Metabolic signatures of adaptation to permanently low temperatures
Algae exhibit the fastest growth rates under optimal conditions, whereas stress can threaten their cellular homeostasis and lead to decreased growth rates and reduced fitness (Borowitzka 2018). The Antarctic alga, UWO241, experiences low but very stable temperatures of 4-6°C year-round, but we found that its growth rate is fastest at 10°C and 15°C (Figure 1). This indicates that the biochemical and metabolic processes operating in UWO241 are better adjusted to higher temperatures than what it experiences in nature (4°C). Nevertheless, we showed that UWO241 is, indeed, adapted for life at 4°C, which is reflected in the fact that light- and CO2-saturated rate of O2 evolution for UWO241 at this temperature are comparable to the photosynthetic rate of C. reinhardtii grown at 28°C (Pocock, Vetterli & Falk 2011). The primary metabolome of 4°C-grown UWO241 did not differ significantly from that of cultures at their optimal growth temperatures of 10°C to 15°C. In contrast, C. reinhardtii showed a strong temperature dependent response at the level of the primary metabolome (Figure 2, Figure 3a). The 10°C-grown C. reinhardtii cultures accumulated increased levels of cryoprotectants and membrane stabilizers than cultures grown at their optimal temperature (28°C), a common response in photosynthetic organisms during cold stress (Wanner & Junttila 1999; Gray & Heath 2005; Kaplanet al. 2007; Guy, Kaplan, Kopka, Selbig & Hincha 2007; Janská, Maršík, Zelenková & Ovesná 2010; Fürtauer, Weiszmann, Weckwerth & Nägele 2019). We interpret this as evidence that the psychrophile UWO241 does not exhibit typical cold stress responses when cultured at 4°C, despite its slow growth rates.
Our data reveal a steady-state re-routing of primary metabolism in UWO241 when compared to the mesophilic model C. reinhardtii . We detected the constitutive accumulation of several metabolites with known stress functions in UWO241, regardless of the growth temperature, while the same metabolites only accumulated at high levels in 10°C-grownC. reinhardtii . First, soluble sugars have known cryoprotectant roles in cold-adapted plants and algae (Tulha et al. 2010; Leya 2013; Su et al. 2016). Most detected soluble sugars (trehalose, sucrose, ribose, maltose, fructose) accumulated at high levels in all UWO241 cultures but only in 10°C-grown C. reinhardtii (Table 1; Supplemental Dataset S1). This suggests that the psychrophile has a re-wired central carbon metabolism and accumulates high amounts of carbohydrates at the expense of other photosynthetic intermediates, consistent with previous studies (Cook et al. 2019; Kalraet al. 2020).
Second, we observed similar patterns with ascorbic acid (AsA) and its oxidized form dehydroascorbic acid (DHA) (Table 1; Supplemental Dataset S1). The ascorbate-glutathione (AsA-GSH) cycle is a fundamental metabolic pathway involved in maintenance of cellular redox homeostasis during cold stress in many photosynthetic species (Dreyer & Dietz 2018; Hasanuzzaman et al. 2019). Thus, UWO241 and other organisms that live in perpetually cold environments may need a robust and constitutively active antioxidant system to cope with high ROS levels. Third, the non-proteinogenic amino acid ornithine also accumulated at high levels in UWO241 at all growth temperatures, but only at 10°C inC. reinhardtii (Table 1). Ornithine plays a pivotal role in polyamine, arginine and proline biosynthesis, and its accumulation has been linked to increased stress tolerance in plants (Kalamaki et al. 2009a; Kalamaki, Merkouropoulos & Kanellis 2009b; Ghahremaniet al. 2014). We also detected the increased amount of the polyamine putrescine in UWO241 compared to C. reinhardtii (and no increases in arginine or proline, Supplemental Dataset S1). High ornithine and polyamine levels may play a role in DNA and RNA protection and stabilization, protein synthesis, and cell cycle progression at low temperatures (Gill & Tuteja 2010; Minocha, Majumdar & Minocha 2014; Chen, Shao, Yin, Younis & Zheng 2019). Taken together, we suggest that in UWO241 the accumulation of stress-related compounds, such as soluble sugars, antioxidants and polyamines, represents a mechanism to ensure efficient metabolism, redox homeostasis and cell division at low temperatures. Unlike C. reinhardtii , where accumulation of stress-metabolites is induced by cold, in UWO241 these protective compounds constitutively accumulate across a range of permissive growth temperatures.