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First published online November 30, 2007; 10.1104/pp.107.112060 Plant Physiology 146:748-761 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Core Genome Responses Involved in Acclimation to High Temperature1,[C],[W],[OA]Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721
Plants can acclimate rapidly to environmental conditions, including high temperatures. To identify molecular events important for acquired thermotolerance, we compared viability and transcript profiles of Arabidopsis thaliana treated to severe heat stress (45°C) without acclimation or following two different acclimation treatments. Notably, a gradual increase to 45°C (22°C to 45°C over 6 h) led to higher survival and to more and higher-fold transcript changes than a step-wise acclimation (90 min at 38°C plus 120 min at 22°C before 45°C). There were significant differences in the total spectrum of transcript changes in the two treatments, but core components of heat acclimation were apparent in the overlap between treatments, emphasizing the importance of performing transcriptome analysis in the context of physiological response. In addition to documenting increases in transcripts of specific genes involved in processes predicted to be required for thermotolerance (i.e. protection of proteins and of translation, limiting oxidative stress), we also found decreases in transcripts (i.e. for programmed cell death, basic metabolism, and biotic stress responses), which are likely equally important for acclimation. Similar protective effects may also be achieved differently, such as prevention of proline accumulation, which is toxic at elevated temperatures and which was reduced by both acclimation treatments but was associated with transcript changes predicted to either reduce proline synthesis or increase degradation in the two acclimation treatments. Finally, phenotypic analysis of T-DNA insertion mutants of genes identified in this analysis defined eight new genes involved in heat acclimation, including cytosolic ascorbate peroxidase and the transcription factors HsfA7a (heat shock transcription factor A7a) and NF-X1.
Abiotic factors, such as temperature and water availability, have a major impact on successful plant establishment, growth, and reproduction. Consequently, plants have evolved mechanisms to monitor their environment and to respond with cellular, physiological, and developmental changes to optimize growth and reproductive success. These environmental response mechanisms include the ability of plants to acclimate rapidly, within hours or days, to extremes in the abiotic environment that would be damaging or lethal without such acclimation (Levitt, 1972  ; Jenks and Hasegawa, 2005).
The ability of plants to acclimate to normally lethal high temperatures (to acquire thermotolerance) has been described for over 40 years (Alexandrov et al., 1961
It is well established that an essential component of acquired thermotolerance in plants, as well as other organisms, is the induction and synthesis of molecular chaperones or heat shock proteins (HSPs; Vierling, 1991
The response of the Arabidopsis (Arabidopsis thaliana) transcriptome to heat stress has been examined by several groups (Rizhsky et al., 2004
We have been interested in defining genes whose expression is causally related to the ability of plants to acclimate to high temperature, i.e. to acquire thermotolerance. In previous work, we used both forward and reverse genetics to identify genes/pathways essential for this process (Larkindale and Knight, 2002
Different Heat Acclimation Treatments Induce Different Degrees of Thermotolerance
To identify changes in gene expression that could be causally related to the acquisition of thermotolerance, we first characterized the effectiveness of two different acclimation treatments in inducing tolerance to an otherwise lethal 45°C stress (Fig. 1
). We reasoned that changes in gene expression common to more than one acclimation treatment are more likely to be required for thermotolerance than changes seen in a single type of acclimation treatment. In one acclimation treatment, typical of treatments we and others have used in the laboratory and designated S for step acclimation, plants were acclimated by heating at a constant, nonlethal temperature (38°C), followed by a 22°C period prior to the 45°C stress (Hong and Vierling, 2000
Thermotolerance Treatments Affect Many Gene Transcripts To identify genes that could be involved in the acquisition of thermotolerance, whole-genome microarrays (Affymetrix At-H1) were used to analyze transcript levels. Plant samples were taken over the time course of the G, S, and D heat treatments as shown in Figure 1 in two separate experiments. The 45°C heat stress was limited to 90 min, conditions that result in similar viability of G- and S-acclimated plants (Fig. 2). Recovery samples were taken when plants were still green and not visibly damaged (G, S, and D 45R). Array data were averaged for duplicate samples and filtered as described in "Materials and Methods." Of the 22,746 genes on the arrays, 4,724 genes passed the filtering criteria and were analyzed further. As compared to unheated samples, large numbers of transcripts increased or decreased during heating, but the G, S, and D treatments affected transcript levels differently. Figure 3A shows the total number of transcripts that increased in abundance either 2- to 5-fold or >5-fold and also the proportions of transcripts that showed different absolute levels of accumulation. At 45°C, many fewer transcripts change in abundance in nonacclimated plants (D45) than in acclimated plants (S45 or G45), consistent with acclimation-protecting processes essential to transcription and RNA stability. Notably, plants subjected to G acclimation showed the largest number of altered transcripts (approximately 1,600 up, approximately 2,500 down), as well as more transcripts with greater fold-changes and higher absolute expression levels than in the S or D treatments. The higher transcript levels in G compared to S samples may contribute to the increased heat tolerance of G-acclimated plants.
The category of transcripts that increased 5-fold or more and accumulated to >5,000 arbitrary units (AU) are likely to represent major effector proteins involved in repair of and recovery from heat damage. There are 185 of these transcripts that are up-regulated by 5-fold or greater in at least one sample (as listed in Supplemental Table S1), approximately one-third of which are HSPs/molecular chaperones. In addition to HSPs and other chaperones, there are five petidyl prolyl isomerases and nine transcripts associated with energy metabolism, in particular, enzymes involved in glycolysis (At4g26270, At2g36460, At5g17310, At5g56630, and At1g79550). There were also 10 non-HSP stress proteins, including two universal stress proteins and several cold- and drought-regulated proteins. Transcripts noted by others as heat regulated are, not surprisingly, part of this list, including HSPs, DREB2A (At5g05410), galactinol synthase (At2g47180), and APX2 (At3g09640).
Significantly more transcripts decreased in abundance under all of the heat treatments than increased in abundance, but again, the greatest change occurred in the most heat tolerant, G-acclimated plants (Fig. 3B). These data suggest RNA turnover and suppression of transcription are also critical for heat tolerance. In contrast to transcripts that increased, 70% to 90% of the decreased transcripts were of low abundance (<500 AU), and there were few transcripts in the >5,000 AU abundance classes. Interestingly, the >5,000 AU expression class included seven pathogen defense-related transcripts (At4g11600, At3g50950, At4g21870, At4g36010, At4g36040, At5g06320, and At5g20630) and four pEARLI-like proteins, which are up-regulated by aluminum and cold stress (Bubier and Schläppi, 2004
We sought to identify genes common to G and S acclimation that were unaffected in plants heated directly to 45°C (D samples), as these may be critical to thermotolerance. We first used statistical clustering of the complete filtered array data sets to compare overall patterns of transcript abundance. Figure 4A shows the result of Euclidean clustering using average linkage of the different array samples (BRB ArrayTools). For reference, biological replicates had similarities of less than 30 AU. Unexpectedly, the G- and S-acclimation treatments showed significant differences. The overall pattern of transcript changes during the G-acclimation treatment is similar for all three G samples (GAcc, G45, and G45R) but distinct from the S and D samples (Euclidian distance of 95). Thus, although both G- and S-acclimated plants are thermotolerant, more and different changes occurred during G acclimation than in S acclimation. The nonacclimated D samples are also more similar to S-acclimated samples than to the unheated samples, emphasizing that large numbers of transcripts are observed to increase even when plants are treated at temperatures that ultimately lead to plant death.
Changes in Specific Sets of Transcripts Are Unique to Thermotolerant Plants To define a subset of transcripts unique to thermotolerant plants, we first compared sets of transcripts that accumulated at least 2-fold more in the three thermotolerant samples, S Acc1, S Acc2, and G Acc, relative to the unheated sample (Fig. 4B). We suggest that transcripts common to the thermotolerant samples are likely critical to the acclimation process. These include 377 transcripts common to all three samples, 240 transcripts increased in both S Acc1 and G Acc, and 220 common to S Acc2 and G Acc. We next compared transcript sets increased during 45°C stress in acclimated (G45 and S45) and nonacclimated plants (D45; Fig. 4C). There were 229 transcripts increased in all heated samples, which not surprisingly included 33 HSPs/molecular chaperones and six heat shock transcription factors (HSFs). We also found 242 transcripts that increased during recovery in thermotolerant plants (S45R, G45R) but not in nonacclimated plants (Fig. 4D).
Using the comparisons above, we defined transcripts that increased uniquely in thermotolerant plants as those genes found in the intersection of the subsets highlighted in bold and underlined in Figure 4, B to D. There were 57 up-regulated genes that fit these criteria (Supplemental Table S2). Using this stringent definition, there were only two transcription factors in this thermotolerance-specific subset: HsfA3 (At5g03720) and the DREB2B transcription factor, which has been linked to drought stress (At3g11020; Nakashima et al., 2000 When the same analysis was performed to identify down-regulated genes unique to thermotolerant samples (Supplemental Fig. S2, A–C), 69 genes were identified (Supplemental Fig. S3; Supplemental Table S3). The down-regulated genes define diverse functions that are notably distinct from those of up-regulated genes (Supplemental Fig. S2, D and E). Genes found are related to pathogenesis or disease, five cytochrome P450 genes, and an unusually large number of protein kinases (nine). There are no genes in the protein fate or energy metabolism categories, but there are five in the cell defense and virulence category (At5g36910, At4g16860, At4g16880, At3g44970, and At5g55990).
While genes expressed uniquely in thermotolerant plants may provide critical functions for optimal survival of heat stress, genes whose transcripts increase in nonthermotolerant plants, as well as differences in absolute transcript level, must also be considered. It is already established that chaperones/HSPs contribute to heat tolerance, and as noted above, transcripts of many chaperones/HSPs increase during heating even in nonthermotolerant plants that subsequently die. Therefore, to define additional groups of genes that might be essential for thermotolerance, we used cluster analysis to identify genes showing similar patterns of regulation. Cluster analysis was performed using Euclidean distance and average linkage of transcript levels of all genes under all of the conditions.
We identified 73 clusters, containing from one to 2,124 genes (for complete list, see Supplemental Table S4). Each cluster was analyzed with respect to known promoter elements, responses in other available array data sets, and expression patterns relative to the other clusters (Fig. 5
; Supplemental Table S5). All clusters with 10 or more genes showed a statistically significant enrichment of specific sequence motifs within 1,000 bp 5' of the known or predicted transcription start site when compared to the whole genome. We focused on clusters that contained putative promoter binding sites of known transcription factors and that in many cases contained genes reported to be coregulated under other stress conditions (Vogel et al., 2005
It is not surprising that six clusters (8, 41–45) included many genes with heat shock elements (HSEs; GAAnnTTC), as this motif binds HSFs (Nover et al., 1996 ). Genes in these clusters, which are up-regulated under all heating conditions (although not necessarily to the same extent), contain the majority of the highest fold-increased transcripts and include more HSPs (28 genes) than any other group of clusters. Transcripts in these clusters have also generally been identified in other microarray studies of heat-treated Arabidopsis plants or cell cultures (Rizhsky et al., 2004; Busch et al., 2005; Lim et al., 2006; Schramm et al., 2006). Cluster 45, which is equally induced in all three heat treatments here (S, G, and D), includes 18 well-characterized HSPs (Hsp101, 14 small HSPs, and three Hsp70s). Genes in clusters 41 to 44 maintain somewhat higher levels in G-acclimated plants than in S-acclimated plants. In D plants, the transcripts generally appear lower than in either acclimation treatment but are still significantly induced. Clusters 41 to 44 comprise approximately 50% unknown proteins but also include stress-related proteins such as chaperonin 60s, organelle Hsp100/ClpB proteins, two DnaJs, petidyl prolyl isomerases, a glutaredoxin, and the cold-regulated protein COR6.6. While many of these transcripts are likely necessary for thermotolerance, their accumulation is not sufficient, as they also accumulate in nonacclimated plants.
The site II motif (TGGGCC; Welchen and Gonzalez, 2005 Three clusters (36, 40, and 51) showed an overrepresentation of the abscisic acid response element (ABRE) sequence, ACGTG. Cluster 40 included 25 transcripts, which were up-regulated during both S and G acclimation. Fifteen of these had ABRE sequences, most of which encoded late embryogenesis abundant proteins and unknown proteins, in addition to the DRE-binding transcription factor DREB2A. Cluster 51 included 76 ABRE-containing sequences (of 163). These included five oxidative stress-related transcripts (At1g77510, At4g15660, At4g15700, At5g36270, and At1g30870), three xyloglucan endotransglycolsylases (At4g14130, At4g03210, and At5g65730), an endoglucan transferase (At4g37800), and two subtilisin-like proteinases (At4g21650 and At1g01900). These transcripts showed a range of expression levels, with the trend being for higher expression in G than in S acclimation. Of the clusters not listed in Figure 5, there were a limited number of up-regulated clusters. One small cluster, cluster 17 (six genes), includes two subunits of NADH dehydrogenase and a cytochrome c oxidase, up-regulated in all heated samples. Genes in clusters 4, 5, 34, and 52 (41 genes total) all increase more in acclimated plants and more in G than in S, but these transcripts are predominantly of unknown function.
Two groups of clusters that were down-regulated exclusively in acclimated plants were identified. One group (clusters 21, 22, 63B, and 71) contained the W-box motif found in disease resistance genes (Raffaele et al., 2006
The other group of down-regulated clusters (46, 62, and 68) contained the sequence TATATA, which may be a form of TATA box (Molina and Grotewold, 2005
We utilized MAPMAN array data visualization software (Thimm et al., 2004
Visualization of changes in stress-related transcripts using MAPMAN also provides insight into potential interactions of stress response networks (Supplemental Fig. S4). Transcripts induced by other abiotic stresses show varied patterns of change. Cold-regulated transcripts accumulate more in S acclimation than in G acclimation, which reinforces the observation that clusters identified as enriched for genes with putative DRE elements are mostly increased in S acclimation only (Fig. 5). Many transcripts associated with oxidative stress are also primarily increased in S acclimation only, although peroxiredoxins and catalase transcripts accumulate under G acclimation as well. These data are consistent with the fact that S-acclimated plants are less thermotolerant than G-acclimated plants and suggest that S-acclimated plants experience greater stress than G-acclimated plants. Another striking transcriptional change in stress transcripts observed in this MAPMAN analysis concerns biotic stress. There is an obvious decrease in large numbers of transcripts associated with biotic stress in acclimated plants only. The magnitude of the decrease is also greatest for G-acclimated plants. This visualization draws together the down-regulation of disease-related transcripts seen in multiple clusters, including those with W-box motifs (Fig. 5).
The decrease in transcripts associated with biotic stress led us to more carefully examine behavior of transcripts associated with programmed cell death (PCD). We found that MYB30 (At3g28910, cluster 46; Raffaele et al., 2006
Transcripts increased in only G or S acclimation cannot themselves be essential for thermotolerance. However, they may control similar protective physiological processes by different mechanisms. Levels of transcripts involved in Pro metabolism illustrate this point. It was recently shown that Pro accumulation is toxic to plants at high temperatures (Rizhsky et al., 2004
To determine if Pro levels were modulated in thermotolerant plants compared to nonacclimated plants, we measured Pro levels at the unheated, S45, D45, and G45 time points (Fig. 6B). Results show that during direct heat stress, leaf Pro content increased on the order of 10-fold or more in the first 30 min of direct heating at 45°C. In S- and G-acclimated plants, the increase in Pro was at least 5 times less than in nonacclimated plants. Thus, control of Pro accumulation appears to be a component of acquired thermotolerance. To further test the importance of Pro regulation for thermotolerance, we studied two T-DNA insertion alleles of the ProOx gene (Supplemental Fig. S5) and tested the ability of the mutants to acquire thermotolerance. Both alleles were unable to acquire thermotolerance normally under either S- or G-acclimation conditions (Fig. 6, B and C), and both alleles accumulated even higher levels of Pro than wild type, both under control and heated conditions (Fig. 6B). These results further confirm the importance of controlling Pro levels during heat stress. We suggest that a different balance in the regulation of Pro synthesis versus degradation prevents Pro accumulation at high temperature in S- and G-acclimated plants.
To test the significance of some of the observed transcript changes, early on in this study we selected 30 genes for mutant analysis (Supplemental Table S6) from the list of 185 genes up-regulated more than 5-fold by heat treatment and showing >5,000 AU of expression (Supplemental Table S1). The criteria for selection included the availability of T-DNA insertion mutant lines and excluded, in general, members of gene families with predicted redundancy (although there are exceptions). This list included the ProOx gene described above (At3g30775), and we also added a putative choline kinase gene (for a total of 31 genes), which showed only >4,000 AU of expression at maximum induction but was over 10-fold induced and known to be regulated by other stresses (Summers and Weretilnyk, 1993
For the other 15 of the 31 lines, at least 12 progeny from each line were tested for phenotype directly, using segregating material obtained from the stock center. Of these 15 mutants, four showed a distinct heat acclimation phenotype (At4g23570, At1g10170, At4g36010, and At1g74320) and were subsequently found to be homozygous insertion lines. The other 11 mutants showed no evidence of heat sensitivity among the seedlings tested. It remains possible that some of these genes may prove important for acclimation, but they have not been further analyzed. In total, those lines analyzed as homozygotes but not having a phenotype were HSFB1 At4g36990, expressed protein At5g67600, LTI78 At5g52310, DREB2A At5g05410, Fer1 At5g01600, immunophilin At4g25340, stress-induced protein At4g12400, ROF1 At3g25230, galactinol synthase At2g47180, and HSFA2 At2g26150 (Table I; Supplemental Table S1). We have not rigorously demonstrated that these homozygous mutants without phenotype are indeed null mutants, although the position of the T-DNA insertions early within the gene structure for all but At5g52310, At4g25340, and At4g12400 is consistent with this interpretation (data not shown).
Of the remaining eight T-DNA insertion mutants with heat acclimation phenotypes, in addition to ProOx, second insertion alleles were obtained for APX2 (At3g09640) and thaumatin (At4g36010), and all mutants were then backcrossed once to wild type and homozygous mutant lines reselected and used to obtain the data in Table I. Absence of detectable full-length RNA in all mutants was confirmed by reverse transcription-PCR (Supplemental Fig. S5), indicating that the mutants most likely are null alleles. These eight mutants represent multiple important functions and had heat sensitive phenotypes ranging from mild to severe, although none was as severe as a mutation in Hsp101 (At1g74310), which was completely unviable after the treatments used (Table I; Supplemental Fig. S6). The most severely defective mutants were APX2, a cytosolic ascorbate peroxidase, and two transcription factors, HsfA7a (At3g51910) and NF-X1 (At1g10170), all showing 20% to 30% viability compared to wild type under both the S- and G-acclimation treatments. The next most severe mutants were ProOx and SGT1a (At4g23570), a factor implicated in Hsp90 function (Takahashi et al., 2003
We have defined transcript profiles associated with plant acclimation to heat stress, revealing the complexity of molecular events that contribute to thermotolerance. We examined two different heat acclimation treatments, a gradual increase to 45°C (G acclimation) and a stepped heat pretreatment (S acclimation), and compared viability and gene expression profiles to nonacclimated, 45°C-treated plants (D treatment). As expected, both acclimation treatments allowed much longer plant survival upon exposure to 45°C than no acclimation. Interestingly, G acclimation, designed to mimic the gradual increase in temperature that would be experienced in the natural environment, induced greater heat tolerance than S acclimation, a treatment typical of those used in heat stress and heat acclimation studies in plants, yeast, bacteria, and mammalian cells (Knop et al., 1985 ; Mackey and Derrick, 1990; Hong and Vierling, 2000). The difference in response to these two treatments indicates the importance of placing transcript profiling results in the context of physiological responses. Our whole-genome transcript profiling defined specific transcripts (Fig. 4; Supplemental Tables S2 and S3), gene clusters (Fig. 5; Supplemental Table S5), and physiological processes (Supplemental Fig. S7; Supplemental Figs. S4 and S5) that are up-regulated as well as down-regulated in thermotolerant plants. In the response to heat and the process of heat acclimation, we suggest that decreases in gene expression may be of more importance than previously recognized, as nonthermotolerant plants fail to make many of these adjustments. Genes whose transcripts specifically increase associated with thermotolerance, as distinct from genes regulated by heat even in plants that fail to acclimate, have also been identified and can now be tested for their role with available mutants. Finally, among the genes with abundant transcripts that are most highly heat induced, we have identified eight new genes required for maximum heat acclimation, including the cytosolic APX2 and the transcription factors HsfA7a and NF-X1.
Several other studies have examined the response of the Arabidopsis transcriptome to heat stress but not as related to heat acclimation. Rizhsky et al. (2004)
Besides the HSE-containing clusters, most of the transcripts in the other clusters showed no simple pattern of change compared to other studies in which the heat stress transcriptome has been examined. As many of these were transiently increased or changed only under specific treatments, it is not surprising that such differences are seen. Interestingly, however, many clusters identified here showed significant correlations with patterns of gene regulation in array studies involving other stresses. Several different clusters of transcripts (HSE clusters; DRE clusters; site II clusters 47, 54, and 56; TATATA clusters; and clusters 4, 59, 65, and 70) showed a similar up- and down-regulation in response to norflurazon and syringolin as they did to heat (Genevestigator; Michel et al., 2006 The greater thermotolerance of G-acclimated compared to S-acclimated plants appears to be due to bona fide differences in the molecular events leading to thermotolerance between the two treatments. Differences in the amount of time plants were exposed to an acclimating temperature do not appear to account for the greater viability of G-acclimated plants. In the treatments analyzed here, plants begin to express HSPs during G acclimation when the temperature reaches approximately 30°C at 225 min prior to 45°C, whereas in S acclimation, HSP expression begins immediately at 38°C, 210 min prior to 45°C (data not shown). Shorter G-acclimation treatments in which the temperature was increased from 22°C to 45°C in 4.5 h resulted in similar protection, while extending the S-acclimation treatment to a total 6 h (90, 180, or 240 min at 38°C, followed by 22°C) did not increase survival compared to the S-acclimation treatment reported here (data not shown). In total, better survival of G-acclimated plants appears due to four general factors: greater expression of heat stress-induced transcripts (including many of the classical HSP/molecular chaperone genes), higher expression of transcripts unique to thermotolerant plants, increases in transcripts unique to the specific heat treatment (gradual heating), and more effective repression during stress of many transcripts presumably not needed until plants have recovered and potentially damaging if expressed during stress. There were a number of transcriptional changes that occurred only during S acclimation. This may be attributed to thermotolerance being induced through different pathways by the two treatments or to differences in the stress being perceived by the plant. Unique S-acclimation transcripts were primarily those in the DRE cluster 63A, which is also up-regulated by cold, drought, hydrogen peroxide, and anoxic stresses (Supplemental Table S5). The significantly greater up-regulation of general stress-related transcripts during S acclimation can be seen clearly in Supplemental Figure S4. This suggests that under S acclimation, the plant is experiencing other stresses secondary to heat stress. S acclimation may be a shock response whereby the plant responds by repairing stress-induced damage. In comparison, the greater numbers and magnitudes of changes in transcript abundance during G acclimation suggest a more adaptive response whereby damage is prevented; the plant reduces its general metabolism and may reduce damage that could occur due to buildup of toxic intermediates. These data further emphasize how the nature of the applied stress significantly affects the outcome of transcriptome studies and supports the need for carefully controlled conditions and documentation of associated physiological responses.
Despite the differences in transcript profiles between the two acclimation treatments, ultimately the same systems must be protected/repaired. We hypothesize that the same physiological outcome might be achieved in different ways. One example of this is the accumulation of Pro, where we suggest that lower levels of this toxic metabolite are achieved by reduction of synthesis in G acclimation and increased degradation in S acclimation. We show that the ability to degrade Pro through Pro oxidase is essential for thermotolerance. Mutants of ProOx showed reduced thermotolerance, had higher Pro levels prior to heat stress, and had similar Pro levels after heat stress in both acclimated and nonacclimated plants. By comparison, wild-type plants subjected to either S or G acclimation did not accumulate high levels of Pro during heat treatment. Previous work has shown that high levels of Pro result in PCD in Arabidopsis at high temperature (Rizhsky et al., 2004
It is well accepted that protection and refolding of cellular proteins through the HSP network of molecular chaperones are important for survival of high temperature stress (Vierling, 1991
Although it has long been known that Hsfs are major transcription factors involved in gene transcription in response to heat stress, the relative roles of the 21 different Arabidopsis Hsfs as well as other transcription factors in heat acclimation are far from resolved. Of the 21 Hsfs, only Hsfs A1e, A2, A3, A7a, B1, and B2b were shown to be heat induced. Charng et al. (2007) The potential importance for stress acclimation of genes with the site II motif has not previously been recognized. Site II clusters up-regulated in both S and G acclimation included splicing factors (At1g36730, At2g18510, and At4g03430), a putative elongation factor (At2g38560), ribosomal proteins (At3g27450 and At3g11120), and translation initiation factors (At1g36730 and At2g04520). Other transcripts within these clusters were associated with ubiquitin and proteasomes (10 transcripts) and with protein folding (20 transcripts, including six chaperonins, three petidyl prolyl isomerases, and two Hsp70s). Importantly, site II cluster 57 (484 genes) was up-regulated exclusively in G-acclimated plants. It includes many additional translation initiation and elongation factors and ribosomal proteins, as well as photosystem subunits. The dramatic increase in components associated with cytosolic protein translation was further emphasized by visualization using MAPMAN (Supplemental Fig. S3). Thus, G-acclimated plants appear well positioned to repair and restart translation and photosynthesis quickly after heat stress and to maintain translation and photosynthesis to higher temperatures, no doubt contributing to their higher survival rate. The heat-induced translation initiation components may account in part for decreased ability to acclimate. Indeed, preliminary analysis of T-DNA insertion mutations in eIF1A-2 (At2g04520) and eIF5-2 (At1g36370) indicates that these factors are dispensable for normal growth but contribute to heat tolerance (data not shown).
Little attention has been paid to transcripts decreased during stress treatments, but results here suggest that decreases in specific transcripts play an important role in stress acclimation. Many transcripts associated with growth and general metabolism were down-regulated during S acclimation and even more so in G acclimation. Transcripts containing TATATA in their promoters also remained down-regulated in G45R samples, when other transcripts had returned to normal. Molina and Grotewold (2005)
Our data further indicate that a decrease in the potential for induction of PCD is important for acclimation to high temperature in plants. High temperatures in nonacclimated plants have been shown to induce a form of PCD (Swidzinski et al., 2002
Plant Growth and Heat Treatment
Wild-type Arabidopsis (Arabidopsis thaliana) plants, ecotype Columbia-0 (Col-0), were grown on plates as described (Larkindale et al., 2005a
RNA samples were prepared with a Qiagen RNeasy plant mini kit. Quality and quantity of RNA was determined by standard methods. RNA was used for cDNA synthesis, labeling, and hybridization of Affymetrix AtH1 arrays (22,746 genes) according to the Affymetrix GeneChip Expression Analysis technical manual (http://www.affymetrix.com) by the Microarray Core Facility, University of Arizona. Duplicate biological replicates were hybridized to separate arrays. Data file quantification was performed as optimized by Affymetrix. Data quality was confirmed by examining standard parameters for Affymetrix arrays with Microarray Suite (MAS 5.0). Data were normalized by scaling the expression of all probe sets to a median expression level of genes on an array to 500 AU. These data were then analyzed with Data Mining Tool (v 3.0, Affymetrix; http://www.affymetrix.com), Microsoft Excel, and BRB ArrayTools (http://linus.nci.nih.gov/BRB-ArrayTools.html). An average expression value for each gene for each experimental condition was generated from the normalized data from the duplicate arrays. Genes were labeled as present only if the probability of detection was P < 0.05, leaving 17,312 transcripts that were detected on at least two duplicate arrays. Data were validated by comparison with real-time PCR for a representative set of genes (Supplemental Fig. S7). For cluster analysis, data were further filtered such that genes were excluded if there was less than a 2-fold change between any individual array and the median value for that gene on all of the arrays, or if the gene was detected on only one or two of the 11 array samples. A total of 4,724 genes passed this filtering and were analyzed further. Clustering of both total transcript accumulation within a specific treatment and of individual genes was done using Euclidean distance and average linkage in BRB array tools. Genes in each cluster were exported into Data Mining Tool and expression of the selected genes across all arrays was compared.
Promoter analysis was performed using 1,000 bp of upstream gene sequence obtained from The Arabidopsis Information Resource (http://www.arabidopsis.org/). cis-Elements were identified by blind searching for common six-letter words using The Arabidopsis Information Resource's motif analysis selecting only those words with a P value of <10–4 and by direct searching for defined non-six-letter cis-element sequences. These words were then compared to known cis-elements using PlantCare (http://oberon.fvms.ugent.be:8080/PlantCARE/index.html) and Google (www.google.com). When similarities to known cis-elements were identified in clusters of interest, the original sequence was searched for the entire element and its variants. Expression was compared to other conditions using Genevestigator (www.genevestigator.ethz.ch; Zimmermann et al., 2004
Pro levels were measured using the method described by Bates et al. (1973)
The following materials are available in the online version of this article.
Thanks to Kevin Keisler (Microarray Core Facility, University of Arizona) for help with processing the microarray data and David Mount (University of Arizona) for help with analysis. We also thank many past members of the Vierling lab who helped with initial screening and testing of many of the T-DNA mutants discussed in this study. Received November 1, 2007; accepted November 19, 2007; published November 30, 2007.
1 This work was supported by the National Science Foundation (grant no. IBN–0213128) and by the U.S. Department of Agriculture (grant no. NRICGP 99–351007618 to E.V.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Elizabeth Vierling (vierling{at}email.arizona.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.112060 * Corresponding author; e-mail vierling{at}email.arizona.edu.
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ABSTRACT
RESULTS
-vacuolar processing enzyme, At4g32940; Clough et al., 2000
-1-pyrroline-5-carboxylate synthetase (P5CS; At3g55610) and Pro oxidase/dehydrogenase (ProOx; At3g30775; Hayashi et al., 2000
0.5. Documentation of the homozygosity of the mutants and absence of full-length RNA is provided in Supplemental Figure S5. Ten other mutants shown to be homozygous but to not have a heat stress phenotype were HSFB1 At4g36990; expressed protein At5g67600; LTI78 At5g52310; DREB2A At5g05410; Fer1 At5g01600; immunophilin At4g25340; stress-induced protein At4g12400; ROF1 At3g25230; galactinol synthase At2g47180; and HSFA2 At2g26150. These mutants were not backcrossed to wild type because of the absence of phenotype.
