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.