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ENVIRONMENTAL STRESS AND ADAPTATION

When Defense Pathways Collide. The Response of Arabidopsis to a Combination of Drought and Heat Stress^1,[w]

Department of Biology, Technion, Israel Institute of Technology, Technion City, Haifa 32000, Israel (L.R.); Department of Botany, Plant Sciences Institute, Iowa State University, Ames, Iowa 50011 (H.L.); Virginia Bioinformatics Institute, Blacksburg, Virginia 24061 (J.S., V.S.); Department of Biochemistry, University of Nevada, Reno, Nevada 89557 (S.D., R.M.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Within their natural habitat, plants are subjected to a combination of abiotic conditions that include stresses such as drought and heat. Drought and heat stress have been extensively studied; however, little is known about how their combination impacts plants. The response of Arabidopsis plants to a combination of drought and heat stress was found to be distinct from that of plants subjected to drought or heat stress. Transcriptome analysis of Arabidopsis plants subjected to a combination of drought and heat stress revealed a new pattern of defense response in plants that includes a partial combination of two multigene defense pathways (i.e. drought and heat stress), as well as 454 transcripts that are specifically expressed in plants during a combination of drought and heat stress. Metabolic profiling of plants subjected to drought, heat stress, or a combination of drought and heat stress revealed that plants subject to a combination of drought and heat stress accumulated sucrose and other sugars such as maltose and gulose. In contrast, Pro that accumulated in plants subjected to drought did not accumulate in plants during a combination of drought and heat stress. Heat stress was found to ameliorate the toxicity of Pro to cells, suggesting that during a combination of drought and heat stress sucrose replaces Pro in plants as the major osmoprotectant. Our results highlight the plasticity of the plant genome and demonstrate its ability to respond to complex environmental conditions that occur in the field.

Drought and heat stress represent an excellent example of two different abiotic stresses that occur in the field simultaneously, especially in semi-arid or drought-stricken areas (Mittler et al., 2001; Moffat, 2002; Rizhsky et al., 2002). Although drought and heat stress have been extensively studied (Vierling, 1991; Ingram and Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki, 1996; Miernyk, 1999; Queitsch et al., 2000), relatively little is known about how their combination impacts plants. A number of studies examined the effect of a combination of drought and heat stress on the growth and productivity of maize, barley, sorghum, and different grasses. It was found that a combination of drought and heat stress had a significantly higher detrimental effect on the growth and productivity of these plants and crops compared to each of the different stresses applied individually (Savage and Jacobson, 1935; Craufurd and Peacock, 1993; Savin and Nicolas, 1996). In maize, resistance to a combination of drought and heat stress is a well-known breeding target (Heyne and Brunson, 1940). Furthermore, a combination of drought and heat stress was found to alter the physiological status of grasses and other plants, to inhibit photosynthesis, and to result in the accumulation of end products of lipid peroxidation (Perdomo et al., 1996; Jagtap et al., 1998; Jiang and Huang, 2001).

Initial studies in tobacco suggested that the molecular response of plants to a combination of drought and heat stress is distinct from that of plants subjected to each of these stresses applied individually. Thus, the steady-state level of a number of transcripts, elevated during drought or heat stress, was reduced during a combination of drought and heat stress, and a small number of transcripts were specifically expressed during a combination of drought and heat stress (Rizhsky et al., 2002). Despite these findings, the number of transcripts tested in tobacco was relatively small (170 transcripts), and the scale of the plant's response to this stress combination remained largely unknown.

In this study we performed an initial analysis of the molecular and metabolic response of Arabidopsis to a combination of drought and heat stress. Our study revealed a new pattern of defense response in plants that includes a partial combination of two multigene defense pathways (drought and heat stress), as well as 454 transcripts that are specifically expressed in cells during a combination of drought and heat stress. In addition, plants subjected to a combination of drought and heat stress accumulated high levels of sucrose and other sugars, but did not accumulate Pro.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Physiological and Molecular Characterization of Arabidopsis Plants Subjected to a Combination of Drought and Heat Stress

A combination of drought and heat stress was imposed on plants according to Rizhsky et al. (2002; Fig. 1A ). As shown in Figure 1, the physiological and molecular response of Arabidopsis to a combination of drought and heat stress was very similar to that of tobacco (Rizhsky et al., 2002). Figure 1, B and C, shows that heat stress was accompanied by enhanced respiration and opening of stomata, whereas drought was accompanied by suppression of photosynthesis and closure of stomata (all measurements were performed with a Li-Cor 6400 [Li-Cor, Lincoln, NE] and refer therefore to rates of CO₂ exchange between the leaf and the LI-6400-leaf chamber). In contrast, a combination of drought and heat stress resulted in the simultaneous enhancement of respiration and suppression of photosynthesis. Compared to tobacco, we could not, however, find a significant difference between the leaf temperature of plants subjected to heat stress or a combination of drought and heat stress (data not shown; Rizhsky et al., 2002). It should, however, be noted that although the same instrument was used to measure leaf temperature in both cases (i.e. a LI-6400), the variability in the measurements obtained with the smaller leaf of Arabidopsis was higher than that obtained with the larger tobacco leaf. In addition, the stomatal conductance of Arabidopsis leaves subjected to a combination of drought and heat stress (Fig. 1C) was not as low as that of tobacco (Rizhsky et al., 2002), suggesting that Arabidopsis plants might have been able to cool their leaves, at least partially, under the conditions used in our assay.

View larger version (35K): [in this window] [in a new window]   Figure 1. Physiological and molecular characterization of Arabidopsis plants subjected to a combination of drought and heat stress. Plants were subjected to heat stress, drought, or a combination of heat stress and drought, as described in "Materials and Methods." Results are presented as mean and standard deviation of three individual measurements. A, The experimental design for applying a combination of drought and heat stress to Arabidopsis. This design attempts to mimic the conditions that occur in the field in which a relatively prolonged period of drought (yellow) is accompanied by a brief period of heat stress (red; usually during midday to early afternoon). B, Photosynthetic activity and dark respiration, measured with a Li-Cor LI-6400 apparatus. C, Stomatal conductance, measured with a Li-Cor LI-6400 apparatus. D, Steady-state level of stress-response transcripts, measured by RNA gel blots. Ribosomal RNA (18S) was used to control for equal loading of RNA.

Transcriptome Profiling of Arabidopsis Plants Subjected to a Combination of Drought and Heat Stress

To examine changes in steady-state transcript level in leaves of Arabidopsis plants subjected to drought, heat stress, or their combination, we performed a transcriptome analysis of leaves using DNA arrays (ATH1 chips; Affymetrix, Santa Clara, CA). As shown in Figure 2 , there was very little similarity between the response of Arabidopsis to drought or heat stress. Out of 1,075 transcripts elevated during drought and 262 transcripts elevated during heat stress (cutoff of 1.5-fold log₂), an overlap of only 29 transcripts was found. Similarly, an overlap of only 48 transcripts was observed between 496 and 279 transcripts decreased during drought or heat stress, respectively (cutoff of 1.5-fold log₂). Compared to nonstressed plants, the steady-state level of 1,057 transcripts was elevated and the steady-state level of 776 transcripts was decreased during a combination of drought and heat stress. Out of the transcripts elevated during a combination of drought and heat stress, 479 were also elevated during drought and 153 were also elevated during heat stress (with a 29-transcript overlap). In addition to transcripts elevated in plants during drought or heat stress, the transcriptome of plants subjected to a combination of drought and heat stress contained 454 transcripts that were specifically elevated by this stress combination (cutoff of 1.5-fold log₂). A similar situation was observed with transcripts decreased in plants during a combination of drought and heat stress, with 318 transcripts specifically decreased during this stress combination (Fig. 2). The transcriptome of plants subjected to a combination of drought and heat stress was therefore different from that of plants subjected to heat or drought stress.

View larger version (26K): [in this window] [in a new window]   Figure 2. Venn diagrams showing the number of transcripts enhanced (left) or decreased (right) in plants in response to drought, heat stress, or a combination of drought and heat stress (compared to control nonstressed plants). Only transcripts with an increase or decrease in steady-state level of 1.5-fold (log2) over control unstressed plants are included. Results are presented as average of three independent experiments. RNA isolation and Affymetrix chip analysis were performed as described in "Materials and Methods."

View larger version (24K): [in this window] [in a new window]   Figure 3. Steady-state transcript level of Arabidopsis transcripts encoding HSPs and HSFs in leaves of plants subjected to drought, heat stress, or a combination of drought and heat stress. Results are presented as average of three independent experiments. A, A Venn diagram showing the number of HSPs elevated during drought, heat stress, or a combination of drought and heat stress (cutoff 1.5-fold log2). B, Expression level of all Arabidopsis HSFs during drought, heat stress, and a combination of drought and heat stress. RNA isolation and Affymetrix chip analysis were performed as described in "Materials and Methods." Cont, Control; D, drought; H, heat stress; D+H, a combination of drought and heat stress.

Transcripts specific for a combination of drought and heat stress (cutoff 2 log₂) belonged to a number of different groups including HSPs, proteases, starch degrading enzymes, and lipid biosynthesis enzymes (Table I; supplemental material). The steady-state level of different transcripts encoding signal transduction proteins was also elevated during a combination of drought and heat stress. These included receptor-like kinases, small GTP-binding proteins, MYB transcription factors, and protein kinases. In addition, the expression of at least five different transcripts encoding membrane channels was elevated in plants subjected to a combination of drought and heat stress (CLC-b chloride channel, aquaporin membrane intrinsic protein (MIP), potassium transporter, Na⁺/Ca²⁺-antiporter, and an ABC-type transporter). Defense transcripts specifically elevated in cells during a combination of drought and heat stress included thioredoxin and 2-peroxiredoxin, important for the prevention of oxidative stress, P450s, and a salt-inducible protein (Table I; supplemental material).

Transcripts elevated by all three treatments are also shown in Table IA (cutoff 2 log₂). They include several HSPs, trehalose-6-phosphate phosphatase, an abscisic acid (ABA)-induced protein phosphatase 2C, a two-component phosphorelay protein, and an ethylene-response transcription coactivator.

Metabolite Profiling of Arabidopsis Plants Subjected to a Combination of Drought and Heat Stress

To examine the accumulation of stress-associated metabolites in leaves of Arabidopsis plants subjected to drought, heat stress, or their combination, we performed a gas chromatography-mass spectrometric (GC-MS) analysis of polar compounds extracted from leaves of plants subjected to the different stresses. For this analysis we used the same batch of leaf tissues used for the physiological and molecular analysis of plants presented in Figures 1 to 3, and Table I. As shown in Figure 4 , the GC profile of plants subjected to a combination of drought and heat stress was more similar to that of plants subjected to drought than to that of control plants or plants subjected to heat stress. Compound identification is shown in Table II. As shown in Table II, plants subjected to a combination of drought and heat stress accumulated high levels of sucrose and other sugars such as maltose, melibiose, gulose, and mannitol. In contrast, Pro that accumulated to a very high level in plants subjected to drought did not accumulate in plants subjected to a combination of drought and heat stress. The level of Gln was specifically elevated in plants subjected to a combination of drought and heat stress (Table II), suggesting that Pro biosynthesis is inhibited and Glu is converted to Gln instead of Pro during the stress combination. Cys, a potential precursor of the antioxidant glutathione, was also elevated in leaves subjected to a combination of drought and heat stress. This accumulation corresponds with the enhancement of different transcripts involved in glutathione biosynthesis (Table I; supplemental material).

View larger version (15K): [in this window] [in a new window]   Figure 4. GC profiles of polar extracts obtained from control plants and plants subjected to heat stress, drought, or a combination of drought and heat stress. C, Control; D, drought; H, heat stress; D+H, a combination of drought and heat stress. Polar compound extraction and GC separation are described in "Materials and Methods."

Amelioration of Pro Toxicity to Cells during Heat Stress

During different abiotic conditions such as cold, salt, and drought, Pro accumulates in cells and functions as an osmoprotectant (Apse and Blumwald, 2002; Zhu, 2002). Moreover, genetically engineering plants to overaccumulate Pro enhances their tolerance to some of these stresses (Kavi Kishor et al., 1995; Nanjo et al., 1999; Nuccio et al., 1999; Rontein et al., 2002). However, Pro can also be toxic to cells if it is not properly removed (Hellmann et al., 2000; Deuschle et al., 2001; Mani et al., 2002; Nanjo et al., 2003). The absence of Pro from cells subjected to a combination of drought and heat stress (Table II) suggested that under these conditions Pro might be toxic to cells. To test whether Pro is toxic to plant cells during heat stress we grew Arabidopsis seedlings on Murashige and Skoog plates that contained different concentrations of Pro and subjected seedlings to a heat stress treatment. Conflicting reports can be found regarding the growth of Arabidopsis seedlings on plates containing Pro. Hellmann et al. (2000) and Mani et al. (2002) reported that Pro is toxic to wild-type seedlings grown on plates. In contrast, Nanjo et al. (2003) reported that the growth of wild-type seedlings is not inhibited by concentrations of up to 25 mM Pro. As shown in Figure 5 , we found that the growth of Arabidopsis seedlings is inhibited in the presence of Pro. Moreover, we found that a heat stress treatment (similar to the treatment used in our analysis of plants subjected to heat stress or a combination of drought and heat stress; Figs. 1–4) ameliorated the toxic effect of Pro.

View larger version (32K): [in this window] [in a new window]   Figure 5. Amelioration of Pro toxicity during heat stress. A, Measurements of root length taken 48 h post a heat stress treatment of seedlings in the presence or absence of Pro. B, Photographs of Arabidopsis seedlings taken 96 h post a heat stress treatment of seedlings in the presence or absence of Pro; the bar in the lower right represents 4 mm. All measurements were performed as described in "Materials and Methods."

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

There was a considerable degree of overlap between transcripts expressed in plants during drought or heat stress and a combination of drought and heat stress (Fig. 2). This overlap suggests that large segments of the defense program of plants against drought or heat stress are coactivated in the same cells during a combination of drought and heat stress. This possibility should be examined in future studies by a comprehensive proteomic approach since it raises a number of interesting questions regarding the co-function of defense proteins such as molecular chaperones and LEA-like proteins in the same cells (see below).

The steady-state level of many different transcripts was specifically elevated during a combination of drought and heat stress (Table I; supplemental material). This group of transcripts included transcripts with an unknown function (over 40%; supplemental material) and a large number of transcripts involved in different defense pathways. Based on changes in steady-state transcript abundance and metabolite levels we could identify the pathways for starch degradation and sucrose biosynthesis as specifically elevated in plants during a combination of drought and heat stress, with some portions of these pathways also expressed during drought (Table I; supplemental material). However, the expression of many other transcripts belonging to different metabolic and defense pathways is also specifically elevated in cells during the stress combination (Table I). It should, however, be noted that our analysis is based upon a single time point (at the end of the heat stress treatment; Fig. 1A), and that a detailed time-course analysis should reveal additional transcripts expressed in cells during drought, heat stress, or their combination.

A considerable overlap was found between transcripts involved in the defense of plants against abiotic conditions such as cold, drought, and salinity (Kreps et al., 2002; Oztur et al., 2002; Seki et al., 2002). In contrast, our findings suggest a relatively small overlap between transcripts induced during drought or heat stress (Fig. 2). Nonetheless, during a combination of drought and heat stress, drought- and heat-stress-specific transcripts are expressed in the same tissues (Table I; supplemental material). Although we can only assume that many of these transcripts are translated, it is interesting that some of these transcripts can be found in the same cells because their function might in some cases be conflicting. For example, some LEA-like proteins and dehydrins can have a helix/random coil structure (Soulages et al., 2002). This structure may interfere with the proper function of some HSPs and molecular chaperones because it may compete with unfolded proteins and enzymes that are the natural substrate of HSPs during stress.

In response to a decrease in leaf water content plants accumulate a variety of compounds that function as osmoprotectants (Bohnert, 2000; Hoekstra et al., 2001). It was suggested that a moderate level of water stress is accompanied by the accumulation of compounds such as Pro and Gly-betaine, whereas a severe level of water stress is accompanied by the accumulation of sugars such as sucrose (Hoekstra et al., 2001). Although the relative water content of plants subjected to drought and plants subjected to a combination of drought and heat stress was not significantly different (Fig. 1B), plants subjected to drought accumulated Pro whereas plants subjected to a combination of drought and heat stress accumulated sucrose (Table II). This difference may suggest that a combination of drought and heat stress imposes on plants a different type of internal stress (compared to drought or heat stress), that requires sucrose rather than Pro as an osmoprotectant. Alternatively, Pro may be toxic to cells during a combination of drought and heat stress. Thus, sucrose may be required to replace Pro as the major osmoprotectant of cells during the stress combination.

At least three different studies suggested that ¹-pyrroline-5-carboxylate (P5C), and perhaps other intermediates in Pro biosynthesis and degradation can be toxic to cells (Hellmann et al., 2000; Deuschle et al., 2001; Mani et al., 2002). It is possible that heat stress alters the balance between Pro biosynthesis and degradation and causes the accumulation of P5C and/or other intermediates. Moreover, an enhanced activity of mitochondria (i.e. enhanced respiration) was found in cells during heat stress and a combination of drought and heat stress (Fig. 1B; Rizhsky et al., 2002). Because Pro biosynthesis and degradation occurs in the mitochondria, the accumulation of toxic compounds such as P5C in this organelle might be more damaging to cells under these conditions (especially in plants subjected to a combination of drought and heat stress that appear to completely depend upon the mitochondria for their energetic metabolism; Fig. 1B; Rizhsky et al., 2002). Our results (Table II; Fig. 5), as well as the results of others (e.g. Deuschle et al., 2001), suggest that plants that were engineered to overaccumulate Pro in order to enhance their tolerance to abiotic stress (Kavi Kishor et al., 1995; Nanjo et al., 1999; Nuccio et al., 1999; Rontein et al., 2002) might not be resistant to field conditions that occur in some areas and include a combination of drought and heat stress, or simply heat stress.

The response of plants to a combination of drought and heat stress highlights the plasticity of the plant genome and its ability to modulate its response to complex environmental conditions that occur in the field. Key to this plasticity is a large network of transcription factors that regulate the response of plants to different stresses (Arabidopsis Genome Initiative, 2000; Chen et al., 2002). Here we show that the complex response of HSPs during heat stress, drought, and their combination (Fig. 3A; Table I; Rizhsky et al., 2002) is reflected in the pattern of expression of HSFs (Fig. 3B). Compared to humans and animals that express, at the most, four different transcripts encoding HSFs, Arabidopsis contains 21 different HSF-encoding genes that belong to at least three different families (Nover et al., 2001). Our results (Fig. 3; Pnueli et al., 2003) suggest that plant HSFs function as a network of transcription factors that controls the expression of HSPs during different stresses. Thus, we identified oxidative- and light-stress-specific HSFs (Pnueli et al., 2003) as well as heat-stress- and drought-specific HSFs (Fig. 3). Future analysis of this gene family, including measurements of HSF activity in cells, might provide an initial insight into how plants compensated during evolution for their sessile nature by developing complex and specialized gene families to control their response to environmental conditions. These were most likely created by gene duplication, however, acquired specific roles related to specific pathways or stresses, as well as their combination (Arabidopsis Genome Initiative, 2000). Similar results were also obtained with several members of the MYB transcription factor gene family (Fig. 1; supplemental material), and MYB-At1g26580 was identified as specifically elevated during a combination of drought and heat stress (Table I).

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material and Growth Conditions

Arabidopsis plants (cv Columbia) were grown under controlled conditions: 21°C to 22°C, 100 µmol m^–2 s^–1, and a relative humidity of 70%. All treatments were performed in parallel. Heat stress was applied by raising the temperature in the growth chamber to 38°C for 6 h. Drought was imposed by withdrawing water from plants until they reached a relative water content (RWC) of 70% to 75% (typically 6–7 d). A combination of drought and heat stress was performed by subjecting drought-stressed plants (RWC of 70%–75%) to a heat stress treatment (38°C for 6 h). All plants, i.e. drought-stressed plants, well-watered plants subjected to heat stress, drought- and heat-stressed plants, and control well-watered plants kept at 21°C to 22°C were sampled at the same time for analysis (Rizhsky et al., 2002; Fig. 1A). All experiments were performed in triplicates and repeated at least three times. All tissues collected were divided and used in parallel for molecular and metabolic analysis of plants.

Molecular and Physiological Analysis

RNA and protein were isolated and analyzed by RNA and protein blots as previously described (Pnueli et al., 2003; Rizhsky et al., 2003). A ribosomal 18S rRNA probe was used to control for RNA loading. Photosynthesis, stomatal conductance, and dark respiration were measured with a Li-Cor LI-6400 apparatus as described by Pnueli et al. (2003) using the Arabidopsis leaf chamber (Li-Cor).

DNA Chip Analysis

In three independent experiments RNA was isolated from control plants and plants subjected to heat stress, drought, and a combination of heat and drought stress (a pool of 80 to 100 plants per treatment in triplicates), as described above. This RNA was used to perform the chip analysis (Arabidopsis ATH1 chips; Affymetrix) at the University of Iowa DNA facility (http://dna-9.int-med.uiowa.edu/microarrays.htm). Conditions for RNA isolation, labeling, hybridization, and data analysis are described in Pnueli et al. (2003) and Rizhsky et al. (2003). Comparative analysis of samples was performed with the GeneChip mining tool version 5.0 and the Silicon Genetics GeneSpring version 5.1. Some of the comparison results were confirmed by RNA blots.

GC-MS Analysis

Extraction and derivatization were performed according to Roessner et al. (2000) and Fiehn et al. (2000). Leaves were harvested, cut into 1- to 2-mm-long pieces, and stored at –80°C. For each sample, a total of 250 mg of frozen leaves was transferred into a 13 x 100 borosilicate culture tube, immediately frozen in liquid nitrogen, and ground to a fine powder with a glass rod. Aliquots of 1.4 mL methanol and 50 µL of ribitol/adonitol (2 mg/mL water stock) were added. Tubes were vortexed, and pH was verified within 5 to 6. The solution was sonicated for 10 min at 42 kHz with a Branson 3510 ultrasonic cleaner (Branson Ultrasonic, Danbury, CT). Extraction was done in a water bath at 70°C for 15 min. Tubes were centrifuged for 20 min at 4,500 rpm and the supernatant was decanted to new culture tubes, and 1.4 mL of water and 0.75 mL of chloroform were added. The mixture was vortexed thoroughly and centrifuged for 5 min at 4,500 rpm. The polar phase (methanol/water) was decanted to 1.5-mL HPLC vials and dried in a Centrivap benchtop centrifugal concentrator (Labconco, Kansas City, MO) overnight. The dried polar phase was methoximated for 90 min at 30°C (80 µL of 20 mg/mL methoxyamine hydrochloride in pyridine), a 40-µL aliquot of a retention time standard mixture was added (Roessner et al., 2000), and the mixture was trimethylsilylated for 30 min at 37°C. Solutions were transferred to glass inserts within the 1.5-mL HPLC vials prior to injection.

Sample volumes of 1 µL were injected at a split ratio of 25:1 into a Trace DSQ GC/MS system (Thermo Finnigan, Austin, TX) equipped with Combi-Pal autosampler (Leap Technologies, Carrboro, NC). Tuning was done using tris(perfluorobutyl)amine (CF43) as a reference gas. Chromatography was performed using a 30-m x 250-µm Alltech AT-5MS column (Alltech Associates, Deerfield, IL). Injection temperature was 230°C, the interface was kept at 250°C, and the ion source was kept at 200°C. Oven temperature program was 5 min at 70°C, followed by a 5°C min^–1 ramp to 310°C, 1 min at 310°C, and a final 6 min at 70°C before the next injection. Carrier gas was helium at a constant flow of 1 mL min^–1. Mass spectra were recorded at two scans per second over a range of 50 to 600 m/z. Compounds were identified based on retention time and comparison with reference spectra in mass spectral libraries. Quantitation of compounds was done using a processing method in Xcalibur version 1.3 (Xcalibur, Herndon, VA) where peak area was integrated with the Genesis algorithm. Statistical analysis of peak area was done using the SAS system version 8.2 (SAS Institute, Cary, NC).

Analysis of Pro Toxicity during Heat Stress

Arabidopsis seedlings (15–20 per plate) were germinated under sterile conditions on Murashige and Skoog plates (0.5x), containing different concentrations of Pro (0–15 mM). Plates were placed vertically, and seedlings were allowed to grow at 21°C to 22°C, 60 µmol m^–2 s^–1. Three-day-old seedlings were subjected to a heat stress treatment as described above and allowed to recover at 21°C to 22°C. Forty-eight hours following the heat stress treatment the root length of seedlings (Rizhsky et al., 2003) was measured and compared between heat-stress-treated and heat-stress-untreated seedlings grown in the presence or absence of Pro. In each experiment six different plates were used for each concentration (three heat stressed and three non-heat stressed).

	ACKNOWLEDGMENTS

 
We thank Drs. Eve Syrkin-Wurtele, Carol Foster, and Hailong Zhang for their help with Affymetrix data analysis.

Received September 16, 2003; returned for revision December 20, 2003; accepted January 2, 2004.

	FOOTNOTES

 
¹ This work was supported by funding from the Plant Sciences Institute at Iowa State University, the Biotechnology Council of Iowa State University, the College of Liberal Arts and Sciences at Iowa State University, the Israeli Academy of Science, the Nevada Agricultural Experimental Station (publication no. 03031333), and the Fund for the Promotion of Research at the Technion.

^[w] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.033431.

^* Corresponding author; e-mail ronm{at}unr.edu; fax 775–784–1419.

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