Multi-Level Interactions Between Heat Shock Factors, Heat Shock Proteins, and the Redox System Regulate Acclimation to Heat

1Plants are continuously exposed to biotic and abiotic stress factors, such as herbivory, pathogen attack, drought, salinity and extreme temperatures. These challenges pose a serious threat to their growth and reproduction and as such affect agricultural yields. With considerable advances in pest and disease management, abiotic factors are now thought to be the primary cause for crop losses worldwide. In case plants cannot prevent an abiotic stress factor from affecting organismal homeostasis (i.e., escape or avoid internal stress), they may adapt their metabolism to acquire a certain level of tolerance.

2Heat stress can be defined as a rise in temperature beyond a threshold level for a period of time, sufficient to cause irreversible damage to plant growth and development. Sudden rises in temperature to high levels may lead to cell death within a few minutes as a consequence of extensive protein denaturation and aggregation and loss of membrane integrity. Furthermore, prolonged exposure to moderately high temperatures can lead to reduced cellular function and overall plant fitness. An important process in this respect is the accumulation of reactive oxygen species (ROS), formed as a by-product in various aerobic metabolic pathways in different cellular compartments such as chloroplasts, mitochondria and peroxisomes and probably also in the apoplast through the activation of NADPH oxidases. Under steady state conditions, ROS molecules are formed as quickly as they are scavenged by anti-oxidative defense mechanisms, but this equilibrium is perturbed by abiotic stress factors such as heat. There is ample evidence that, when plants are exposed to heat, ROS production rapidly becomes excessive. This causes cellular damage to membranes, proteins, lipids, organelles, and DNA. In order to prevent cell damage and regain redox homeostasis, one of the responses to heat is the hyper-activation of the ROS scavenging machinery. The expression and protein level of genes responsible for ROS scavenging are induced under heat stress in many different plant species and has been associated to basal heat tolerance. Furthermore, the induction of scavenging genes was significantly stronger in heat tolerant genotypes than that of sensitive ones, and improvement of plant heat stress tolerance has been achieved by increasing antioxidant enzymes activities. Taken together, this shows the importance of ROS scavenging in the heat-stress response.

3In contrast to their harmful character, however, ROS are also considered as important signal molecules. Cells are capable of rapid and dynamic production and control of several forms of ROS, enabling a tight local control in the cell as well as more holistic control of the entire plant. Therefore, they are thought to be involved in the transduction of intracellular and intercellular signals controlling gene expression and activity of anti-stress systems. Indeed, NADPH oxidase activity is rapidly induced upon heat and the mutation of RBOHB makes Arabidopsis seedlings more sensitive to heat.

4One of the best studied anti-stress mechanisms is the production of heat shock proteins (HSPs) upon exposure to high temperatures. By acting as molecular chaperones, HSPs prevent deleterious protein conformations and eliminate non-native aggregations, which are formed during stress. The expression of HSPs and other heat-responsive genes is regulated by heat shock factors (HSFs; ) through their association to a palindromic binding motif (5′-nAGAAnnTTCTn-3′) in the promoter region of the heat-responsive genes: the heat shock element (HSE;; ). Activation of HSFs upon stress occurs via a multistep process involving homotrimer formation and acquisition of transcriptional competence for target gene induction.

5Clearly, both the activation and production of HSFs/HSPs and the increase in ROS/scavenging activity belong to the major responses of plants to heat stress and play important roles in acclimation. A number of recent genetic and biochemical studies, however, indicate that there are complex interactions between these responses. This review describes the evidence for crosstalk between HSFs, HSPs, ROS, and ROS scavenging enzymes at various points in the heat stress response pathway and presents a model with a timing component.

6In non-stressed situations, the HSFs are located in the cytoplasm for most eukaryotes, in an inactive monomeric form due to association with HSP70, HSP90, and potentially other proteins. According to the chaperone titration model, heat results in a higher load of denatured proteins, which pulls HSPs away from HSF complexes through competitions to act as molecular chaperones. This then leads to the release of HSFs, which form trimers and relocate to the nucleus to activate expression of HSP and other heat-responsive genes.

7A number of studies, however, report that expression of heat-responsive genes is also increased upon application of the ROS H2O2. For example, AtHSP17.6 and AtHSP18.6 achieved similar expression levels through heat treatment as they do through H2O2 application at room temperature. Several hypotheses have been formulated that suggest that heat can indirectly activate HSFs via the action of ROS.

8Firstly, damaging amounts of heat-induced ROS also induce protein denaturation. In this way ROS enhances dissociation of the HSP–HSF complex, as described by the titration model. Secondly, and similar to what was found for mammalian and Drosophila HSFs, it has been proposed that certain plant HSFs act as H2O2 sensors. Among all the ROS molecules, H2O2 plays a key role in signaling due to its moderate reactivity and thus relatively long lifetime. In addition, H2O2 can bypass membranes easily, making it a good candidate to function as a signaling molecule. suggested that H2O2 might directly modify HSFs and induce HSF trimerization. Indeed, both heat and oxidative stresses result in the formation of high molecular weight HSE-binding complexes and the formation of these complexes has been shown to be a signature of early HSFA1a/A1b-dependent gene expression in heat-stressed leaves of Arabidopsis. In vitro and in vivo studies confirmed activation of AtHSFA1a via trimerization in response to heat and H2O2 stress but also via pH alterations. HSFA1a, purified from E. coli, sensed the different stresses directly in a redox dependent fashion. In vitro stress treatments caused monomer-to-trimer transitions of HSFA1a, while the presence of the reducing agent dithiothreitol reversed this action. Although the study suggested a redox dependent fashion for HSF trimerization for all three stresses, the exact mechanism of action is still unclear. There is empirical evidence that the transcription factors may be sensitive to H2O2 via “single-Cys” or “two-Cys” redox sensory mechanisms. These cysteine residues are typically responsive to oxidative stress. HSFA1a contains one Cys residue located at the N-terminal portion of the trimerization domain. N-terminal deletions of HSFA1a negatively affected the sensing of H2O2 and pH changes, which suggests that trimerizations were induced by HSF conformational changes. In addition, recently showed that an HSFA8 Cys residue is responsible for translocation to the nucleus upon oxidative stress: H2O2 treated protoplasts showed cytosol-to-nucleus translocations of the wild-type HSFA8, but not of the HSFA8C24S mutant variant. Interestingly, however, the N-terminal deletion of HSFA1a did not inhibit heat sensing. This shows that activation of this particular transcription factor is stress-specifically regulated despite a common dependency on oxidative activity. Notably, all stress treatments induced of HSFA1a binding to the HSP18.2 and HSP70 promoter, as detected by both formaldehyde cross-linking and chromatin immunoprecipitation, which paralleled the mRNA expression of these HSFA1a target genes.

9In addition to the above two processes, cellular communication between ROS and HSFs may involve mitogen-activated protein kinases (MAPK). HSF phosphorylation has been observed in yeasts and mammals and might thus occur in plants as well. Indeed, Arabidopsis HSFA2 was found to be phosphorylated by MPK6 on T249 after heat treatment, and this was associated with subsequent intracellular localization changes. Furthermore, MPK3- and MPK6-dependent phosphorylation of AtHSFA4A Ser309 and physical interaction between the proteins was reported recently. Activated HSFA4A in turn controlled the transcription of HSP17.6A. In tomato, heat-induced MAPKs were shown to transduce heat stress signals via HSFA3. In Arabidopsis, the same MAPKs that phosphorylate HSFs, namely MAPK3 and MAPK6, have been shown to be activated by H2O2. However, despite the presence of putative phosphorylation sites in tomato HSFA1, no heat-induced phosphorylation of this HSF was observed. Also, the phosphorylation site in AtHSFA4 was not conserved in HSFA4A proteins of citrus, grapevine and poplar. Taken together, this implies that both HSF oxidation and ROS-dependent phosphorylation can play a role in HSF activation, but that the latter is not a general signaling mechanism.

10In addition to activation of HSFs by ROS signaling, evidence for interaction between HSFs and ROS scavenging genes has also been obtained. The expression of APX1 was found to be regulated by HSFA2: overexpression of HSFA2 resulted in increased expression of APX1, while AthsfA2 knock out mutants showed a reduced expression of APX1. In agreement with this, AtHSFA2 overexpression lines showed increased heat and oxidative stress tolerance. Expression of a dominant-negative construct for AtHSFA4a prevented the accumulation of APX1 transcripts. Interestingly, the AtHSFA4a dominant-negative construct also prevented accumulation of the H2O2-responsive zinc-finger protein ZAT12, which is required for APX1 expression during oxidative stress. The ZAT12 promoter contains HSE binding sites and therefore, HSFA4a might directly interact with the ZAT12 promoter. However, HSEs are also present in the promoter region of the APX1 gene itself, suggesting that direct activation via HSFs is also possible. Using Pennisetum glaucum APX1 and a PgHSFA, a specific binding interaction between the APX1 HSE and HSF was confirmed, via in vitro gel shift assays as well as their expression patterns over time.

11Although APX1 has been shown to be a central component of the Arabidopsis ROS network, APX2, another isoform also localized in the cytosol, revealed a stronger induction by heat stress. AtHSFA2 has also been found to act as an APX2 activator. Transcription level comparison between wild-type and athsfa2 knock out plants revealed that transcripts of APX2 were absent in heat shock induced leaves of the knock out background, but present in the wild-type plants. Deletion analyses of the promoter region of APX2 functionally mapped the HSFA2 binding sites to HSEs near the transcription start site.

12In addition, and found strongly enhanced expression of galactinol synthase (GolS1 and GolS2) ROS scavenging genes in an HSFA2 overexpressing line.

13Combining these results, HSFA2 seems to play a central role in ROS scavenger expression and thus constitute an important link between heat shock and oxidative stress responses.

14Heat shock proteins function as molecular chaperones and play an important role in stress tolerance. In tomato, overexpression of the LeCDJ1 DnaJ protein coding gene (also known as J-protein or HSP40; ) resulted in improved thermotolerance, accompanied by increased APX and superoxide dismutase (SOD) activity after heat stress and reduced accumulation of O2– and H2O2. Despite the higher APX and SOD activity, transcription of the corresponding genes was not enhanced in the transgenic plants. Therefore, the influence of DnaJ proteins on APX and SOD activity was proposed to be post-transcriptional, due to their functionality as chaperones. Other studies have found similar effects of HSPs on ROS scavenging proteins upon heat stress. In Arabidopsis, overexpression of RcHSP17.8 enhanced SOD activity whereas overexpression of ZmHSP16.9 in tobacco enhanced POD, CAT, and SOD activity. Altogether, it may be hypothesized that the HSP proteins positively affect thermotolerance by protecting ROS scavenging protein conformation and activity, resulting in a lower ROS concentration.

15An alternative link between DnaJ proteins and ROS scavenging was suggested by. They showed that Arabidopsis AtDjB1 knockout plants (atj1-1) were more sensitive to heat stress than wild-type plants. After heat shock, the knockout plants showed an increased concentration of H2O2 and other oxidative products as well as a decreased concentration of the antioxidant ascorbate (ASC;; ). The viability of atj1-1 knockout seedlings after heat stress was rescued by exogenous ASC application. This suggests that lower concentrations of the antioxidant in atj1-1 knockout plants resulted in increased H2O2 concentrations leading to a decreased thermotolerance. As the underlying cause, the authors hypothesize a link with the electron transport chain (ETC). AtDjB1 directly interacts with a mitochondrial HSP70 and stimulates ATPase activity, a mechanism which is conserved among several kingdoms. AtDjB1 knockout potentially leads to the accumulation of cellular ATP, which feedback inhibits ETC. Because the last step of ASC synthesis is linked to the ETC, decreased ETC results in decreased ASC concentration and, consequently, the accumulation of H2O2.

16Although it is unclear whether there is a specific interaction between HSPs and the ROS scavenging machinery or that HSPs generally maintain protein functions, these results indicate that upon heat stress, accumulation of ROS is reduced via HSP supported ROS scavenger activity.

17In contrast to the positive effects of ROS reducing mechanisms on heat stress tolerance, an Arabidopsis study provided evidence linking enhanced ROS accumulation to higher stress tolerance. The research indicated the existence of a positive a feedback loop, whereby heat and ROS allow for further ROS accumulation, depending on the actions of microRNA398 (miRNA398). miRNA398 expression was found to be induced within 1 h and reach its peak 2 h after heat stress. The miRNA398 promoter region contains a putative HSE, and chromatin immune-precipitation assays revealed direct binding of HSFA1b and HSFA7b to the HSE promoter region under heat stress. Thus, association of these HSFs to the promoter region seems to be responsible for the induction of this miRNA upon heat stress. miRNA398 negatively regulates the expression of three target genes: CSD1, CSD2, and CCS. CSD1 and CSD2 genes are isoforms of copper/zinc-SOD scavenging genes which are located in the cytoplasm and chloroplasts, respectively and CCS is a copper chaperone encoding gene, which delivers copper to both CSD genes. Consequently, CSD1, CSD2, and CCS are down-regulated during heat stress, allowing further ROS accumulation. This pathway acts in an autocatalytic manner, as H2O2 in turn promotes expression of various HSFs, including HSFA7b. Accumulation of ROS seems to be an unfavorable response for the plant to survive heat stress. However, comparison of wild-type and csd1, csd2, and ccs mutants plants revealed higher heat tolerance in mutant plants while transgenic plants over-expressing miR398-resistant versions of CSD1, CSD2, or CCS were hypersensitive to heat stress.

18These unexpected outcomes may be explained by the increases in oxidative power for helping activate the primary set of HSFs at the start of the heat response. In contrast to; found that lack of miRNA398 enhances tolerance to some other stress factors, high light and chemically induced ROS, via enhanced expression of CSD1 and CSD2. Therefore, it seems that the benefit of reduced SOD activity is heat-specific, potentially due to the importance of high HSF activity in the first hours of the response to this stress.

19A number of recent studies have provided evidence for connections between HSFs, HSPs, ROS, and ROS scavengers upon heat stress. Here, we propose a comprehensive model on the relations between the various components to explain a large proportion of the observations (Figure 1). Through contrasting effects on ROS scavenging activity, heat shock induces a short-term positive (roughly, within the first few hours of heat stress) and a long-term negative feedback loop (after the first few hours of heat stress) on the HSF signaling pathway. The proposed complexity of the heat-stress response network is mirrored in some of the counter-intuitive observations, such as enhanced heat tolerance in certain scavenger mutants. However, analogous to the proposed miRNA398 mechanism, constitutively, slightly elevated ROS levels in such mutants may result in a primed state and, as a consequence, a stronger and/or faster response to a heat treatment. indeed showed that knockout mutations in CSD1 and CSD2 were accompanied by constitutively higher levels of HSF and HSP transcripts. In accordance, the importance of ROS at early heat response was shown by: a rapid oxidative burst of ROS during the first 15 min of the heat shock stimulates HSF DNA-binding and is essential for the induction of heat responsive gene expression, e.g., of HSPs and APX2. The typical “late” high mobility HSE-binding complexes, formed after 2 h, were shown to be ROS independent, which is in accordance with the production of anti-oxidants and ROS scavengers reducing the ROS overload after the early onset of the HSR. Nevertheless, if plants are continuously exposed to heat stress, the activity of some antioxidants and scavengers, e.g., APX and CAT, decreased after 3 days of heat stress in tomato, alfalfa and tobacco cell cultures. The changes of these components of the antioxidant system were ascribed to the impaired health and growth of plants under long term heat stress and are different from short term heat stress.

20Importantly, the model described here refers specifically to the complexity of events after a short term heat shock; its applicability to other types of heat stress, e.g., mild levels of heat stress, which only affect plant physiology in the long term, is not evident and more research will be necessary in order to clarify how the HSF/HSP and ROS systems behave under those circumstances. Also, it should be noted that the proposed model is not stand-alone and will interact with other factors, such as phytohormones. Abscisic acid (ABA), salicylic acid (SA) and ethylene have all been implicated in the heat response and can induce the production of ROS. While a number of phytohormone-related mutants show impaired tolerance to heat, application of these hormones may enhance thermotolerance via an effect of ROS. SA application, for example, enhanced SOD activity and HSP expression during heat stress. Dedicated analysis of the role of hormones during the first hours of heat treatment should clarify their putative positions in the response model.

21The model may well have broader applicability then to the heat response only. Not only are ROS accumulation, signaling and scavenging thought to occur and play a role in myriad other stress responses, but so is HSP activity. HSPs are also induced upon water stress, salinity and osmotic stress, cold, anoxia, UV-B light, and oxidative stress. Furthermore, overexpression of various HSFs enhanced tolerance to abiotic stresses other than heat, including salt, drought, osmotic, and anoxic stress. Also, tomato plants overexpressing the DnaJ/HSP40 LeCDJ1 showed both higher heat and chilling tolerance and overexpression of BRZ-INSENSITIVE-LONG HYPOCOTYLS 2 (BIL2), a mitochondrial-localized DnaJ/HSP40 family member, enhanced resistance against salinity and high light stress. The role of both the oxidative stress and HSF/HSP systems in multiple stress responses might explain the phenomenon of cross-acclimation, where exposure to a certain stress factor improves tolerance to a subsequent different stress factor.

22The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.