Plant Science 172 (2007) 756–762
www.elsevier.com/locate/plantsci
A deficiency in salicylic acid alters isoprenoid accumulation in
water-stressed NahG transgenic Arabidopsis plants
Sergi Munné-Bosch a, Josep Peñuelas b, Joan Llusià b,*
a
Departament de Biologia Vegetal, Universitat de Barcelona, Facultat de Biologia, Avinguda Diagonal 645, 08028 Barcelona, Spain
b
Unitat d’Ecofisiologia CSIC-CEAB-CREAF, CREAF (Center for Ecological Research and Forestry Applications), Edifici C,
Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
Received 28 June 2006; received in revised form 7 December 2006; accepted 8 December 2006
Available online 17 December 2006
Abstract
Previous studies have shown that salicylic acid is an essential component of the plant resistance to pathogens and participates in the plant
response to adverse environmental conditions. The present study was aimed at better understanding the response of salicylic acid-deficient
transgenic NahG Arabidopsis plants to water deficit conditions, with an especial emphasis on the effects of salicylic acid on isoprenoid
accumulation. Total monoterpene contents, chlorophylls and a-tocopherol (which contain a diterpene phytyl moiety in their molecule) and the
tetraterpenes, carotenoids, were measured in wild type and NahG Arabidopsis plants exposed to 2 weeks of water deficit. The results show that a
salicylic acid deficiency in NahG tansgenic lines suppressed the water stress-induced loss of chlorophylls and carotenoids observed in the wild
type. a-Tocopherol accumulation was also affected in NahG plants, which showed lower levels of this antioxidant both under irrigated and water
stress conditions. In addition, accumulation of monoterpenes was suppressed in NahG plants both under irrigated and water stress conditions,
although the effects were more apparent under stress and, as this was more severe. We conclude that salicylic acid affects isoprenoid accumulation
in leaves specially under water stress conditions.
# 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: a-Tocopherol; Antioxidants; Carotenoids; Diterpenes; Monoterpenes; Salicylic acid; Water deficit
1. Introduction
Environmental stresses such as drought are among the
factors most limiting to plant productivity. Therefore, elucidation of the mechanisms by which plants perceive and transduce
these stresses is critical if we are to understand the plant
response and introduce genetic or environmental improvement
to stress tolerance. Drought stress triggers responses ranging
from altered gene expression to changes in plant metabolism
and growth. One of the early responses to drought is the
reduction in leaf expansion, which is usually followed by
stomatal closure and reductions in photosynthesis. Exposure to
prolonged drought may lead to an inhibition of PSII, which may
Abbreviations: IR, irrigated; RWC, relative leaf water content; SA,
salicylic acid; WS, water stress (or water-stressed)
* Corresponding author. Tel.: +34 935812934; fax: +34 935814151.
E-mail address: [email protected] (J. Llusià).
0168-9452/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2006.12.005
result in an excess of excitation energy in chloroplasts
[reviewed by 1,2].
Plants have evolved several mechanisms to get rid of this
excess energy in photosynthetic membranes, some of which
involve isoprenoid compounds. a-Tocopherol and carotenoids
represent a conserved mechanism of photoprotection, while
other isoprenoids, such as monoterpenes, represent an
additional or alternative photoprotection mechanism [reviewed
by 3]. a-Tocopherol and carotenoids display antioxidant
activity and therefore contribute to keep thylakoid membrane
structure and function under stress [4,5]. The levels of atocopherol increase in tolerant plant species exposed to
drought, which linked to their antioxidant properties, suggests
a protective role for this compound from water deficit, when
reactive oxygen species are likely to increase as a result of
excess energy in thylakoids [6]. The production of monoterpenes has been linked to an increased thermo- and waterstress (WS) tolerance in some species [7–11]. Stress protection
of monoterpenes may be attributed to their capacity to increase
S. Munné-Bosch et al. / Plant Science 172 (2007) 756–762
membrane fluidity and stability due to their lipophility and
antioxidant capacity similarly to what occurs with other
isoprenoids [12–18].
In addition to its role in plant pathogenesis [19], salicylic
acid (SA) is also believed to play a role in plant responses to
abiotic stresses such as ozone and UV light [20–22], heat stress
[23–25], chilling and drought [25], and salt and osmotic stresses
[26]. These studies suggest that while moderate doses of SA
enhance the antioxidant status and induce stress resistance,
higher concentrations activate a hypersensitive cell death
pathway and increase stress sensitivity. Besides, parallel
increases in SA and pathogenesis-related proteins have been
reported in plants exposed to UV-C light and ozone, which
suggests a common signal transduction pathway in plant
responses to biotic and abiotic stresses [22].
Although both SA and isoprenoids seem to be involved in
plant responses to water deficit, little is still known about the
interaction between them, and about the regulation of
isoprenoid accumulation by SA under WS. In a previous study
we showed that endogenous SA accumulates in WS Phillyrea
angustifolia plants, which paralleled increases in a-tocopherol
and decreases in b-carotene and chlorophylls [27]. Furthermore, we showed that a sustained accumulation of methyl
salicylate suppresses heat-induced accumulation of a-tocopherol in holm oak leaves, while it increases oxidative damage
[28]. A regulatory link between SA-mediated disease resistance
and the methyl-erythritol 4-phosphate pathway in the
Arabidopsis csb3 mutant has recently been found [29], but
further studies are needed to get further insight into the role SA
may play in plant responses to abiotic stress and on regulation
of isoprenoid accumulation. With this aim, we evaluate in the
present study the pattern of isoprenoid accumulation (total
monoterpenes, chlorophylls, a-tocopherol and carotenoids
levels) in wild type and SA-deficient transgenic NahG
Arabidopsis plants exposed to irrigated (IR) and WS
conditions.
2. Materials and methods
2.1. Plant material and water deficit treatment
We used the Arabidopsis thaliana ecotype Columbia (Col-0)
as wild type and the transgenic NahG line, which expresses a
salicylate hydroxylase (NahG) gene. The 35S-NahG line was
donated by Luis A.J. Mur (Institute of Biological Sciences,
University of Wales). Seedlings were grown in pots containing
a mixture of peat/perlite/vermiculite (1:1:1, v/v/v) in a
controlled environment chamber (8 h photoperiod, 90–
110 mmol quanta m 2 s 1, air temperature 21–3 8C). After
12 weeks of growth, the experiment started and two water
regimes were imposed for 2 weeks on both Col-0 and NahG
plants: (i) plants watered with Hoagland’s solution at saturation
(IR plants), and (ii) plants not watered at all (WS plants).
Measurements were taken at the start of the experiment and
after 7, 11 and 14 days of treatment. Plant water status,
photosynthetic pigments (chlorophylls and carotenoids), atocopherol and monoterpene concentrations, were measured
757
simultaneously in rosette leaves in the middle of the light
period. For measurements of photosynthetic pigments, atocopherol and monoterpenes, whole rosette leaves were
collected, frozen in liquid nitrogen, and stored at 30 8C for
later analysis. Lipid peroxidation measurements were conducted at the end of the experiment, 14 days after treatment
started.
2.2. Plant water status
Plant water status was determined by measuring the relative
leaf water content (RWC) as follows. Leaves were weighed, rehydrated for 24 h at 4 8C in darkness and subsequently ovendried for 24 h at 80 8C. RWC was determined as
100 (FW DW)/(TW DW), where FW is the fresh
weight, TW is the turgid weight after re-hydrating the leaves,
and DW is the dry weight after oven-drying the leaves.
2.3. Photosynthetic pigments and a-tocopherol
For measurement of chlorophylls and carotenoids, leaves
were ground in liquid nitrogen and repeatedly extracted (three
times) with ice-cold 80% (v/v) acetone using ultrasonication
(Vibra-Cell Ultrasonic Processor, Sonics and Materials Inc.,
Danbury, CT, USA). Chlorophylls and carotenoids were
estimated spectrophotometrically and specific absorption
coefficients for chlorophyll a, chlorophyll b, and total
carotenoids reported by [30] were used. A molecular weight
of 570 for carotenoids was used for calculations.
The extraction and HPLC analysis of a-tocopherol were
carried out as described by [31]. In short, leaves were
repeatedly extracted (three times) with ice-cold n-hexane
containing 1 mL L 1 butylated hydroxytoluene (BHT) using
ultrasonication (Vibra-Cell Ultrasonic Processor). a-Tocopherol was separated on a Partisil 10 ODS-3 column
(250 mm 4.6 mm, Scharlau, Barcelona) at a flow rate of
1 mL min 1. The solvents consisted of (A) methanol:water
(95:5, v/v) and (B) methanol. The gradient used was: 0–10 min,
100% A; 10–15 min, decreasing to 0% A; 15–20 min, 0% A; 20
to 23 min, increasing to 100% A; and 23–28 min, 100% A. aTocopherol was quantified through its absorbance at 285 nm
(UV–vis Detector Kontron 535, Bio-Tek Instruments Inc.,
Winooski, VT, USA) and it was identified by co-elution with an
authentic standard provided by Sigma (Steinheim, Germany).
2.4. Monoterpene contents
For measurement of monoterpenes, leaves were ground in
liquid nitrogen and repeatedly extracted (three times) with
pentane. Before chromatographical analysis, we centrifuged
extracted leaves with pentane at 10,000 rpm for 5 min. Extracts
were then concentrated with a stream of nitrogen, given their
low concentrations.
Monoterpene separation and analyses were conducted in a
GC-MS system (Hewlett Packard HP59822B, Palo Alto, CA,
USA). Extracts (4 mL) were injected in to the GC-MS system
and passed into a 30 m 0.25 mm 0.25 mm film thickness
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S. Munné-Bosch et al. / Plant Science 172 (2007) 756–762
capillary column (Supelco HP-5, Crosslinked 5% Me Silicone,
Supelco Inc., Bellefonte, PE, USA). After sample injection, the
initial temperature (40 8C) was increased at 30 8C min 1 up to
60 8C, and thereafter at 10 8C min 1 up to 150 8C, maintained
for 3 min, and thereafter raised at 70 8C min 1 up to 250 8C,
which was maintained for another 5 min. Helium flow was
1 mL min 1. The identification of monoterpenes was conducted by GC-MS and comparison with standards from Fluka
(Buchs, Switzerland), literature spectra, and GCD Chemstation
G1074A HP. Internal standard dodecane, which did not mask
any terpene, together with frequent calibration with common
terpene a-pinene, 3-carene, b-pinene, b-myrcene, p–cymene,
limonene and sabinene standards once every five analyses were
used for quantification. Terpene calibration curves (n = 4
different terpene concentrations) were always highly significant
(r2 > 0.99) in the relationship between signal and terpene
concentration. The most abundant terpenes had very similar
sensitivity (differences were less than 5%). We conducted
recovery tests that resulted in better recoveries than 90% in all
cases (one test every day of analyses).
2.5. Lipid peroxidation measurements
The extent of lipid peroxidation was estimated by measuring
the amount of malondialdehyde (MDA) in leaves by the method
described by [32], which takes into account the possible
influence of interfering compounds in the thiobarbituric acidreactive substances (TBARS) assay. Leaf samples were
extracted with 80:20 (by vol.) ethanol/water using ultrasonication (Vibra-Cell Ultrasonic Processor, Sonics & Materials Inc.,
Canbury, CT). After centrifuging at 3000 g for 10 min at
48 C, the pellet was re-extracted twice with the same solvent.
Supernatants were pooled and an aliquot of appropriately
diluted sample was added to a test tube with an equal volume of
either (i) TBA solution comprised of 20% (w/v) trichloroacetic acid and 0.01% (w/v) BHT, or (ii) +TBA solution
containing the above plus 0.65% (w/v) TBA. Samples were
heated at 95 8C for 25 min, and after cooling, absorbance was
read at 440, 532, and 600 nm. MDA equivalents (nmol mL 1)
were calculated as 106 ((A B)/157,000), where A = ((Abs
532+TBA) (Abs
600+TBA) (Abs
532 TBA Abs
600 TBA)), and B = ((Abs 440+TBA Abs 600+TBA) 0.0571)
0.0571) ([32]).
Fig. 1. Time course evolution of relative leaf water content (RWC) in irrigated
(IR) and water-stressed (WS) wild type (black symbols) and NahG transgenic
(white symbols) Arabidopsis plants. Error bars indicate S.E.M.; n = 4–5 plants.
The symbol (*) indicates statistically significant difference between wild type
and NahG plants (Student’s t-test, P < 0.05). Significance of time course
evolution, NahG-generated changes and interaction between both are depicted
inside panel (results of ANOVA).
chlorophylls and a-tocopherol (which contain a diterpene
phytyl moiety in their molecule) and carotenoids (tetraterpenes), in parallel with the RWC in wild type and SA-deficient
NahG Arabidopsis plants both under IR and WS conditions.
RWC was kept above 80% throughout the experiment in both
wild type and NahG plants under IR conditions. In contrast,
RWC decreased progressively under water deficit, attaining
minimum values below 20% after 2 weeks of stress (Fig. 1). No
significant differences in RWC were observed between wild
type and SA-deficient plants, except at the beginning of the
experiment, when RWC was slightly higher in NahG plants.
Despite the fact that both plant groups showed similar
RWCs, SA-deficient plants retained more chlorophylls under
water deficit conditions. While chlorophyll a + b levels kept
above 10 mmol [g DW] 1 in WS NahG plants, water deficit
induced a chlorophyll loss of up to 60% in the wild type (Fig. 2).
The chlorophyll a/b ratio was the same in both plant groups,
2.6. Statistical analyses
Analysis of variance (ANOVA) with Bonferroni post hoc
tests for all studied dependent variables and Student’s t-tests
between NahG and wild type plants in each measuring date and
water treatment were conducted using STATISTICA Version
5.0 for Windows (StatSoft Inc. Tulsa, OK, USA). Differences
were considered significant at a probability level of P < 0.05.
3. Results
To evaluate the effects of SA on isoprenoid accumulation,
we measured simultaneously the levels of total monoterpenes,
Fig. 2. Time course evolution of chlorophyll (Chl) a + b levels in irrigated (IR)
and water-stressed (WS) wild type (black symbols) and NahG transgenic (white
symbols) Arabidopsis plants. Error bars indicate S.E.M.; n = 4–5 plants. The
symbol (*) indicates statistically significant difference between wild type and
NahG plants (Student’s t-test, P < 0.05). Significance of time course evolution,
NahG-generated changes and interaction between both are depicted inside
panel (results of ANOVA).
S. Munné-Bosch et al. / Plant Science 172 (2007) 756–762
759
Fig. 3. Time course evolution of a-tocopherol contents, given per dry weight
and per chlorophyll unit, in irrigated (IR) and water-stressed (WS) wild type
(black symbols) and NahG transgenic (white symbols) Arabidopsis plants.
Error bars indicate S.E.M.; n = 4–5 plants. The symbol (*) indicates statistically
significant difference between wild type and NahG plants (Student’s t-test,
P < 0.05). Significance of time course evolution, NahG-generated changes and
interaction between both are depicted inside panel (results of ANOVA).
Fig. 4. Time course evolution of total carotenoid contents, given per dry weight
and per chlorophyll unit, in irrigated (IR) and water-stressed (WS) wild type
(black symbols) and NahG transgenic (white symbols) Arabidopsis plants.
Error bars indicate S.E.M.; n = 4–5 plants. The symbol (*) indicates statistically
significant difference between wild type and NahG plants (Student’s t-test,
P < 0.05). Significance of time course evolution, NahG-generated changes and
interaction between both are depicted inside panel (results of ANOVA).
both wild type and NahG plants showing values of 2.6 and 2.3
under IR and WS conditions, respectively (data not shown).
a-Tocopherol accumulation differed in wild type and NahG
plants both under IR and WS conditions. Either given per dry
weight or chlorophyll unit, a-tocopherol levels were significantly lower in SA-deficient plants compared to the wild
type. a-Tocopherol levels, expressed on a dry weight basis,
increased slightly as plants aged in the wild type, while they did
not increase, or even tended to decrease, in the SA-deficient
plants (Fig. 3). This differential pattern of a-tocopherol
accumulation in both plant groups was very similar both
under IR and WS conditions. When given on a chlorophyll
basis, differences in a-tocopherol levels between both plant
groups were more evident, particularly under WS conditions
and as the stress was more severe. Maximum differences
between plant groups were observed after 2 weeks of water
deficit, when a-tocopherol levels per unit of chlorophyll were
five times lower in SA-deficient plants than in the wild type
(Fig. 3).
The accumulation of carotenoids differed in wild type and
NahG plants under WS conditions only. While carotenoid
levels, given per dry weight, increased progressively up to 52%
as WS progressed in NahG plants, they decreased up to 50% in
WS wild type plants (Fig. 4). Maximum differences between
both plant groups were attained after 2 weeks of water deficit,
when carotenoid levels, on a dry weight basis, were more than
twice larger in SA-deficient plants than in the wild type (Fig. 4).
Differences in carotenoids between both plant groups
disappeared when given on a chlorophyll basis. Carotenoids
per unit of chlorophyll increased slightly as plants aged in both
plant groups, and no significant differences were observed
between both plant groups under neither IR nor WS conditions.
Both wild type and NahG plants accumulated several
monoterpenes, being a-pinene and limonene the most abundant
ones, the former predominantly in the first days of the
experimental period and the latter with higher prevalence in the
final days of this experimental period (data not shown). The
amount of accumulated total monoterpenes differed in wild
type and NahG plants both under IR and WS conditions, major
Fig. 5. Time course evolution of total monoterpene contents in irrigated (IR)
and water-stressed (WS) wild type (black symbols) and NahG transgenic (white
symbols) Arabidopsis plants. Error bars indicate S.E.M.; n = 4–5 plants. The
symbol (*) indicates statistically significant difference between wild type and
NahG plants (Student’s t-test, P < 0.05). Significance of time course evolution,
NahG-generated changes and interaction between both are depicted inside
panel (results of ANOVA).
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S. Munné-Bosch et al. / Plant Science 172 (2007) 756–762
Fig. 6. The extent of lipid peroxidation in leaves, estimated as malondialdehyde
(MDA) concentrations in wild type and NahG transgenic plants of Arabidopsis
after 14 days treatment under irrigated (IR) and water stress (WS) conditions.
Data represent the mean S.E. of three measurements. Different letters
indicate statistically significant differences (ANOVA, post hoc Bonferroni tests,
P < 0.05).
differences being observed under WS and as this was more
intense. Monoterpene contents increased as plants aged in both
plant groups, although to a lower extent under IR conditions
than under WS conditions (Fig. 5). Differences between wild
type and SA-deficient plants were already observed after 7 days
of water deficit, but maximum differences were attained after 2
weeks of stress, when monoterpene levels were 2.5-fold higher
in wild type than NahG plants in WS conditions and ca. 2 fold
higher in IR conditions (Fig. 5).
At the end of the experiment, after 14 days of treatment, the
extent of lipid peroxidation estimated as malondialdehyde
(MDA) concentrations in leaves of SA-deficient NahG
transgenic plants was half that in wild type plants, both under
irrigated and water stressed conditions (Fig. 6).
4. Discussion
Changes in isoprenoid metabolism occur in response to
several abiotic stresses, including water deficit [8,31,33]. In
addition, increases in SA have been reported to occur in WS
plants and such increases could play a role, among other
factors, in plant stress resistance [27]. However, the interaction
among these components in plant response to stress is still
poorly understood. Our results now show that SA may be
involved in the changes taking place in the plant under water
deficit and that SA may directly or indirectly modulate the
accumulation of isoprenoids under stress.
A deficiency in SA in the NahG transgenic line clearly
prevented WS-induced chlorophyll loss in the present study.
Chlorophyll loss is one of the early symptoms of leaf senescence
and, in the present study it led to enhanced a-tocopherol levels
per chlorophyll unit in the wild type. Our results therefore
indicate that SA may be involved in accelerating water-stress
induced chlorophyll loss, which may be probably linked with an
accelerated senescence under stress. At the end of the
experiment, after 14 days of treatment, the lower extent of lipid
peroxidation in SA-deficient NahG transgenic plants (lower
MDA concentrations; Fig. 6) also indicates a delay on the
senescence of SA-deficient plants. All this is in agreement with
[34], who demonstrated that SA has a key role in regulating gene
expression during senescence. Furthermore, it has been shown
that some genes implicated in SA signaling are upregulated in
response to singlet oxygen and that Arabidopsis flu mutants
expressing transgenic NahG are partially protected from the
death provoked by the release of singlet oxygen [35]. Aside from
chlorophylls, loss of carotenoids was also prevented in NahG
transgenic plants. Furthermore, it has been shown that SA
accumulates in WS Phillyrea angustifolia plants, in parallel with
increases in a-tocopherol and malondialdehyde, and decreases in
b-carotene and chlorophylls [27]. Lack of SA therefore leads to
reductions in a-tocopherol, and higher accumulation of
photosynthetic pigments (chlorophylls and carotenoids) in the
NahG transgenic lines.
It is interesting to note that while the effect of SA on
photosynthetic pigments specifically occurred under WS,
regulation of a-tocopherol levels by SA occurred irrespective
of plant water status and probably linked to plant aging, due to the
advanced developmental stage of plants. Several studies have
shown an increase in the levels of a-tocopherol as leaves age [36–
39], thus our results suggest that the increase of this antioxidant
as leaves age may be mediated, at least in part, by SA.
The accumulation of monoterpenes was also highly
influenced by SA. The deficiency in SA in the NahG transgenic
lines prevented accumulation of monoterpenes both under IR
and WS conditions, although major effects were observed
under stress. Aging and WS-induced increases in monoterpene
contents may therefore be mediated, at least in part, by SA. In
fact, in a previous study conducted at temperatures between 25
and 45 8C, we observed that MeSA-fumigated plants showed
ca. 3–4-fold greater monoterpene leaf concentrations and
monoterpene emission rates than the control plants [40],
showing that at least airborne MeSA may drive plants to
produce and emit monoterpenes in a similar way to what occurs
with the expression of defense-related genes in neighboring
plants [19], with the expression of genes involved in defense
against oxidative stress during chilling [41] or with the increase
in antioxidant enzymes activity [42].
Although a regulatory link between SA-mediated disease
resistance and the methyl-erythritol 4-phosphate pathway in the
Arabidopsis csb3 mutant has recently been found [29], further
studies are needed to elucidate the mechanism(s) for these SAmediated changes in isoprenoid contents including both
biosynthetic and degradation pathways.
We conclude that SA may affect isoprenoid accumulation in
leaves both under IR and WS conditions, and that changes in
isoprenoid metabolism mediated by SA are more apparent
under stress and as this is more intense. Further research is
required to better understand how SA mediates changes in
isoprenoid metabolism, whether biosynthetic or degradation
pathways are affected and how isoprenoid building blocks are
diverted to specific biosynthetic routes.
S. Munné-Bosch et al. / Plant Science 172 (2007) 756–762
Acknowledgements
This research was supported by REN2003-04871,
CGL2004-01402/BOS, CGL2006-04025/BOS and BFU200601127 grants from the Spanish Government, grant ISONET
(Marie Curie network contract MC-RTN-CT-2003 504720)
from the European Union, a 2004 grant from the Fundación
BBVA, and by a SGR2005-00312 grant from Catalan
Government. We are indebted to Luis A. J. Mur (Institute of
Biological Sciences, University of Wales) for kindly providing
the NahG transgenic Arabidopsis line. We also thank the
Serveis Cientı́fico-Tècnics and Serveis dels Camps Experimentals de la Universitat de Barcelona for technical assistance.
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