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 used for ROS detoxification 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 AsA-GSH pathway is particularly important for antioxidative defense in plants but appears to play a lesser role in algae and cyanobacteria (Foyer & Halliwell, 1976; Foyer, Lopez-Delgado, Dat, & Scott, 1997; Hu et al., 2008).
Some photosynthetic organisms have evolved to survive and grow under permanent 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 xanothophyll 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 forms 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). 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 UWO 241 utilizes CEF and ROS detoxification as long-term stress acclimation mechanisms to maintain photostasis and protect 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). 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.