Metabolic signatures of adaptation to permanently low
temperatures
A key observation stemming from our work is that despite low growth
rates, the primary metabolome of UWO241 cultures grown at 4°C did not
differ significantly from those grown nearer their optimal growth
temperatures of 10°C to 15°C (Figure 1). In contrast, C.
reinhardtii showed a strong temperature dependent response at the level
of the primary metabolome (Figure 2, Figure 3A), with 10°C-grown
cultures accumulating increased levels of cryoprotectants and membrane
stabilizers as compared to cultures grown near their optimal temperature
(28°C). These compounds are typically present in photosynthetic
organisms exposed to cold stress (Wanner & Junttila 1999; Gray & Heath
2005; Kaplan et al. 2007; Guy, Kaplan, Kopka, Selbig & Hincha
2007; Janská, Maršík, Zelenková & Ovesná 2010; Fürtauer, Weiszmann,
Weckwerth & Nägele 2019). We interpret this as evidence that when the
psychrophile UWO241 is cultured at 4°C it does not experience cold
stress, despite this temperature being well below its growth optimum
(10-15°C). C. reinhardtii , on the other hand, exhibits typical
cold-stress responses at the level of the primary metabolome when
cultured at temperatures that lead to slow growth rates.
We suggest that our metabolomic data reveal a constitutive re-routing of
primary metabolism in UWO241 when compared to the mesophilic modelC. reinhardtii . First, our study indicates that constitutively
high accumulation of soluble sugars is a low-temperature adaptation in
UWO241. It appears that this alga has a re-wired central carbon
metabolism and accumulates high amounts of soluble sugars at the expense
of other photosynthetic intermediates, consistent with previous results
for UWO241 (Cook et al. 2019; Kalra et al. 2020).
Second, amino acids and their derivatives accumulated at high levels in
low temperature-grown C. reinhardtii, but this response was
absent in UWO241 (Figure 3B, Table 2). High amino acid levels could be a
protective cold stress response, but it could also be the consequence of
decreased efficiency of protein synthesis at low temperatures inC. reinhardtii (Valledor et al . 2013). The fact that
UWO241 does not accumulate amino acids may indicate an efficient protein
synthesis machinery that is not negatively affected by low temperatures.
One exception was the increased amount of ornithine in UWO241 at all
growth temperatures, but only at 10°C in C. reinhardtii (Table
2). Ornithine is a non-proteinogenic amino acid with a pivotal role in
polyamine, arginine and proline biosynthesis, and its accumulation has
been linked to increased stress tolerance in plants (Ghahremani et
al . 2014; Kalamaki et al . 2009b, 2009a). We also detected
increased amounts of the polyamine putrescine in UWO241 compared toC. reinhardtii (and no increases in arginine or proline;
Supplemental Dataset S1). Polyamines play important roles in DNA and RNA
protection and stabilization, protein synthesis and cell cycle
progression (Gill & Tuteja 2010; Minocha, Majumdar & Minocha 2014;
Chen, Shao, Yin, Younis & Zheng 2019). Our data suggest that in UWO241,
constitutive accumulation of ornithine and polyamines is not a cold
stress response, but a mechanism to ensure cell division and growth at
low temperatures. Ensuring nucleic acid protection and efficient protein
synthesis could be key psychrophilic adaptations to permanently cold
environments.
Third, ascorbic acid (AsA) and its oxidized form dehydroascorbic acid
(DHA) accumulate at high levels in UWO241 at all growth temperatures but
only at 10°C in C. reinhardtii (Table 2; Supplemental Dataset
S1). Photosynthesis creates an oxic intracellular environment, further
exacerbated by reactive oxygen species (ROS) formation due to metabolic
imbalances caused by low temperatures (Dreyer & Dietz 2018). The depth
at which UWO241 is found in Lake Bonney (17 meters below the surface) is
a hyperoxic environment (200% air saturation) due to oxygen having a
higher solubility at low temperatures and poor diffusion in the presence
of permanent ice cover (Morgan-Kiss et al. 2006). Thus, UWO241
and other organisms that live in such environments need a robust and
constitutively active antioxidant system to cope with high intra- and
extracellular ROS. The ascorbate-glutathione (AsA-GSH) cycle is a
fundamental metabolic pathway involved in maintenance of cellular redox
homeostasis (Sakhno, Yemets & Blume 2019; Hasanuzzaman et al.2019). Constitutively high antioxidant levels and increased amounts of
AsA-GSH enzymes have been reported previously in polar diatoms (Janknegt
et al., 2008). We propose that the AsA-GSH cycle is constitutively
active in the psychrophile UWO241 as an adaptation to low temperatures.