High accumulation of HSPs in UWO241 could have adaptive roles at
low temperature.
HSPs are one of the first hallmarks of the HSR and key component of
protein synthesis, folding, and the prevention of protein aggregates
during heat stress (Schroda et al. 2015). Screening of the UWO241
genome revealed more putative HSP genes than any other green alga
examined to date (Table 4, Supplementary Figure S4). This expansion is
mostly driven by multiple copies of a few genes, including six (6) ClpB3
and HSP70A genes, three (3) CPN60A genes and no less than ten (10)
HSP22A genes homologous to those in C. reinhardtii . The genome of
psychrophilic Antarctic sea-ice green alga ICE-L also encodes for a
significantly more HSP genes as compared to non-psychrophilic species
(Table 4). UWO241 belongs to the Moewusinia clade of the order
Chlamydomonadales (Possmayer et al. 2016), while ICE-L is a
member of the closely affiliated Monadinia clade (Zhang et al.2020). This suggests that an expanded HSP gene family may be a
consequence of life in the permanent cold, and not a feature common to a
particular algal clade.
Sequencing of the UWO241 genome revealed an unusually large (212 Mb)
nuclear genome with hundreds of highly similar duplicate (HSD) genes
from diverse cellular pathways (particularly in protein translation, DNA
packaging and photosynthesis), more than any other algal species with a
sequenced genome (Zhang et al, 2021). Its genome has been termed “a
genome in upheaval” due to the presence of multiple partial duplicates,
gene fragments and pseudogenes (Zhang et al. 2021a). It was
postulated that this rampant gene duplication, likely
retrotransposon-mediated, is random and thus neutral or even
maladaptive. But if the presence of a duplicated gene confers an
increase in fitness, that gene will be fixed in the genome (Qian &
Zhang 2014). But why would multiple HSP gene copies be fixed in the
UWO241 genome, and what would be the fitness advantage? HSPs in C.
reinhardtii have been studied mostly for their role during stress
responses (reviewed in Nordhues et al., 2010) but do they have an
equivalent role in UWO241, an obligate psychrophile released from the
challenges of a variable environment?
Detailed RNA-Seq analysis revealed that UWO241 induced the expression of
some, but not all HSPs, when exposed to heat stress, regardless of the
initial culturing temperature (Figure 8). This response was particularly
strong for sHSP genes, and most of the HSP22A homologs were strongly
upregulated by exposure to heat. The strong heat-induction of sHSP
transcripts has been shown before in UWO241 under short-term heat stress
(Possmayer et al. 2011), as well as in C. reinhardtii and
other algae (Mühlhaus et al. 2011; Kobayashi et al. 2014;
Uji, Gondaira, Fukuda, Mizuta & Saga 2019; Liang et al. 2020).
Curiously, UWO241 failed to accumulate increased protein amounts when
challenged with heat stress, despite increased HSP transcript levels
(Figure 7).
In a direct comparison with C. reinhardtii , we showed that UWO241
accumulates significantly higher protein amounts of major HSPs (HSP70A,
HSP90A, CPN60A) under a range of steady-state growth temperatures in the
absence of heat stress (Figure 9). Furthermore, we also observed high
HSP levels in C. reinhardtii acclimated to 10°C (Figure 10). Cold
induction of HSP expression has been previously observed in C.
reinhardtii (Maikova, Zalutskaya, Lapina & Ermilova 2016) and land
plants (Renaut, Hausman & Wisniewski 2006; Timperio, Egidi & Zolla
2008), suggesting a role of chaperones during low-temperature growth.
Unlike UWO241, C. reinhardtii cultured at 10°C retained the
ability to induce further accumulation of most HSPs, including HSF1,
when heat stressed despite having high levels of HSPs during
steady-state growth (Figure 11). Thus, while high levels of HSPs are
common between the two species at low temperature, only the mesophile
could significantly increase HSP amounts when challenged with heat.
Whether constitutively high HSP levels confer resistance to heat is
still a matter of debate. Thermotolerance in HSP over-expressors in
plants, yeast and bacteria (Fragkostefanakis, Röth, Schleiff & Scharf
2015; Santhanagopalan, Basha, Ballard, Bopp & Vierling 2015) is often
limited to a narrow range of conditions or a specific developmental
stage (Fragkostefanakis et al. 2015; Waters & Vierling 2020).
Considering the increased evidence for broader roles of chaperones in
cellular protein homeostasis (Lindquist & Craig 1988; Vierling 2003;
Wang et al. 2004; Gupta et al. 2010; Park & Seo 2015), it
appears that the ability of an organism to tightly regulate its central
defense network plays a more prominent role in thermotolerance rather
than the amount of chaperone proteins themselves. This certainly appears
to be the case in UWO241, which cannot survive moderate temperatures
(Figure 4a) despite constitutively accumulating high quantities of HSPs
(Figure 9). Similarly, increased levels of HSPs in cold-grown C.
reinhardtii did not confer greater survival under prolonged heat stress
when compared to those grown at 28°C (Figure 4b). The significance of
high HSP expression but not protein accumulation during heat stress is
currently not clear. UWO241 does not experience temperature fluctuations
in its natural environment, but inducible HSP expression could be a
remnant of its distant past before its arrival in Lake Bonney. We do not
know the evolutionary history of UWO241, but it is closely related to
the marine species Chlamydomonas parkeae (Possmayer et al.2016), indicating an ancestral lifestyle in a variable environment where
stress-induced protective pathways would be beneficial.
We propose that the constitutively high accumulation of HSPs in UWO241
plays an important role in protein quality control and ensures a robust
capacity for protein folding at low temperatures, rather than protection
from heat stress. Protein synthesis and the folding of nascent proteins
are temperature-sensitive cellular processes (Hebraud, Dubois, Potier &
Labadie 1994; Phadtare 2004; Piette, Struvay & Feller 2011; Rosa,
Roberts & Rodrigues 2017), and it has been suggested that the
involvement of molecular chaperones is a crucial component of cold
adaptation in psychrophilic bacteria (Piette et al. 2011; Feller
2013). We propose a similar role for HSPs in UWO241. One of the main
differences between the primary metabolomes of UWO241 and C.
reinhardtii were that 10°C-grown C. reinhardtii showed high
levels of amino acids, but UWO241 did not, at any growth temperature
(Figure 3b, Table 1). Amino acid accumulation could be a protective cold
stress response, or the consequence of decreased efficiency of protein
synthesis at low temperatures in C. reinhardtii (Valledor,
Furuhashi, Hanak & Weckwerth 2013). The lack of a large free amino acid
pool in UWO241 may indicate an efficient protein synthesis machinery
that is not negatively affected by low temperatures.
Zhang et al . (2021) proposed that HSDs in the UWO241 genome aid
in survival at extreme environments by contributing increased protein
amounts via gene dosage (Kondrashov 2012; Qian & Zhang 2014). Indeed,
UWO241 was shown to have increased protein accumulation of
photosynthetic ferredoxin (PETF) when compared to C. reinhardtii .
Unlike other green algae, PETF in UWO241 is encoded by two near
identical genes (Fd-1A and Fd-1B) (Cvetkovska et al. 2018). The
increased PETF protein levels may contribute to the higher capacity for
photosynthetic electron transport and maintenance of photostasis in
UWO241 (Szyszka, Ivanov & Hüner 2007; Szyszka-Mroz et al. 2019;
Kalra et al. 2020). We propose that the expansion and
duplicate-gene retention of the HSP gene family in UWO241 confers an
adaptive advantage for life in the cold by increasing the capacity for
protein folding at low temperatures.