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).