Progress in Lipid Research 51 (2012) 208–220

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Review

Heat shock response in photosynthetic organisms: Membrane and lipid connections Ibolya Horváth a, Attila Glatz a, Hitoshi Nakamoto b,c, Michael L. Mishkind d, Teun Munnik e, Yonousse Saidi f, Pierre Goloubinoff f, John L. Harwood g,⇑, László Vigh a,⇑ a

Institute of Biochemistry, Biol. Res. Centre, Hungarian Acad. Sci., Temesvári krt. 62, H-6734 Szeged, Hungary Department of Biochemistry and Molecular Biology, Saitama University, Saitama 338-8570, Japan c Institute for Environmental Science and Technology (IEST), Saitama University, Saitama 338-8570, Japan d National Science Foundation, Division of Integrative Organismal Systems, 4201 Wilson Boulevard, Arlington, VA 22230, USA e Section of Plant Physiology, Swammerdam Institute for Life Science, University of Amsterdam, Science Park 904, 098 XH, Amsterdam, The Netherlands f Department of Plant Molecular Biology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland g School of Biosciences, Cardiff University, Cardiff CF10 3AF, Wales, UK b

a r t i c l e

i n f o

Article history: Received 16 September 2011 Received in revised form 31 January 2012 Accepted 1 February 2012 Available online xxxx Keywords: Heat shock response Small heat shock proteins Molecular chaperones Membrane sensor hypothesis Transient Ca2+ influx Membrane fluidizer Signaling lipids Phosphatidylinositol 4,5-bisphosphate Phosphatidic acid

a b s t r a c t The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membrane’s physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membrane’s physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress. Ó 2012 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant and cyanobacterial heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. ch-sHSP and cyanobacterial sHSP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. ch-cpn60, and cyanobacterial GroEL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. ch-HSP70 and cyanobacterial DnaK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. ch-HSP90 and cyanobacterial HtpG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. ch-HSP100 and cyanobacterial ClpB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Synechocystis model of stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: BA, benzyl alcohol; DAG, diacylglycerol; DPI, diphenylene iodonium; HSP, heat-shock protein; HSR, heat-shock response; IP3, inositol-1,4,5trisphosphate; IP6, inositol hexaphosphate (phytic acid); JDP, J-domain protein; NBD, nucleotide binding domain; MD, middle domain; MG1cDG, monoglucosyldiacylglycerol; NEF, nucleotide exchange factor; PA, phosphatidic acid; PIP2, Phosphatidylinositol 4, 5-bisphosphate; PIPK, phosphatidylinositol 4-phosphate kinase; PLC, phospholipase C; PLD, phospholipase D; sHSP, small heat-shock protein (generally less than 45 kDa). ⇑ Corresponding authors. Tel.: +44 2920 874108; fax: +44 2920 874116 (J.L. Harwood), tel./fax: +36 62 432048 (L. Vigh). E-mail addresses: [email protected] (J.L. Harwood), [email protected] (L. Vigh). 0163-7827/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plipres.2012.02.002

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3.1. From cold sensing to heat sensing: evolution of the membrane sensor hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. A subclass of HSP-chaperones is membrane-associated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane lipids and heat stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Heat shock activates phospholipid-based signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Heat sensors and upstream signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. A role for PIP2 in the nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct evidence for a role of the plasma membrane in heat sensing in moss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. The heat shock response is transient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Calcium entry across the plasma membrane is essential to induce HSPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Membrane fluidizers activate heat shock genes in a calcium-dependent manner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Temperature is continually changing within the environment, both during the day and with the season. Photosynthetic organisms, such as plants and cyanobacteria are directly exposed to acute temperature alterations as well as climate change. Photosynthesis is considered among the most heat sensitive cell functions and, among its various components, the oxygen-evolving complex of photosystem II (PSII) is the most heat sensitive [1]. A mild sublethal temperature stress causes the rapid reprogramming of cellular activity to ensure survival during a subsequent exposure to noxious heat stress. This phenomenon called acquired thermotolerance depends on a prior priming mild heat treatment which is essential to induce the timely accumulation of cell components that reduce heat damage and mediate a rapid recovery of the cellular function during a subsequent recovery period. Many data indicate that, following a sub-lethal priming mild heat stress, there is an induction of heat-shock proteins (HSPs) that correlates with the development of acquired thermotolerance. Here we briefly mention five highly-conserved ubiquitously distributed HSP-families – all molecular chaperones – that are responsible in a major way for the onset of thermotolerance in plants. We then consider these results in the context of the growing appreciation that membrane lipids also play key roles in the acclimation of plants and cyanobacteria to stressful high temperatures. The early demonstration that formation in heat-stressed cells of damaged, non-native proteins alone is necessary and sufficient to activate hsp genes provided a conceptual framework for the view that HSPs (like, HSP90 and HSP70) may become recruited by heat-denatured proteins thereby acting as part of a ‘‘cellular thermometer’’ [3]. But several lines of evidence suggest that high temperature, cold and osmotic stress might be perceived by plants and cyanobacteria also via changes in the fluidity of their cell membranes [4–7]. It has been suggested that the elevation of membrane fluidity caused by the increased temperatures can activate the expression of hsp genes. Evidence to support this concept for photosynthetic cells was obtained first in Synechocystis PCC6803 [5]. At non-heat-shock temperatures, the membrane fluidizer benzyl alcohol (BA) activated the transcription of some hsp genes apparently as efficiently as heat stress. The ‘‘membrane sensor’’ hypothesis outlined here is by no means exclusive. Besides thermally-induced changes in membrane organization and protein conformation, the hypothetical list of possible thermometers includes alteration of RNA and histones as suggested recently [8]. Nevertheless, the central dogma in this field has been that the primary heat shock signal occurs as a feedback reaction to the accumulation of damaged proteins. Another possibility, which intuitively appears more appropriate for cell homeostasis under non-damaging mild warming conditions, is that a signal is generated before any damage has occurred: mild heat causing small

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incremental changes in membrane fluidity would be perfectly optimally capable of alerting the cell to an upcoming noxious heat stress in order to generate a timely molecular response of defense. Upon exposure to heat, a subset of HSPs, in particular the small HSPs (sHSP) become membrane-associated, localizing within specific membrane domains, thereby correcting in part the lipid order and phase-state of the membranes [9]. As also discussed below, such crosstalk between the primary heat-sensor, the membranes and the HSPs indicate a possible feedback mechanism to regulate their gene expression. Heat shock, like many other stresses, induces specific and highly-regulated signaling cascades leading to the transcriptional up-regulation of hsp genes. Here, we present new data on the operation of heat shock-activated phospholipid-based sensing and signaling pathways. It is suggested that in plants, a small Gprotein coordinates the activity of phosphatidylinositol (4)-phosphate kinase (PIPK) and phospholipase D (PLD) during the response to heat stress, responsible for the accumulation of the two key signaling lipids, phosphatidylinositol (4,5)-bisphosphate (PIP2) and phosphatidic acid (PA). Finally, in support of a central heat-sensing role of membranes, we will discuss recent experimental evidence that both heat and chemical manipulation of membrane fluidity can trigger a specific, transient Ca2+ influx from outside of the cell. Calcium entry across the plasma membrane is essential for heat to induce hsp genes. Remarkably, membrane fluidizers also activate hsp genes at constant low temperature in a strict calcium-dependent manner [6]. Taken together, these data suggest that early sensing of mild temperature increases occurs at the plasma membrane of plant cells independently from cytosolic protein unfolding.

2. Plant and cyanobacterial heat shock proteins Higher plant genomes contain about 300 genes encoding for molecular chaperones, co-chaperones and foldases. Whereas a non-lethal heat shock in Arabidopsis can up-regulate about 1300 genes, only 90 of them belong to the highly conserved families of molecular chaperones. The other 210 chaperones, co-chaperones and foldases which are not induced by heat and therefore cannot be called HSPs, may instead be induced by other stresses or are developmentally expressed in specific organs or remain constitutively expressed to carry out house keeping functions in protein homeostasis [2]. Thus, molecular chaperones take part both in many physiological as well as stress-related cellular processes. Ample evidence from transgenic plants has shown that HSP-chaperones are important for plant development and response to various stresses. All the five representative HSPs (below) are members of highly-conserved ubiquitously-distributed protein families. They are classified on the basis of their sequence similarity and named according to their approximate molecular masses on SDS

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gels [10,11]: HSP100/ClpB, HSP90/HtpG, HSP70/DnaK, HSP60 (chaperonin or cpn60)/GroEL and sHSP/IbpA/B. ClpB, HtpG, DnaK, GroEL and IbpA/B are the terms used for Escherichia coli members. In this section we describe briefly the chloroplast HSP-chaperones (ch-HSPs), a group of molecular chaperones that are unique to plants and are responsible in a major way for the onset of thermotolerance in plants. Included in this group are the evolutionarily-related chaperones of the cyanobacteria. Cyanobacteria are thought to be ancestors of chloroplasts, and have been used as model organisms to elucidate chloroplast functions. Thus, studies with cyanobacteria are useful in revealing the importance of molecular chaperones in chloroplasts. 2.1. ch-sHSP and cyanobacterial sHSP The Arabidopsis thaliana and Oryza sativa genomes each have one chloroplast sHSP (ch-sHSP) [12,13]. The ch-sHSP plays a role under stress since transgenic Arabidopsis plants which constitutively overexpress ch-sHSP acquire higher levels of thermotolerance than wild-type plants [14]. Similarly, acquisition of PSII thermotolerance in tomato plants constitutively expressing chsHSP was observed when heat stress or cold stress was administered in the presence of high light [15]. These results suggest that ch-sHSP protects Arabidopsis and tomato PSII from temperaturedependent oxidative stress. Sundby et al. [16] proposed that chsHSP could act as an antioxidant under oxidative stress using the conserved methionines to quench reactive oxygen species. Synechococcus elongatus PCC 7942 constitutively overexpressing a sHSP also acquired higher thermotolerance in the light than a reference strain [17]. Furthermore constitutive expression of a sHSP increased thermostability of PSII [17], while inactivation of a sHSP gene resulted in a reduced activity of photosynthetic oxygen evolution in heat-stressed cyanobacterial cells [18]. Interestingly, immuno-cytochemical studies showed that the main localization of sHSP in cyanobacterial cells shifts from the thylakoid area to the cytoplasm, then back to thylakoids during heat stress [19]. In vivo evidence has been obtained that lipid-mediated interaction of sHSPs with thylakoid membranes directly affects the PSII complex and leads to a greatly enhanced resistance to UV-induced PSII inactivation via facilitating PSII repair [20]. 2.2. ch-cpn60, and cyanobacterial GroEL The chloroplast type I chaperonin (ch-cpn60) is a cylindrical 14mer comprised of two stacked rings with sevenfold symmetry [21]. Ch-cpn60s from various higher plants consist of stoichiometric amounts of two divergent subunits, a and b [22], which reside in the same tetradecamer [23,24]. A ch-cpn60a mutant showed a defect in chloroplast/embryo development [25], while a ch-cpn60b developed lesions and is less thermotolerant than the wild type [26]. Recently, it was shown that both ch-cpn60a and ch-cpn60b are required for plastid division in Arabidopsis [27]. Cyanobacterial genomes generally contain two groEL genes [28– 32]. One of them, groEL1, forms an operon with groES and is essential in Synechococcus elongatus PCC 7942 [33,34]. GroEL1 forms a tetradecamer, while GroEL2 forms a heptamer or dimer, but the GroEL1/GroEL2 oligomers are extremely unstable [32]. 2.3. ch-HSP70 and cyanobacterial DnaK A. thaliana and O. sativa genomes each have two chloroplast stromal HSP70s [35], which are essential for plant development [36]. In addition to the import of nuclear-encoded polypeptides [37], a role for ch-HSP70 in protection and repair of PSII during and after photoinhibition has been shown in the green algae Chlamydomonas reinhardtii over- or underexpressing ch-HSP70 [38].

Cyanobacterial genomes also contain multiple genes encoding for DnaK. DnaK2 and DnaK3 of the three DnaKs are essential in Synechococcus elongatus PCC7942 [39]. 2.4. ch-HSP90 and cyanobacterial HtpG The Arabidopsis genome contains one copy of a gene encoding an HSP90 targeted to chloroplast (ch-HSP90) [40]; which is constitutively expressed and induced further by heat or light [41]. A htpG knockout mutant of Synechococcus is sensitive to high light and cold stresses [42,43] and mutants of both Synechococcus and Synechocystis exhibited significant loss of thermotolerance as compared with the wild type strain [44,45]. High sensitivity to methyl viologen was observed [43], indicating that HtpG is involved in responses to oxidative stress. 2.5. ch-HSP100 and cyanobacterial ClpB Three genes encoding HSP100 have been identified in the Arabidopsis genome [46]. There are cytosolic, chloroplastic, and mitochondrial homologs. The cytosolic one is required for acclimation to high temperatures [46,47]. The T-DNA insertion mutants of chloroplast and mitochondrial homologs showed no evidence for heat stress phenotypes [48], however, the chloroplast homolog was shown to be essential for chloroplast development [48]. In the S. elongatus PCC 7942 genome, there are two clpB genes. The gene encoding for ClpBI is not essential under normal conditions, but is heat-induced and required for thermotolerance [49]. On the other hand, the other gene (clpBII) is not induced by heat shock, but is essential [50]. 3. The Synechocystis model of stress 3.1. From cold sensing to heat sensing: evolution of the membrane sensor hypothesis The two physiologically important membranes in the cyanobacterial cells are the plasma membrane and the thylakoid membrane. The former is the boundary membrane responsible for controlling the influx and efflux of ions and metabolites, whereas the latter is the site of energy-related metabolism, such as photosynthesis, respiration and ATP synthesis. Based on the results obtained by using surface membrane selective catalytic lipid hydrogenation of cyanobacterial cells [51], it has been shown that modification of the membrane’s physical state induces the cold-responsive D12 desaturase (desA) gene in Synechocystis PCC 6803 under isothermal conditions [4]. Catalytic hydrogenation of a small pool of plasma membrane fatty acids in live Synechocystis cells stimulated the transcription of the desA gene in the same way as chilling. In addition, hydrogenated cells displayed an elevated sensitivity to cold stress, indicating that hydrogenation and cooling have ‘‘additive effects’’ on desA induction and that cold perception is mediated through changes in membrane fluidity. These and other findings led to the hypothesis that the physical state of cellular membranes might be involved in temperature sensing in Synechocystis PCC 6803 (for reviews see [1,52]). A membrane-associated histidine kinase, Hik33, and a soluble histidine kinase, Hik19, appear to be involved in the perception and transduction of the low-temperature signal. Using a reporter construct driven by another Synechocystis desaturase (desB::lux) [53] it was shown that deletion of any of the two histidine kinases (Hik19 and Hik33) strongly reduced the cold inducibility of desB and desD genes (but not desA). Microarray analysis of Hik33 mutant revealed that the expression of several genes have been changed in the mutant strain [54]. Interestingly, the desB and desD

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desaturases genes were not among the genes whose expression was significantly suppressed by the Hik33 inactivation under cold stress. Similarly, the cold-induced expression of several members of the protein synthesis machinery (which are thought to have an important role in cold stress adaptation) remained unchanged in the Hik33 mutant. Also, the cold-protective role of the genes, whose expression displayed marked alteration in Hik33 mutants, remained to be established [55]. Further microarray measurements in fatty acid composition mutants (and desA/desD) revealed that gene-engineered removal of polyunsaturated fatty acids led to the increase of the cold inducibility of certain chaperone genes (hspA, dnaK2, clpB1), some sulfate-transport genes (sbpA, cysA, cysT, cysW) and Hik34 (suggested to be involved in thermotolerance) as well [56]. Interestingly, most of the induction levels of these genes did not change significantly in Hik33/desA/desD mutant. Rather, the cold inducibility of high light-inducible proteins (hliA and hliB; [57], in desA/desB background was moderately diminished by Hik33 inactivation [56]. It was suggested that these genes might play a role in protection against high light [57]. Interestingly, it has also been shown that the Hik33 (also called as dspA) mutant is sensitive to high light stress under normal temperature (30 °C). The authors suggested that Hik33 is able to perceive light, redox, and/or reactive oxygen species [58]. Combining the above data with results reporting that Hik33 is involved in sensing of salt[59] hyperosmotic- [60] and hydrogen peroxide stress [61] as well, Hik33 might be considered as a general stress sensor and the existence of additional sensors more specific to cold stress (like Hik19) can be expected [62]. According to the classic model, during thermal stress, proteins tend to lose their native conformation and serve as a signal to induce the heat shock response (HSR) [63]. In Synechocystis PCC 6803, changes in the thylakoid membrane’s physical properties alter the temperature threshold of the induction of all the major heat shock genes [1,5]. In analogy to cold stress, if heat shock is truly transduced into an intracellular signal at the level of membranes, then any manipulation that modulates membrane physical parameters (fluidity, lipid domain organization) should alter the profile and temperature threshold of HSP response. To confirm this working hypothesis, Northern blot analysis was performed on cells acclimated to contrasting temperatures (22 °C vs. 36 °C), treated by the membrane fluidizer benzyl alcohol (BA) or which had been catalytically saturated selectively in their cytoplasmic (plasma) membrane before various heat exposures [5]. The findings clearly showed that in cells acclimated to 36 °C, all the hsp-genes tested (groESL, groEL2, dnaK2 and especially hsp17 also known as hspA) were activated at significantly elevated temperatures in comparison with the low temperature-grown counterparts [5]. The threshold temperature for the maximal activation was 44 °C in 36 °Cadapted samples, whereas this value downshifted to 38–40 °C in 22 °C-grown cells. Moreover, unlike dnaK, groEL, and cpn60, the hsp17 gene is transcribed exclusively at 44 °C and above in samples of 36 °C-grown cells [5]. These results also demonstrated that the temperature set point for the activation of the hsp-genes is not a fixed value but is clearly affected by the growth temperature. The main factors governing the long-term acclimation in cyanobacteria to ambient temperatures are changes in the level of lipid unsaturation and ratio of proteins to lipids in the membrane [3,64–66], which in turn, cause alterations in the membrane physical state. To test whether correlation between growth temperature, thylakoid thermosensitivity, and activation of hsp-genes are truly mediated via changes in the membrane physical state, the membrane fluidity of isothermally grown cells was modulated before temperature stress. Administration of BA to 36 °C-grown cells, causing reduced molecular order in both the cytoplasmic and thylakoid membranes, dramatically lowered the threshold

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temperature of heat shock gene activation [5] Further supporting its unique feature, the administration of the fluidizer increased the hsp17 either at mRNA or protein level for the whole temperature range tested. The selective modulation of plasma membrane physical state achieved by catalytic hydrogenation of 22 °C-grown cells resulted in no significant effect on the temperature profile of the induction of hsp-genes [5]. Since the in vivo catalytic saturation was shown to be confined mostly to one lipid class (phosphatidylglycerol) in the plasma membrane [4], at that stage we concluded that alterations both in plasma- and thylakoid membrane fluidity are involved in sensing high temperature stress and can control the expression of hsp-genes. Elevation of temperature resulted in a fast, transient stimulation of H2O2 production in the tobacco cells [67]. Moreover, a similar H2O2 burst could be induced by membrane fluidization using BA. Diphenylene iodonium (DPI), a NADPH oxidase inhibitor, prevented both the heat- and BA-triggered H2O2 rise. Likewise with Synechocystis, the synthesis of sHSPs was shifted to lower temperatures by BA application and was suppressed by DPI treatment [67]. Heat stress-induced H2O2 was shown to be required for the effective expression of heat shock genes in Arabidopsis [68]. Taken together, these data indicate that formation of H2O2, an early component of the heat-signaling pathway, which responds rapidly to changes in membrane fluidity is required for the activation of sHSP synthesis [67,68]. Other factors involved in the regulation of the heat shock genes of Synechocystis include light conditions and photosynthetic electron transport. These have been implicated in case of the chaperonin genes (groESL1 operon, groEL2). In addition, a role for a HrcAtype repressing mechanism has been suggested [69] and reported [70]. Recently a novel, HrcA independent positive regulatory mechanism was also demonstrated together with increased survival rate of the HrcA mutant compared to wild-type cells upon applying lethal temperature stress [71]. Although under different growth and stress conditions, the histidine kinase hik34 mutant also survived lethal heat treatment [72]. Based on microarray data demonstrate that the hik34 mutation has no significant effect on heat induced gene expression, but when overexpressed, heat shock gene expression is repressed. These results indicate that Hik34 negatively regulates the heat shock genes [72]. Synechocystis PCC 6803 contains nine RNA polymerase sigma factors of which SigB proved to be heat inducible at the protein level [73]. Inactivation of sigB was shown to decrease the steady state mRNA level of different heat shock genes after shifting the cells from 32 to 43 °C for 60 min, but whether the rate of induction or the stability of the corresponding mRNAs are changed, remains to be established [74]. Recently, another sigma factor, SigC was suggested to be involved in the heat acclimation of Synechocystis PCC 6803 in a carbon dioxide concentration dependent manner [75]. Recent microarray analysis has shown that the topology of DNA is also involved in the regulation of genes induced by cold, heat, and salt stress [76]. Since the structure and function of cellular macromolecules (proteins, lipids, RNA, DNA, etc.) are sensitive to the temperature changes, the emergence of new sensor candidates can be expected (for review see [77,78]). Moreover, Kortmann et al. [78] demonstrated that the hsp17 mRNA in Synechocystis might also serve as a thermosensor. These authors have identified a short 50 untranslated region, which inhibits the translation of the mRNA when cells are kept at normal growth temperatures but allows the synthesis of the protein upon heat stress. Although HSP17 is present at normal growth temperature when cells are subjected to membrane fluidizers [5] or high light [1,78] further studies are required to clarify this interesting regulatory mechanism. It should be noted that most of the data discussed above are based on microarray experiments. It should be emphasized (see [79]) that to exert their protective function, the induced genes

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must be translated and/or their products be modified post-translationally. Microarray data do not provide any information about the fate of the detected transcript. On the other hand, if a regulatory protein needs only post-translational modification to fulfill its signaling function, its gene need not necessarily be induced by the given stress. Therefore future efforts will be required to identify the effector proteins linking primary temperature-sensing event to stress defense. 3.2. A subclass of HSP-chaperones is membrane-associated Changes in membrane composition and/or fluidity in yeast, E. coli, and plant and mammalian cells uniformly alter the ‘‘set

point’’ for HSP induction, with expression initiated at lower temperatures in cells with more fluid membranes [6,7,80–82]. In addition a lipid-selective association of a subpopulation of cyanobacterial HSPs (first shown for GroEL and thereafter for HSP17) with membranes can cause an increased molecular order and reduced propensity to form membrane-disrupting non-bilayer lipid phases [9,83–86]. It was proposed that HSP-membrane interactions may lead to down-regulation of the heat shock gene expression [87,88]. The association of certain amphitropic HSPchaperones and lipid molecular species thus may remodel the pre-existing hyper structure of membranes [83]. Lipid-mediated membrane association of HSP-chaperones is not confined to cyanobacterial HSP17 and GroEL/GroES but it is also a

Fig. 1. Thylakoid membranes acting as high temperature sensors in cyanobacteria. Upon membrane perturbation caused by heat or membrane fluidizers, the membrane fluidity is increasing. It leads to the induction of a specific set of HSPs, activation of MGlcDG synthase and likely to the inhibition of MGlcDG epimerase which is a committed step in MGDG synthesis. As the desaturation takes place on MGDG molecules, the above changes in enzyme activities lead to the accumulation of highly saturated ‘‘heat shock lipid’’, MGlcDG. MGlcDGs and a subfraction of sHSPs translocated to membranes via their lipid anchors readjust fluidity and attenuate the stress response.

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feature of additional bacterial chaperones, such as E. coli sHSPs (IbpA and IbpB) [89] and cyanobacterial DnaK, a homolog of mammalian HSP70 [90]. Likewise, HSP17 in Synechocystis, E. coli IbpA and IbpB (separately and together) can also regulate the membrane fluidity and modulate membrane lipid polymorphism, as well [89]. The association of the sHSP HSP16.3 with the plasma membrane of Mycobacterium tuberculosis was also documented: dissociation of the sHSP oligomers was a prerequisite of the membrane localization [91]. It is tempting to speculate, that being either residents or visitors [92], a specific subset of these HSP-chaperones may also participate in homeostatic controlling mechanisms, which may ultimately retailor the lipid composition and consequently control basic functions of HSP-hosting membranes. To our knowledge, the possible participation of HSP-chaperones in the active regulation of the lipidome of cells and membranes is has, up-to-now, been completely overlooked. The rapidly formed monoglucosyldiacylglycerol (MGlcDG) may act as a novel ‘‘heat-shock lipid’’ that can stabilize membranes at the early phase of thermal stress via its highly saturated fatty acyl chains and by its unique capability to interact selectively with (and thereby anchor) HSP17 in Synechocystis thylakoids [86]. Recently, Ohta and co-workers further examined the underlying mechanism of such an increase of MGlcDG following heat shock in Synechocystis by measuring MGlcDG synthase (Sll1377) activity [93]. Whereas the Sll1377 protein level remained unchanged, a high temperature dependent activation of Sll1377 was observed exclusively in the membrane fraction of Synechocystis. Surprisingly, if expressed in E. coli the recombinant MGlcDG synthase was also found primarily in the membrane fraction and its activity increased upon temperature elevation. In this context, Sll1377 could be a ‘‘heat-sensing’’ protein playing a key role in regulating cell signaling at high temperature and its membrane fluidity-controlled activation may be a more rapid and efficient response than transcriptional/translational up regulation or covalent modification. These results suggest that elevated temperature first rapidly activates MGlcDG synthase in a membrane fluidity-mediated manner, which is followed by an increased level of MGlcDG that ultimately stabilizes the membranes via binding to HSP17, thereby promoting cell survival (Fig.1). The possibility that MGlcDG epimerase inhibition can also play role in the accumulation of MGlcDG cannot be ruled out. An additional role of the high temperature-dependent increase in MGlcDG could be to preserve membrane integrity via its highly saturated fatty acids with strong bilayer stabilizing propensity, as shown by in vitro catalytic hydrogenation of thylakoids more than 20 years ago [94,95]. A high temperature-dependent hyper-activation of the heat shock lipid synthase has also been observed in the membrane fraction of another cyanobacteria, Anabaena variabilis [96]. It has been established that of the three dnaK genes, mostly one (dnak2) is transcribed and exhibits a typical heat stress response in Synechocystis and, most importantly, a subset of this gene product is thylakoid-associated. Earlier attempts to disrupt dnaK2 by insertional mutagenesis led to the formation of merodiploids, thereby confirming that this gene is essential [90]. It should be crucial to understand the complexity of ‘‘cross-talk’’ between DnaK2 and rest of the major, membrane fluidity controlled chaperones (HSP17, GroEL/GroES). To our knowledge, there are no studies available concerning the factors which govern the association of prokaryotic DnaK with membranes via lipids. However, there is evidence, for specific and high-affinity interactions between mammalian HSP70 and certain lipid classes (such as the lysosomal membrane lipid lysobisphosphatidic acid, (LBPA) and globotriaoslyceramide (Gb3) [97–99]). Induction of HSP-chaperones in cyanobacteria occurs also after exposure to UV-B radiation or strong visible light [100]. These phenomena indicate that HSP-chaperones may have an important role

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in protection against photo-oxidative stress. The thylakoid-associated pool of the sHSP HSP17 can provide protection against UV-B stress in Synechocystis, with a specific mutation of HSP17 (Q16R) dramatically altering its membrane/lipid association properties. In a systematic study the HSP17 mutant Q16R was found to have greatly enhanced lipid-mediated interaction with thylakoid membrane, which directly affected the PSII complex and led to a much increased resistance to UV-induced PSII inactivation through facilitating PSII repair. This was the first documentation of the significant improvement of UV-B stress tolerance attained by targeting a mutant HSP to the thylakoid membrane [20]. Further underlying the importance and future potential of membrane-sHSP interaction, introduction of the carrot sHSP HSP17.7 into potato through gene transfer was shown to enhance its thermotolerance via affecting cellular membrane stability [101]. The sHSP Lo18 from lactic acid bacteria Oenococcus oeni reduced in vitro thermal aggregation of proteins and modulated the membrane fluidity of native liposomes, as well. It was shown, that an alanine 123 to serine substitution induces a decrease in chaperone activity, and that tyrosine 107 is required for membrane lipid rigidification, suggesting that different amino acids are involved in the thermo stabilization of proteins and in fluidity regulation of membranes [102]. Inactivation of a sHSP (HSP 18.55) affected both the cell morphology and membrane fluidity in Lactobacillus plantarum [103]. Taken together: the primary composition and physical state of the lipid phase of membranes can control both the expression and membrane-association of pre-existing HSP-chaperones at the early phase of heat stress. Interaction of newly formed HSP-chaperones with membranes (together with the hitherto unexplored cross-talk of stress protein and membrane lipid homeostasis) may represent an additional layer of this control. These findings urge a substantial reevaluation of the basic cellular functions ascribed to the ‘‘moonlighting’’ stress protein molecular chaperones [104]. The properties of membranes are modulated not only by changes in the physical state of the lipid phase, but also by chemical modifications of the hydrophilic lipid head groups. In the case of phospholipids, such changes have been implicated in a diversity of key cellular functions, including signaling events that lead to rapid modulation of ion channels, gene expression patterns and the cytoskeleton. Recent evidence demonstrates that in addition to its effects reviewed above, heat stress also initiates rapid, large scale changes in the pattern of lipid signaling. The view of heat stress from the vantage point of phospholipid-based signaling is discussed in the next section.

4. Membrane lipids and heat stress 4.1. Heat shock activates phospholipid-based signaling pathways Two key signaling lipids, phosphatidylinositol (4,5)-bisphosphate (PIP2) and phosphatidic acid (PA), rapidly accumulate in plant cell membranes after the onset of a temperature stress [105]. PIP2 is an extensively characterized participant in an astounding diversity of processes throughout the cell [106–111]. PA, although less well understood than PIP2, is also known to play essential roles in a diversity of cellular processes [112–114]. The participation of these lipids across the entire cell physiological landscape suggests their role in the HSR will likely be far from unitary. PIP2 is one of the best studied phosphoinositides, originally gaining interest as the substrate of phospholipase C (PLC), which cleaves it to generate inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG) (for historical overview see [115]). In animal cells (but likely not in plants, see below) these products are major

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second messengers, with IP3 binding to receptors on the endoplasmic reticulum, thereby releasing Ca2+ from that organelle into the cytoplasm and DAG activating protein kinase C (PKC). The manifold downstream events following PLC catalyzed cleavage of PIP2 are now known to be only one aspect of the role of this lipid in orchestrating cellular functions. By acting as a highly localized docking site for effector proteins that can be rapidly constructed and disassembled [116], PIP2 nucleates domains on the cytoplasmic surfaces of cellular membranes that modulate the structure of the actin cytoskeleton, drive endocytosis, and regulate the activity of ion channels [108,109,117,118]. Under non-stressful conditions PIP2 is a minor component of cellular lipids, especially in plants where it represents 0.1% of total phospholipid pools. Heat stress induces rapid PIP2 accumulation, with significant increases evident within two minutes after the onset of a temperature increase from 22 °C to 40 °C in tobacco BY-2 cells metabolically pre-labeled with 32P-phosphate to detect new lipid synthesis. Increases observed in experiments such as these are in the range of 5- to 6-fold after a 30 min stress period. The increase occurs at temperatures as low as 35 °C, a mild heat stress with no effect on viability of the cell cultures or seedlings [105]. The cellular location of the heat-induced PIP2 accumulation suggests potential functions. PIP2 was imaged by confocal microscopy in living BY-2 cells expressing the PIP2 biosensor YFP–PHPLCd1. With the very low PIP2 levels in cells at control temperatures, the marker distributes uniformly throughout the cytoplasm. A jump to 40 °C initiates the following progression: the plasma membrane (identified by its co-labeling with the lipophylic dye, FM4-64) becomes brightly fluorescent within 15 min of the temperature increase. Over the next 45 min, labeling of the plasma membrane decreases in intensity, accompanied by a gradual accumulation of fluorescent punctuate structures in the cytoplasm. In addition, beginning at 30 minutes of heat stress, the nuclear envelope and later the nucleolus display the PIP reporter [105]. The numerous well-defined functions of PIP2 at the plasma membrane, in light of the heat-triggered accumulation of that lipid at that location, suggest that survival during heat shock requires adjustment in one or more of these processes as part of the response and adaptation to this stress. PIP2 is a major orchestrator of endocytosis, with its synthesis at the site of vesicle formation and its role in the recruitment of the AP-2 complex [108] to the membrane. In addition, the targeted synthesis and hydrolysis of PIP2 during vesicle budding is thought to provide the ‘pinching force’ that releases newly formed coated vesicles from the membrane [117]. The accumulation of PIP2- containing punctuate structures in the cytoplasm as heat stress continues, may be an indication that elevated levels of endocytosis are part of the overall heat stress response. Elevated rates of endocytosis may be a component of stress responses in general, given that salt-stress induces the formation of PIP2-enriched clathrin coated vesicles in plants [119]. Other well-characterized roles of PIP2 at the plasma membrane include modulating actin dynamics by way of its interaction with several actin-binding proteins [108] and its regulation of a large number of channels, ion pumps and other integral membrane proteins (reviewed in [111]). In plants massive remodeling of the cytoskeleton is an early consequence of heat stress. For example, the F-actin cortical arrays throughout the plant completely disappear within a few minutes of the onset of a 41 °C heat stress, recovering over several hours upon return to 20 °C [120,121]. Whether PIP2 plays a role in these dramatic heat-induced remodeling events remains to be determined. 4.2. Heat sensors and upstream signaling The immediate upstream cause of PIP2 and PA accumulation after a temperature increase is heat-induced new synthesis of

these lipids rather than a reduction in their turnover rates. Pulselabeling of tobacco BY-2 cells with 32P-phosphate revealed that the PIP(2) response is due to the activation of a PIPK rather than inhibition of a lipase or a PIP(2) phosphatase. [105]. Thus, heat stress rapidly activates a phosphatidylinositol-phosphate kinase (PIPK). Genes encoding this enzyme in plants comprise a large multi-gene family (11 members in Arabidopsis) with different substrate specificities and tissue localization patterns [122,123]. The family member(s) responsible for heat-induced PIP2 accumulation has not yet been identified. PA synthesis occurs either through diacyglycerol kinase (DGK), which catalyzes phosphorylation of diacyglycerol (DAG), or by the action of phospholipase D (PLD), which transfers the phosphatidyl group of a structural phospholipid to water to generate PA and a free head group. Using 1-butanol as an alternative substrate to water, PLD activity can be monitored by assaying the accumulation of the non-biological lipid phosphatidylbutanol [124–126]. Experiments using this protocol show increases in PLD activity beginning within one to two minutes after the onset of a temperature increase. Consistent with these results, in the pulse-chase experiments described above, PA incorporated only small amount of radioactivity, thereby indicating that structural phospholipids, rather than DAG, are the source of the PA which accumulates during heat stress [105]. The rapid induction of PIPK and PLD after the onset of heat shock suggests that the regulation of activity of these enzymes in response to temperature increases occurs post-translationally. Thus it comes as no surprise that PIPKs and PLDs are not among the genes whose transcript levels have been reported to increase substantially during temperature stress [127,128]. Although links between heat perception and enzyme activation remain unknown, observations derived from the study of these enzymes in animals suggest that small G-proteins may play a role in this process. Moreover, PA activates a member of the PIPK family of enzymes, while PIP2 is known to activate PLD. Furthermore, the small G-protein ARF6 activates both a PLD and a PIPK. The links between enzymes, substrates and products suggested that activation of ARF6 generates a feed-forward loop that stimulates both PIPK and PLD, leading to potentially large and coordinate activation of both enzymes and increased synthesis of PIP2 and PA [108]. Over-expression studies in plants of heterotrimeric G-protein subunits [129–131] are consistent with a role for G-protein signaling in mediating stress responses but direct observations from plants to support a mechanism such as this is lacking. Complementing the genetic approaches, inhibitor data also suggest that further research into the role of G-proteins in the response to heat stress is merited. Aluminum fluoride binds small G-proteins (and ARF6 in particular), stabilizing their confirmation in active, signaling-competent conformations. It thereby prevents the cycling between GTP- and GDP-bound states necessary for its function. When included in the metabolic labeling assays used for monitoring changes in phospholipid levels, aluminum fluoride completely blocks heat-induced increases in PIP2 and PA [105]. It is thus conceivable that a small G-protein in plants coordinates the activity of PIPK and PLD during the response to heat stress. A candidate for this role is the Arabidopsis ARF6 homolog, ARFB1a (At2g15310), which is expressed throughout the plant [132] and is localized to the plasma membrane [133]. A link between a primary heat sensor and the phospholipid signaling events that follow a temperature stress remains to be determined. The signaling cascade initiated by the heat-generated Ca2+ influx at the plasma membrane includes many critical components of the HSR, namely activation of kinases and calmodulin, synthesis of HSPs and the induction of thermotolerance [6]. It is not known whether the activation of PIPK and PLD is among these. The Ca2+ influx that accompanies heat stress appears due to the activation

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of a non-selective Ca2+ permeable channel, with the increased membrane fluidity that accompanies a temperature rise [134] causing the channel to open. Increases in PIPK and PLD activity may be a secondary consequence of events initiated by the rise in cytoplasmic Ca2+ concentrations. Alternately, phospholipid signaling may occur in parallel with these events, with heat-induced membrane changes other than the Ca2+ influx, (activation of small G-proteins?) bringing about PIP2 and PA accumulation. In either case, the localized membrane remodeling that accompanies the synthesis of these two lipids may contribute to the specificity that appears to be required for the incoming Ca2+ to generate the heat signal. Among the many properties of PIP2 is its capacity to activate Ca2+ channels in the plasma membrane [116]. In plants, the relationship of PLC to Ca2+ mobilization remains uncertain [107,118,135]. Since two canonical components of the PLC signaling pathway, IP3 gated Ca2+ channels and DAG-dependent PKC appear to be absent in the lineage leading to the vascular plants, alternate modes of action for this enzyme are likely [118]. Recent evidence suggests that plants efficiently phosphorylate IP3 to generate IP6 (phytic acid), with this polyanion serving the role as second messenger for Ca2+ release and other functions [136,137], reviewed in [118]. Since PLC activity is required to sustain a HSR (see below), IP6 might be a component of this system. It is perhaps noteworthy that heat treatment of Arabidopsis seedlings leads to a rapid six-fold increase in transcript levels for myo-inositol 3-phosphate synthase, which catalyzes the first step in the lipid-independent pathway for IP6 synthesis [118,128]. These observations thus suggest that heat stress brings about increased levels of IP6 through both the lipiddependent and lipid-independent routes. A growing body of evidence implicates the activity of PLC in the heat stress response. Although indirect, studies with the PLC inhibitor U73122 suggest that an active PLC is required for the HSR to proceed. This inhibitor blocks the HSR in Physcomitrella patens [6] as well as heat induced PIP2 and PA accumulation in BY-2 cells [105]. This raises the possibility that heat stress, by activating both PIPK and PLC, generates the futile cycling of PIP2. Rather than a wasteful use of ATP, however, a cycle such as this may be a mechanism to obtain spatio-temporal control over PIP2 levels. Indeed, it has recently been pointed out that PIP2 cycling may be instrumental in regulating signaling complexes at the plasma membrane [138] and in generating sufficient levels of signaling metabolites upon cell stimulation [139]. 4.3. A role for PIP2 in the nucleus The important events in the nucleus of course center on DNA replication and transcription, and indeed, a recent report demonstrates that a particular histone variant (H2A.Z) mediates transcriptional changes in response to temperature [140]. It is thus particularly intriguing that, as discussed above, PIP2 accumulates in the nucleus during heat stress. A rapidly growing literature (reviewed in [141]) implicates PIP2 in various aspects of RNA processing and transport and regulation of nuclear actin. Furthermore, nuclear IP6 appears to play a role in transcriptional regulation with the discovery that it is a co-factor of the auxin receptor TIR1 [142]). The nucleus contains its own enzymatic machinery for the synthesis and turnover of phosphoinositides; whether these are activated in response to heat stress, as well as the function of nuclear PIP2 remain exciting avenues for further study. 5. Direct evidence for a role of the plasma membrane in heat sensing in moss For plant cells it is vital to accurately sense early signs of temperature change, before cellular damage occurs and to trigger

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appropriate cellular defences in anticipation of more severe damaging conditions. For their colonization of terrestrial environments, former aquatic plants had to adapt to dry lands, and successfully acquire sophisticated protective mechanisms to withstand dehydration, excess light and extreme temperature variations [143– 146]. Among photosynthetic organisms, bryophytes have a unique phylogenetic position between algae and vascular plants. They can tolerate extreme abiotic stresses and show high ability to recover from them [147,148]. Mosses are thus attractive models to investigate the evolution of temperature sensing and heat signaling in plants. Earlier studies in mammalian cells and higher plants suggested that HSP70 and HSP90 regulate the HSR by interacting, consequently inactivating, heat shock factors (HSFs) [63,149,150]. Recently, however, the earliest events of thermal perception in the moss P. patens were shown to involve a specific calcium influx initiating at the plasma membrane [6].

5.1. The heat shock response is transient When submitted to a temperature rise, a rapid and transient elevation of cytosolic calcium is observed in moss cells. The amplitude of this calcium influx was proportional to the intensity of the applied heat shock and determined the extent of the subsequent HSP synthesis [6]. A similar Ca2+-signature was also observed in heat-treated tobacco cells [151], suggesting a highly conserved mechanism for temperature sensing among land plants. The transient elevation of Ca2+ under heat shock was followed by a refractory period during which no further heat-induced Ca2+-influx was observed despite the ongoing heat shock [6,151]. Previous studies in A. thaliana showed that mRNA synthesis of major classes of HSPchaperones, such as HSP101, HSP90, HSP70 and sHSPs, was also transient [152,153]. In P. patens exposed to a continuous mild heat shock, HSP synthesis was found to be induced only during the first hours [6,154]. As in the case of Ca2+ influx, despite the maintenance of elevated temperatures, de novo synthesis of HSPs became rapidly repressed and a re-setting period of 5 h at non-inducing temperature was needed to restore the full potency of another heat shock [6]. In P. patens, the observed refractory period was independent of the levels of cytosolic chaperones. After 5 h recovery postheat shock, moss cells responded to a second heat shock by inducing a strong HSP expression, despite the presence of elevated amounts of HSP70 [6]. This suggests that chaperones play a less important role in the early events of heat perception than initially reported. The response to heat shock is thus transient and sequential. An immediate Ca2+-influx is followed by transient mRNA synthesis that resulted in transient protein expression (Fig. 2).

Fig. 2. Schematic representation of the sequential events occurring in the cell following continuous exposure to a mild temperature increment. A temperature increase immediately triggers a rapid and transient elevation of cytosolic Ca2+ concentration. Subsequently, a transient synthesis of different HSP mRNAs occurring and results in transient protein expression, which takes place within hours.

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Interestingly, the amount of HSPs that were induced in P. patens by a specific heat shock did not depend only on the inducing temperature, but also on the basal growth temperature [6,155]. When mosses which had been pre-acclimated at 22 °C were treated 1 h at 34 °C they showed a four fold higher HSR compared to mosses preacclimated at 30 °C. This correlated with a massive increase in the lipid saturation of their membranes at 30 °C as compared to 22 °C. This substantiated earlier reports in other organisms [5,80–82] and suggested that membranes, which are capable of adapting their lipid composition to the growth temperature, might well be involved in the thermo sensory response.

5.2. Calcium entry across the plasma membrane is essential to induce HSPs Several reports showed the involvement of calcium in higher plants’ response to elevated temperatures [151,156]. In moss, depletion of extracellular calcium inhibited the heat-induced Ca2+ influx, prevented the induction of HSPs and negatively affected the acquired thermotolerance [6]. Precise dose response analysis showed that micromolar amounts of Ca2+ ions (or Sr2+, or Ba2+), but not of Mg2+, Na+, or K+, could specifically drive the plant HSR. Electrophysiology experiments confirmed the existence of a temperature-sensitive calcium channel in P. patens plasma membrane, which was transiently activated following a temperature increase [6]. This showed that the heat-activated calcium channel was similar to other voltage- or ion-gated channels as it transiently responds to a temperature stimulus and become inactivated as long as the stimulus persists [157]. Additional experiments confirmed that the first minutes of a temperature increase are particularly crucial [6]. The passage of Ca2+ through the plasma membrane during this initial phase was central to the proper induction of HSPs. A ten minutes delay in Ca2+ availability during the first 30 min of a heat shock at 38 °C reduced by half the Ca2+ signature and the subsequent HSR and thermotolerance (Fig. 3), while other Ca2+-dependent stress signaling pathways remained unaffected. These experiments elucidated the transient nature of the HSR and explained the need for a minimal period at non-inducing temperature before an additional heat signal can effectively trigger the synthesis of another dose of HSPs [6,151]. When

extracellular calcium was depleted, the denaturation of cytosolic luciferase, a well-known chaperone substrate, did not suffice to trigger the moss HSR [6]. In contrast to previous findings in other organisms, where cytosolic protein unfolding was suggested to be the heat sensor [63,149] this [6] strongly suggested that heatunfolded proteins cannot serve as the primary heat sensor and that Ca2+ has an upstream position in the heat shock signaling pathway. 5.3. Membrane fluidizers activate heat shock genes in a calciumdependent manner Several observations in different organisms suggest that perception of the thermal stimulus initially occurs at the level of the cellular membrane [5,51,158]. The subtle changes in the membrane’s physical state and its lipid composition following temperature increase is indeed well documented [52,159]. The exposure of moss cells to compounds that increase membrane fluidity has led to a significant activation of HSPs at non-inducing temperatures [6,160], thus confirming previous reports in animal cells and bacteria [47,51,161]. Membrane fluidizers like BA induced a major (albeit transient) elevation of cytosolic Ca2+, activated HSP promoters and improved P. patens thermotolerance [6,154]. Exposure to BA also evoked the opening of a non-selective Ca2+ channel in excised plasma membrane patches, which was very similar, in conductance and ion selectivity, to the heat-activated channel. Moreover, BA-mediated HSR was strictly dependent on extracellular calcium and addition of Ca2+ chelators abolished the BA-mediated thermotolerance [6]. A chemical screen of inhibitors of the HSR in P. patens pointed to two calcium channel blockers already known to block store-operated calcium entry and TRP channels in animal cells [6]. Micromolar amounts of 2-aminoethyldiphenyl borate (2-APB) and flufenamic acid inhibited the moss HSR in a highly specific manner. The activation of osmotic stress genes, which also occur via Ca2+ signaling, remained unaffected by these inhibitors. The specificity of these inhibitors suggests that the heat-induced Ca2+ signal is distinct from other stress signaling pathways that use Ca2+ as a second messenger. In Arabidopsis and maize, calmodulin proteins were shown to be involved in the HSR [156,162,163]. Moreover, previous work in tobacco and alfalfa demonstrated that specific mitogen activated protein kinases were phosphorylated just minutes following exposure to heat [164,165]. Recently, a calmodulin-binding kinase was reported to be central for the heat shock signal transduction in Arabidopsis [166]. In line with these reports, dicoumarol, a MAP kinase inhibitor, was shown to be capable of considerably reducing the HSR and negatively affecting thermotolerance in P. patens [6]. Many similarities in the reaction, of P. patens and other plants, to drugs and various membrane-interacting compounds suggest the existence of a conserved heat-signaling pathway involving a Ca2+/calmodulin-dependent activation of a MAP kinase and leading to the transcription of HSPs. The plasma membrane is likely to play an early role in the perception of temperature elevation and the regulation of Ca2+-signature leading to timely HSR and allowing the establishment of acquired thermotolerance [7]. 6. Conclusions Photosynthetic organisms need to resist, tolerate and acclimate to various environmental stresses. The initial step in the stress acclimation is the perception of stress signal by the sensor(s). Changes in ambient temperature would cause fluctuations in membrane fluidity that in turn could trigger regulatory functions like the up or down regulation of genes. A membrane location for temperature sensing seems reasonable given that fluidity and

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Fig. 4. Plasma membrane-associated key players of the heat-signaling pathway leading to the expression of HSPs. Heat triggers an increase in the fluidity of the plasma membrane that elicits a transient increase in the cytosolic Ca2+ concentration via non-selective calcium permeable channels. This Ca2+ influx regulates the activation of calmodulin binding kinase (CBK), on a calmodulin (CaM)-dependent manner. Active CBK promotes the phosphorylation of heat shock transcription factors (HSFs), which in turn modulate the expression of HSPs. High temperature also induces the accumulation of PIP2 via action of PIPK, which may regulate small G-protein coupled signaling or activate ion channels directly. PIP2 can also be hydrolyzed by PLC to generate InsP3, which can be phosphorylated to generate InsP6. The latter may act as a messenger and modulate cytosolic Ca2+ increase. Temperature elevation also triggers PLD activity, generating the second messenger PA. The latter may be responsible for the increase in H2O2 level, which also induces the influx of Ca2+. Similarly, PLD and PA maybe related to the elevation of NO level and the rearrangement of the cytoskeleton. Solid arrows indicate pathways supported by evidence in the literature and dotted arrows refer to hypothetical processes.

microdomain organization of membranes are highly sensitive to changes in even nondenaturing physiological temperatures and can be adjusted by the cell, thereby explaining the importance of relative, rather than absolute, temperature for HSRs. The change in the phase behavior of the membrane could bring about conformational changes in membrane protein(s) and that, in turn, would lead to signal transduction involving events such as protein phosphorylation and de-phosphorylation. This review underscores the complex nature of the membrane connection to the heat shock response in photosynthetic organisms. As summarized on Fig. 4, multiple plasma membrane-associated primary events lead ultimately to the expression of HSPs. These range from heat-induced modulation of the membrane physical state, to changes in membrane properties brought about by its interaction with HSPs, to induction of lipid-based signaling cascades and Ca-channels that respond to temperature-induced changes in the membranes. At present these diverse processes remain mechanistically unconnected. The task that lies ahead is to integrate these membrane-located events with each other as well as with protein and gene level responses. Only then will a sophisticated understanding of the physiological response to heat stress emerge. | Acknowledgements This work was supported in part by Grant-in-aids for Scientific Research (C) (No. 16570028) to H.N. from the Ministry of Education, Science, Sports and Culture of Japan, by Hungarian Basic Research Fund to I.H. A.G. and L.V. (OTKA, No. K84257 and No. 82097), by MTA-JSPS (No. 122) and by the Hungarian National Development Agency TAMOP-4.2.2/08/1-2008-0014. References [1] Glatz A, Vass I, Los DA, Vigh L. The Synechocystis model of stress: from molecular chaperones to membranes. Plant Physiol Biochem 1999;37:1–12. [2] Finka A, Mattoo RU, Goloubinoff P. Meta-analysis of heat chemically upregulated chaperone genes in plant and human cells. Cell Stress Chaperon 2011;16:15–31. [3] Bukau B, Weissman J, Horwich A. Molecular chaperones and protein quality control. Cell 2006;125:443–51. [4] Vigh L, Los DA, Horváth I, Murata N. The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC 6803. Proc Natl Acad Sci USA 1993;90:9090–4. [5] Horváth I, Glatz A, Varvasovszki V, Török Z, Páli T, Balogh G, et al. Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: identification of hsp17 as a ‘‘fluidity gene’’. Proc Natl Acad Sci USA 1998;95:3513–8.

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