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.