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First published online May 27, 2005; 10.1104/pp.105.062257 Plant Physiology 138:882-897 (2005) © 2005 American Society of Plant Biologists
Heat Stress Phenotypes of Arabidopsis Mutants Implicate Multiple Signaling Pathways in the Acquisition of Thermotolerance1,[w]Department of Biochemistry and Molecular Biophysics (J.L., E.V.) and Department of Molecular and Cellular Biology (J.D.H., E.V.), University of Arizona, Tucson, Arizona 85721; and Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom (M.R.K.)
To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress.
Plants and other organisms have both an inherent ability to survive exposure to temperatures above the optimal for growth (basal thermotolerance) and an ability to acquire tolerance to otherwise lethal heat stress (acquired thermotolerance). Acquired thermotolerance is induced by a short acclimation period at moderately high (but survivable) temperatures or by treatment with other nonlethal stress prior to heat stress (Kapoor et al., 1990  ; Vierling, 1991; Flahaut et al., 1996; Burke et al., 2000; Hong and Vierling, 2000; Massie et al., 2003; Larkindale et al., 2005). The ability to withstand and to acclimate to supra-optimal temperatures results from both prevention of heat damage and repair of heat-sensitive components. Organisms must also maintain metabolic homeostasis during stress or be able to reestablish homeostasis subsequent to the stress period. Although plants are frequently subjected to dramatic heating to above the optimal growth temperature, relatively little is known about the critical genes controlling either basal or acquired thermotolerance in plants.
Heat stress has a complex impact on cell function, suggesting that many processes are involved in thermotolerance. Some processes may be specific to basal thermotolerance, others may be induced during acquired thermotolerance, and many may be involved in both. High temperatures are known to affect membrane-linked processes due to alterations in membrane fluidity and permeability (Alfonso et al., 2001
The best-characterized aspect of acquired thermotolerance is the production of heat shock proteins (HSPs; Vierling, 1991
Several lines of evidence indicate, however, that HSP synthesis is only one aspect of protection against heat-induced damage. Using an assay for acquired thermotolerance of hypocotyl elongation of 2.5-d dark-grown seedlings, we identified seven loci, designated hot1 through 7, with reduced acquired thermotolerance (Hong and Vierling, 2000
Genetic data also indicate that different genes contribute to heat tolerance at different stages of the plant life cycle and that different genes may be essential for basal and acquired thermotolerance. Several of the hot mutants are defective in acquired thermotolerance only when assayed as 2.5-d seedlings but not at later growth stages (Hong et al., 2003 As described above, in our previous efforts to identify genes involved in acquired thermotolerance, we screened for mutants defective in acquired thermotolerance of hypocotyl elongation of 2.5-d dark-grown seedlings. Although the screen was not carried out to saturation (second alleles were found for three of seven loci), the small number of loci identified was surprising, given the diverse processes perturbed by heat. Therefore, to determine the contribution of additional genes to acquired and basal thermotolerance, in this study we examined the heat stress phenotypes of 45 existing Arabidopsis mutants and one transgenic line (NahG). As described in Table I, the mutants selected were mutants that disrupt processes that could be predicted, based on previous data, to play a role in responses to heat. These included mutants defective in signaling pathways (abscisic acid [ABA], salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or mutants previously found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Although some of these mutants have been shown to be defective in heat tolerance, their responses to heat at different growth stages had not been systematically tested nor had their basal and acquired thermotolerance responses been compared. We tested each mutant for basal and acquired thermotolerance at five growth stages and assessed levels of thiobarbituric acid reactive substances and HSPs. The data indicate that, separate from HSP induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that the uvh6 gene plays a significant role in temperature responses in addition to its previously described role in UV light stress. The results further support the conclusions that thermotolerance is a complex multigenic process, with different gene sets involved in acquired and basal thermotolerance and in thermotolerance at different plant growth stages.
Identification of Mutants with Altered Heat Tolerance as 7-d Seedlings The rationale for choosing each of the 45 mutants used in this study is summarized in Table I. Further information about each mutant, including the nature of the gene product and the specific gene locus (when available), is provided in Table II (see also Supplemental Table I). In most cases, only a single allele at each locus was examined, although multiple mutants in the same process (e.g. ABA signaling) were studied. The number of the allele used is provided in Table I, but will otherwise not be used in the text. Most of the mutations are point mutations generated by ethyl methanesulfonate mutagenesis (Supplemental Table I), and therefore it should be kept in mind that they may not represent complete loss-of-function alleles.
All of the mutants were initially tested for their responses to a heat stress applied to 7-d seedlings grown on agar plates in the light. We chose this growth stage with an interest in evaluating heat tolerance of photosynthetically active, autotrophic seedlings. Furthermore, we had already performed a forward genetic screen for thermotolerance in 2.5-d-old dark-grown seedlings as described above and found that only a subset of the mutants identified showed a phenotype after growth in the light (Hong et al., 2003 ). Plants were tested either for basal thermotolerance by heating directly to 45°C for 60 min or for acquired thermotolerance by acclimation at 38°C for 90 min plus 2 h at room temperature, followed by the challenge heat treatment of 45°C for 180 min. As controls, each assay plate also included wild-type seedlings of the corresponding Arabidopsis accession (Supplemental Table I) and hot1 (Hsp101) mutant seedlings. Both heat treatments represent the limits of survival for wild-type plants and are lethal to the hot1 mutant. The hot1 mutant was chosen as the heat-sensitive control because it is the most heat sensitive of all the thermotolerance (hot) mutants that we have characterized at all growth stages (Hong et al., 2003). None of the mutants suffer any damage from the 38°C heat acclimation treatment alone (data not shown). Five days after heat treatment, the percentage of seedlings that survived (i.e. remained green and growing) was determined and compared to the number of surviving wild-type plants on the same plate. As shown in Figure 1, 15 of the 46 mutants/transgenic lines tested showed significant phenotypes in these thermotolerance assays. Mutants with some of the stronger phenotypes included plants with mutations in genes required for ABA biosynthesis (aba1, aba2, and aba3; Fig. 1A) and plants with mutations in genes involved in ABA signaling (abi1 and abi2; Fig. 1B). These mutants all showed reduced basal thermotolerance (approximately 40%–55% wild-type) and a more severe reduction in acquired thermotolerance (approximately 30%–40% wild-type). Of the ABA mutants we tested, only abi3 (Fig. 1B) and ade1 (data not shown) showed no significant reduction in thermotolerance as compared to wild-type plants. These results implicate ABA in thermotolerance responses.
Three other mutants link additional signaling pathways to thermotolerance. NahG transgenic plants, unable to accumulate salicylic acid (SA), and two respiratory burst oxidase mutants (atrbohB and atrbohD), proposed to be involved in active oxygen species signaling (Torres et al., 1998 , 2002), showed similar reductions in acquired thermotolerance (approximately 40% wild-type) as well as statistically similar reductions in basal thermotolerance (Fig. 1, C and E). In contrast, npr1, which is defective further downstream in SA-induced defense responses, showed only limited heat sensitivity in either assay. Mutations in six other Atrboh genes (see Tables I and II) did not result in any significant thermotolerance defects (data not shown). The final group of signaling mutants, those defective in ethylene signaling (etr1 and ein2), also showed reduced basal thermotolerance (40% wild-type) but only a very minor defect in acquired thermotolerance (approximately 70%–85% wild-type; Fig. 1D). The ethylene overaccumulating mutants eto1, 2, and 3 did not show any significant defects (data not shown). Four mutants with defects in genes relating to antioxidant metabolism (vtc1, vtc2, npq1, and cad2) showed only moderate thermotolerance defects (Fig. 1F). These mutants showed a small (approximately 20%) decrease in acquired thermotolerance and a somewhat stronger decrease in basal thermotolerance, with vtc2 showing the strongest phenotype in both assays. The catalase defective mutant tested (cat1/3) showed no phenotype in either assay (data not shown).
Finally, the strongest defect detected in our screen was exhibited by the UV-sensitive mutant uvh6, which carries a defect in a DNA repair helicase and presumed transcription factor gene (Liu et al., 2003 All the other mutants tested, as listed in Tables I and II, showed no decreased thermotolerance. That is, their survival was >80% of the wild type under the assay conditions.
As an additional test of heat sensitivity, as well as a measure of oxidative damage, mutants found to have a phenotype as shown in Figure 1 were assayed for accumulation of thiobarbituric acid reactive substances (TBARS). High levels of TBARS correlate with high levels of oxidative damage to lipid membranes (Heath and Packer, 1968 As shown in Figure 2, all of the mutants with significant phenotypes in the heat stress assays also had higher levels of TBARS than wild-type plants (50%–250% higher), indicative of increased oxidative damage. However, while in the survival assays it was clear that hot1 and uvh6 showed a stronger phenotype than the other mutants, in the TBARS assay, the NahG transgenic line and atrbohB and atrbohD also showed a similar degree of oxidative damage to hot1, under both basal and acquired thermotolerance treatments. It is possible that this represents the maximum level of TBARS that can be produced prior to death of the seedling. The abi1 and 2 mutants also had very high TBARS but cannot be directly compared to hot1 because of their different genetic backgrounds (Columbia versus Landsberg erecta; Supplemental Table I).
We also observed that the TBARS levels for the ethylene (Fig. 2D) and oxidative stress mutants (Fig. 2F) were higher after basal thermotolerance tests than after acquired thermotolerance tests, as was expected from the survival levels seen in these mutants (survival was lower after basal treatments, Fig. 1, D and F). In contrast, this relationship does not hold for the aba1, 2, and 3 mutants (Fig. 2A). These mutants show no significant difference in their ability to survive acquired versus basal thermotolerance treatments but show slightly higher levels of oxidative damage in the basal thermotolerance tests. These data confirm that oxidative stress is a significant component of heat-induced damage for all of these mutants but that other processes also contribute to determine overall survival of heat stress.
Oxidative stress is increased by high light (Niyogi, 1999
Parallel assays using high light during recovery were performed after the acquired thermotolerance heat treatment (38°C for 90 min, 2 h room temperature, 45°C for 180 min). In these assays, survival of the mutants shown to have a phenotype in Figure 1 (aba1, aba2, aba3, abi1, abi2, NahG, atrbohB, atrbohD, and uvh6) was <20%, while survival of wild-type plants was 75% ± 20%. Increased light levels do not, therefore, affect the defect in acquired thermotolerance in these plants. The experiments above were all done with both the 38°C pretreatment and the 45°C heat treatment occurring in the dark. The same assays were done with the aba1, aba2, aba3, abi1, abi2, NahG, atrbohB, atrbohD, uvh6, and hot1 mutants using heat treatments performed under normal growth light conditions. No significant differences in viability compared to results with treatments in the dark were found (data not shown). We did not test the effects of high light during heat treatment.
While the mutants depicted in Figures 1 and 2 were defective in thermotolerance when assayed as 7-d plants, previous work has shown that thermotolerance phenotypes can vary at different stages of growth (Hong and Vierling, 2000
We then extended our assays to consider other growth stages, testing only the mutants found to have a phenotype in the 7-d-old seedling assay, along with uvh3 because of its heat-sensitive hypocotyl elongation phenotype. The assays used tested basal thermotolerance during germination, acquired thermotolerance of root growth of 4-d-old plants, or chlorophyll retention (extent of bleaching) in leaves from 25-d plants. Averages for the results from these assays, along with the hypocotyl and 7-d seedlings assays already discussed, are presented for all of these mutants in Table III. Because of its strong phenotypes, pictures from representative experiments along with the quantitative data for uvh6 compared to hot1 are also presented in Figure 4. It is important to note that a line of the uvh6 mutant complemented by transformation with the wild-type Uvh6 gene (Liu et al., 2003
Although only hot1, uvh3, and uvh6 showed a phenotype in the germination or hypocotyl elongation assays, after 4 d of growth and exposure to light, all of the mutants except uvh3 showed defects in heat tolerance. The relative degree of heat sensitivity for each mutant appeared to be consistent across the assays performed in older plants, with npr1 showing the least severe phenotype and hot1, uvh6, and the abi mutants showing the strongest phenotypes (Table III; Fig. 4).
Since the expression of HSPs is known to contribute to thermotolerance, it was of interest to determine if any of the mutants with thermotolerance phenotypes as 7-d seedlings were defective in HSP accumulation. To assess HSP levels, protein was extracted from 7-d seedlings that had been heat treated at 38°C for 90 min followed by 30 min at 22°C, conditions similar to the acclimation treatment used to induce acquired thermotolerance. Levels of Hsp101 and class I and class II small heat shock proteins (sHsps) were then examined by western blotting. Results for a subset of the mutants (one from each functional group: npq1, aba3, ein2, NahG, atrbohB, and uvh6) are shown in Figure 5. Equivalent results were obtained from the other mutants tested (aba1, aba2, abi1, abi2, npr1, etr1, atrbohD, cad2, vtc1, and vtc2; data not shown). All of the mutants showed normal levels of these HSPs compared to wild-type plants at this time point. HSPs also accumulated to wild-type levels in 2.5-d dark-grown uvh6 seedlings subjected to the same heat stress (data not shown). Therefore, the observed heat sensitivities of these mutants do not correlate with defects in HSP accumulation during the acquisition of thermotolerance, consistent with the conclusion that these mutants are defective in other processes involved in the acquisition of thermotolerance.
Basal versus Acquired Thermotolerance Evidence from Figures 1 and 2 suggested that the ethylene signaling mutants and the antioxidant mutants were more impaired in basal thermotolerance than in acquired thermotolerance. This observation indicates that different pathways could be involved in basal and acquired thermotolerance. It might therefore be possible to elucidate the relative importance of different signaling pathways in basal versus acquired thermotolerance by looking at the relative severity of the mutant phenotypes under different heat stress conditions. That is, a mutant that showed a phenotype greater than the average phenotype in the acquired assay but merely average in the basal assay might be expected to play some role in events specific to acquired thermotolerance. A mutant that showed a less defective phenotype in the acquired than in the basal thermotolerance assays would be anticipated to be involved in processes made redundant during acquired thermotolerance. Such processes, although critical to basal thermotolerance, are not necessary for survival after acclimation. The mutants were therefore tested in the 4-d root growth and 7-d survival assays for both basal and acquired thermotolerance, with 45°C heating periods from 30 to 120 min without pretreatment (basal) or 90 to 250 min with pretreatment (acquired). Since longer heating periods impose greater stress on the plant, the most heat-sensitive mutants would be expected to show a phenotype after the shortest period of heating. Figure 6 compares the length of heat treatment required to reduce survival or root growth to 50% of unheated samples in comparison to the hot1 mutant. Results for both basal and acquired thermotolerance of seedling survival (Fig. 6A) or of root growth (Fig. 6B) are graphed for each mutant. Note that abi1 and abi2 are in the Landsberg erecta background (Ler), while the other mutants are in a Columbia background (Col), so these cannot be directly compared.
As expected, in both the root growth and survival assays, hot1 and uvh6 showed the strongest phenotypes of all of the mutants. Interestingly, all the other mutants performed similarly in the basal thermotolerance tests, requiring approximately the same additional time of heating compared to hot1 to show 50% reduction in survival (approximately 30 min) or root growth (approximately 15 min). By contrast, in the acquired thermotolerance tests, aba1, aba2, and aba3 performed essentially as poorly as hot1 and uvh6, as did abi1 and abi2, although it should be noted that the Ler control was also more heat sensitive than Col. The phenotype of the NahG transgenic line and the atrbohD and atrbohB mutants were intermediate, and ein2, etr1, cad2, vtc, vtc2, and npq1 were only slightly worse than the wild type in acquiring thermotolerance. These data, which are consistent with the results in Figures 1 and 2, suggest that the ABA signaling mutants are most likely to be involved in processes specifically required for acquired thermotolerance and that the NahG transgenic line and the atrbohD and atrbohB mutants play some role in acquired thermotolerance but that the ethylene and antioxidant mutants are more likely to be critical in basal heat tolerance, with less of a role in processes required for acquired thermotolerance.
We have evaluated the importance of different processes and pathways to heat stress tolerance by testing 45 Arabidopsis mutants and one transgenic line for sensitivity to heat (basal thermotolerance) and ability to acquire thermotolerance at different stages of growth. Mutants were chosen on the basis of previous data implicating the mutant gene in responses to high temperature (Table I) and included mutants in signaling pathways, mutants with defects in the production of antioxidants, and other mutants with defects in genes previously associated with heat stress or heat-induced pathways (Larkindale et al., 2005 ). Of the 46 plants tested, the most significant phenotypes in both basal and acquired thermotolerance were seen in several ABA biosynthesis or signaling mutants, in plants unable to accumulate SA, in two mutants of respiratory burst oxidase homologs, and in the uvh6 mutant, which is defective in a DNA helicase. All of these mutants accumulated HSP101 and class I and II sHsps normally, suggesting that the processes affected by the mutant genes are independent of the heat shock response. This indicates that there are multiple processes involved in the acquisition of thermotolerance, of which HSP synthesis is only one critical aspect. Although some of these mutant plants are small (e.g. vtc1) or show other unrelated phenotypes (uvh6 is yellow-green), we argue that the heat phenotypes seen in this study are not due to inherent weaknesses in the mutants. We base this assumption on the fact that some of the mutants tested that showed no phenotype in any assay were also small (e.g. axr1) and that the mutants that did show a phenotype showed it only under specific conditions, i.e. at specific stages of development, more so during basal than acquired thermotolerance, or only after specific heat treatments. Therefore, we conclude that the selected mutants show genuine heat-defective phenotypes.
Results of this study demonstrate that there are many protective pathways that contribute to survival of plants at high temperatures and suggest that the relative importance of the different pathways changes throughout plant development. This conclusion is supported by the observation that mutants showed differential heat sensitivity depending on the stage of growth at which they were assayed. Strikingly, of the mutants tested, only uvh6 and uvh3 (and hot1 as described previously) showed heat sensitivity during seed germination or when tested as 2.5-d dark-grown seedlings. The other mutants were sensitive only after 4 or more d of growth in the light, growth stages at which uvh6 and hot1 (but not uvh3) were also sensitive. Our results also agree with those of Clarke et al. (2004) ABA signaling appears to be more critical for acquired thermotolerance than for basal thermotolerance (while playing a part in both), while the converse is true for ethylene signaling and antioxidant protection. The ABA mutants aba1, aba2, aba3, abi1, and abi2 were unique in showing significantly stronger phenotypes in the acquired thermotolerance assays than in the basal thermotolerance assays. In contrast, the ethylene and antioxidant pathway mutants all showed significantly stronger basal than acquired thermotolerance phenotypes, especially under high light conditions, suggesting that control of oxidative damage is more critical in nonacclimated plants. Together, these data support the hypothesis that heat tolerance involves multiple processes, some of which are primarily of importance during basal thermotolerance (ethylene and antioxidant pathways), some of which are more critical during the acquisition of thermotolerance (ABA pathways), and a number of which are required for survival of any heat treatment (involving an oxidative burst, SA, UVH6, and HSPs). These components may function in multiple pathways acting together to allow plant survival at high temperatures, and the exact balance of components needed for survival depends both on the plant growth stage and on the duration and severity of the heat stress.
The abi3 mutant was unique among the ABA signaling mutants in that it did not appear to be involved in the acquisition of thermotolerance at any growth stage. This transcription factor has previously been associated with developmental control of HSP accumulation in seeds (Rojas et al., 1999
Given that both ABA biosynthesis mutants (aba mutants) and ABA-insensitive mutants (abi1 and 2) were found to be sensitive to heat stress, it can be hypothesized that ABA is likely to accumulate during heat stress, triggering downstream responses. In Arabidopsis, pretreatment of plants with exogenous ABA has been shown to improve basal thermotolerance (Larkindale and Knight, 2002
ABA appears to play a part in some pathway distinct to the induction of HSPs that is nonetheless essential to the acquisition of thermotolerance in light-grown plants. At present, little is known about what a heat-stress ABA pathway might involve, although it is reasonable to hypothesize that similar genes might be induced by ABA during heat stress as during drought stress. Large numbers of stress- and ABA-regulated genes have been implicated in drought tolerance (Zhu, 2002
The NahG transgenic line and atrbohB and atrbohD mutants all showed a significant phenotype both after a direct heat treatment and after acquisition of thermotolerance. It is difficult to determine to what extent these plants are defective in processes specifically involved in acquired thermotolerance, since their defect in basal thermotolerance may also affect plants after acquisition of thermotolerance. Heat pretreatment does not compensate for the effects of the mutations, however, as the plants do not survive as well as wild-type plants after acclimation. These results suggest that signaling pathways involving SA and active oxygen species (AOS) are critical for events during both basal and acquired thermotolerance. The involvement of SA and AOS signals in the acquisition of thermotolerance requires further investigation, as end points for these signaling pathways during heat stress have not been determined. SA and AOS signaling pathways during biotic stresses have been well established (Sandermann, 2000
In contrast to the other mutants, the ethylene and the antioxidant pathway mutants showed almost wild-type acquired thermotolerance but a severe defect in basal thermotolerance (Figs. 1, 2, and 6). Thus, these mutants are defective in some pathway that is essential to basal thermotolerance but that is made redundant by processes induced during the acquisition of thermotolerance. The affect of heat on the ein2 and etr1 mutants and on the mutants compromised in antioxidant defenses was dramatically enhanced by increasing the light intensity during recovery from heat stress (Fig. 3), and these mutants show no phenotype in assays performed during early seedling growth in the dark. Photooxidative damage has been shown to be a critical aspect of 10-d-old seedling survival during basal thermotolerance treatments (Larkindale and Knight, 2002
Of the mutants in this study, only uvh6 showed a strong defect in both basal thermotolerance and acquired thermotolerance at all stages of its life cycle from germination through to 25 d, similar to the Hsp101 mutant hot1. Therefore, this DNA helicase appears to control some function essential to both basal and acquired heat tolerance that is neither age nor light dependent. The ability of uvh6 to accumulate Hsp101 and sHsps indicates that its essential function is distinct from the induction of HSPs or the function of Hsp101. This DNA helicase is believed to have two cellular functions based on the known functions of human and yeast (Saccharomyces cerevisiae) homologs. These homologs act both as a DNA helicase involved in DNA damage repair by the nuclear excision pathway and as a core component of the TFIIH transcription factor complex (Svejstrup et al., 1996
We also tested 30 mutants that showed no heat sensitivity under any of the conditions used, including mutants that had previously been documented as sensitive to high temperatures, e.g. tsl4 and some of the fad mutants (Hugly et al., 1991 Based on our data, we can now diagram a framework for understanding how different signaling pathways contribute to both basal and acquired thermotolerance as shown in Figure 7. Depending on the stress applied and the age of the plant, it appears that different aspects of heat-induced damage impact plant survival, and different types of heat-induced damage are prevented or repaired through different cellular systems. The signaling molecules AOS, SA, and ABA and the transcription component uvh6 potentially activate or modulate other pathways involved in the plant response to heat that are different from the HSP pathway. Because they affect both basal and acquired thermotolerance, they must have effects distinct from the antioxidant pathway. What kind of damage is being prevented or repaired by such pathways has yet to be determined. The SA transgenic line and the AOS mutants showed similar phenotypes and affected basal and acquired thermotolerance equally; therefore, in Figure 7, they are depicted as potentially part of the same pathway. ABA is depicted as part of a separate signaling pathway to the other components, although some aspects of ABA signaling may be common to a pathway involving SA and AOS. The effect of uvh6 is different from the effects of the other mutants in that the gene is essential for thermotolerance in plants of all ages. Although UVH6 function may impact other signaling pathways, it is clear that at least some aspect of UVH6 function is unique and essential for survival in plants prior to exposure to light.
In Figure 7, five separate pathways are therefore illustrated, which, acting together, produce the thermotolerant phenotypes in light-grown plants. Only some of these pathways are critical in 2.5-d dark-grown plants. Heat is a complex stress causing damage to a range of cellular components, so it should not be surprising that a large number of different protective pathways are required in order to survive. Induction of any one of these pathways allows the plant to acquire some measure of thermotolerance, and the loss of any specific pathway merely limits the extent of that tolerance.
Plant Growth Conditions
Arabidopsis (Arabidopsis thaliana) plants of the different mutant lines and their associated wild-type accessions were used as shown in Table I, and the seed source/donor is shown in Supplemental Table I. Mutants were obtained in the homozygous state from the stock center or individual donor and were propagated directly to obtain sufficient material for experimentation (in some cases through three generations). The homozygosity of all mutants with easily scorable phenotypes, e.g. ABA insensitivity or presence of a transgene, was also directly verified. Seeds were surface sterilized, plated on nutrient medium (Haughn and Somerville, 1986
Thermotolerance assays of seeds, 2.5-d dark-grown, 4-d light-grown, and 7-d light-grown seedlings were performed according to Hong and Vierling (2000)
Analysis of TBARS was performed according to Heath and Packer (1968)
Seedlings were treated at 38°C for 90 min and cooled to room temperature for 30 min prior to harvesting the tissue for protein extraction. Total protein from seedlings was extracted in SDS sample buffer (60 mM Tris-HCl [pH 8.0], 60 mM dithiothreitol, 2.0% [w/v] SDS, 15% [w/v] Suc, 5 mM
We thank the many individuals who provided seeds of mutants not available in the stock center, Dr. Suk-Whan Hong (Department of Applied Plant Sciences, Chonnam National University, Korea) for initially identifying the thermotolerance phenotype of uvh6, and Dr. Nicola Evans (Department of Plant Sciences, University of Oxford) for genotyping the Atrboh mutants. Received March 4, 2005; returned for revision March 23, 2005; accepted March 25, 2005.
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). 
[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.105.062257. * Corresponding author; e-mail vierling{at}email.arizona.edu; fax 520–621–3709.
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RESULTS
-amino-N-caproic acid, and 1.0 mM benzamidine). Protein concentration was determined using a Coomassie Brilliant Blue dye-binding assay (Ghosh et al., 1988
