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).