First published online October 15, 2002; 10.1104/pp.006858
Plant Physiol, November 2002, Vol. 130, pp. 1143-1151
The Combined Effect of Drought Stress and Heat
Shock on Gene Expression in Tobacco1
Ludmila
Rizhsky,
Hongjian
Liang, and
Ron
Mittler*
Department of Biology, Technion-Israel Institute of Technology,
Technion City, Haifa 32000, Israel (L.R.); and Department of Botany,
Plant Sciences Institute, Iowa State University, Room 353 Bessey Hall,
Ames, Iowa 50011 (H.L., R.M.)
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ABSTRACT |
In nature, plants encounter a combination of environmental
conditions that may include stresses such as drought or heat shock. Although drought and heat shock have been extensively studied, little
is known about how their combination affect plants. We used cDNA
arrays, coupled with physiological measurements, to study the effect of
drought and heat shock on tobacco (Nicotiana tabacum)
plants. A combination of drought and heat shock resulted in the closure
of stomata, suppression of photosynthesis, enhancement of respiration,
and increased leaf temperature. Some transcripts induced during
drought, e.g. those encoding dehydrin, catalase, and glycolate oxidase,
and some transcripts induced during heat shock, e.g. thioredoxin
peroxidase, and ascorbate peroxidase, were suppressed during a
combination of drought and heat shock. In contrast, the expression of
other transcripts, including alternative oxidase, glutathione
peroxidase, phenylalanine ammonia lyase, pathogenesis-related proteins,
a WRKY transcription factor, and an ethylene response transcriptional
co-activator, was specifically induced during a combination of drought
and heat shock. Photosynthetic genes were suppressed, whereas
transcripts encoding some glycolysis and pentose phosphate pathway
enzymes were induced, suggesting the utilization of sugars through
these pathways during stress. Our results demonstrate that the response
of plants to a combination of drought and heat shock, similar to the
conditions in many natural environments, is different from the response
of plants to each of these stresses applied individually, as typically
tested in the laboratory. This response was also different from the
response of plants to other stresses such as cold, salt, or pathogen
attack. Therefore, improving stress tolerance of plants and crops may require a reevaluation, taking into account the effect of multiple stresses on plant metabolism and defense.
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INTRODUCTION |
Under optimal conditions, cellular
homeostasis is achieved by the coordinated action of many biochemical
pathways. However, different pathways may have different molecular and
biophysical properties, making them different in their dependence upon
external conditions. Thus, during events of suboptimal conditions
(stress), different pathways can be affected differently, and their
coupling, which makes cellular homeostasis possible, is disrupted. This process is usually accompanied by the formation of reactive oxygen intermediates (ROIs) because of an increased flow of electrons from the
disrupted pathways to the reduction of oxygen (Halliwell and
Gutteridge, 1989 ; Noctor and Foyer, 1998; Asada, 1999; Dat et al.,
2000; Mittler, 2002). One example for this process is the effect of
heat shock on mitochondrial electron transfer. It was shown that during
heat shock, membrane-bound complexes at the inner mitochondrial
membrane are uncoupled or disrupted. Electrons from NADH produced by
the soluble, and less temperature-sensitive, Krebs cycle enzymes are
then channeled to the reduction of O2 to ROI by
different components of the uncoupled electron transport chain
(Davidson and Schiestl, 2001).
To counter the effects of stress, plants undergo a process of stress
acclimation. This process may require changes in the flow of
metabolites through different pathways, the suppression of pathways
that may be involved in the production of ROI during stress, and the
induction of various defense genes such as heat shock proteins (HSPs)
and ROI-scavenging enzymes (Vierling, 1991; Dat et al., 2000; Mittler,
2002).
The complexity of signaling events associated with the sensing of
stress and the activation of defense and acclimation pathways is
believed to involve ROI, calcium, calcium-regulated proteins, mitogen-activated protein kinase cascades, and cross talk
between different transcription factors (Liu et al., 1998; Xiong et
al., 1999; Bowler and Fluhr, 2000; Knight and Knight, 2001; Kovtun et
al., 2000; Chen et al., 2002). Interestingly, different stress conditions such as drought and cold can result in the activation of
similar stress response pathways (Seki et al., 2001; Chen et al.,
2002). Thus, a high degree of overlap may exist between gene clusters
activated by different stresses. This overlap may explain the
well-documented phenomena of "cross tolerance," in which a particular stress can induce in plants resistance to a subsequent stress that is different from the initial one (Bowler and Fluhr, 2000).
Although the study of abiotic stress response has advanced considerably
in recent years, analyzing the effect of a single stress on plants can
be very different from the conditions encountered by plants in the
field in which a number of different stresses may occur simultaneously
(Merquiol et al., 2001; Mittler et al., 2001). These can alter
plant metabolism in a novel manner that may be different from that
caused by each of the different stresses applied individually, and may
require a new type of response that would not have been induced by each
of the individual stresses.
To characterize some of the mechanisms involved in the response of
plants to a combination of stresses, applied simultaneously, we studied
the effect of drought and heat shock on tobacco (Nicotiana tabacum) plants. A combination of drought and heat shock can
represent the conditions encountered by many plants and crops growing
within arid and semiarid environments (Mittler et al., 2001);
therefore, its understanding may be critical for the development of new
strategies and tools to enhance stress tolerance via genetic manipulations.
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RESULTS |
Physiological Characterization of Drought Stress, Heat Shock, and a
Combination of Drought Stress and Heat Shock in Tobacco
To mimic the conditions encountered by plants during extended
periods of drought, accompanied by brief exposures to heat shock (typically occurring between midday to late afternoon; Merquiol et al.,
2001), we subjected tobacco plants to drought stress until they
reached a relative water content (RWC) of 65% to 70%. Plants were
then exposed to a heat shock treatment and sampled. As controls, we
used well-watered plants (control), drought-stressed plants that were
not subjected to heat shock (drought), and well-watered plants that
were subjected to heat shock (heat shock). All plants were analyzed and
sampled at the same time (after the heat shock treatment). Recovery
tests indicated that plants subjected to a combination of drought
stress and heat shock could recover within a few days upon watering and
changing of temperature to 23°C (not shown). The conditions used in
our study, therefore, were not lethal to plants.
As shown in Figure 1, drought
stress resulted in the suppression of respiration and photosynthesis.
In contrast, heat shock resulted in the enhancement of respiration, but
did not significantly alter photosynthesis. Interestingly, the
combination of drought stress and heat shock resulted in the
suppression of photosynthesis, similar to drought stress, but the
enhancement of respiration to levels that were comparable with those
measured in plants after heat shock. Measurements of stomatal
conductance, shown in Figure 2A,
indicated that heat shock is accompanied by opening of stomata, probably to enable the cooling of leaves via an enhanced transpiration stream. In contrast, stomata remained closed after drought or a
combination of drought and heat shock, suggesting that plants subjected
to a combination of drought and heat shock may be unable to cool their
leaves by enhanced transpiration. Measurements of leaf temperature,
shown in Figure 2B, revealed that the leaf temperature of plants
subjected to a combination of drought and heat shock was higher by
2°C to 3°C compared with that of plants subjected to heat shock
without drought. In addition, measurements of leaf transpiration
confirmed that during heat shock transpiration is enhanced, whereas
during a combination of drought and heat shock, transpiration is almost
completely abolished (not shown). The results presented in Figures 1
and 2 suggest that a combination of drought and heat shock affects
plants differently from drought or heat shock applied individually. The
differences included changes in photosynthesis, respiration, stomatal
conductance, and leaf temperature.
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Figure 1.
Measurements of photosynthesis and respiration in
plants subjected to heat shock, drought stress, and a combination of
heat shock and drought stress. Plants were subjected to stresses as
described in "Materials and Methods," and photosynthetic activity
and dark respiration were measured with an LI-6400 apparatus (LI-COR,
Lincoln, NE). Photosynthetic activity is shown to be suppressed
after drought stress or a combination of drought and heat shock,
whereas respiration is enhanced after heat shock and a combination of
drought and heat shock. A combination of drought and heat shock,
therefore, is different from drought or heat shock by having a high
rate of respiration and a low rate of photosynthetic activity. Results
are presented as mean and SD of five individual
measurements.
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Figure 2.
Stomatal conductance (A) and leaf temperature (B)
of plants subjected to heat shock, drought stress, and a combination of
heat shock and drought stress. Measurements were performed as described
in "Materials and Methods." The temperature of leaves subjected to
a combination of drought and heat shock is shown to be higher than that
of plants subjected to heat shock in the absence of drought. This
difference may result from the inability of plants, subjected to the
stress combination, to cool their leaves by transpiration because their
stomata are closed.
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Molecular Characterization of Gene Expression during Drought
Stress, Heat Shock, and a Combination of Drought Stress and Heat Shock
in Tobacco
To examine the effect of drought and heat shock on gene
expression in tobacco, we designed and used cDNA arrays composed of 170 cDNA clones encoding different defense and metabolic genes. These were
spotted in duplicates on nylon filters and used to assay changes in the
steady state level of their corresponding transcripts during drought,
heat shock, and a combination of drought and heat shock. Identical
filters were hybridized with radiolabeled cDNAs obtained from total RNA
isolated from plants subjected to the different stresses. The overall
pattern of gene expression detected by the filter arrays was different
among control, drought stress, heat shock, and a combination of drought
stress and heat shock (not shown). A summary of the changes in gene
expression calculated as percent of control and averaged over five
different experiments, each analyzed
individually, is shown in Tables I through
III. To compare the changes in expression
during heat shock, drought stress, and a
combination of drought stress and heat shock with other stresses, we
subjected plants to salt stress, cold stress, PQ application, TMV
infection, treatment with MJ, or to the expression of bO (Mittler et
al., 1995). A summary of the changes in gene expression during these
stresses is also shown in Tables I through III. As described
previously, TMV infection and bO expression result in the activation of
the hypersensitive response and the enhanced generation of ROI (Mittler
et al., 1998). Because each of these additional stresses requires a
different treatment, e.g. spraying with Tween 20 for PQ, mock infection for TMV, or growth in liquid culture for salt stress, an adequate control was designed for each treatment. These were critical because, as shown in Figures 3 and 4, and as
described previously (Mittler and
Zilinskas, 1992), different control
treatments alter the expression of key genes such as APX and catalase.
To confirm that these results, obtained with the cDNA arrays,
adequately represent changes in steady-state transcript levels, we
tested the expression of nine different cDNAs by RNA blots. These,
shown in Figures 3 and 4, were found to be in good agreement with the
results presented in Tables I through III (the results shown in Figs. 3
and 4 are from one experiment, whereas the results shown in Tables I
through III are the average and SD of five different
experiments, for the drought and heat experiments
[n = 5], and the average of two different experiments
each repeated twice for the other stresses [n = 4], including the experiments shown in Figs. 3 and 4). Because the leaf
temperature of plants subjected to drought and heat shock was higher
than that of plants subjected to heat shock in the absence of drought
(Fig. 2), we performed additional experiments of heat shock at a higher
temperature (i.e. 46°C); however, we did not find a significant
difference between the induction of HSPs in tobacco plants subjected to
heat shock at 46°C or heat shock at 44°C (not shown).
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Figure 3.
Changes in the steady-state level of transcripts
encoding stress response and metabolic proteins and enzymes during a
combination of drought and heat shock. RNA gel blots were used to assay
the steady-state level of selected transcripts during a combination of
drought and heat shock. Many of the transcripts shown in this figure
have a distinct expression pattern during a combination of drought and
heat shock. RNA isolation, blots, and analysis are described in
"Materials and Methods."
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Figure 4.
Changes in the steady-state level of transcripts
encoding stress response and metabolic proteins and enzymes after
different environmental stresses. RNA gel blots were used to assay the
steady-state level of selected transcripts during different stresses.
RNA isolation, blots, and analysis are described in "Materials and
Methods."
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Table I summarizes results obtained for cDNAs encoding different
HSPs, and different ROI removal enzymes. As shown in Table I, a number
of HSPs were induced during a combination of drought and heat shock.
These included cytosolic HSP90, HSP70, and HSP100, and sHSPs
(cytosolic, mitochondrial, and chloroplastic). Overall, the induction
of HSPs was higher in drought and heat shock compared with heat shock
or drought. Analyzing the changes in ROI removal enzymes revealed
interesting differences among the different stresses. During heat
shock, cytosolic APX and thioredoxin peroxidase appeared to be
dominant. In contrast, during drought stress, CAT and GPX appeared to
be specifically induced. During a combination of drought and heat
shock, however, AOX, GPX, glutathione reductase, CuZn-SOD, and
glutathione-S-transferase were induced. Thus, the panel of transcripts encoding ROI-detoxifying enzymes induced during each of the
different stresses appeared to be different, and ROI detoxification may
occur via different routes during the different stresses. The induction
of cytosolic APX during heat shock was in agreement with previous
reports on the presence of a heat shock factor-binding sequence at the
promoter of ApxI (Mittler and Zilinskas, 1992; Storozhenko et al., 1998).
Changes in the steady-state transcript level of different
metabolic genes are shown in Table II. As shown in Table II, many of
the photosynthetic genes were suppressed during stress. Exceptions were
transcripts encoding a PSI reaction center protein, the large subunit
of Rubisco, and a subunit of cytochrome B6F. Because cyclic electron
flow involves PSI and cytochrome B6F, it is possible that during stress
some energy dissipation is obtained via this pathway. Glycolate
oxidase, a key enzyme of the photorespiratory pathway induced during
drought, was suppressed during a combination of drought and heat shock.
In contrast to the suppression of photosynthetic genes, some
transcripts encoding enzymes of the pentose phosphate pathway and
glycolysis were induced during a combination of drought and heat shock.
These included Glc-6-phosphate dehydrogenase and pyruvate kinase. The
induction of these transcripts may suggest that during a combination of
drought and heat shock, the flow of sugars through these pathways is
enhanced, possibly for the production of reducing energy, such as
NAD(P) H, in the absence of photosynthesis. In contrast to the
suppression of transcripts involved in photosynthesis during a
combination of drought and heat shock, transcripts encoding different
components of the mitochondrial respiration pathway were not suppressed
during a combination of drought stress and heat shock (Table
II).
Table III summarizes changes in the expression pattern of
different stress response genes. In contrast to drought or heat shock, a combination of drought and heat shock resulted in the induction of a
number of different stress response transcripts. These included transcripts encoding PR proteins and PAL. In contrast to PR proteins that were not induced to the same extent as during TMV infection, PAL
was induced to levels that were similar to or even higher than those
found during pathogen infection. DHN, highly induced during drought
stress, was only moderately induced during a combination of drought and
heat shock (Table III; see also Fig. 3). In contrast, the induction of
a different drought-induced protein (DI-19) was augmented by the
combination of drought and heat shock. However, unlike DHN, this
transcript was also induced during heat shock. The induction of
transcripts encoding different components of the UB protein degradation
pathway was also elevated during a combination of drought and heat
shock. The induction of the different stress and pathogen response
transcripts during a combination of drought and heat shock suggests
that this combination may have activated a signal transduction pathway
that is also activated during wounding or pathogen infection. This
activation might have resulted from the combined synthesis of different
plant hormones such as abscisic acid, ethylene, and MJ. The expression
of lipoxygenase, involved in jasmonic acid synthesis, was elevated
during drought and heat shock.
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Table III.
Changes in the steady-state level of transcripts
encoding general stress, ubiquitin, and "housekeeping"
proteins
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Expression of Stress Response Transcripts with Homology to
Transcripts Isolated from the Desert Plant Retama raetam
during a Combination of Drought Stress and Heat Shock in
Tobacco
We recently cloned, by a subtraction cDNA cloning method, a number
of stress response cDNAs induced in the desert plant R. raetam in response to a combination of different naturally
occurring stresses, of which drought and heat shock appear to be the
most prominent (Pnueli et al., 2002). To test whether homologs of these transcripts are also involved in the response of laboratory-grown plants to a combination of stresses, we studied their expression in
tobacco plants subjected to drought, heat shock, and a combination of
drought and heat shock.
As shown in Figure 5, the expression of
two transcripts with a high degree of homology to transcripts induced
in the desert plant, i.e. those encoding a WRKY transcription factor,
and an ethylene response transcriptional co-activator (ERTCA), was
specifically induced during a combination of drought and heat shock in
tobacco. The specific induction of these transcription factor homologs during a combination of drought and heat shock may suggest that this
combination is accompanied by the activation of a unique genetic
program different from the programs activated in plants during drought
or heat shock. The expression of another transcript, i.e. a homolog
PR-10, induced in the desert plant (Pnueli et al., 2002), was also
induced during a combination of drought and heat shock. However, this
transcript was also induced during heat shock in the absence of
drought. In contrast, a homolog of a novel transcript corresponding to
the Arabidopsis gene AC007508.2, induced in the desert plant (Pnueli et
al., 2002), was not specifically induced during a combination of
drought and heat shock in tobacco (Fig. 5).
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Figure 5.
Expression of transcripts with homology to stress
response cDNAs isolated from the desert plant R. raetam. RNA
gel blots were used to study the expression of different transcripts
that hybridized to cDNAs isolated from the desert plant R. raetam subjected to a combination of drought and heat shock in its
natural environment. Hybridizations were performed at a high stringency
(60°C) using full-length R. raetam cDNA clones as
described in "Materials and Methods."
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DISCUSSION |
We performed an initial characterization of the response of
tobacco plants to a combination of drought stress and heat shock. Our
results strongly suggest that the effect of this combination on plants
is very different from that of drought or heat shock applied
individually. Because in the field or in nature plants are often
subjected to a combination of stresses such as drought and heat shock,
studying the response of plants to a combination of different stresses
may be critical to our understanding of stress tolerance in plants.
Thus, stress combinations such as drought and cold, heat shock and high
light, or drought and heat shock should be studied before a successful
manipulation of plant metabolism can be achieved, to artificially
enhance stress tolerance. Future studies using full-scale genome arrays
conducted on Arabidopsis plants subjected to similar stress
combinations may reveal key regulators of gene clusters activated
during a combination of stresses. The identification of two transcripts
encoding homologs of proteins involved in the transcriptional
regulation of gene expression, i.e. WRKY and ERTCA, specifically
induced during a combination of drought and heat shock (Fig. 5),
supports the presence of key regulators involved in this response. The
finding that a combination of drought and heat shock results in the
activation of wound and pathogen response pathways, not activated by
each of these stresses applied individually, can also be viewed as an
evidence for the induction of a unique genetic program upon stress
combination. Our results, therefore, may provide an entry point and a
reference to future analysis of gene expression during a combination of
stresses. In addition, our results can suggest possible targets for the
enhancement of stress tolerance in crops by genetic engineering. Thus,
it may be possible to enhance the tolerance of plants to multiple
stresses by manipulating the expression of different enzymes of the
pentose phosphate pathway, AOX, GPX, and/or homologs of the
transcription factors identified by our study (i.e. WRKY and ERTCA).
A number of new findings were uncovered by our analysis. For example, a
role for mitochondrial AOX and GPX in the protection of cells from
ROI-related damage during a combination of stresses can be suggested.
In addition, the finding that the expression of DHN is suppressed
during a combination of drought and heat shock may suggest that during
this combination, HSPs can replace the stabilizing function of DHN, and
it is no longer required for drought-related cellular protection. The
source of NAD(P) H used for the removal of ROI during stress is mostly
unknown. Our results suggest that the reduction of NAD(P)+ to NAD(P) H during stress, in the absence of photosynthesis, may occur via the
pentose phosphate pathway. This suggestion is supported by a number of
studies in animal cells and yeast (Saccharomyces
cerevisiae), linking the pentose phosphate pathway to the removal
of ROI during normal metabolism and stress (Pandolfi et al., 1995;
Juhnke et al., 1996), and by our recent findings that plants with
suppressed expression of APX and CAT have enhanced expression of
transcripts encoding enzymes of the pentose phosphate pathway (Rizhsky
et al., 2002). The expression of transcripts encoding enzymes of the
pentose phosphate pathway was also elevated during other stresses such
as PQ and salt (Table II).
Drought stress and heat shock may affect plant metabolism in a
different manner when applied individually. However, it is not entirely
clear how they affect plant metabolism when occurring simultaneously.
Our analysis suggests that the mitochondria may be critical during a
combination of drought and heat shock. During this combination
photosynthesis is suppressed, whereas respiration is enhanced (Fig. 1).
In addition, the expression of photosynthetic genes is suppressed,
whereas the expression of genes involved in respiration is unchanged or
induced (Table II). Moreover, the expression of mitochondrial AOX,
implicated in the defense of plants from mitochondria-generated ROI
during stress (Maxwell et al., 1999), is specifically elevated during a
combination of drought and heat shock (Table I; Fig. 3). However, the
exact role of the mitochondria, aside from energy supply in the absence of photosynthesis, is unknown.
The response of plants to a combination of drought and heat shock is
composed of suppression of photosynthesis, enhancement of respiration,
induction of a large number of defense genes, including genes induced
during pathogen defense, and changes in genes involved in sugar
metabolism. The overall balance between the expression of transcripts
encoding different ROI removal enzymes and HSPs is also altered during
a combination of drought and heat shock. These changes strongly suggest
that the combination of drought and heat shock results in the
activation of a unique genetic program that is different from that
activated during drought or heat shock. Comparing the expression
pattern of the different transcripts shown in Tables I through III
between the combination of drought and heat shock and other stresses,
such as cold, salt, PQ, or pathogen attack, suggests that the response
of plants to the stress combination is also different from the response
of plants to these stresses.
Drought and heat shock combination resulted in the induction of at
least one senescence-associated transcript (SAG12; Table III). An
overlap in the activation of at least 28 different transcription factors was recently reported between senescence and environmental stresses such as cold, salt, and pathogen attack (Chen et al., 2002).
Therefore, it is possible that some overlap may also exist between
senescence and a combination of drought and heat shock. Interestingly,
the study of Chen et al. (2002), although very comprehensive, could not
assign a function to a specific WRKY protein, identified as the
Arabidopsis homolog of NtWRKY4, also a homolog of the R. raetam WRKY used for the hybridizations shown in Figure 5. From
our results (Pnueli et al., 2002; Fig. 5), it is possible that this
WRKY is involved in the response of plants to a combination of stresses
such as drought and heat shock, or drought and cold stress.
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MATERIALS AND METHODS |
Growth Conditions and Physiological Measurements
Growth of tobacco (Nicotiana tabacum cv Xanthi-nc
NN) plants and experiments were conducted under controlled
environmental conditions at 23°C or 44°C. Plants were individually
potted in equal amounts of Pro-Mix, and watered with 0.5× Hoagland
solution. Continuous illumination was provided by cool-white
fluorescent lamps (150 µmol m 2 s11).
Photosynthetic activity, dark respiration, leaf temperature, and
stomatal conductance were measured with a LI-COR LI-6400 apparatus using the following measuring cell (6 cm2) parameters:
23°C or 44°C, 150 µmol photons m2 s1,
and an air flow of 300 µL s1, as previously described
(Mittler et al., 2001). RWC was determined as described by Mittler and
Zilinskas (1994).
Stress Treatments
Heat shock was applied by raising the temperature in the growth
chamber to 37°C for 1 h, followed by another increase to 44°C for 6 h. Drought stress was imposed by withdrawing water from plants until they reached a RWC of 65% to 70% (typically 6-7 d). A
combination of drought and heat shock was performed by subjecting drought-stressed plants (RWC of 65%-70%) to the heat hock treatment. All plants, i.e. drought-stressed plants, well-watered plants subjected
to heat shock, drought- and heat-shocked plants, and control
well-watered plants kept at 23°C were sampled at the same time for
analysis. Cold stress was imposed by changing the temperature in the
growth chamber to 4°C for 48 h. Control plants were kept at
23°C. Mock TMV infection plants expressing the bO gene and treatment
of plants with MJ were performed as described previously (Mittler et
al., 1998). PQ treatment was performed as described by Mittler and
Zilinskas (1992). Salt stress was induced by subjecting 7-d-old tobacco
seedlings, grown in culture in a medium containing 0.5× Hoagland, to
250 mM NaCl for 3 d. Control seedlings were grown in
the same culture media without NaCl. For all stresses, control and
stressed tissue were sampled at the same time.
RNA Isolation and RNA Gel Blots
Total RNA was isolated as previously described (Mittler et al.,
1998) and subjected to RNA gel-blot analysis (Mittler and Zilinskas,
1992). A probe for 18S rRNA was used to ensure equal loading of RNA.
Hybridization conditions were as follows: 0.25 M
Na2HPO4, 1 mM EDTA, 7%
(w/v) SDS, and 1% (w/v) casein (pH 7.4) at 60°C to
65°C, overnight, and washes were at 1× SSC and 0.1× SSC in the
presence of 0.1% (w/v) SDS.
Filter Array Hybridization
Clones for the production of filter arrays were ordered from the
tomato (Lycopersicon esculentum) expressed
sequence tag library at Clemson University (SC), or obtained
from the laboratories of Drs. Dirk Inzé (University of Gent,
Belgium), Barbara A. Zilinskas (Rutgers University, NJ), Pierre
Goloubinoff (Hebrew University, Jerusalem, Israel), and Gadi Schuster
(Technion, Haifa, Israel). Filter cDNA arrays were prepared from
the clones by spotting PCR products in duplicates on nylon membranes at
the Hadassah Medical School DNA Facility of the Hebrew University.
Filters were hybridized with radiolabeled cDNAs prepared from total RNA
isolated from the different plants using oligo-dT and Superscript
reverse transcriptase (Life Technologies/Gibco-BRL, Cleveland)
as suggested by the manufacturer. Hybridization conditions were as
follows: 57°C, 5× SSC, 5× Denhart, 0.5% (w/v) SDS,
and 100 µg mL1 salmon sperm DNA, overnight. Washing
conditions were as follows: 57°C, 2× SSC, and 0.1% (w/v) SDS
for 20 min, followed by 0.2× SSC and 0.1% (w/v) SDS, 57°C, for 20 min. After hybridization and washes, the signals were assayed with a
phosphor imager (BAS1000, Fuji Photo Film, Tokyo) and analyzed
with TINA software (Raytest, Pittsburgh). A number of control
"housekeeping" genes, animal-specific genes (as negative controls),
and empty spots (for background) were also spotted on the membrane.
These were used to normalize the intensity of signals between the
different filters and calculate the changes in gene expression
presented in Tables I through III. When pertinent, the expression level
of specific genes was verified by RNA blots.
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ACKNOWLEDGMENTS |
We thank Drs. Dirk Inzé, Barbara A. Zilinskas, Pierre
Goloubinoff, and Gadi Schuster for gift of cDNA clones. We also thank Dr. Daniel Goldenberg for his help with preparing filter arrays.
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FOOTNOTES |
Received April 7, 2002; returned for revision May 17, 2002; accepted July 5, 2002.
1
This work was supported by the Israeli Academy
of Science, by the Hebrew University Minerva Arid Ecosystem Research
Center, by The Biotechnology Council (Iowa State University), and by
the fund for the promotion of research at Technion.
*
Corresponding author; e-mail rmittler{at}iastate.edu; fax
515- 294-1337.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.006858.
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