INTRODUCTION
Photostasis is a phenomenon common to all photosynthetic organisms: it
encompasses processes which contribute to balancing rates of
photosynthetic energy absorbed with energy consumed by metabolism
(Öquist & Hüner, 2003). Disruption of photostasis is manifested as an
accumulation of a reduced pool of the mobile electron acceptor,
plastoquinone (PQ), leading to photooxidative stress. This phenomenon
occurs under excessive light conditions; however, any environmental
condition which impacts an organism’s ability to use absorbed light
energy can lead to an over-reduction of the PQ pool (Hüner et al., 2012;
Morgan-Kiss, Priscu, Pocock, Gudynaite-Savitch, & Hüner, 2006). Thus,
any alteration in an organism’s environment can exacerbate disruption to
photostasis and enhance the probability of photooxidative stress,
including day/night cycle, salinity, drought, heat, chilling, and
nutrient status (Bartels & Sunkar, 2005; Ensminger, Busch, & Hüner,
2006; Liu, Qi, & Li, 2012; Sharma, Jha, Dubey, & Pessarakli, 2012;
Takahashi & Murata, 2008).
A major biproduct of unbalanced photosynthesis is the production of
reactive oxygen species (ROS). ROS accumulates when the photosynthetic
electron transport chain becomes over-reduced, causing oxidative injury
and damage to proteins, lipids, nucleic acids, and many components of
the photosynthetic apparatus (Apel & Hirt, 2004; Asada, 1996; Møller,
Jensen, & Hansson, 2007; Sirikhachornkit & Niyogi, 2010). Oxidative
stress responses are distinct at the level of time scale and can be
classified into mechanisms for short-term, acute oxidative stress
occurring over seconds to minutes, or long-term, constitutive stress
occurring over hours to years (Niyogi, 1999; Suzuki, Koussevitzky,
Mittler, & Miller, 2012). Short-term responses are non-heritable
adjustments to physiology and biochemistry which avoid ROS production
(Ledford, Chin, & Niyogi, 2007; Sirikhachornkit & Niyogi, 2010).
Common short-term stress response mechanisms are phototaxis, state
transitions, nonphotochemical quenching (NPQ), and alternative electron
transport pathways, such as the water-water cycle and PSI-associated CEF
(Kozi Asada, 2000; Cournac et al., 2002; Minagawa, 2011; Müller, Li, &
Niyogi, 2001; Witman, 1993). Changes in gene expression and protein
translation aid in maintenance of photostasis over longer time scales.
Long-term responses can involve minimizing ROS production and/or
increasing ROS detoxification, and include changes to antenna size or
PSI/PSII stoichiometry increased CO2 fixation capacity,
and activation of antioxidant pathways (Kozi Asada, 2006; Falk et al.,
1994; Falk, Maxwell, Gray, Rezansoff, & Hüner, 1993; Lucker & Kramer,
2013; Tanaka & Melis, 1997; Yamori, Makino, & Shikanai, 2016).
Enzymatic antioxidants include superoxide dismutase (SOD), catalase
(CAT), and enzymes of the ascorbate-glutathione (AsA-GSH) cycle (Noctor
& Foyer, 1998). Maintenance of high antioxidant capacity has been
associated with tolerance to environmental stress in plants and algae
(Aldesuquy, Baka, El-Shehaby, & Ghanem, 2013; Chen, Zhang, & Shen,
2011; Van Alstyne, Sutton, & Gifford, 2020). The metabolite ascorbate
is particularly versatile in ROS defense, possessing the ability to
directly react with several ROS molecules, including singlet oxygen,
superoxide and hydroxyl radicals. Plants accumulate milli-molar levels
of ascorbate; however, algae and cyanobacteria contain significantly
lower levels of the antioxidant (Foyer & Halliwell, 1976; Foyer,
Lopez-Delgado, Dat, & Scott, 1997; Hu et al., 2008).
Some photosynthetic organisms have evolved to survive and grow in
permanently stressful environments. Relative to the well-studied
processes of short- and long-term stress acclimation, strategies of
photosynthetic adaptation to permanent abiotic stress are significantly
less understood. Low temperature environments are abundant at high
latitudes (Young & Schmidt, 2020): photopsychrophiles are
photosynthetic organisms which are physiologically adapted to permanent
low temperatures (Morgan-Kiss et al., 2006). The AntarcticChlamydomonas sp. UWO 241 (UWO 241) was isolated from a
permanently ice-covered, hypersaline lake and represents one of the few
models for photosynthetic adaptation to combined low temperatures and
high salinity (Cvetkovska, Hüner, & Smith, 2017). Early studies
reported that UWO 241 exhibits minimal capacity for short-term
acclimatory mechanisms, such as the xanthophyll cycle and state
transitions (Morgan-Kiss, Ivanov, & Hüner, 2002; Morgan, Ivanov,
Priscu, Maxwell, & Hüner, 1998), and sensitivity to short-term thermal
or high light stress (Morgan-Kiss, Ivanov, Williams, Mobashsher, &
Hüner, 2002; T. Pocock, Koziak, Rosso, Falk, & Hüner, 2007). In lieu of
short-term acclimation, UWO 241 has evolved to rely on constitutive
mechanisms as a consequence of adaptation to permanent low temperatures
and high salinity (Morgan-Kiss et al., 2006). While UWO 241 exhibits
high susceptibility to high light stress, it also possesses the ability
to rapidly recover from photoinhibition (Pocock et al., 2007). Despite
the presence of cold-active thylakoid kinases, energy transfer from PSII
to PSI uses a poorly understood spill-over mechanism (Szyszka-Mroz et
al., 2019). In addition, under native low temperature and high salinity
conditions, UWO 241 assembles a novel PSI supercomplex which allows the
organism to maintain a strong capacity for PSI-driven CEF (Cook et al.,
2019; Szyszka-Mroz, Pittock, Ivanov, Lajoie, & Hüner, 2015). The
additional proton motive force (pmf) derived from CEF is used for
constitutive capacity for NPQ and production of additional ATP in cells
grown under high salinity (Kalra et al., 2020). High ATP production may
suggest a lower requirement for reductant under permanent environmental
stress where growth and assimilation rates could be slow. The
adjustments to the photosynthetic apparatus are accompanied by
alterations in carbon metabolism, including upregulation of several
enzymes within the Calvin Benson Bassham cycle (CBB), and key enzymes of
the shikimate pathway, a high carbon flux pathway which synthesizes
precursors for aromatic metabolites (Julkowska, 2020; Kalra et al.,
2020). Together, these novel adaptive strategies allow UWO 241 to
maintain robust growth and photosynthesis under the combined stress of
permanent low temperature and high salinity.
While activation of CEF is known to be essential in plants and algae
exposed to short-term stress, the discovery of a strong CEF capacity in
a psychrophilic, halotolerant alga suggests that there is a previously
unappreciated role for CEF during long-term adaptation to environmental
stress. We hypothesized that constitutive CEF and ROS detoxification
allows UWO 241 to survive multiple stresses by maintaining photostasis
and protecting the photosynthetic apparatus from photooxidative damage.
We tested this hypothesis by comparing growth physiology as well as PSII
and PSI photochemistry in UWO 241 and a related mesophilic species,Chlamydomonas raudensis SAG 49.72, acclimated to long-term stress
conditions (high light, low temperature, high salinity). We also
monitored production of a major ROS
(O2-) as well as activity of two key
enzymes of the AsA-GSH pathway (Ascorbate Peroxidase, APX; Glutathione
Reductase, GR) and levels of AsA-GSH substrate, ascorbate. Our study
shows that UWO 241 possesses robust ability to acclimate to long-term
photooxidative stress by both avoiding ROS production by maintaining
photostasis through CEF and relying on constitutive ROS detoxification.
We suggest that this reliance on the redundant systems allow the
organism to withstand long-term exposure to multiple stressors in its
native habitat while minimizing energy expenditures for repair
processes.