0111).
Atrazine levels in the MS-agar medium at the end of the
experiments corresponded to the nominal values of treatment. Atrazine levels in 0.5 ␮M and 10 ␮M atrazine treatments were found to be, respectively, 0.50 (±0.05 S.E.M.)
pmol per mg FW and 10.5 (±1.5 S.E.M.) pmol per mg
FW, thus indicating that there was no significant decrease
of atrazine concentration in the surrounding medium during
the course of the experiments. Moreover, whereas atrazine
levels of 3 (±2 S.E.M.) pmol atrazine per mg FW were measured in control seedlings subjected to 0.5 ␮M atrazine and
showing complete inhibition, sucrose-treated plantlets were
found to contain 3.7 (±0.3 S.E.M.) pmol atrazine per mg
FW in the presence of 0.5 ␮M atrazine, and 23 (±3 S.E.M.)
pmol atrazine per mg FW in the presence of 10 ␮M atrazine.
It thus seemed that sucrose-treated plantlets could accumulate atrazine at levels which were lethal for seedlings in the
absence of sucrose.
Under the same growth conditions as those of Fig. 1(E),
atrazine produced a decrease of chlorophyll and carotenoid
levels in leaves in the presence of glucose or sucrose
(Fig. 1F). However, even at 40 ␮M atrazine, chlorophyll and
carotenoid accumulation was not abolished, whereas in the
absence of exogenous sugars, 500 nM atrazine was sufficient
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Fig. 1. Effects of atrazine on Arabidopsis seedling development. Development state (A, D), primary root length (B, E), and chlorophyll and carotenoid
contents (C, F) are given. Inhibition treatment consisted of atrazine concentrations from 5 nM to 1000 nM (A–C). Treatments with exogenous sugars
consisted of 80 mM sucrose (Suc) or glucose (Glc) and atrazine concentrations from 1 to 40 ␮M (D–F). Values are the mean (±S.E.M.) of measurements
on at least 12 (A–C) and 33 (D–F) 15-day-old plantlets. Means of measurements and associated regression curve are given in (B). These experiments
were carried out five (A–C) and three (D–F) times and trends were similar.
to obtain complete loss of chlorophylls and carotenoids
(Fig. 1C). Moreover, in the presence of sucrose, but not in
the presence of glucose, 40 ␮M atrazine treatment appeared
to give higher chlorophyll and carotenoid levels than those
at 5 ␮M atrazine (Fig. 1F).
The apparent differential effects of glucose and sucrose
were further investigated. Seedlings were grown for 15 days
on MS-agar medium containing 1 ␮M atrazine, which gave
complete growth inhibition in the absence of exogenous sugars (Fig. 1). Root growth inhibition in the presence of 1 ␮M
C. Sulmon et al. / Plant Science 167 (2004) 913–923
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Fig. 2. Concentration-dependent effects of soluble sugars on Arabidopsis seedling sensitivity to atrazine. Treatments consisted of 1 ␮M atrazine and
sucrose (Suc) or glucose (Glc) concentrations from 2.5 mM to 80 mM. Values are the mean (±S.E.M.) of measurements of primary root length on at
least twelve 15-day-old seedlings. This experiment was carried out three times and trends were similar.
atrazine was found to be significantly lifted by addition of
sucrose or glucose to the growth medium, which resulted
in an increase of primary root length compared to control
(P < 10−4 ) (Fig. 2). This lifting of atrazine inhibition was
concentration-dependent from 2.5 to 80 mM of exogenous
sugar, and sucrose provided stronger increase of root growth
than glucose did. For the same carbon-equivalent level, such
as 40 mM sucrose and 80 mM glucose, primary root length
of seedlings grown in the presence of glucose was significantly lower than that in the presence of sucrose (P < 10−4 ).
Similarly, increasing concentrations of exogenous glucose or
sucrose restored chlorophyll and carotenoid accumulation in
the presence of 1 ␮M atrazine (data not shown). This effect
of exogenous glucose and sucrose on preventing leaf bleaching was concentration-dependent from 2.5 to 60 mM, with
sucrose being five times more efficient than glucose. The
lowest levels of exogenous glucose and sucrose that were
tested, i.e. 2.5 mM, gave a significant lifting of atrazine inhibition whether on primary root growth or on chlorophyll and
carotenoid accumulation (P < 10−4 ). This effect of low levels of exogenous sugars and the marked difference between
glucose and sucrose treatments strongly suggested that lifting of atrazine inhibition could not be ascribed solely to
carbon supply compensating the inhibition of photosynthesis. Moreover, inhibition by lethal concentrations of DCMU
was also lifted by addition of 10–80 mM exogenous sugars
(data not shown). Since DCMU is also a PSII-bound herbicide affecting the site of the exchangeable quinone [2], the
protection effects of exogenous sugars were investigated in
relation to the PSII target rather than to the specific chemical
structure of atrazine.
3.2. Sucrose-induced protection of PSII and D1 protein
against atrazine-mediated injury
Plants were grown for 15 days on MS-agar medium containing varying concentrations of sucrose and atrazine. Increasing concentrations of sucrose were found to result in
maintenance of PSII potential activity in the presence of
sublethal and lethal doses of atrazine (Fig. 3A). Whereas decrease of Fv /Fm values in atrazine-inhibited seedlings was
correlated with increase of F0 , as described in other studies
[15], sucrose treatment in the presence of atrazine was found
to maintain both Fv /Fm (Fig. 3A) and F0, which remained
at the same value (34 ± 2 S.E.M.) as that of sucrose-grown
plantlets in the absence of atrazine (33 ± 3 S.E.M.). Moreover, plantlets that had been grown in the presence of 80 mM
sucrose and 1 ␮M atrazine showed a photosynthetic oxygen
evolution of 320 (±30 S.E.M.) nmol h−1 per plantlet. This
rate was not affected by the presence of 1 ␮M atrazine, and
was similar to the rate of 270 (±40 S.E.M.) nmol h−1 per
plantlet measured on sucrose-grown plantlets in the absence
of atrazine.
Since increase of F0 and decrease of Fv /Fm have been
associated with degradation of D1 protein [16], D1 protein
levels in membrane-associated proteins were estimated by
dot blot and Western blot. Antibodies raised against the Cterminal region of D1 protein [17] were found to recognize,
in Arabidopsis membrane proteins, monomeric D1 protein
as a band of 31 kDa, D1 aggregates of higher molecular
weight, and products of proteolytic cleavage, as described
by Lupinkova et al. [18] and Shipton and Barber [19] in
other species. Dot blot, which detected all of these D1
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Fig. 3. Effects of sucrose on the protection of PSII and D1 protein in
Arabidopsis seedlings in the presence of atrazine. Values of Fv /Fm (A)
are the mean (±S.E.M.) of measurements on at least 10 plantlets, which
had been grown for 15 days in the presence of the various concentrations
of sucrose (0, 10, or 80 mM) and atrazine (0, 0.25, or 1 ␮M) that are
indicated in the figure. Membrane proteins (1 ␮g per dot) from 15-day-old
plantlets were loaded for dot blot analysis of D1 protein with anti-D1
antibodies (B). Treatments consisted of 10 or 80 mM sucrose in the
absence of atrazine or in the presence of 0.25 or 1 ␮M atrazine. These
experiments were carried out three times and trends were similar.
protein products, showed that 1 ␮M atrazine induced disappearance of D1 products in unprotected seedlings, whereas
sucrose treatment resulted in the maintenance of D1 protein
levels (Fig. 3B).
The effects of sucrose were further investigated by Western blot analysis of monomeric D1 protein and proteolytic
products (Fig. 4A). Under the conditions of light and mineral nutrition used in the present study, sugar feeding up to
80 mM was found to enhance seedling growth, carotenoid
accumulation, and chlorophyll accumulation in the absence
of atrazine (data not shown). Such positive effects of sugar
feeding on seedling growth have been reported in a number of cases [20,21,22]. However, 80 mM exogenous sucrose
was found to result in a decrease of D1 protein levels in the
absence of atrazine treatment. In contrast, sucrose treatment
in the presence of 0.25 ␮M atrazine gave markedly greater
accumulation of D1 and also increase of D1 breakdown
products. In the presence of 1 ␮M atrazine, a similar pattern
was obtained when seedlings were treated with 10 mM exogenous sucrose. In contrast, in the presence of 80 mM sucrose and 1 ␮M atrazine, seedlings presented lower levels of
both monomeric D1 protein and related products. However,
Fig. 4. Effects of soluble sugars on D1 protein and psbA mRNA levels in
the absence and in the presence of atrazine. Membrane proteins (A) and
total RNA (B) were isolated from 15-day-old plantlets, which had been
treated with 10 or 80 mM glucose or sucrose in the absence or presence
of atrazine. Membrane proteins (10 ␮g per track) were separated by
SDS–PAGE and blotted onto a nitrocellulose membrane. Immunodetection
was carried out with anti-D1 antibodies (A). Total RNA (0.2 ␮g per track)
was separated by agarose-gel electrophoresis and blotted onto a nylon
membrane. Hybridisation was carried out with the specific DIG-labelled
probe of the Arabidopsis psbA gene (B). Ethidium bromide staining was
used to ensure equal RNA loading per lane in the gel.
80 mM sucrose treatment maintained a substantial level of
D1 monomer in atrazine-treated seedlings, which otherwise
would have been depleted of all of the different D1 protein species (Figs. 3B and 4A). Steady-state levels of psbA
mRNA were also found to be significantly modified by soluble sugars and by atrazine (Fig. 4B). Both glucose and
sucrose at 80 mM exerted a repressing effect on the levels
of psbA mRNA in the absence of atrazine. This repression
of psbA mRNA levels by glucose and sucrose appeared to
be lifted in the presence of 0.25 and 1 ␮M atrazine. Thus,
glucose- and sucrose-grown plantlets paradoxically showed
much higher levels of psbA mRNA in the presence than in
the absence of atrazine.
3.3. Sensitivity to atrazine of Arabidopsis sugar-response
mutants
Fig. 5(A) shows seedling primary root growth and
chlorophyll content in the presence of 1 ␮M atrazine, when
various carbon sources were added to MS-culture medium.
Exogenous supply of malate or glycerol was found to con-
C. Sulmon et al. / Plant Science 167 (2004) 913–923
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Fig. 5. Effects of carbon supply and sugar signalling pathways on Arabidopsis seedling sensitivity to atrazine. The effects of carbon sources (A), of
glucose analogues (B, C), and of sugar-signalling mutations (D) are described. The percentage of seedling growth (A), primary root length (B), chlorophyll
content (C), and percentage of chlorophyll content (D) were measured. Sense-AtHXK and anti-AtHXK transgenic lines, respectively, overexpress HXK
and express antisense HXK [26]. The sis1 mutant is insensitive to high sucrose concentrations [21]. Treatments consisted of 1 ␮M atrazine (A–D) in
the presence of 40 mM mannitol, malate, glycerol, glucose (Glc), or sucrose (Suc) (A–C), 40 mM 3-methylglucose or 2-deoxyglucose (B, C), or 80 mM
sucrose (D). In the study of glucose analogues (B, C), germination and early development were carried out on MS medium, seedlings were then transferred
to treatment media, and measurements were done after 14 days of further growth. Values are the mean of measurements on at least twelve 15-day-old
seedlings (A), eleven (B, C), and eight (D) 23-day-old seedlings (B, C). These experiments were carried out at least twice and trends were similar.
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fer partial levels of tolerance to atrazine (Fig. 5A). Thus,
glycerol was found to exert positive effects on both primary
root growth and chlorophyll accumulation in the presence
of 1 ␮M atrazine. Mannitol had no effect on seedling sensitivity to atrazine. Glucose analogues, 3-methylglucose and
2-deoxyglucose, were used to test the potential involvement
of sugar signalling effects [23,24] in the lifting of atrazine
inhibition. In order to avoid inhibition of seed germination
induced by glucose analogues [25], seedlings were first
grown on MS-agar medium for 9 days before transferring
to MS-agar medium containing 40 mM of glucose analogue and 1 ␮M atrazine. After 14 days, arrest of primary
root growth (Fig. 5B) and complete loss of chlorophyll
(Fig. 5C) induced by 1 ␮M atrazine were not lifted by the
presence of 3-methylglucose or 2-deoxyglucose. Treatment
with 2-deoxyglucose had a strong negative effect on development of the 9-day-old seedlings and resulted in loss of
chlorophylls even in the absence of atrazine. Surprisingly, 3methylglucose treatment was found to enhance chlorophyll
accumulation relative to control seedlings grown on MS
medium (P = 0.0122), but this enhancement of chlorophyll
accumulation was not correlated with greater tolerance to
atrazine.
Arabidopsis mutant and transgenic plants affected in the
sugar response were tested for their sensitivity to atrazine
in the presence of sucrose (Fig. 5D). The sugar-insensitive
1 (sis1) mutant of Arabidopsis, which is insensitive to inhibitory effects of high sucrose concentrations on seedling
development [21], was found to show sensitivity to atrazine
in the presence of sucrose. This sis1 mutant has been reported to have similar patterns of chlorophyll accumulation
as those of the wild type in the absence and in the presence
of 150 mM sucrose [21]. In the absence of sucrose, this mutant was as sensitive to atrazine as the wild type (data not
shown). In the present study, under conditions of 80 mM sucrose feeding, the sis1 mutant showed a marked difference
of chlorophyll accumulation relative to wild type (Fig. 5D).
Moreover, treatment of sis1 mutants with 1 ␮M atrazine
in the presence of 80 mM sucrose resulted in decrease of
growth and chlorophyll levels (Fig. 5D), as compared to
wild-type seedlings. Transgenic Arabidopsis plants of the
Bensheim ecotype expressing antisense hexokinase (HXK)
or overexpressing HXK [26] were also tested for their sensitivity to atrazine (Fig. 5D). The Bensheim ecotype showed
the same growth response to sucrose and the same sucroseinduced tolerance to atrazine as the Ws ecotype. Both types
of Arabidopsis transgenic lines were found to behave like
wild-type seedlings in terms of response to sucrose and
of tolerance to atrazine in the presence of 80 mM sucrose
(Fig. 5D).
4. Discussion
As pointed out by Rolland et al. [27], sugar control of
metabolism, growth, and development has long been thought
to be a metabolic effect. Similarly, sugar-induced lifting of
atrazine inhibition, as shown in Fig. 1, might have been
interpreted as mere compensation of photosynthesis impairment by carbon feeding. However, as stated by Rutherford
and Krieger–Liszkay [2], cell and plant death ensuing treatment with PSII-bound herbicides is not a result of starvation.
Moreover, the striking differential effects of glucose and
sucrose (Fig. 1) indicated that lifting of atrazine inhibition
could not be ascribed to mere carbon feeding. Finally, glycerol, which can relieve some aspects of carbon starvation
[28], did not fully relieve bleaching by atrazine (Fig. 5).
Besides carbon feeding of primary metabolism, sugars are
also the precursors of antioxidant compounds which may be
efficient for protection against atrazine-mediated oxidative
stress. A variety of metabolic pathways may be involved,
such as production of NADPH by the pentose phosphate
pathway [29,30], ascorbate synthesis [31], carotenoid synthesis [32], and conjugation-based detoxification pathways,
such as that of UDPG–glucosyltransferase, which is involved in atrazine detoxification [33]. Thus, glucose has
been shown to enhance cellular defences against cytotoxicity of hydrogen peroxide in animal cells [29]. In all of the
above cases, glucose should be as efficient as sucrose as
metabolic precursor. Thus, no major difference of pentose
phosphate pathway activity in higher plant cells [34] has
been reported for glucose and sucrose feeding. Therefore,
the hypothesis of metabolic feeding of antioxidant processes did not agree with the striking differential effects of
glucose and sucrose on induction of atrazine tolerance.
Significant lifting of atrazine inhibition by glucose and
sucrose started at sugar concentrations of 5 mM, which have
been shown to be effective on gene expression [23]. The
hexokinase-dependent pathway, which is a major route for
sugar signalling [26], can be activated by both glucose and
sucrose, and appears to be primarily involved in gene repression [27]. In the absence of atrazine, D1 protein accumulation was enhanced by low sucrose feeding and decreased by
higher (80 mM) sucrose concentrations. A number of genes
encoding photosynthesis components have been reported
to be repressed by sugars [35]. Moreover, in the cyanobacterium Synechocystis, glucose feeding, which is used to
increase the reducing power of the cell, has been shown to
depress the steady-state mRNA levels of PSII genes [36]
and, under dark conditions, to induce the destabilisation
of psbA transcripts [37]. In accordance with these studies,
the present work shows that 80 mM glucose or sucrose
feeding results in depressed levels of psbA mRNA levels in
Arabidopsis plantlets. Moreover, 2-deoxyglucose treatment,
which activates the hexokinase signalling pathway [23,27],
was found to result in the loss of chlorophylls (Fig. 5C).
In other words, activation of the hexokinase signalling
pathway by either glucose or sucrose would be likely to enhance, rather than alleviate, the inhibitory effects of atrazine
by decreasing the levels of the protein target. This likely
effect of the hexokinase-dependent signalling pathway and
the differential effects of sucrose and glucose on atrazine
C. Sulmon et al. / Plant Science 167 (2004) 913–923
tolerance strongly suggested that sugar-induced tolerance
to atrazine must involve hexokinase-independent pathways
[20,27]. Indeed, Arabidopsis transgenic lines modified in the
expression of hexokinase [26] were found to retain sugarinduced enhancement of growth and tolerance to atrazine
(Fig. 5). In contrast, the sis1 Arabidopsis mutant, which is
insensitive to high levels of sucrose [21], was affected in
both enhancement of chlorophyll accumulation by sucrose
and sucrose-induced tolerance to atrazine (Fig. 5). The sis1
gene is allelic to the ethylene-pathway ctr1 gene [21], and
the glucose/hexokinase pathway has been shown to be antagonistic to the ethylene/CTR1 pathway [38]. Moreover, the
sis1 mutant has been shown to remain sensitive to 300 mM
glucose, whereas it is insensitive to 300 mM sucrose [21].
In other words, the sucrose-based enhancement of chlorophyll accumulation and tolerance to atrazine probably uses
a pathway antagonistic to the glucose-hexokinase pathway,
which may be connected to the ethylene/CTR1 pathway.
Treatment with 3-Me-Glc, which does not activate the hexokinase pathway and may activate a sugar carrier pathway
[39], was found to promote a certain level of chlorophyll accumulation (Fig. 5), thus also suggesting that enhancement
of chlorophyll accumulation by sucrose was, at least partially, hexokinase-independent. However, 3-Me-Glc did not
confer atrazine tolerance, thus confirming the hypothesis of
the involvement of a sucrose-specific pathway. Such sucrosespecific pathways remain to be identified [27]. Further characterisation of sucrose-induced tolerance to atrazine should
therefore contribute to the identification of these sucrosespecific sensing mechanisms.
A number of studies on the involvement of sugars in the
response to abiotic stress have reported protective effects
on the photosynthetic machinery. Thus, the positive effects
of trehalose on abiotic stress tolerance have been related to
modifications of sugar sensing and protection of PSII [40].
Enhancement of sugar levels in the chloroplasts of mesophyll cells by manipulating invertase activity also results in
protection of PSII under salt stress [41]. Sucrose has also
been involved in the thermostability of plant proteins [42].
Sucrose treatment was found to maintain chlorophyll levels (Fig. 1; Fig. 5), D1 protein levels, PSII potential activity
(Fig. 3), and photosynthetic oxygen evolution in the presence of atrazine in the growth medium and in the plantlet. Thus, in planta in the presence of sucrose, PSII was
insensititive to or escaped inhibition by atrazine. Sucrose
treatment was therefore likely to induce protection mechanisms as reported in the response to drought and salt stress
[40,41]. The molecular mechanisms of these protective effects, which have not yet been elucidated [40,41], may be
related to the general protective effects of compatible solutes, such as removal of free radicals and stabilisation of
the hydrated structure of proteins [41], or to carbohydratemodulated induction of defence-related genes [24]. Further
work should also determine whether the protection towards
atrazine operates not only in whole plantlets and whole cells,
but also in isolated chloroplasts and thylakoids.
921
Moreover, in the presence of protective concentrations of
sucrose, increasing atrazine concentration was unexpectedly
found to enhance markedly root growth (Fig. 1), to improve
chlorophyll and carotenoid levels relative to plantlets treated
with 5 ␮M atrazine (Fig. 1), and to increase D1 protein and
psbA mRNA levels (Fig. 4). In line with the differential
effects of glucose and sucrose described above, increasing
concentrations of atrazine in the presence of glucose did not
improve chlorophyll and carotenoid contents (Fig. 1), and
the levels of psbA mRNA in the presence of 1 ␮M atrazine
were greater in the presence of 80 mM sucrose than in the
presence of 80 mM glucose (Fig. 4). This striking enhancement of D1 protein and psbA mRNA levels in the presence
of both soluble sugar and atrazine (Fig. 4) clearly showed
that atrazine was also able to affect photosynthetic development positively under certain physiological conditions.
This may bear resemblance to the fact that atrazine-tolerant
Pseudomonas strains are able to utilize atrazine as carbon
and nitrogen source [43]. However, in the present study, the
positive effect on plant development was obtained at low
atrazine concentrations that would give negligible carbon
and nitrogen contribution. Furthermore, in the present study,
seedling growth was carried out in the presence of MS salts,
which have been shown to provide optimal nitrogen supply [44]. Alternatively, atrazine may generate interacting
nitrogen-related or reactive oxygen species signals, which
have been shown to interfere, respectively, with glucose repression of photosynthesis gene expression [45,46] and with
expression of the psbA gene [47]. Herbicide tolerance in
higher plants can result from different mechanisms, including overexpression of the wild-type target [48]. Both the
effects on psbA and D1 protein overexpression and on protection of D1 protein may contribute to the tolerance against
atrazine. Further work on the effects of sucrose and atrazine
on psbA expression and D1 accumulation is in progress in
order to understand how hexokinase-independent sucrose
signalling and atrazine-related regulatory mechanisms interact to optimise D1 expression in response to xenobiotic
stress. However, the wide range of carbohydrate-modulated
gene expression includes induction of genes involved in
stress response through hexokinase-dependent [24,27] or
hexokinase-independent [49] signalling. The possible induction by soluble sugars of genes involved in the defence
against xenobiotic toxicity is also under investigation.
Acknowledgements
This work was supported by the programme Environnement-Vie-Société (CNRS, France). We are very grateful to
Anne Kersanté for help with atrazine analysis, to Dr. Bruce
A. Diner (Central Research and Development Department,
du Pont de Nemours and Company) for the kind gift of
anti-D1 antibodies, and to Dr. Jen Sheen (Harvard Medical School, Boston) for kindly providing hexokinase-related
Arabidopsis transgenic lines. We also thank the Nottingham
922
C. Sulmon et al. / Plant Science 167 (2004) 913–923
Arabidopsis Stock Centre for providing the sis1 Arabidopsis
mutant.
[20]
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