INTRODUCTION
Perennially cold environments, such as polar and alpine regions, are one of the world’s largest ecosystems. Phototrophic microbes, many of which are obligate cold extremophiles (psychrophiles), are the dominant primary producers in these habitats and the base of virtually all low temperature food webs (Morgan-Kiss, Priscu, Pocock, Gudynaite-Savitch & Huner 2006; Margesin 2008; Lyon & Mock 2014; Chrismas, Anesio & Sánchez-Baracaldo 2015). Most studies on psychrophily focus on the biochemical characteristics that enable these organisms to thrive at permanently low temperatures, including cold-adapted proteins and increases in membrane fluidity, as well as the presence of ice-binding proteins, antifreeze proteins and cryoprotectants (reviewed in Siddiquiet al. 2013; De Maayer, Anderson, Cary & Cowan 2014). But a distinguishing feature of psychrophiles is not an exceptional ability to grow at low temperatures. Indeed, many land plants, green algae and cyanobacteria survive and grow at both cold and warm temperatures and are therefore not psychrophilic (Huner, Öquist & Sarhan 1998; Tang & Vincent 1999; Öquist & Huner 2003; Hüner et al. 2012; Yamori, Hikosaka & Way 2014; Chang, Bräutigam, Hüner & Ensminger 2021). Rather, it is the inability of psychrophiles to survive at moderate (mesophilic) temperatures (≥20°C) that distinguishes them from cold-tolerant species (Morita 1975).
Chlamydomonas sp. UWO241 is one of the most comprehensively studied algal psychrophiles and an up-and-coming model for photosynthetic adaptation to extreme environments (Morgan-Kiss et al. 2006; Dolhi, Maxwell & Morgan-Kiss 2013; Cvetkovska, Hüner & Smith 2017). This alga resides at a depth of 17 meters within the water column of the perennially ice-covered Antarctic Lake Bonney (Neale & Priscu 1995), where it faces several environmental challenges including constantly low temperatures (~5°C), high salinity (0.7M), extreme shading (5-15 µmol m-2s-1), high oxygen concentrations (200% air saturation levels), low phosphorus levels (N:P ~1000), and seasonal extremes in photoperiod. While this environment is extreme in many aspects, it is also very stable. The perennial ice-cover prevents wind-driven water mixing and promotes a vertically stratified environment where the salinity, nutrient levels and temperature profiles remain extraordinarily constant year-round (Obryk, Doran, Hicks, McKay & Priscu 2016; Spigel, Priscu, Obryk, Stone & Doran 2018).
In green algae, heat stress affects most cellular processes, including the fluidity of biological membranes, metabolism, enzyme activities and protein homeostasis. To maintain cellular function and prevent irreversible damage, green algae induce a heat stress response (HSR) that involves arrest of active cell growth, a switch from regular to stress metabolism, compositional remodelling of membrane lipids, and maintenance of protein homeostasis (Schroda, Hemme & Mühlhaus 2015). One of the first responses to heat stress is the increased transcription of genes encoding Heat Shock Proteins (HSPs), highly conserved molecular chaperones that engage in nascent protein synthesis as well as folding and transport (Lindquist & Craig 1988; Vierling 2003; Wang, Vinocur, Shoseyov & Altman 2004; Gupta, Sharma, Mishra, Mishra & Chowdhuri 2010). HSPs were first described in relation to heat shock inDrosophila (Ritossa 1962), but are now known to be important in both normal homeostatic growth (Lindquist & Craig 1988) as well as during several abiotic and biotic stresses (Vierling 2003; Wang et al. 2004; Park & Seo 2015). Their function has been extensively studied in the model green alga Chlamydomonas reinhardtii(Schroda & Vallon 2009; Nordhues, Miller, Mühlhaus & Schroda 2010; Schroda et al. 2015), where their expression is regulated by a single heat shock transcription factor (HSF1) (Schmollinger et al. 2013). The HSR in C. reinhardtii is rapidly initiated, with increased HSP expression seen within the first 25 minutes of exposure to 42°C and robustly maintained for at least 24 hours (Mühlhaus, Weiss, Hemme, Sommer & Schroda 2011; Hemme et al. 2014; Légeretet al. 2016).
Life in the perpetual cold has shaped the physiological make-up of UWO241, which has been mostly studied at the level of its photosynthetic machinery, as reviewed in (Cvetkovska et al. 2017) and subsequently discussed in (Szyszka-Mroz et al. 2019; Cooket al. 2019; Kalra et al. 2020). This alga is unable to grow above 18°C, but little is known how exposure to temperatures above this upper growth limit affect its physiology. It been shown that exposure to 24ºC is lethal but cell death occurs slowly, and the effects are reversible in the first 12 hours (Possmayer et al. 2011). Short-term exposure to 24ºC resulted in cessation of cell growth, inhibition of PSII efficiency and expression of the molecular chaperone HSP22A, and longer exposures led to cell death (Possmayer et al.2011). In addition, two key photosynthetic proteins in UWO241, ferredoxin (Cvetkovska et al. 2018) and the chloroplast kinase STT7 (Szyszka-Mroz et al. 2019) were shown to be specifically adapted to low temperatures, with higher activities in the cold but at the expense of increased sensitivity and loss of activity at more moderate temperatures when compared to their mesophilic homologs fromC. reinhardtii .
While it is clear that UWO241 experiences stress at moderate temperatures, it is currently unknown whether this psychrophile mounts an HSR in a manner comparable to its mesophilic relatives. To gain insight into the systemic response of UWO241 to temperature stress, we examined the growth, primary metabolome and transcriptome of UWO241 under steady-state low temperature and heat stress conditions. The recent sequencing of the UWO241 genome (Zhang, Cvetkovska, Morgan-Kiss, Hüner & Smith 2021a) has placed UWO241 in an excellent position for comparative sequence analysis since it is phylogenetically closely related to a number of model green algae models, including C. reinhardtii (Possmayer et al. 2016; Cvetkovska et al.2017). Using this new resource, we investigated the presence of HSP genes in the UWO241 genome and their responsiveness to heat stress using RNA-Seq and protein immunoblotting. Our work contributes to a better understanding of psychrophilic stress biology, a question that is gaining in importance since polar environments are particularly threatened by current patterns of global climate change (Xavier et al. 2016; Kennicutt et al. 2019).