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).