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. SAG 49.72 was originally
isolated from a temperate lake: it’s 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 stark contrast,
within the deep photic zone of Lake Bonney, Antarctica, 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 (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 to both PSII and PSI
under long-term stress.
Long-term stress acclimation in 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 Chl a 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 decrease in the size of LHCII (Maxwell et al., 1994;
Smith et al., 1990; Wilson & Hüner, 2000). Decreases 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). On the other hand, UWO 241 maintained very
low Chl a/b ratios across all treatments, indicating that it does not
need to adjust LHCII antenna size even when exposed to either growth
temperatures approaching 0oC or hypersalinity. These
results support previous observations that UWO 241 maintains a
relatively large oligomeric LHCII (Morgan et al. 1998). Szyszka et al.
(2007) also observed that UWO 241 does not modulate abundance of two
major LHCII polypeptides in response to variable light intensity.
Morgan-Kiss et al. (2002) demonstrated that UWO 241 is also unable to
undergo state transitions. More recently, Szyszka-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
relative to PSI content under long-term stress. Despite an apparent lack
of some classic acclimatory mechanisms, stress-acclimated cells of UWO
241 maintained a high qL and comparable energy partitioning relative to
control conditions, suggesting that the psychrophile deals with high
excitation pressure other strategies.
CEF is an essential process in plants and algae for balancing ATP/NADPH
and photoprotection; although, most studies have been restricted to
considering CEF during 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 (Iwai et
al., 2010) 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
(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 sustained CEF
as a general long-term acclimatory strategy.
CEF generates additional transthylakoid proton motive force which is
used 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. In UWO
241, CEF rates exhibited a strong correlation with higher capacity for
NPQ compared. In contrast, SAG 49.72 had low CEF under all conditions
and no correlation between NPQ and CEF (Figure 6). This suggests
constitutive capacity for PSII protection in the psychrophile owing to
enhanced CEF-generated pmf.
High CEF also 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 possesses 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). Similarly, elevated CEF protects PSI from low
temperature-associated photoinhibition during the winter-spring
transition in Scots pine (Yang et al. 2020), which fits well with an
earlier hypothesis that UWO 241 is an ‘evergreen alga’ during the polar
night (Morgan-Kiss et al. 2006). Thus, PSI photoinhibition is a
deleterious consequence for survival under long-term stress. We suggest
that constitutive CEF serves multiple roles, 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,
Gautier, & Stevens, 2013). For example, the model C. reinhardtiiappears 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 S2). These genes were
also detected at the level of the genome, with four APX genes present as
highly similar tandem duplicates (Table S3; 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. Similarly, Zhang
and colleagues (Zhang et al. 2020) recently reported that the Antarctica
sea ice alga, Chlamydomonas sp. ICE-L, exhibited gene expansion
of the APX enzyme and increased APX activity. We did not identify an
isoform for MDHAR in the genome or transcriptome. This enzyme is
prevalent in plant genomes, but often missing from algae which can also
regenerate Asc by non-enzymatic dismutation of MDHA to (Gest, Gautier,
& Stevens, 2013). 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).
Massive expansion of multiple gene families involved in photoprotection,
DNA repair, ROS detoxification, and several other essential processes
for environmental adaptation, were also detected in the genome ofC. sp. ICE-L (Zhang et al. 2020).