- Top of page
- Experimental procedures
Salicylic acid (SA) is reported to protect plants from heat shock (HS), but insufficient is known about its role in thermotolerance or how this relates to SA signaling in pathogen resistance. We tested thermotolerance and expression of pathogenesis-related (PR) and HS proteins (HSPs) in Arabidopsis thaliana genotypes with modified SA signaling: plants with the SA hydroxylase NahG transgene, the nonexpresser of PR proteins (npr1) mutant, and the constitutive expressers of PR proteins (cpr1 and cpr5) mutants. At all growth stages from seeds to 3-week-old plants, we found evidence for SA-dependent signaling in basal thermotolerance (i.e. tolerance of HS without prior heat acclimation). Endogenous SA correlated with basal thermotolerance, with the SA-deficient NahG and SA-accumulating cpr5 genotypes having lowest and highest thermotolerance, respectively. SA promoted thermotolerance during the HS itself and subsequent recovery. Recovery from HS apparently involved an NPR1-dependent pathway but thermotolerance during HS did not. SA reduced electrolyte leakage, indicating that it induced membrane thermoprotection. PR-1 and Hsp17.6 were induced by SA or HS, indicating common factors in pathogen and HS responses. SA-induced Hsp17.6 expression had a different dose–response to PR-1 expression. HS-induced Hsp17.6 protein appeared more slowly in NahG. However, SA only partially induced HSPs. Hsp17.6 induction by HS was more substantial than by SA, and we found no SA effect on Hsp101 expression. All genotypes, including NahG and npr1, were capable of expression of HSPs and acquisition of HS tolerance by prior heat acclimation. Although SA promotes basal thermotolerance, it is not essential for acquired thermotolerance.
- Top of page
- Experimental procedures
Capacity to survive heat shock (HS) varies with plant species and genotype (Martineau et al., 1979; Srinivasan et al., 1996; Tal and Shannon, 1983), as well as developmental stage (Hong and Vierling, 2001; Nieto-Sotelo et al., 2002). HS tolerance in the absence of pre-adaptation has been termed basal or basic thermotolerance (Hong and Vierling, 2001; Lee et al., 1995; Nieto-Sotelo et al., 2002). In addition, plants subjected to a milder heat stress can transiently acquire tolerance to previously lethal high temperatures: this phenomenon is known as acquired thermotolerance or heat acclimation (Dat et al., 1998a; Hong and Vierling, 2000, 2001; Hong et al., 2003; Howarth et al., 1997; Lee et al., 1995; Nieto-Sotelo et al., 2002), and is probably an adaptation to the gradual increases in temperature in the natural environment.
Relatively little is known about the signaling pathways that control basal and acquired thermotolerance, although hormonal, calcium, and redox signals have been implicated (Foyer et al., 1997; Larkindale and Knight, 2002; Pastori and Foyer, 2002). In recent years, salicylic acid (SA) has been found to protect potato, mustard, tobacco, tomato, bean, and Arabidopsis from heat stress (Dat et al., 1998a, 2000; Larkindale and Knight, 2002; Lopez-Delgado et al., 1998; Senaratna et al., 2000). However, major issues concerning the role of SA in thermotolerance remain unresolved, and this paper addresses the following details. First, although transient accumulation of endogenous SA was found in mustard seedlings subjected to heat acclimation treatment (Dat et al., 1998b), it has not yet been established whether SA might be an essential signal for acquired thermotolerance, or rather one component of a multifactorial regulation of plant thermotolerance. We therefore compared basal and acquired thermotolerance in plants with altered SA signaling, including plants constitutively expressing the Pseudomonas putida SA hydroxylase transgene NahG, which are unable to accumulate SA (Delaney et al., 1994).
Second, further insights into the thermoprotective cellular effects of SA are needed. SA has been found to induce changes in the antioxidant system (Dat et al., 1998a,b, 2000; Lopez-Delgado et al., 1998), and to protect against heat-induced oxidative damage (Larkindale and Knight, 2002). However, protection against oxidative damage was incompletely correlated with survival in Arabidopsis, so thermotolerance probably also involves protection against additional kinds of heat damage (Larkindale and Knight, 2002).
Much evidence supports the involvement of plant HS proteins (HSPs) in thermotolerance (Gurley, 2000; Hong and Vierling, 2000, 2001; Malik et al., 1999; Prändl et al., 1998; Queitsch et al., 2000), and we investigated whether SA affects expression of Arabidopsis HSPs from two families. First, Hsp17.6 belongs to the class I cytosolic family of plant small HSPs (sHSPs; Wehmeyer et al., 1996). Hsp17.6 assembles into 200–300 kDa oligomeric complexes and has protein-refolding activity (Sun et al., 2001). A Synechocystis sHSP has also been shown to act as a stabilizing factor for heat-stressed membranes (Török et al., 2001). Malik et al. (1999) obtained evidence for the physiological importance of carrot Hsp17.7, as modified expression of its gene could increase or decrease thermotolerance. Second, Hsp101 belongs to the Hsp100/ClpB family of ATP-dependent chaperones thought to disaggregate heat-induced protein complexes (Hong and Vierling, 2001; Mogk et al., 1999). Hsp101 has been shown to be essential in thermotolerance by mutation and antisense RNA inhibition (Hong and Vierling, 2000, 2001; Queitsch et al., 2000).
The physical state of membranes is highly sensitive to heat stress, and has attracted interest as a possible cellular temperature sensor (Carratu et al., 1996). Electrolyte leakage has been an experimental screen for heat-tolerant crop plants for a number of years (Howarth et al., 1997; Martineau et al., 1979; Srinivasan et al., 1996; Tal and Shannon, 1983). New evidence for the importance of membrane properties in thermotolerance has been obtained by Hong et al. (2003), whose hot2 Arabidopsis mutant fails to acquire thermotolerance and shows increased ion leakage, despite apparently normal HSP levels. In the present study, we found that protection of membrane integrity is an important effect of SA in heat stress.
Third, the signal transduction pathway(s) of the thermotolerance effects of SA remain unknown, as does the relationship with signaling in the better known effects of SA in systemic acquired resistance (SAR) and the hypersensitive response (Delaney et al., 1994; Mur et al., 1997). There are common pathways and components in different biotic and abiotic stress responses (Pastori and Foyer, 2002), so interactions between SA signaling in thermotolerance and SAR could occur. To investigate this, we utilized Arabidopsis mutants with defective or constitutive SAR, and modified SA signaling, which have been discovered in disease resistance research but not previously tested for thermotolerance. We used mutants defined in relation to pathogenesis-related (PR) proteins, which are SA-inducible markers of SAR. The nonexpresser of PR proteins (npr1) mutant has enhanced susceptibility to pathogens and lacks SA-responsive expression of PR genes, despite wild-type SA levels on infection (Cao et al., 1994). The recessive mutation suggests a positive regulator of the SA signal pathway. NPR1 is a nuclear-localized protein, which may interact with basic Leu zipper transcription factors that bind the PR-1 promoter (Després et al., 2000; Fan and Dong, 2002; Kinkema et al., 2000; Zhou et al., 2000). We also tested recessive mutants that are constitutive expressers of PR proteins (cpr). The cpr1 and cpr5 mutants have elevated levels of SA and constitutive expression of SAR; both gene products seem to act upstream of SA, in contrast to NPR1 (Bowling et al., 1994, 1997; Clarke et al., 2000). The cpr1 gene maps to a Resistance gene cluster on chromosome 4 (Stokes and Richards, 2002). CPR5 encodes a novel protein with a potential nuclear localization motif in its amino-terminal region, and five putative transmembrane domains in its carboxyl-terminal region (Yoshida et al., 2002).
Fourth, we sought more evidence on the stage(s) of heat stress in which SA acts. In addition to the stress during the period of high temperature, thermotolerance studies have recognized events during a distinct recovery period when plants return to non-stressful temperatures. Recovery after heat stress is characterized by restoration of cellular protein activity and/or synthesis (Hong et al., 2003), and transient elevations of cytosolic calcium ions (Larkindale and Knight, 2002).
Fifth, we tested the effects of SA at several plant growth stages, as thermotolerance can show developmental variation (Nieto-Sotelo et al., 2002). Thus, four hot mutant loci were identified by defective hypocotyl thermotolerance, but only the hot1 mutant has defective seed thermotolerance, and only hot1 and hot2 show defective thermotolerance as 10-day-old seedlings (Hong et al., 2003).
This paper thus provides new evidence to clarify the role of SA in thermotolerance and in the interaction between the thermotolerance and SAR signal pathways.
- Top of page
- Experimental procedures
This study substantiated the thermoprotective effects of SA in Arabidopsis using multiple criteria. Extending demonstrations of enhanced survival and protection against oxidative stress in various species (Dat et al., 1998a, 2000; Larkindale and Knight, 2002; Lopez-Delgado et al., 1998; Senaratna et al., 2000), we showed that membrane properties are stabilized in heat-stressed plants by SA, whether applied externally or as an endogenous metabolite that is depleted in plants expressing the NahG transgene. Hong et al. (2003) have shown genetically that thermotolerance mechanisms can differ during seedling growth, but we found endogenous SA-promoted tolerance of HS at all growth stages tested. Thus, the NahG genotype showed greater propensity to leaf necrosis and electrolyte leakage in 3-week-old plants, and poorer survival in 5-day-old seedlings and imbibed seeds. Additionally, 10-day-old NahG seedlings show poorer survival and greater oxidative damage after HS (Larkindale and Knight, 2002).
Electrolyte leakage, as a rapidly measured parameter, enabled us to demonstrate that SA protected membranes in both the HS and subsequent recovery periods. A 24-h pre-treatment with SA substantially reduced leakage during both HS and recovery, while NahG plants showed greater leakage in both periods. While the increased leakage in NahG plants during HS may seem less dramatic (Figure 2f), these plants would have had no prior period for induction of protective mechanisms like those recovering from HS or pre-treated with SA.
We could find no evidence that these effects of heat on NahG Arabidopsis were because of catechol production by the transgene product, as recently suggested for non-host resistance to P. syringae (Van Wees and Glazebrook, 2003). We found no effects of exogenous catechol on thermotolerance, unlike their non-host resistance system, and we detected no accumulation of catechol in NahG plants. Another difference from their system is that our findings with SAR mutants were also consistent with the involvement of SA.
While NahG was the least thermotolerant genotype and 3-week-old NahG plants had nearly 90% less total (free + glucosylated) SA than Col-0, cpr5 as the most thermotolerant genotype had about 14-fold more SA than Col-0. It is interesting that the electrolyte leakage for Col-0 treated with 1 mm SA (Figure 1b) was similar to that for untreated cpr5 (Figure 5b). Thermotolerance intermediate between these extremes was displayed by cpr1 and npr1 mutants. Three-week-old cpr1 mutants accumulated total SA to 2.7-fold the Col-0 levels, which was apparently insufficient to enhance thermotolerance in these plants, although cpr1 seeds were highly thermotolerant. In npr1 mutants at 3 weeks, total SA level was nearly twice that of the Col-0 levels, but the ability to recover from HS was impaired, and 5-day-old npr1 seedlings were also less thermotolerant: this would be explained if the npr1 mutation compromises a thermotolerance response downstream of SA accumulation, as in SAR (Cao et al., 1994). The greater amounts of SA in npr1 mutants have been noted by others, and it may be a function of NPR1 to downregulate SA accumulation (Clarke et al., 2000).
We thus obtained a range of evidence that SA-dependent signaling promotes tolerance of HS in plants not given a prior heat acclimation treatment, i.e. basal thermotolerance. This indicates that SA content and the state of SA-inducible thermotolerance mechanisms could be important determinants of basal thermotolerance in plants. In a similar way, we found clearly that plants are also able to acquire thermotolerance by mechanisms other than SA signaling. All tested genotypes, including NahG and npr1 with deficient SA content or signaling, when given a mild heat treatment were subsequently able to tolerate a previously lethal HS, whether tested as 3-week-old plants or as 2.5-day-old hypocotyls. SA content remained low in NahG during heat treatment, so its ability to acclimate was not because of heat-induced loss of activity of the transgene product.
It may be that SA is dispensable because heat acclimation is always initiated by some other key factor(s), or because SA is only one of multiple alternative acclimation signals. If SA were a potential mediator of a heat-induced acclimation response, SA levels should be heat inducible. As in the study by Dat et al. (1998b) on mustard, we found a moderate and transient but statistically significant increase in glucosylated SA during heat treatment in Col-0 plants. Given the significant basal levels of SA in Arabidopsis and mustard, the extent to which such changes in SA metabolism provide additional thermotolerance is uncertain, but they suggest that despite the metabolic stress of heat treatment, the plant actively maintains biosynthesis of this hormone. In plants, SA is subject to glucosylation (Dat et al., 1998b, 2000; Dean et al., 2003), which might be involved in transport (Seo et al., 1995) or vacuolar localization (Dean et al., 2003). Heat-induced SA increases were not apparent in the other Arabidopsis genotypes, although this might be because of their altered SA metabolism or signaling.
Our mutant studies indicate that SA-signaling pathways involved in SAR overlap with those promoting basal thermotolerance. In SAR, a key regulator of SA-inducible gene expression is the NPR1 protein, which interacts with members of the TGA subclass of basic Leu zipper transcription factors (Després et al., 2000; Zhou et al., 2000). SA promotes formation of the TGA–NPR1 complex, and both SA and NPR1 are required for the DNA binding of the transcriptional factor (Fan and Dong, 2002). There are seven known TGA factors in Arabidopsis, with differing affinities for NPR1 (Després et al., 2000; Zhou et al., 2000), and binding sites for the TGA factors occur in gene promoters responding to diverse stress signals (Chen and Singh, 1999; Pascuzzi et al., 1998). In view of this versatility of the transcription factors with potential to interact with NPR1, it is not surprising that evidence is emerging that the npr1 mutation affects diverse physiological processes in which SA is implicated, including senescence (Morris et al., 2000) as well as our new results on thermotolerance.
NPR1-related signal transduction could therefore be a target for improvement of thermotolerance, but did not exclusively determine thermotolerance. While the ability of npr1 mutants to recover from HS was impaired (although less severely than NahG), wild-type tolerance during HS was found in this mutant. It may be that an NPR1-dependent pathway is partially involved in recovery from HS, but an NPR1-independent pathway predominates during HS. There is evidence for SA-mediated, NPR1-independent responses in disease resistance and senescence (Clarke et al., 2000; Morris et al., 2000).
In the case of cpr5, it is interesting to find a gene whose mutation enhances thermotolerance. The cpr mutants have pleiotropic effects in pathogen defence and plant development, although it is still unproven that CPR proteins are normal components of pathogen resistance signaling (Clarke et al., 2000). Clarke et al. (2000) obtained evidence that elevated SA content is the primary determinant of pathogen resistance in cpr mutants. Yoshida et al. (2002) selected early senescence hys1 mutants allelic to cpr5, and isolated the gene by map-based cloning. Although the deduced HYS1/CPR5 sequence revealed no homology to known sequences, it has features of a signal transduction protein with a possible nuclear localization signal and five putative transmembrane domains. One interpretation of the recessive mutant phenotypes is that HYS1/CPR5 plays a repressive role in early processes for pathogen defence and senescence (Yoshida et al., 2002). Our data are consistent with a further repressive role for CPR5, in thermotolerance signaling, possibly mediated by SA. It remains to be seen whether CPR5 is an integral component of thermotolerance signaling, or whether thermotolerance in cpr5 is an abnormal pleiotropic effect, e.g. because of excessive SA levels.
A further uncertainty in thermotolerance signaling is the extent of interaction between different hormonal signaling pathways. Such cross-talk might be expected by analogy with pathogen defence responses, in which a complex of signaling pathways mediated by SA, jasmonic acid (JA), and ethylene is now recognized (Clarke et al., 2000). These pathways do not function independently, but influence each other through positive and negative regulatory interactions (Kunkel and Brooks, 2002). Recent studies with Arabidopsis mutants implicate ethylene and abscisic acid alongside SA in protection against HS (Larkindale and Knight, 2002), while induction of HSPs by methyl-JA has been reported (Ding et al., 2001; Hamilton and Coleman, 2001). It is therefore likely that the chemical and genetic effects we report do not involve SA action in isolation.
Our analyses of gene expression support our mutant studies in suggesting that thermotolerance and SAR share common features. Thus, genes associated with these two defense responses had overlapping expression patterns. We found either heat- or SA-induced expression of certain PR protein and HSP genes and their promoters as mentioned below.
HS induced PR-1 expression
We tested PR-1 expression because this group of pathogen- and SA-induced PR proteins are common markers for SAR (Malamy et al., 1992; Uknes et al., 1992). Although now there is evidence for an antifungal role, however, their biochemical action remains uncertain (Rauscher et al., 1999). We found that HS induced PR-1 expression. Although in certain cases, PR proteins are induced by abiotic stresses (Van Loon and Van Strien, 1999), there are relatively few reports of heat induction of PR genes. Margis-Pinheiro et al. (1994) reported that a bean acidic class IV chitinase is induced by heat and SA, although class III chitinase, β-1,3-glucanase, and thaumatin-like protein were not heat inducible. In Arabidopsis, the loss-of-thermotolerance hot2 mutation (Hong et al., 2003) maps close to the chitinase-like AtCTL1 gene (Zhong et al., 2002). PR-1 proteins, however, have been reported not to be heat inducible in tobacco (Ohashi and Matsuoka, 1985; Pfitzner et al., 1988). This may somehow reflect the temperature sensitivity of the disease resistance response in tobacco (Malamy et al., 1992), because in Arabidopsis, we found that not only the native PR-1 transcript but also the introduced tobacco PR-1a promoter was heat inducible.
SA-induced sHSP expression
Salicylic acid treatment of Arabidopsis induced expression of the native Hsp17.6 gene and an introduced soybean Hsp17.3B promoter, while Hsp17.6 was constitutively expressed in the SA-accumulating cpr5 mutant. The SA-concentration dependence of Hsp17.6 induction differed from that of PR-1 and indeed appeared more sensitive. Expression of Hsp17.6 was optimal at lower applied SA concentrations in the cpr mutants, possibly because of their higher endogenous SA levels. We also compared heat induction of this sHSP in genotypes with altered SA signaling. Interestingly, although heat induction of Hsp17.6 appeared similar, Hsp17.6 protein was reproducibly slower to appear during HS in NahG. However, Hsp17.6 had accumulated to similar levels in all genotypes by 5 h. SA treatment only partially induced the HSP response, however. SA-induced levels of Hsp17.6 were lower than those rapidly accumulating in HS. Moreover, SA did not induce Hsp101 expression, and expression of Hsp101 protein was unaltered in the mutants.
Few other plant studies have looked at SA effects on HSP expression. Cronjé and Bornman (1999) reported that SA enhanced Hsp70 expression in tomato cells, although effects were relatively small as this HSP is constitutively expressed. Methyl-SA increased accumulation of HSP mRNAs in tomato fruit (Ding et al., 2001). Interestingly, the human HS response can also be partially induced by SA, which induces a DNA-binding state of the human HS transcription factor, but apparently via a different phosphorylation pattern to heat, and SA alone does not induce HS gene expression (Jurivich et al., 1995).
The fact that both NahG and npr1 were capable of heat-induced Hsp17.6 and Hsp101 expression indicates that factors other than SA can induce HSPs. This is consistent with our conclusion that SA is not essential for acquired thermotolerance, in which Hsp101 in particular has been proven to play a key role (Gurley, 2000; Hong and Vierling, 2000; Queitsch et al., 2000). On the other hand, our observations that NahG and npr1 show poor basal thermotolerance, despite being capable of Hsp17.6 and Hsp101 expression, indicate that HSPs are not the sole determinants of basal thermotolerance. This accords with the recent genetic evidence provided by Hong et al. (2003), who found that the hot2 and hot4 mutants produced normal levels of Hsp101 and sHSPs, despite their failure to develop thermotolerance, and moreover that the map positions of these mutations fall outside intervals containing HSP genes.
Although our experiments therefore suggest a multiplicity of factors determining HSP expression and thermotolerance, it is possible that SA-induced sHSPs do contribute to thermotolerance. While the effects of SA on Arabidopsis Hsp17.6 expression were considerably less than those of HS, minor levels of sHSP induction can enhance basal thermotolerance. In plants whose HSP genes were de-repressed by a modified HS transcription factor, constitutive sHSP levels, only 20% of those reached in HS, were correlated with increased basal thermotolerance, although acquired thermotolerance was unchanged (Lee et al., 1995). sHSPs may preserve both protein and membrane integrity in HS. Arabidopsis Hsp17.6 has been shown to have protein chaperone activity (Sun et al., 2001), while Synechocystis sHSP may regulate membrane fluidity to preserve membrane structure during HS (Török et al., 2001) and a soybean sHSP appears to become membrane associated in HS (Lin et al., 1985). Therefore, expression of Hsp17.6 in SA-treated plants and the cpr5 mutant might at least partially contribute to their decreased electrolyte leakage during HS.
Decreased electrolyte leakage indicates a maintenance of physical order of cell membranes. It is well established that many physiological responses to environmental stress are caused by modifications of membrane structures (Carratu et al., 1996). Electrolyte leakage is a sensitive indicator of membrane damage and has been widely used to assess thermotolerance in plants (Howarth et al., 1997; Martineau et al., 1979; Srinivasan et al., 1996; Tal and Shannon, 1983). The phenotype of the hot2 mutant has recently confirmed that membrane properties are an essential aspect of thermotolerance (Hong et al., 2003). As apparently normal HSP levels failed to protect hot2 from increased ion leakage in HS, Hong et al. (2003) proposed that sHSPs cannot be the sole factor protecting membranes during HS. SA-induced changes in antioxidants and reduced oxidative damage (Dat et al., 1998a,b, 2000; Larkindale and Knight, 2002; Lopez-Delgado et al., 1998) may also protect cell membranes, but mutations such as hot2 could reveal further membrane-protective mechanisms. What seems clear from the present study is that maintenance of membrane properties is a major factor in the thermoprotective effects of SA.
In conclusion, SA promotes basal thermotolerance in Arabidopsis plants, hypocotyls and seeds, although SA is not essential for acquired thermotolerance. SA protects during HS and in the subsequent recovery. Mutations known to affect SA signaling in pathogen defences also affect thermotolerance. Common features of these defences are further suggested by induction of certain PR and HSP genes by both SA and HS. SA induces thermoprotective effects, possibly including certain HSPs in protection against membrane dysfunction and other damage. It is likely that SA represents one of the many factors regulating heat stress physiology in plants. As SA affects basal thermotolerance, its associated signaling pathways and thermoprotective mechanisms could be targets for genetic improvement of crop thermotolerance.