The growth and metabolic make-up of UWO241 and C. reinhardtii at low temperature
In its native environment, UWO241 is exposed to year-round low temperatures (4°C-6°C). A culturing temperature of 4°C supported a robust growth (~0.22 day-1), but maximal growth rates were achieved at 10°-15°C (0. 25-0.30 day-1). As an obligate psychrophile, UWO241 did not grow at 20°C (Figure 1a). In accordance with previous reports (Schrodaet al. 2015), C. reinhardtii exhibited maximal growth rates at 20°C-28°C (~0.6 day-1), lower rates at temperatures both higher and lower than this range (Figure 1b) and was unable to grow <10°C. C. reinhardtii cultures were able to grow at 10° and 15°C, albeit at a decreased rate (0.25-0.32 day-1) compared to growth at 28°C (Figure 1b), but notably the rates were similar to those of UWO241 between 10°-15°C (Figure 1a).
Untargeted analysis of the primary metabolome of both species acclimated to different temperatures (4°C, 10°C, 15°C – UWO241; 10°C, 15°C, 28°C – C. reinhardtii ) detected 771 unique metabolites, 163 of which were positively identified based on their mass spectra and retention times (Kind et al. 2009). PCA analysis revealed that the metabolic status of C. reinhardtiiwas dependent on the culturing temperature and differed between both components. This contrasted with UWO241 where temperature had minimal effects on the overall metabolic status, and we observed only nominal separation between the cultures grown at different temperatures (Figure 2). Differentially accumulated metabolites (DAMs) were defined as those exhibiting a 2-fold change (FC) in accumulation between treatments (p<0.01, ANOVA, Tukey’s post-hoc). The metabolome of C. reinhardtii responded strongly to growth temperature with 273 (35%) DAMs at 10°C compared to the optimal temperature of 28°C. This response was temperature dependent, with cultures at 15°C exhibiting a similar metabolite profile but with a decreased magnitude of the response (72 DAMs, 9%) (Figure 3a). The metabolic profile of UWO241 was similar regardless of the culturing temperature. Cultures at 10°C and 15°C had only 78 (10%) and 48 (6%) DAMs when compared to 4°C (Figure 3a). These findings indicate that the mesophile grown at low temperatures adjusts its metabolome as an acclimation to cold, while the primary metabolome of the psychrophile is less affected by culturing temperature.
Next, we analyzed the 163 primary metabolites with positively identified chemical signatures by comparing all samples to the metabolome ofC. reinhardtii grown at 28°C (Figure 3b; Supplemental Dataset S1). We present the 20 metabolites that have the largest differences in abundance between the treatments (Table 1). Carbohydrates and glycerol are well-known cryoprotectants in cold-adapted plants and algae (Roser, Melick, Ling & Seppelt 1992; Tulha, Lima, Lucas & Ferreira 2010; Leya 2013; Su et al. 2016). We observed high accumulation of several carbohydrates (trehalose, maltose, and fructose) and glycerol metabolism intermediates in C. reinhardtii grown at 10°C. UWO241 accumulated these compounds constitutively, regardless of culturing temperature (Figure 3b; Supplemental Dataset S1). Carboxylic acids and TCA cycle intermediate accumulation showed a strong dependence on growth temperature and was increased at the lowest temperature for both algae. Notably, α-ketoglutarate (α-KG), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP) showed the highest increases in abundance in UWO241 grown at 4°C (FC 48.1, 39.8, and 18.0 respectively). Lactic acid is the exception: its abundance is significantly increased in the mesophile (FC 70.2 at 10°C) but decreased in the psychrophile (FC 6.7 at 4°C). We also detected high accumulation of dehydroascorbic acid in bothC. reinhardtii at 10°C (FC 19.7) and UWO241 at all temperatures (FC 12.4-22.5) (Table 1). Threonic acid, a product of ascorbate catabolism (Debolt, Melino & Ford 2007), showed a similar pattern, suggesting an important role for the ascorbate pathway during low temperature growth.
We observed species-specific differences in the primary metabolomes. Glucose, which has known roles in osmotolerance and cold stress (Demmig-Adams, Garab, Adams III, & Govindjee 2014; Taïbi et al.2018), was present at lower levels in UWO241 compared to C. reinhardtii , regardless of the temperature (FC 24-81). Sugar alcohols are important molecules in cold-stress tolerance in plants and algae (Roser et al. 1992; Leya 2013); their accumulation was increased in C. reinhardtii but not in UWO241 (Figure 3b), although both species showed a significant decrease in several sugar alcohols (mannitol and galactinol; Table 1) at low temperatures. Amino acid metabolism was significantly affected by low temperature in C. reinhardtii and nearly all detected amino acids increased in abundance. Again, we did not observe this in UWO241, and amino acid abundance was largely unchanged or decreased (Figure 3b; Table 1; Supplemental Dataset S1). An exception is the non-proteinogenic amino acid ornithine, which accumulated at low temperatures in both species. In C. reinhardti i, this accumulation was temperature dependent (higher at 10°C than at 28°C, FC 9.7), whereas in UWO241 it is constitutively high at all temperatures (FC 33.5 – 34.7; Table 1). N-containing compounds, including those involved in purine and pyrimidine metabolism, exhibited cold-dependent accumulation in C. reinhardtii ; however, we observed the opposite trend in UWO241 where N-compounds accumulated at higher levels at 15°C when compared to 4°C (thymidine, Figure 3b, Table 1; Supplementary Dataset S1). Altogether, we suggest that these data reflect a metabolic switch in the primary metabolism of UWO241 due to life in a perennially cold environment. UWO241 appears to retain an active energy metabolism at low temperatures, which fuels the constitutive accumulation of stress-related compounds across a range of growth temperatures.