DISCUSSION
This study examined whether two Chlamydomonas species adapted to extreme contrasts in their native environments rely upon comparable strategies for survival under long-term stress conditions. SAG 49.72 was originally isolated from a temperate lake: it is a mesophilic species and possesses limited ability to acclimate to either salinity or low temperature stress (Pocock et al., 2011; Szyszka et al., 2007). In contrast, in its native Antarctic lake environment, UWO 241 has survived under permanent low temperature and hypersalinity stress for at least 1000 years, based on estimates of the last occurrence of ice-free conditions in Lake Bonney (Morgan-Kiss et al., 2006). Our results confirmed that although both the mesophilic SAG 49.72 and the psychrophilic UWO 241 exhibited the ability to grow robustly under high light, low temperature or high salinity, their tolerance levels and long-term acclimatory strategies to these environmental stressors were markedly different. For the mesophilic SAG 49.72, long-term acclimation could be summarized into maintenance of photostasis by adjustments in PSII antenna size and PSII-PSI energy distribution, both classic long-term acclimatory mechanisms described for other model algal species (Maxwell, Falk, Trick, & Hüner, 1994; Tanaka & Melis, 1997). In contrast, the psychrophilic UWO 241 relies upon constitutive CEF and continuous ROS detoxification capacity to provide photoprotection of PSII and PSI under long-term stress.
Long-term stress acclimation in the mesophile SAG 49.72 involved an increase in the ratio of Chl a/b and a concomitant decrease in PSII/PSI at the level of 77K fluorescence emission. Higher Chl a/b ratios in response to long-term stress have been reported across many algae and plants and coincides with a reduction in the size of LHCII (Maxwell et al., 1994; Smith et al., 1990; Wilson & Hüner, 2000). Reductions in PSII/PSI stoichiometry under either high light or low temperature stress are also well documented and reflects enhanced distribution of absorbed light energy in favor of PSI (Smith et al., 1990; Velitchkova, Popova, Faik, Gerganova, & Ivanov, 2020). In contrast with SAG 49.72, UWO 241 does not appear to rely on either of these classic acclimatory mechanisms to survive long-term stress. Morgan-Kiss et al. (2002) demonstrated that UWO 241 is also unable to undergo state transitions. More recently, Szyska-Mroz and colleagues reported that the psychrophile relies instead on a poorly understood constitutive spill-over mechanism under HS growth conditions (Szyszka-Mroz et al., 2019). Thus, UWO 241 is a natural variant lacking state transitions that maintains a relatively large LHCII and high PSII content under long-term stress. Despite the lack of these well-characterized mechanisms, UWO 241 maintained a high qL and comparable energy partitioning relative to control conditions, suggesting that the psychrophile relies on alternative processes to avoid high excitation pressure.
CEF is an essential process in plants and algae for balancing ATP/NADPH and photoprotection; although, most studies have considered the role of CEF under short-term stress. Early reports identified that UWO 241 exhibits relatively high rates of PSI-driven CEF compared with mesophilic strains (Morgan-Kiss et al., 2002b; Morgan-Kiss et al., 2006). Maximal CEF requires restructuring of the photosynthetic apparatus and assembly of a novel PSI supercomplex (Kalra et al., 2020; Szyszka-Mroz et al., 2015). The UWO 241 supercomplex is distinct from that of previously described complex from C. reinhardtii because the former is not associated with state-transition-inducing treatments and it lacks typical PSI 77K fluorescence emission despite the presence of many PSI core proteins (Iwai et al., 2010; Kalra et al., 2020; Szyszka-Mroz et al., 2015). Here we show that UWO 241 exhibits faster CEF rates under salinity stress, high light or low temperatures, suggesting that the extremophile relies on CEF as a general long-term acclimatory strategy.
CEF generates additional transthylakoid proton motive force which has been proposed to be utilized for several purposes, including balancing ATP/NADPH production and photoprotection of both PSII and PSI (Bulte, Gans, Febeille, & Wollman, 1990; Chaux, Peltier, & Johnson, 2015; He et al., 2015; Lucker & Kramer, 2013; Yamori et al., 2016). Kalra and colleagues showed that under long-term HS stress CEF serves multiple purposes in UWO 241, including additional ATP production as well as constitutive photoprotection (Kalra et al., 2020). Higher ATP levels are used in part to support enhanced CBB pathway activity which supplies substrates for storage compounds (starch), osmoregulants (glycerol), as well as the shikimate pathway (Kalra et al., 2020). It is likely that CEF is utilized for similar processes when UWO 241 is acclimated to HL or LT. This current study provides evidence that high CEF in all three stress conditions is associated with enhanced photoprotection of PSII. Increased CEF rates in cells of UWO 241 acclimated to HL, LT or HS all exhibited a higher capacity for NPQ compared with control cells. This suggests constitutive capacity for PSII protection owing to enhanced CEF-generated pmf.
High CEF provides PSI photoprotection. UWO 241 cultures acclimated to all three long-term stress conditions were associated with reduced levels of ɸNA relative to control-grown UWO 241 cells. These data suggest that CEF also contributes to protection of PSI by preventing accumulation of reduced Fd and minimizing acceptor side limitation. Over-reduction of PSI manifests as production of the ROS, O2- (Asada, 1999). We show that UWO 241 appears to possess remarkable ability to avoid O2- accumulation: cells exposed to either short-term LT or HL stress exhibited minimal accumulation of this ROS. This ability to keep O2- levels lows is likely due to CEF-associated prevention of PSI acceptor side limitation. In contrast the mesophile exhibited significant levels of O2- when exposed to the same conditions. While PSII is typically considered sensitive to all environmental stresses, PSI photodamage occurs under specific environmental conditions, including drought, high salinity and low temperature, and repair of PSI is slow and inefficient (Huang, Yang, Zhang, Zhang, & Cao, 2012; Huang, Yang, Hu, & Zhang, 2016; Huang, Zhang, Xu, & Liu, 2017; A. G. Ivanov et al., 1998; Yamori et al., 2016; Zhang & Scheller, 2004). Thus, PSI photoinhibition is a deleterious consequence for survival under long-term stress. We suggest that constitutive CEF simultaneously plays critical roles in protecting both PSII and PSI from photo-damage in UWO 241 for survival under long-term environmental stress.
UUWO 241 exhibits constitutive protection of PSII and PSI in UWO 241 by minimizing ROS production; however, there is also evidence that the psychrophile possesses enhanced ability for ROS detoxification. The AsA-GSH pathway is a major ROS detoxification pathway in plants, and is responsible for regeneration of the antioxidant ascorbate (Foyer & Noctor, 2012; Foyer & Shigeoka, 2011). The AsA-GSH pathway involves four enzymes, ascorbate peroxidase (APX), monohydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) (Noctor & Foyer, 1998). Plants express multiple isoforms of each enzyme, in particular APX (Pitsch et al. 2010; Teixeira, Menezes-Benavente, Margis, & Margis-Pinheiro, 2004). High concentrations of ascorbate accumulate in plants, particularly under stress conditions, including high light, low temperatures and high salinity (Bartoli, Buet, Grozeff, Galatro, & Simontacchi, 2017; Maruta & Ishikawa, 2017; Wildi & Lütz, 1996; Zechmann, Stumpe, & Mauch, 2011; Zhang et al., 2011). On the other hand, cyanobacteria and algae exhibit significantly lower levels of ascorbate and possess only one isoform or are missing one or more enzymes of the AsA-GSH pathway (Gest et al., 2013). For example, the model C. reinhardtii appears to lack the thylakoid-bound APX found in plants, expressing only a single isoform of APX which is localized to the stroma (Pitsch et al., 2010). A second APX2 isoform has been predicted to localize to the chloroplast, but its function has not been studied (Wu and Wang, 2019). Three pieces of evidence indicate that UWO 241 may rely on the AsA-GSH pathway to a greater extent than previously appreciated in other algal species. First, activity of two enzymes, APX and GR, are constitutively high in UWO 241 relative to the mesophile SAG 49.72 under both control and all long-term stress conditions. Second, UWO 241 cells accumulated millimolar levels of the substrate ascorbate. Last, unlike other algae studied thus far, UWO 241 appears to possess more isoforms of several enzymes necessary for ascorbate cycling. A search of a previously published transcriptome of UWO 241 (Raymond & Morgan-Kiss, 2013) revealed multiple potential homologues for enzymes of the AsA pathway, including 3 APX, 3 DHAR, and 3 GR genes (Table S1). These genes were also detected at the level of the genome, with four APX genes present as highly similar tandem duplicates (Table S2; Figure S3). In addition, one of the putative UWO 241 APX proteins is related to a plant thylakoid-bound isoform from Triticum aestivum . APX catalyzes the oxidation of ascorbate by H2O2, while DHAR and GR work in concert to regenerate glutathione. We did not identify an isoform for MDHAR in the genome or transcriptome, which is needed for recycling of ascorbate. The additional isoforms may be localized to different cellular compartments, as in plants, or may contribute to constitutively high AsA-GSH pathway activity. Gene duplications have been shown for several other UWO 241 genes including photosynthetic ferredoxin (Cvetkovska et al., 2018), chlorophyllide a oxygenase (Cvetkovska et al., 2019) and the chloroplast kinase Stl-1 (Szyszka-Mroz et al., 2019).