The Chlamydomonas heat stress response.

The Plant Journal (2015) 82, 466–480

doi: 10.1111/tpj.12816

SI CHLAMYDOMONAS

The Chlamydomonas heat stress response € hlhaus Michael Schroda*, Dorothea Hemme and Timo Mu Molecular Biotechnology & Systems Biology, TU Kaiserslautern, Paul-Ehrlich-Straße 23, 67663 Kaiserslautern, Germany Received 24 November 2014; revised 25 February 2015; accepted 26 February 2015; published online 6 March 2015. *For correspondence (e-mail [email protected]).

SUMMARY Heat waves occurring at increased frequency as a consequence of global warming jeopardize crop yield safety. One way to encounter this problem is to genetically engineer crop plants toward increased thermotolerance. To identify entry points for genetic engineering, a thorough understanding of how plant cells perceive heat stress and respond to it is required. Using the unicellular green alga Chlamydomonas reinhardtii as a model system to study the fundamental mechanisms of the plant heat stress response has several advantages. Most prominent among them is the suitability of Chlamydomonas for studying stress responses systemwide and in a time-resolved manner under controlled conditions. Here we review current knowledge on how heat is sensed and signaled to trigger temporally and functionally grouped sub-responses termed response elements to prevent damage and to maintain cellular homeostasis in plant cells. Keywords: Chlamydomonas reinhardtii, molecular chaperones, compatible solutes, membrane fluidity, cell cycle, photosynthesis, lipid bodies.

WHY STUDY THE HEAT STRESS RESPONSE IN CHLAMYDOMONAS? Occasions of extreme heat stress (HS) as a consequence of global warming may severely reduce crop yield, in particular when HS occurs during anthesis (Lobell et al., 2011; Deryng et al., 2014). To identify entry points for metabolic engineering or for the application of chemicals aiming at improving crop plant resistance to HS, a comprehensive understanding of how plants sense HS and respond to it is required. Although the heat stress response (HSR) has been studied in considerable detail in bacteria, yeast, fruit fly, and mammals (Voellmy and Boellmann, 2007; Akerfelt et al., 2010; Guertin et al., 2010; Richter et al., 2010; Verghese et al., 2012; Velichko et al., 2013), not all concepts elucidated there may apply to plant systems (Mittler et al., 2012). To a large part this is due to plant cells housing plastids, which with their at least 1300 proteins and central role in metabolism add another level of complexity to eukaryotic cells (Zybailov et al., 2008; Ferro et al., 2010). Hence, there is no doubt that the HSR needs to be studied directly in plants – but why use Chlamydomonas for this? In fact, as a model system to study the plant HSR, Chlamydomonas has some advantages over land plants: (i) cells can be grown under highly defined conditions; (ii) HS 466

can be separated from drought stress; (iii) HS can be applied homogeneously to all cells in a culture; (iv) all cells in a culture are of the same type and differences in cell cycle stage can be averaged out by growing cells asynchronously; and (v) in general, gene families in Chlamydomonas are smaller than in land plants. For example, two heat shock factor (HSF) genes in Chlamydomonas compare with at least 18 in land plants (Schulz-Raffelt et al., 2007; Scharf et al., 2012), and roughly twice as many genes encode basically the same set of molecular chaperones in land plants as compared with Chlamydomonas (Schroda, 2004). Finally, studying the HSR in Chlamydomonas and land plants may allow drawing conclusions on the evolution of the plant HSR. In this review, we give an overview of our current understanding of the HSR in Chlamydomonas. When data on Chlamydomonas are scarce, as is the case regarding the sensing and signaling of HS, we place a larger focus on data from land plants. TEMPERATURES INDUCING A HSR IN CHLAMYDOMONAS In many experiments, an increased expression of heat shock protein (HSP) genes was used as a marker for the © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd

The Chlamydomonas heat stress response 467 HSR, although this is more a marker for a disturbance of protein homeostasis as one effect of HS rather than a marker for the complex HSR. Nevertheless, based on increased HSP expression as marker, a HSR was shown to be elicited in Chlamydomonas when cells were shifted from 20°C to 39–41°C (Tanaka et al., 2000). Kobayashi et al. (2014) reported a HSR to be induced when Chlamydomonas cells were shifted from 24°C to at least 36°C, and found cell survival to be compromised when the temperature shift went beyond 42.4°C. We found HSP genes to be induced when Chlamydomonas cells were shifted from 25°C to at least 37°C and cells to tolerate a maximum temperature of 43.5°C. The variations observed are best explained by differences in growth conditions and/or variations between the strains employed. WHAT TRIGGERS A HSR IN PLANT CELLS? Heat affects many cellular processes, including: (i) protein folding and protein complex assembly; (ii) the function of biological membranes when membranes become too fluid; (iii) cellular metabolism when enzyme activities change; (iv) cell division; and (v) DNA replication and repair (Richter et al., 2010; Velichko et al., 2012). Hence, to ensure survival, cells need to take measures to control the disturbances inflicted by heat on each of these processes. Conceptually, each disturbance might be sensed individually to trigger a process-specific response. For example, unfolded proteins accumulating under HS would be sensed and a signaling cascade unleashed to trigger the production of compatible solutes and the expression of molecular chaperones that cooperatively act to reestablish protein homeostasis. Likewise, an increased fluidity of cell membranes under HS would be sensed individually and a signaling cascade activated that increases the production of saturated fatty acids and their incorporation into membrane lipids to restore normal membrane viscosity. Likewise, any other process affected by HS would be sensed individually to trigger a specific response, with the ensemble of all responses representing the cell’s HSR. Alternatively, few primary sensors may sense heat and elicit several responses to control disturbances of a variety of processes affected by heat. The latter concept was proposed to hold true for land plants, with a primary heat sensor situated in the plasma membrane. For example, under elevated temperatures this sensor would not only trigger responses leading to the restoration of normal membrane viscosity, but also responses that increase the cell’s capacity to cope with problems in protein homeostasis (Saidi et al., 2011; Mittler et al., 2012). In the following we will briefly describe some of the cellular processes affected by HS in Chlamydomonas and land plants and how disturbances of these processes might be sensed.

Disturbances of protein homeostasis The expression of genes encoding HSPs is induced when misfolded proteins accumulate in the cell. This was first demonstrated in Escherichia coli when the production of misfolded proteins was induced by feeding cells with the arginine analog canavanine or with antibiotics that cause translation errors or premature translation arrest (Goff and Goldberg, 1985). In Xenopus laevis, the simple injection of denatured proteins into oocytes induced HSP gene expression (Ananthan et al., 1986). HSP gene expression in E. coli is tightly regulated by the activity of the r32 factor, which again is regulated by DnaK (the bacterial Hsp70 homolog) and its co-chaperone DnaJ: both chaperones bind r32 and keep it in an inactive state. When the chaperones are attracted to misfolded proteins, they release r32 which assumes DNA-binding competence and mediates the transcription of HSP genes (Tomoyasu et al., 1998). A similar model was suggested for mammalian cells, with Hsp90 binding to HSF to repress its activity. Once Hsp90 is attracted to misfolded proteins HSF is released and, following trimerization and phosphorylation, able to mediate HSP gene transcription (Ali et al., 1998; Zou et al., 1998). Also in land plants and Chlamydomonas the accumulation of unfolded proteins was shown to trigger HSP gene expression. This was demonstrated by feeding plants/cells with the arginine analog canavanine or the proline analog L-azetidine-2-carboxylic acid (AZC) (Kurepa et al., 2003; Sugio et al., 2009; Schmollinger et al., 2013) (Figure 2). Also feeding with Hsp90 inhibitors geldanamycin and radicicol induced the expression of HSP genes in Arabidopsis and Chlamydomonas (Yamada et al., 2007; Schmollinger et al., 2013), which might be explained by the inactivation of cytosolic Hsp90 as a repressor of HSF activity (Figure 1). However, Hsp90s are abundant chaperones, with cytosolic HSP90A, ER HSP90B, and plastid HSP90C on ranks 85, 285, and 290 respectively in a list of 1207 soluble proteins quantified in Chlamydomonas by unbiased shotgun proteomics (Table 1 and Table S1) (Hemme et al., 2014). In Chlamydomonas cells exposed to HS for 24 h, levels of these Hsp90s increased strongly, catapulting HSP90A, HSP90B and HSP90C to ranks 2, 95, and 52 respectively (for comparison, cytosolic HSP70A, ER BIP1 and plastid HSP70B rank at positions 60, 200, and 114 respectively and move to ranks 11, 87, and 19 after 24 h HS). Hence, similar to the situation in yeast, Hsp90s in Chlamydomonas are likely to play a prominent role in maintaining protein homeostasis, presumably by keeping inherently unstable proteins in a close-to-native conformation (Nathan et al., 1997). Consequently, when cellular Hsp90s are inactivated by geldanamycin/radicicol, misfolded Hsp90 clients may accumulate to concentrations that are sufficient to elicit an unfolded-protein response (UPR) (Schmollinger et al., 2013) (Figure 2).

© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd, The Plant Journal, (2015), 82, 466–480

468 Michael Schroda et al.

Figure 1. Overview of proposed HS signal transduction chains in land plants. HS induces the activation of phospholipase D (PLD) and phosphatidylinositolphosphate (PIP) kinase, leading to the rapid accumulation of phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP2). HS also activates phospholipase C (PLC) that converts PIP2 into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 may be further phosphorylated to IP6 which binds to calcium channels in ER membranes leading to the release of calcium from intracellular stores. HS also induces the opening of cyclic nucleotide gated channels (CNGC) in the plasma membrane leading to the influx of extracellular calcium into the cell. CNGCs may be activated by increased membrane fluidity and/or cAMP produced by HS-activated adenylyl cyclase. While G proteins were implicated in the activation of PIP kinase, they are also known to activate PLC/PLD and adenylyl cyclase. Increased levels of calcium, potentially together with increased levels of DAG, activate protein kinase C (PKC), which via phosphorylation activates MAP kinase (MAPK). Calcium via calmodulin also activates calmodulin-binding protein kinase 3 (CBK3) and calmodulin-dependent phosphatase 7 (PP7). While PP7 might remove inhibitory phosphate groups (light green) from class A heat shock factors (HsfA), CBK3 and MAPK phosphorylate HsfAs at sites required for their activation (dark green). Also cytoskeleton disassembly via an unknown pathway triggers the activation of stress kinases. Unfolded cytosolic proteins may sequester HSP90 from monomeric HsfAs to allow them to trimerize. Hyperphosphorylated, trimeric HsfAs are competent of driving HSP gene transcription. While an unfolded protein response (UPR) in the chloroplast appears to act at the level of HsfA activation, the UPR of the ER drives stress gene expression directly via the bZIP28 transcription factor. Its mobilization from the ER membrane to the nucleus is initiated when ER Hsp70 homolog BiP becomes sequestered to unfolded proteins. Finally, a NADPH oxidase (respiratory burst oxidase homolog RBOH) in the plasma membrane becomes activated by HS via an increased membrane fluidity and/or via calcium, activated kinases, phosphatidic acid, or G proteins to eventually produce H2O2, which by an unknown pathway activates HsfAs. Levels of intracellular H2O2 may also rise by the HS-induced inactivation of catalase and ascorbate peroxidase, which under non-stress conditions remove H2O2 constantly produced e.g. by mitochondria (Mt) and chloroplasts (Cp). All protein activities and processes activated by HS are shown in red. See main text for references.

In mammals and other organisms it is established that misfolded proteins are not only sensed in the cytosol, but also in the ER and in mitochondria to elicit UPRs (Richter et al., 2010; Walter and Ron, 2011; Haynes et al., 2013). Multiple lines of evidence support the existence of an ER UPR in Arabidopsis (Srivastava et al., 2014) (Figure 1), and compromising protein folding in the ER by exposing Chlamydomonas cells to DTT or tunicamycin also has been shown to induce the expression of genes encoding ER resident disulfide isomerases (Perez-Martin et al., 2014). Recent studies also point to the existence of a chloroplast UPR: when exposed to HS, Chlamydomonas cells expressing antisense RNA directed against transcripts encoding chloroplast HSP70B showed a delayed induction

of other plastid HSPs and of HSPs targeted to cytosol and mitochondria (Schmollinger et al., 2013). As levels of HSP70B in non-stressed antisense lines were only mildly affected, chloroplast HSP70B might be tightly regulating the activity of a factor involved in retrograde stress signaling, similar to the regulation of r32 by DnaK/DnaJ in E. coli (Tomoyasu et al., 1998). If levels of chloroplast HSP70B drop below a threshold in a subfraction of cells in a culture, these cells via the chloroplast UPR might trigger a mild HSR that leads to the desensitizing of HS signal transduction chains in these cells. Note that Chlamydomonas cells exposed to a 30-min HS require approximately 5 h to regain the competence for another full HSR (Schroda et al., 2000). The desensitized HS signal


The Chlamydomonas heat stress response 469 Table 1 The 20 most abundant soluble proteins in heat-stressed Chlamydomonas cells

Protein name PCY1 HSP90A EEF1a3 PSBP1 PSBO PSBQ HSP22A FBA3 HFO18 rbcL HSP70A PSAG RBCS2 FAP103 RPS28 RPS18 CPN60B2 TUB2 HSP70B HTR16

Protein name in full Plastocyanin Heat shock protein 90A Eukaryotic translation elongation factor 1 a Oxygen-evolving enhancer protein 2 Oxygen-evolving enhancer protein 1 Oxygen-evolving enhancer protein 3 Heat shock protein 22A Fructose-1,6-bisphosphate aldolase 1 Replication linked H4 cluster RuBisCo large subunit Heat shock protein 70A Photosystem I - G (P35 protein) RuBisCo small subunit 2 Flagellar associated protein 103 Cytosolic ribosomal protein S28 Cytosolic 40S small ribosomal subunit protein S18 Chaperonin 60B2 Beta-tubulin 2 Heat shock protein 70B Replication linked H3 cluster

Rank in 24 h heat-stressed C. reinhardtii (rank variance)a,b

3 h HS (fold change)c

24 h HS (fold change)b

1 (0) 85 (6) 7 (1) 4 (1.5) 3 (1.5) 13 (4) – 9 (2.5) 5 (4.5) 2 (1) 60 (1.5) 20 (3.5) 6 (1) 23 (2) 12 (9.5) 10 (1)

1 (0) 2 (1.5) 3 (0.5) 4 (1) 5 (2) 6 (0.5) 7 (1.5) 8 (2.5) 9 (10) 10 (1) 11 (3) 12 (0) 13 (4.5) 14 (4) 15 (10) 16 (3)

n.d. 5.17 n.d. 0.69 0.81 n.d. 21.41 0.92 n.d. 0.89 2.85 n.d. n.d. 0.89 0.87 0.90

1.15 3.26 1.12 1.14 1.64 1.21 8.78 1.12 0.58 0.63 2.99 1.24 0.66 1.15 0.89 0.92

157 (14) 11 (3) 114 (15) 15 (11.5)

17 (2.5) 18 (5.5) 19 (5) 20 (14.5)

3.99 n.d. 2.38 n.d.

3.88 0.78 2.64 0.72

Rank in non-stressed C. reinhardtii (rank variance)a,b

n.d., not detected, no change, or change not significant. a Shown is the rank among 1207 quantified soluble proteins from non-stressed Chlamydomonas cells and cells exposed to HS for 24 h, ordered according to the protein’s rank in heat-stressed cells. For a full list, see Table S1. Protein abundance was determined by intensity based absolute quantification (iBAQ) (Schwanhausser et al., 2011) excluding 15N-labeled peptides. The rank variance is derived from three independent biological replicates. b Data from Hemme et al. (2014). c € hlhaus et al. (2011). Data from Mu

transduction chains in many cells of a culture would prevent them from eliciting a full HSR when the culture is exposed to HS, leading to an on average reduced response (Schmollinger et al., 2013). The perturbation of the HSF-dependent HSR in Arabidopsis mutants with reduced levels of the heat-responsive chloroplast ribosomal RPS1 protein may similarly be explained by the desensitizing of a retrograde stress signal transduction chain caused by translation defects in chloroplasts of rps1 mutant lines (Yu et al., 2012). Further evidence for a chloroplast UPR came from the finding that the conditional downregulation of the chloroplast-encoded ClpP protease in Chlamydomonas induced the expression of several nucleus-encoded HSPs (Ramundo et al., 2014). Increased membrane fluidity Changes in temperature alter the fluidity of biological membranes, which may impair their function in barrier formation between cellular compartments and may affect the activity of integral membrane proteins. To reestablish normal membrane viscosity after a temperature change, cells adjust fatty acid (FA) length and the ratio of saturated versus unsaturated FAs in membrane lipids in a process termed homeoviscous adaptation (Sinensky, 1974). This

process requires sensitive membrane sensors that are able to detect subtle changes in membrane viscosity and to control the activity of FA desaturases/elongases and the de novo synthesis of saturated FAs. The interdependence between membrane viscosity and temperature would render sensors of membrane viscosity also as ideal sensors for temperature changes, which is the fundamental concept of the idea that primary sensors for heat are located in the plasma membrane of plant cells (Saidi et al., 2009, 2011). This concept was supported by the finding that land plant cells whose membrane fluidity was increased at ambient temperatures by the addition of membrane fluidizers benzyl alcohol or pentachlorophenol reacted by an increased expression of HSP genes and the activation of a heat stress kinase (HAMK) (Sangwan et al., 2002; Saidi et al., 2007, 2009; Konigshofer et al., 2008; Suri and Dhindsa, 2008; Wu et al., 2012; Finka and Goloubinoff, 2014). In contrast, heat-stressed cells supplemented with the membrane rigidifier DMSO did not elicit a HSR (Sangwan et al., 2002; Suri and Dhindsa, 2008). Moreover, the higher the growth temperature and degree of membrane lipid saturation prior to exposure to a fixed HS temperature, the smaller the extent of the HSR in moss cells (Saidi et al., 2010).


470 Michael Schroda et al.

Figure 2. Hypothetical model of the HSR in Chlamydomonas. Heat directly and rapidly leads to the thermal inactivation of Rubisco activase and generally increases the concentration of unfolded proteins, the fluidity of membranes, and the solubility of O2 versus CO2. Via unknown pathways heat rapidly leads to a reduction in bulk protein synthesis, to a shift from general metabolism to a specific stress metabolism, and to arrests of DNA replication and cell division (black lines). Accumulating unfolded proteins activate one or more stress kinases that hyperphosphorylate(s) HSF1, leading to the increased transcription of HSP genes that are translated to molecular chaperones. Within approximately 3 h the combined actions of reduced bulk protein synthesis and accumulating molecular chaperones, compatible solutes, and proteases restore protein homeostasis, thereby leading to the inactivation of stress kinase(s) and to HSF1 dephosphorylation (blue lines). Increased membrane fluidity by an unknown pathway triggers the exchange of polyunsaturated fatty acids (FAs) in membrane lipids by de novo synthesized saturated FAs to restore normal membrane viscosity. Released polyunsaturated FAs are deposited as triacylglycerols (TAGs), while de novo synthesis of saturated FAs is supported by NADPH and ATP from photosynthetic light reactions. NADPH and ATP become amply available by the arrest of CO2 fixation caused by the rapid inactivation of Rubisco activase. In the time period between 3 h and 24 h of HS, when normal membrane viscosity is restored and TAG accumulation is at its limit, Rubisco activase via the action of CPN60 regains activity to activate Rubisco (dotted red line). The low CO2/O2 ratio at elevated temperatures favors the incorporation of O2 into ribulose-1,5-bisphophate by Rubisco, leading to high rates of photorespiration. The depletion of sinks for NADPH and ATP results in an overreduction of the photosynthetic electron transport chain, triggering antenna uncoupling and increased Mehler reactions (red lines). Superoxide produced via Mehler reactions and H2O2 produced as side reaction from the oxygenation reaction of Rubisco by an unknown pathway induce the expression of ROS scavengers as a late response to heat stress.

The primary heat sensors in the plasma membrane of plant cells were suggested to be calcium channels that open when membrane fluidity increases, thereby allowing the influx of extracellular calcium to trigger the HSR (Saidi et al., 2009, 2010) (Figure 1 and see below). Also a NADPH

oxidase located in the plasma membrane was suggested to represent a heat sensor. This enzyme might be activated by an increased membrane fluidity at elevated temperatures as well, thereby leading to the generation of reactive oxygen species (ROS) that may act as second messengers


The Chlamydomonas heat stress response 471 in HS signaling (Konigshofer et al., 2008) (Figure 1). Finally, an increased membrane fluidity at HS temperatures was also suggested to induce changes in the organization of membrane-associated cytoskeleton components that activate stress kinases (Sangwan et al., 2002) (Figure 1). Whether in Chlamydomonas HS is sensed via changes in membrane fluidity remains to be elucidated. Metabolic imbalances In addition to an increase in the activity of a NADPH oxidase in the plasma membrane, HS may promote ROS production also by changing the activities of other enzymes. For example, reduced activities of catalase (Dat et al., 1998) or ascorbate peroxidase (Panchuk et al., 2002) may impair the removal of H2O2 constantly produced e.g. by mitochondrial and chloroplast electron transport chains (Figure 1). ROS have been shown to play a role in the HSR (see below). Moreover, an altered carbon status observed in heat-stressed Arabidopsis plants might be sensed (Vasseur et al., 2011). Whether metabolic imbalances play roles in HS sensing in Chlamydomonas needs yet to be investigated. Impaired DNA replication and DNA damage repair In S-phase Chinese hamster ovary cells, hyperthermia resulted in persisting single-stranded DNA regions, which may be the source of chromosomal aberrations presumably responsible for the high thermosensitivity of S-phase cells (Wong et al., 1988). HS also impairs DNA repair mechanisms in mammals (Velichko et al., 2013). Effects of HS on DNA replication and repair in plants are generally poorly understood. HEAT STRESS SIGNAL TRANSDUCTION Calcium as a second messenger for HS signaling Considerable evidence has accumulated for a role of calcium in HS signal transduction in plants: upon application of HS calcium concentrations were found to transiently increase in the plant cell cytosol (Gong et al., 1998; Liu et al., 2003, 2006; Saidi et al., 2009; Gao et al., 2012; Zheng et al., 2012). In particular the influx of extracellular calcium was found to be crucial, as the HSR was diminished/abolished when calcium chelators EGTA or BAPTA, or the calcium ion channel blockers lanthanum, gadolinium or verapamil were applied prior to HS (Link et al., 2002; Sangwan et al., 2002; Liu et al., 2003; Suri and Dhindsa, 2008; Saidi et al., 2009; Gao et al., 2012; Wu et al., 2012). While results from some studies indicate that calcium accumulating in the cytosol of heat-stressed cells is only derived from extracellular stores (Sangwan et al., 2002; Saidi et al., 2009; Wu et al., 2012), those from other studies also point to intracellular calcium stores as a source (Gong et al., 1998; Zheng et al., 2012) (Figure 1). The influx of extracel-

lular calcium was suggested to be mediated by the opening of specific calcium channels as a consequence of an increased membrane fluidity at elevated temperatures (Saidi et al., 2009). Good candidates for calcium channels are cyclic nucleotide-gated channels (CNGC) located in the plasma membrane, which are non-selective inward cation channels that mediate the transport of cations (including calcium), and contain domains for the binding of cyclic nucleotides and calmodulin (Kohler et al., 1999) (Figure 1). Accordingly, the HS-induced calcium influx was abolished in Arabidopsis cngc6 knock-out plants and correlated with a 60–70% reduced expression of HSP genes and reduced thermotolerance (Gao et al., 2012). Surprisingly, however, Physcomitrella ΔCNGCb and ΔCNGCd mutants were hypersensitive to HS and showed a faster and stronger calcium influx compared with wild-type plants (Finka et al., 2012; Finka and Goloubinoff, 2014). In agreement with a role of calcium in HS signal transduction, also calmodulin was found to play an important role (Liu et al., 2003, 2005, 2008; Li et al., 2004; Zhang et al., 2009a) (Figure 1). Results obtained with Chlamydomonas regarding a role of extracellular calcium in mediating the HSR were inconsistent. While washed cells treated with calcium chelator BAPTA displayed a delayed and less pronounced induction of HSP gene expression and reduced thermotolerance under HS, washed cells supplemented with EGTA behaved like controls (Schmollinger et al., 2013). These inconsistent results might be due to effects caused by BAPTA that are independent of its calcium-chelating properties and interfere with HS sensing/signal transduction (Lancaster and Batchelor, 2000; Saoudi et al., 2004). Other second messengers H2O2 was shown to transiently accumulate in heat-stressed land plant cell cultures. In one study performed with tobacco BY2 cells, H2O2 levels were found to increase approximately threefold after 3 min of HS and to return to basal levels after 20 min (Konigshofer et al., 2008). In another study performed with Arabidopsis cells H2O2 accumulated 2.3-fold within 15 min of HS, but the return to basal levels took hours (Volkov et al., 2006). Supplementing cells with membrane fluidizer benzyl alcohol under ambient temperatures also induced a transient, two to threefold accumulation of H2O2. Administration of the NADPH oxidase inhibitor diphenyleneiodonium or of ascorbate abolished the induction of HSP gene expression by both, benzyl alcohol and HS. These findings indicate an important role for H2O2 in HS signaling and for plasma membrane NADPH oxidase as a source for H2O2 produced under HS (Volkov et al., 2006; Konigshofer et al., 2008) (Figure 1). Here it is important to distinguish between H2O2 produced rapidly after the onset of HS in the frame of an oxidative burst with a putative role in signaling, and H2O2 accumulating late as a result of Rubisco side reactions and


472 Michael Schroda et al. Mehler reactions, when sinks for electrons from photosynthetic light reactions become limited (see below). Other second messengers were proposed to intertwine with calcium responses to mediate HS signal transduction in plants. Gao et al. (2012) detected a 2.9-fold increase in intracellular cAMP levels within 2 min after the onset of HS in Arabidopsis. Moreover, application of a membrane permeable cAMP analog or inhibition of cNMP phosphodiesterase led to a CNGC6-dependent influx of calcium resulting in a 1.7-fold increase in HSP transcript levels. Liu et al. (2006) and Zheng et al. (2012) demonstrated a phospholipase C (PLC) dependent approximately threefold increase of inositol 1,4,5-trisphosphate (IP3) levels within approximately 3 min after the onset of HS in Arabidopsis. Wild-type plants supplemented with a PLC inhibitor and plc9 mutant plants displayed a reduced induction of HSP gene expression upon HS, which came along with a reduced increase in intracellular calcium concentrations. However, the influx of extracellular calcium was not disturbed when the plc9 mutant was exposed to HS, suggesting that PLC acts on internal calcium stores (Zheng et al., 2012) (Figure 1). Also levels of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid were found to rapidly accumulate in heat-stressed plants, presumably because HS induced the activation of PIP kinase and phospholipase D (Mishkind et al., 2009) (Figure 1). The activation of PIP kinase apparently depended on the activity of a G protein (Mishkind et al., 2009). As (heterotrimeric) G proteins are also known to activate adenylyl cyclases and phospholipases, it appears attractive to hypothesize that all cascades leading to the increase of intracellular calcium concentrations and to kinase activation are initiated by activated G proteins (Figure 1). As plant NADPH oxidases were shown to be regulated by calcium, calmodulin-dependent kinases, phosphatidic acid, and monomeric G proteins (Kobayashi et al., 2007; Wong et al., 2007; Ogasawara et al., 2008; Zhang et al., 2009b; Suzuki et al., 2011), also the HS-induced H2O2 burst might be triggered directly by G proteins, or indirectly by second messengers produced via activated G proteins (Figure 1). Potential roles for second messengers in HS signaling in Chlamydomonas have not yet been explored. Protein kinases and phosphatases In alfalfa, tomato, and tobacco BY cells, HS was shown to activate MAP kinases in a calcium-dependent manner (Link et al., 2002; Sangwan et al., 2002; Suri and Dhindsa, 2008). MAP kinase activation by HS was abolished when alfalfa cells had been fed with calcium-dependent protein kinase antagonist W7 or protein kinase C inhibitor H7, suggesting that these kinases lie upstream of MAP kinases in HS signal transduction (Sangwan et al., 2002) (Figure 1). A direct link between the calcium signal and kinase activation apparently is realized by calmodulin-binding protein kinase

3 (CBK3), as thermotolerance and HSP gene expression were impaired in cbk3 mutants, while they were enhanced in CBK3 overexpressors (Liu et al., 2008). Also calmodulindependent phosphatase 7 (PP7) appears to directly relay the calcium signal to downstream responses, as thermotolerance and HSP gene expression were impaired in pp7 mutants, while they were enhanced in PP7 overexpressors (Liu et al., 2007b) (Figure 1). A role of kinases in HS signaling in Chlamydomonas was demonstrated by the delayed accumulation of HSP gene transcripts upon supplementing cells with protein kinase inhibitor staurosporine (Schmollinger et al., 2013) (Figure 2). The identity of the stress kinases in Chlamydomonas is elusive. Targets of signal transduction chains Important targets of kinase cascades triggered by HS are HSFs. Land plants contain between 18 and 52 HSFs (Scharf et al., 2012), which are grouped into classes A, B, and C depending on the length of amino acid sequence insertions between hydrophobic regions A and B of their oligomerization domains (Nover et al., 1996). Induced thermotolerance was shown to be mediated only by class A HSFs (Mishra et al., 2002; Liu et al., 2011), while class B and C members modulate the activities of class A HSFs (Bharti et al., 2004; Kumar et al., 2009). Chlamydomonas encodes two HSFs (HSF1 and 2), of which only HSF1 is a canonical HSF that in phylogenetic analyses clusters with plant class A HSFs. Accordingly, Chlamydomonas cells in which HSF1 was depleted by RNAi or amiRNA were unable to induce HSP gene expression and thermotolerance under HS conditions (Schulz-Raffelt et al., 2007; Schmollinger et al., 2010). While HSFs in animals and land plants are monomeric under non-stress conditions and trimerize under HS conditions (Rabindran et al., 1993; Lee et al., 1995) (Figure 1), HSFs in yeast and Chlamydomonas are constitutively trimeric (Sorger and Nelson, 1989; Schulz-Raffelt et al., 2007). In addition to trimerization, hyperphosphorylation is a prerequisite for HSF activation in all eukaryotes (Sorger and Pelham, 1988; Cotto et al., 1996). Accordingly, a HSactivated MAP kinase was shown to phosphorylate HsfA3 in tomato (Link et al., 2002), and calmodulin-binding protein kinase 3 was shown to phosphorylate HsfA1 in the presence of calcium and calmodulin in Arabidopsis (Liu et al., 2008) (Figure 1). Also Chlamydomonas HSF1 was shown to become hyperphosphorylated during HS, with the degree of phosphorylation in the HS time course correlating with the accumulation of HSP gene transcripts (Schulz-Raffelt et al., 2007). HSF1 hyperphosphorylation and HSP gene expression during HS were strongly delayed in cells treated with protein kinase inhibitor staurosporine (Schmollinger et al., 2013), suggesting that HSF1 activity also in Chlamydomonas is regulated by stress kinases (Figure 2). Interestingly, the level of histone H2B ubiquitination in Chlamydomonas was found to decline during HS and


The Chlamydomonas heat stress response 473 the kinetic of H2B deubiquitination correlated well with that of HSP transcript accumulation (Shimogawara and Muto, 1989, 1992; Schulz-Raffelt et al., 2007). This suggests that H2B deubiquitination is either a consequence of HSP gene transcription, or regulated by a HS signal e.g. as a prerequisite for HSF1 binding. The observation that Chlamydomonas HSF1-RNAi lines exposed to HS failed to induce the expression of genes encoding plastid-targeted chaperones suggests that HSF1 is also the target of a retrograde signal postulated for the chloroplast UPR (SchulzRaffelt et al., 2007) (Figures 1 and 2). As mentioned above, none of the components of the cytosolic or chloroplast UPR have yet been identified in Chlamydomonas. The transcriptional response to HS is fast in Chlamydomonas, as judged from the finding that binding of HSF1 to the HSP22E/F promoter and accumulation of HSP22F transcripts were detectable already 30 sec after the onset of HS (Strenkert et al., 2011). Microarray analysis revealed 3988 genes to be differentially expressed in Chlamydomonas cells exposed to HS for 45 min, the vast majority of them being upregulated (Voss et al., 2011). Which of these genes are regulated by HSF1 (directly or indirectly), or regulated by other transcription factors activated by HS remains to be elucidated. An interesting feature of Chlamydomonas HSF1 is that it acts as a strong activator. HSF1 was shown to have a high affinity for the HSP70A promoter and to bind to this promoter also under non-stress conditions, while the HSP22E/ F promoter was only occupied under HS conditions (Strenkert et al., 2011). When bound to the HSP70A promoter, HSF1 appears to efficiently recruit RNA polymerase II to mediate transcription from promoters fused downstream of HSP70A. This results in increased levels of histone H3/4 acetylation and H3K4 trimethylation at the HSP70A promoter and at downstream promoters and to a partial overriding of repressive effects mediated by H3K9 monomethylation often associated with transgene promoters in Chlamydomonas (Strenkert et al., 2013). This particular feature of HSF1 as a strong activator therefore explains why gene constructs containing fusions of the HSP70A promoter upstream of other promoters results in high frequencies of transgene expression (Schroda et al., 2000, 2002; Lodha et al., 2008). As outlined in the next section, other targets of HS signal transduction chains than HSFs are likely proteins controlling the cell cycle, DNA replication, metabolic remodeling, FA synthesis and lipid remodeling (Figure 2). RESPONSES TRIGGERED BY HS IN CHLAMYDOMONAS From recent work using top-down Systems Biology approaches it became clear that the Chlamydomonas HSR consists of sub-responses that can be temporally and functionally grouped into response elements. These are implemented in order to prevent irreversible damage and to

maintain cellular homeostasis, thereby allowing cells to € hlhaus et al., 2011; acclimate to HS and to survive (Mu Hemme et al., 2014). Five response elements could be distinguished: (i) the arresting of DNA replication and cell division; (ii) the switching from regular metabolism to stress metabolism; (iii) the maintenance of protein homeostasis; (iv) the restoration of normal membrane viscosity (homeoviscous adaptation); and (v) the control of photosynthetic light reactions and the funneling of electrons between carbon fixation and FA synthesis (Figure 2). Some of these response elements could also be identified in other stress responses. For example, a cell cycle arrest and the funneling of electrons toward triacylglycerol (TAG) synthesis were also found in the Chlamydomonas N starvation response (Li et al., 2012; Schmollinger et al., 2014). Whether in Chlamydomonas these response elements are coordinately triggered by one or few primary heat sensors, or by many different sensors specific for each process disturbed by heat remains to be elucidated. In the following we will briefly discuss the response elements identified in heat-stressed Chlamydomonas cells and how they are temporally coordinated. Cell cycle arrest When exposed to HS, Chlamydomonas cells rapidly arrested cell cycle progression in G1 and G2 phases, coming along with an arrest of DNA replication and cell division (Hemme et al., 2014) (Figure 2). This most likely happens to avoid DNA damage occurring during replication under HS conditions (Velichko et al., 2013), and to avoid mitotic catastrophe resulting from the heat-induced disassembly of the mitotic spindle (Coss et al., 1982). When cells were placed back to 25°C following a 24-h exposure to 42°C, DNA replication resumed after approximately 3 h while it took approximately 8 h until cell division resumed (Mu€ hlhaus et al., 2011; Hemme et al., 2014). Switching from regular metabolism to a specialized metabolism for stress protection Rapidly after exposing Chlamydomonas cells to HS central metabolites from glycolysis and from the TCA, glyoxylate, and Calvin cycles got depleted (Hemme et al., 2014). While a similar response was observed in heat-stressed E. coli cells and interpreted as an energy conservation strategy (Jozefczuk et al., 2010), levels of many central metabolites were found to increase in heat-stressed Arabidopsis plants (Kaplan et al., 2004; Rizhsky et al., 2004). Why these organisms react so differently is not clear. A possible reason may lie in the trophic state of the cells, as E. coli and Chlamydomonas cells in these experiments were grown hetero/mixotrophically, while Arabidopsis plants were grown photoautotrophically. While central metabolites got depleted, other metabolites accumulated rapidly after exposing Chlamydomonas


474 Michael Schroda et al. cells to HS (Figure 2). Many of them appear to be generated via the catabolism of larger molecules, e.g. b-alanine from the degradation of uracil, myo-inositol, glycerophosphoglycerol (GPG), and phosphoethanolamine from the hydrolysis of phospholipid head groups, and ornithine and putrescine from the degradation of arginine (Hemme et al., 2014). These metabolites may play roles in stress signal transduction (see above), may directly act as compatible solutes, or may serve as precursors for the synthesis of molecules with roles in stress protection (see below). Maintenance of protein homeostasis The toxicity of unfolded/misfolded proteins accumulating in heat-stressed Chlamydomonas cells apparently is counteracted by four different means: (i) the accumulation of compatible solutes; (ii) an increased expression of molecular chaperones; (iii) a reduced synthesis of bulk proteins; and (iv) protein degradation (Figure 2). Compatible solutes are metabolites that stabilize membranes and prevent protein unfolding and aggregation. For this, they need to accumulate rapidly after the onset of HS, but also need to be degraded rapidly upon return to regular growth conditions to not interfere with protein folding (Singer and Lindquist, 1998; Yancey, 2005). Metabolites meeting these criteria in Chlamydomonas are GPG, trehalose, alanine, isoleucine, asparagine, glutamine, and guanosine (Hemme et al., 2014). Particularly interesting is GPG, because among the 43 metabolites found to change in abundance during HS it was the one exhibiting the largest change (a 39-fold increase). As levels of membrane lipid phosphatidylglycerol (PG) rapidly decreased in heatstressed Chlamydomonas cells, GPG appears to be derived from the action of phospholipase A/lipid acyl hydrolase that split PG into GPG and free FAs. In fact, GPG has been shown to accumulate in response to high salt and heat in Archaeoglobus fulgidus and to thermostabilize proteins in vitro (Martins et al., 1997; Lamosa et al., 2000). As PG levels did not change in heat-stressed tobacco BY2 cells (Mishkind et al., 2009), GPG is unlikely to play a role as a potential compatible solute in land plants. Pulse-labeling experiments revealed that the translation capacity in heat-stressed Chlamydomonas cells is shifted toward the synthesis of a select group of HSPs at the expense of bulk protein synthesis (Kloppstech et al., 1985; Schmollinger et al., 2013). While in heat-stressed HSF1RNAi lines the drop in bulk protein synthesis was still observed, HSP synthesis was largely abolished, suggesting that under HS conditions translation is directed toward HSP gene transcripts which do not accumulate in HSF1RNAi lines (Schulz-Raffelt et al., 2007). In wild-type cells, levels of most molecular chaperones plateaued after € hlhaus et al., 2011). The most approximately 3 h of HS (Mu abundant chaperones after 24 h HS were (their rank among 1207 soluble Chlamydomonas proteins is given in

parentheses): cytosolic chaperones HSP90A (2), HSP22A (7), and HSP70A (11) followed by plastid CPN60B2 (17), HSP70B (19), CPN10 (24), CGE1 (34), CPN23 (42), and HSP90C (52) (Table 1 and Table S1). The most abundant mitochondrial chaperones were CPN60C (55) and HSP70C (124), and the most abundant ER lumenal ones BIP1 (87) and HSP90B (95). The rank order of representatives from these compartments was the same also under non-stress conditions. This suggests that the sizes of soluble Chlamydomonas subcompartment proteomes are in the order cytosol > stroma > matrix ≥ ER lumen, and/or that the demand for assistance in protein folding under non-stress and HS conditions is in that order. Levels of several chaperones were modulated between 3 and 24 h of HS. In particular chaperones specialized in dealing with protein aggregates (small HSPs and ClpBs) were actively degraded (Hemme et al., 2014). This suggests that the problem of aggregate formation is alleviated during long-term HS, presumably because concentrations of compatible solutes and/or levels of Hsp70 and Hsp90 systems have increased and these are effective in protecting misfolded proteins from aggregation (Nathan et al., 1997; Singer and Lindquist, 1998). Such a role is particularly likely for Hsp90s, as the addition of Hsp90 inhibitors geldanamycin and radicicol resulted in an increased amplitude of HSP gene expression and a delayed attenuation of the response (Schmollinger et al., 2013) (Figure 2). Levels of other chaperones (further) increased between 3 and 24 h HS, like those of ER chaperones BIP1 and HSP90B, or of mitochondrial chaperones HSP70C and CPN60C/CPN10, suggesting proteotoxic effects to increase during long-term HS in mitochondria and ER (Hemme et al., 2014). In line with the pulse-labeling data (Kloppstech et al., 1985; Schulz-Raffelt et al., 2007; Schmollinger et al., 2013), bulk protein per cell culture volume increased by only approximately 57% during a 24-h HS period while an approximately eightfold increase was expected in this time from the approximately 8 h generation time of Chlamydomonas cells. This reduction in protein synthesis rates came along with an approximately 15% decrease in levels of € hlhaus et al., 2011; Hemme et al., cytosolic ribosomes (Mu 2014). This is certainly a consequence of the cell cycle arrest, but also reduces the risk of generating misfolded proteins during translation at elevated temperatures. Interestingly, proteins accumulating during HS had a higher content of branched chain amino acids, potentially pointing to a more elaborated hydrophobic core that might facilitate their folding at elevated temperatures (Hemme et al., 2014). Proteins conjugated with ubiquitin were found to rapidly accumulate after the onset of HS and to migrate on SDSPAGE with an apparent MW less than approximately 60 kDa (Shimogawara and Muto, 1989; Wettern et al., 1990). This suggests that several misfolded proteins get


The Chlamydomonas heat stress response 475 ubiquitinated during HS to be marked for degradation. Indeed, among the 688 proteins found to be changing during a 24-h HS and 8-h recovery time course, 29 were actively degraded, among them proteins like METE that are known to be thermolabile (Mogk et al., 1999; Hemme et al., 2014). Homeoviscous adaptation During the first approximately 2 h after the onset of HS, Chlamydomonas cells were found to remodel the composition of membrane lipids by decreasing their content in polyunsaturated FAs and increasing their content in saturated FAs. At the same time, TAGs enriched with polyunsaturated FAs accumulated within lipid bodies, suggesting that polyunsaturated FAs from membrane lipids were deposited in TAGs, while newly synthesized, saturated FAs were incorporated into membrane lipids to restore normal membrane viscosity (Hemme et al., 2014). Hence, during the first approximately 2 h of HS lipid bodies appear to constitute a buffer for polyunsaturated FAs, presumably because the cell cycle arrest precludes that membrane lipids with polyunsaturated FAs are simply diluted out by the incorporation of de novo synthesized lipids with saturated FAs during growth and division (Figure 2). The finding that Arabidopsis plants exposed to HS accumulated plastoglobules in their chloroplasts (Zhang et al., 2010) suggests that land plants might follow a similar strategy. As mentioned above, one of the rapidly accumulating metabolites in Chlamydomonas cells and Arabidopsis plants exposed to HS was b-alanine (Kaplan et al., 2004; Rizhsky et al., 2004; Hemme et al., 2014). Although b-alanine was suggested to act as a compatible solute (Kaplan et al., 2004), the kinetic of its accumulation in Chlamydomonas does not support this idea (b-alanine levels peaked 30 min after the onset of HS, remained only approximately threefold elevated after long-term HS, and did not decline during recovery). Hence, in light of the massive de novo synthesis of FAs and lipid rearrangements taking place early after onset of HS, b-alanine is more likely to serve as a precursor for the synthesis of pantothenate, which is required for the synthesis of the 40 -phosphopantetheine moiety of coenzyme A and the acyl carrier protein (Ottenhof et al., 2004). The restoration of normal membrane viscosity during HS might not only be mediated by an increase in the content of lipids with more saturated FAs, but also by membrane-associated small HSPs (Tsvetkova et al., 2002). Accordingly, in Chlamydomonas a 22-kDa small HSP was reported to associate with the outer chloroplast envelope at temperatures above 38°C and to be released again at temperatures below 38°C (Eisenberg-Domovich et al., 1994). This small HSP was originally suggested to be imported into the chloroplast and to protect photosystem (PS) II from photoinhibition during HS (Schuster et al.,

1988). It later turned out to be cytosolic HSP22A and its localization to thylakoid membranes likely to be due to their contamination with chloroplast envelopes (Grimm et al., 1989; Eisenberg-Domovich et al., 1994; Schroda, 2004). Controlling photosynthetic light reactions and the funneling of electrons between carbon fixation and FA synthesis CO2 fixation in spinach leaves and chloroplasts exposed to HS was reduced by 80% which was shown to be due to the inactivation of Rubisco (Weis, 1981). Rubisco inactivation was attributed to the thermolability of Rubisco activase (RCA) (Feller et al., 1998). RCA catalyses the release of ribulose-1,5-bisphosphate and other sugar phosphates from the Rubisco active site. If sugar phosphates occupy the Rubisco active site prior to its carbamylation and Mg2+ binding, the enzyme is rendered inactive (Jensen, 2000). In Chlamydomonas cells exposed to HS for 3 h, the abundance of RCA1 protein dropped by 30% while that of RCA1 transcript increased, indicating that also Chlamydomonas € hlhaus et al., 2011). An RCA1 might be thermolabile (Mu open question is whether the inactivation of RCA by HS is an undesired effect or rather a mechanism implemented deliberately as part of a regulated response to moderate HS (Sharkey, 2005; Sage et al., 2008; Sharkey and Zhang, 2010). Given the high demand for NADPH and ATP for the de novo synthesis of saturated FAs in the first hours after the onset of HS, the rapid arrest of CO2 fixation by the thermolability of RCA might indeed be intended to allow for a rapid restoration of membrane viscosity (Figure 2) (Hemme et al., 2014). In fact, FA synthesis generally appears to be an important sink for electrons from the photosynthetic light reactions in growth-limited Chlamydomonas cells, thus explaining the accumulation of lipid droplets also under N- and S-starvation conditions (Cakmak et al., 2012; Li et al., 2012; Schmollinger et al., 2014). PS II, and particularly its oxygen-evolving complex, was considered to be the most thermosensitive of the protein complexes driving photosynthetic light reactions (Berry and Bjorkman, 1980). However, it was pointed out recently that significant thermosensitivity of PS II in land plants is only observed at unphysiologically high temperatures (Sharkey, 2005; Sharkey and Zhang, 2010). This view is also supported by data from Chlamydomonas cells, which under mixotrophic growth conditions needed to be shifted from 25 to 44.5°C for a 50% inactivation of oxygen evolution (Tanaka et al., 2000)–a temperature shift that is likely to be lethal (Hema et al., 2007; Kobayashi et al., 2014). Accordingly, oxygen evolution was not impaired and PSII maximum quantum efficiency was only slightly reduced in Chlamydomonas cells shifted from a growth temperature of 25°C to non-lethal 40–42°C for 3 h (Nordhues et al., 2012; Hemme et al., 2014). This also indicated that at this


476 Michael Schroda et al. time sufficient sinks for NADPH and ATP still exist, presumably because lipid rearrangements are still ongoing. However, HS in combination with light was reported to foster the cross-linking of the D1 protein of PSII with other proteins, with the extent of cross-linking being proportional to the light intensity applied (Schuster et al., 1988). The fraction of saturated FAs in TAGs was found to increase in Chlamydomonas cells exposed to long-term HS, suggesting that once membrane viscosity is restored the de novo synthesis of saturated FAs and their incorporation into TAGs continues to a small extent (Hemme et al., 2014). Nevertheless, between approximately 3 and 24 h of HS exposure, FA synthesis will no longer serve as a major sink for NADPH and ATP. However, it was at this time that RCA1 protein levels increased again by 1.6-fold. That some of this RCA1 protein might be active is suggested by the finding that Arabidopsis Cpn60b appears to protect RCA from thermal denaturation (Salvucci, 2008) and that in Chlamydomonas levels of CPN60B1/2 were 1.8- to 3.9-fold upregulated after 24 h of HS (Hemme et al., 2014). The increase in RCA1 abundance came along with a 2.4-fold increase in the abundance of phosphoglycolate phosphatase (PGP1), which removes 2-phosphoglycolate, the toxic product of O2 incorporation into ribulose-1,5-bisphophate by Rubisco. Moreover, levels of starch and of Calvin cycle and photorespiration metabolites also increased (Hemme et al., 2014). These data suggest a reactivation of Rubisco in the time between 3 and 24 h of HS exposure, presumably by the action of CPN60 chaperonins (Salvucci, 2008) (Figure 2). The apparently high rate of O2 incorporation into ribulose-1,5-bisphosphate by Rubisco under HS conditions can be explained by the increased ratio of dissolved O2 versus CO2 at 42°C and the reduced selectivity of Rubisco for CO2 at elevated temperatures (Jordan and Ogren, 1984). Most likely, Chlamydomonas cells reactivate CO2 fixation after long-term HS to reestablish a sink for NADPH and ATP from photosynthetic light reactions once the sink provided by de novo FA synthesis is depleted. In this respect, RCA appears to represent a thermoswitch that regulates the distribution of electrons from photosynthetic light reactions between carbon fixation and other sinks like de novo FA synthesis. A side product of the oxygenation reaction by Rubisco at elevated temperatures is the formation of H2O2 (Kim and Portis, 2004). Moreover, the reduced availability of strong sinks for electrons produced by the photosynthetic light reactions between 3 h and 24 h of HS is likely to increase Mehler reactions, that is the transfer of electrons from photosystem I to O2 instead of ferredoxin, leading to the formation of superoxide (Mehler, 1951; Li et al., 2012) (Figure 2). In Chlamydomonas, ROS apparently generated by these pathways induced the synthesis of several ROS scavenging enzymes, including superoxide dismutases, hybrid cluster proteins, peroxiredoxins, thioredoxins, and

enzymes involved in the biosynthesis of ascorbate and glutathione. Abundances of these ROS scavengers did not change or even declined in cells exposed to HS for 3 h, but increased between 3 and 24 h of HS, thus representing a late response to HS (Mu€ hlhaus et al., 2011; Hemme et al., 2014). Also in higher plant species exposed to HS for more than 6 h, abundances of proteins involved in ROS scavenging like superoxide dismutase, thioredoxin h, ascorbate peroxidase, and dehydroascorbate reductase have been found to increase (Ferreira et al., 2006; Lee et al., 2007; Xu and Huang, 2008; Scafaro et al., 2010). Another way to cope with the depletion of sinks for electrons from photosynthetic light reactions is to reduce the efficiency of light harvesting. Also this strategy is apparently used in Chlamydomonas, as judged from the uncoupling of antenna from photosystems II and I taking place mainly in the time between 3 and 24 h of HS, which was accompanied with a decrease in oxygen evolution rates by two-thirds (Hemme et al., 2014). Hence, once normal membrane viscosity is restored and TAG accumulation has reached a plateau, heat-stressed Chlamydomonas cells appear to cope with the depletion of electron sinks by antenna uncoupling and by reactivating CO2 fixation (Figure 2). LHCII uncoupling as a response to moderate HS was also observed earlier in the desert shrub Larrea divaricata (Armond et al., 1978). An uncontrolled response to HS–the apparently futile accumulation of photosystem subunits Almost all responses triggered in heat-stressed Chlamydomonas cells appear to be highly controlled–except for the mysterious approximately 1.3-fold accumulation of subunits of both photosystems in cells exposed to HS for 24 h (Hemme et al., 2014). As by then rates of oxygen evolution had decreased by two-thirds (certainly also as a consequence of antenna uncoupling), and also ATPase subunits had decreased by approximately 15%, it is unlikely that the additional photosystem subunits contribute to functional photosystems. Strikingly, photosystem subunit accumulation came along with the formation of prolamellar bodylike structures (PLBs) in regions from which multiple thylakoid membrane lamellae emerge. PLBs already have been observed in Chlamydomonas strains depleted of the vesicleinducing protein in plastids (VIPP1) (Nordhues et al., 2012). VIPP1 was proposed to be involved in the organization of so-called thylakoid centers, at which the biogenesis/ repair at least of PS II might occur (Rutgers and Schroda, 2013). VIPP1 was shown to form dimers, rings and rods and the interconversion between these assembly states was demonstrated to be controlled by plastid Hsp70 and Hsp90 (Liu et al., 2007a; Heide et al., 2009; Feng et al., 2014). If the function of VIPP1 depends on the dynamic assembly and disassembly of its oligomeric structures by these chaperones, their sequestration to unfolded proteins


The Chlamydomonas heat stress response 477 might impair VIPP1 function, thereby leading to the accumulation of unassembled photosystem subunits in PLBs. Alternatively, proper photosystem assembly might have been disturbed by a reduced availability of PG and SQDG, which are structural lipids of both photosystems and whose levels were found to decrease during HS (Jordan et al., 2001; Guskov et al., 2009; Hemme et al., 2014). As the abundances of enzymes involved in chlorophyll biosynthesis decreased during HS, also the insufficient incorporation of pigments might have interfered with proper € hlhaus et al., 2011; Hemme photosystem assembly (Mu et al., 2014). Why the apparently superfluous synthesis of photosystem subunits is not negatively controlled during long-term HS remains enigmatic.

(vi) How are the diverse response elements coordinated? (vii) Do responses differ if cells are grown hetero-, mixo-, or autotrophically? (viii) How does the applied light intensity influence the responses? (ix) As HS was identified as another trigger for the accumulation of TAGs in Chlamydomonas, can HS be exploited for biodiesel production in Chlamydomonas and other algae? ACKNOWLEDGEMENTS This work was supported by the Deutsche Forschungsgemeinschaft (Schr 617/6-1; Schr 617/9-1) and the Forschungsschwerpunkt BioComp. The authors declare no conflict of interests.

RECOVERY FROM HS

SUPPORTING INFORMATION

The perhaps most interesting aspect regarding the recovery of Chlamydomonas cells exposed to HS for 24 h is that within an 8-h recovery period cells failed to reestablish the cellular state present before the onset of HS (Hemme et al., 2014). This was found to be true for every system level investigated, i.e., proteins, lipids, and even polar metabolites. Despite this failure, cells had resumed their ability to divide, which reflects the high plasticity of the system. It is possible that during recovery cells aim at resuming cell division as fast as possible to simply restore the original growth state by dilution through a few rounds of division. Alternatively, maintaining the HS acclimated cellular state to some extent might be part of a precautionary concept to be prepared for repeated instances of HS.

Additional Supporting Information may be found in the online version of this article. Table S1. The 1207 most abundant soluble proteins in heatstressed Chlamydomonas.

OUTLOOK The suitability of Chlamydomonas to study the HSR system-wide and in a time-resolved manner under controlled conditions and the amenability of Chlamydomonas to pharmacological and RNA silencing approaches have allowed deep insights into how a plant cell responds to HS. However, many questions are still open and need to be addressed in order to obtain a comprehensive picture of a plant cell’s HSR that eventually may allow the engineering of crop plants toward higher thermotolerance. Crucial questions are: (i) Is HS indeed sensed by one or few primary sensors that coordinately trigger multiple responses, or is each process affected by HS sensed individually to trigger a response specific for that process? (ii) What is the molecular nature of the sensors and downstream components of HS signal transduction chains? (iii) How are disturbances of protein homeostasis linked to calcium responses? (iv) How does the chloroplast UPR work? (v) Which targets other than HSFs do HS signal transduction chains have?

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Plant Heat Adaptation: priming in response to heat stress.

The heat shock response. Cells coping with transient stress.

Sir2 links the unfolded protein response and the heat shock response in a stress response network.

Overexpression of ferredoxin, PETF, enhances tolerance to heat stress in Chlamydomonas reinhardtii.

TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules.

Stress response and virulence of heat-stressed Campylobacter jejuni.

Expression and interaction of small heat shock proteins (sHsps) in rice in response to heat stress.

Light stress and photoprotection in Chlamydomonas reinhardtii.

Spatial and temporal specificity of Ca2+ signalling in Chlamydomonas reinhardtii in response to osmotic stress.

ATF1 modulates the heat shock response by regulating the stress-inducible heat shock factor 1 transcription complex.

Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling.

Chronic Heat Stress Induces Immune Response, Oxidative Stress Response, and Apoptosis of Finishing Pig Liver: A Proteomic Approach.

An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

Expression profiles of the heat shock protein 70 gene in response to heat stress in Agrotis c-nigrum (Lepidoptera: Noctuidae).

Canonical modeling of the multi-scale regulation of the heat stress response in yeast.

Gene Expression Profile in the Long-Living Lotus: Insights into the Heat Stress Response Mechanism.

The interplay of light and oxygen in the reactive oxygen stress response of Chlamydomonas reinhardtii dissected by quantitative mass spectrometry.

Two strategies for the acute response to cold exposure but one strategy for the response to heat stress.

Phenotypic analysis of the Arabidopsis heat stress response during germination and early seedling development.

Heat shock factor and the heat shock response.

Proteomic changes of the porcine small intestine in response to chronic heat stress.

Experience Modulates the Reproductive Response to Heat Stress in C. elegans via Multiple Physiological Processes.

How a retrotransposon exploits the plant's heat stress response for its activation.

Age and hypohydration independently influence the peripheral vascular response to heat stress.