INTRODUCTION
Green algae have a plastic metabolism and physiology, which allows them to cope with many environmental variables. Changes in temperature affect basic cellular functions by modifying the fluidity of biological membranes (Los & Murata 2004; Morgan-Kiss, Priscu, Pocock, Gudynaite-Savitch & Hüner 2006; Horváth et al. 2012; Los, Mironov & Allakhverdiev 2013) and the rate of enzymatic reactions (Feller 2013, 2018; Isaksen, Åqvist & Brandsdal 2016), and can influence a wide array of biological, chemical, and physiological processes. It is generally accepted that an optimal temperature results in highest rates of metabolism and growth of algal populations, whereas temperatures above and below this optimum inhibit metabolic processes, which leads to lower growth rates (Borowitzka 2018). At non-permissive temperatures (below the minimum or above the maximum for growth), these processes can fail and may ultimately cause cell death.
Perennially cold environments, such as polar and alpine regions, are among the world’s largest ecosystems. Phototrophic microbes, including eukaryotic algae, are the dominant primary producers in polar habitats and are at the base of virtually all low temperature food webs (Morgan-Kiss et al. 2006; Margesin 2008; Lyon & Mock 2014; Chrismas, Anesio & Sánchez-Baracaldo 2015). Many of these organisms are obligate cold extremophiles (psychrophiles) which grow optimally at temperatures close to the freezing point of water but are unable to survive at moderate temperatures ≥ 20ºC (Casanueva, Tuffin, Cary & Cowan 2010; Cvetkovska, Hüner & Smith 2017). The green algaChlamydomonas sp. UWO241 is among the best-studied eukaryotic algal psychrophiles. Its close phylogenetic relationships with model green algae, including Chlamydomonas reinhardtii , make it an ideal candidate for comparative studies (Possmayer et al. 2016).
UWO241 was isolated 17 m below the surface of the perennially ice-covered Lake Bonney (McMudro Dry Valleys, Antarctica), where it thrives under many different extremes, including low but stable temperatures (~5°C), high salinity (0.7 M NaCl), low light (<10 μmol m-2 s-1) enriched in blue-green wavelengths (450-550 nm), and extremes in photoperiod (e.g., long-term darkness during the polar night). Despite originating from such a severe habitat, UWO241 shows strong physiological plasticity, and can successfully grow under a variety of laboratory conditions (white light; intensity 10-250 µmol m-2 s-1; salinity 0.01-1.2M) (Morgan-Kiss, Ivanov & Huner 2002; Morgan-Kiss et al. 2005; Pocock et al. 2004; Pocock; Szyszka, Ivanov & Hüner 2007; Takizawa, Takahashi, Hüner & Minagawa 2009; Szyszka-Mroz, Pittock, Ivanov, Lajoie & Hüner 2015). UWO241 is an obligate psyschrophile with an upper temperature limit of 18ºC, above which population growth ceases and cells eventually die (Possmayer et al. 2011).
Previous studies of UWO241 focused on understanding the photosynthetic mechanisms behind psychrophilic adaptations (reviewed in (Morgan-Kisset al. 2006; Dolhi, Maxwell & Morgan-Kiss 2013). Studies on UWO241 revealed novel adaptations to extreme conditions, including a remodeled photosynthetic machinery. UWO241 is the only known natural algal variant that does not undergo typical photosynthetic state transitions, which balance the energy distribution between photosystems I and II (PSI and PSII) via a reversible LHCII phosphorylation and migration (Minagawa 2011; Rochaix 2014). Instead, UWO241 balances the energy budget of its electron transport chain by re-organizing the photosynthetic apparatus, resulting in direct energy spillover between the two photosystems and increased cyclic electron flow (CEF) around PSI (Szyszka et al. 2007; Szyszka-Mroz et al. 2015, 2019; Kalra et al. 2020). This unique mechanism of energy balance was recently linked to the presence of cold-adapted photosynthetic proteins. Ferredoxin (Fd-1) is involved in the transfer of electrons from PSI to various metabolic pathways, including carbon fixation and CEF (Hanke & Mulo 2013). The UWO241 genome, which was recently sequenced (Zhang, Cvetkovska, Morgan-Kiss, Hüner & Smith 2021), encodes two Fd-1 isoforms, which accumulate at higher amounts compared to mesophilic Fd-1 from C. reinhardtii . In UWO241, Fd-1 is most active at 10ºC but has increased structural sensitivity and lower activity when incubated at temperatures >40°C (Cvetkovska et al ., 2018). The chloroplast protein kinase Stt7 phosphorylates the major light-harvesting proteins associated with PSII (LHCII), facilitating their movement from PSII to PSI during state transitions in C. reinhardtii (Lemeille et al . 2010; Rochaix, 2014). In contrast to C. reinhardtii , Stt7 in UWO241 has higher LHCII-phosphorylation activity at 8ºC than at 23ºC, which appears to reflect the reorganization of the photosynthetic apparatus required for energy spillover (Szyszka-Mroz et al. 2019). Thus, these two key photosynthetic proteins appear to have evolved to optimize photosynthetic light harvesting, energy transfer, and carbon fixation at low temperatures at the expense of lower activities at moderate and high temperatures.
The underlying assumption of most studies on psychrophily is that cold-adapted organisms have inherent biochemical characteristics enabling them to thrive at low temperatures. However, the distinguishing feature of a psychrophile is not necessarily an exceptional ability to grow at low temperatures, but rather an inability to survive at moderate, seemingly innocuous temperatures. Indeed, many photosynthetic species, including crop plants, evergreen conifers, green algae and cyanobacteria, survive and grow at both cold and warm temperatures and are therefore not psychrophilic (Chang et al. 2020; Adamset al ., 1995; Hüner et al. , 1998; Tang and Vincent, 1999; Öquist and Hüner, 2003; Hüner et al ., 2012; Yamori et al ., 2014). UWO241 is one of the few psychrophilic chlamydomonadalean algae that have been studied in relation to heat stress. It has been shown that exposure to 24ºC can be lethal but cell death occurs slowly, and the effects are reversible in the first 12 hours (Possmayer et al. 2011). Moreover, short-term exposure to 24ºC resulted in a myriad of physiological changes, including cessation of cell growth, inhibition of PSII efficiency and accumulation of the molecular chaperone HSP22A (Possmayer et al. 2011). Long-term exposure of UWO241 to 24ºC leads to cell death.
Can UWO241, which likely never experiences temperatures above 5°C in its natural environment, mount a heat stress response comparable to a mesophile? And can it acquire thermotolerance, whereby small, non-lethal increases in temperature prime the cell to respond more rapidly and strongly to subsequent heat stress? Indeed, exposure to higher but non-lethal temperatures can have a protective effect in mesophiles (Song, Jiang, Zhao & Hou 2012; Horváth et al. 2012; Yeh, Kaplinsky, Hu & Charng 2012; Kishimoto, Ariga, Itabashi & Mikami 2019) but this topic, to the best of our knowledge, has never been addressed in a psychrophile. Here, we examine the growth and metabolic profiles of UWO241 under steady-state low temperature and heat stress conditions. We analyze and describe its adaptive strategies to cold, as well as its heat stress response upon exposure to non-permissive temperatures. This work contributes towards the understanding environmental stress responses in a psychrophilic Antarctic alga — a contribution made more important by the fact that polar environments and organisms that thrive in the cold are particularly threatened by current patterns of global climate change (Kennicutt et al. 2015, 2019; Xavier et al.2016).