Resolving the contribution of specific metabolites to cold acclimation in green algae
We analyzed the subset of 163 primary metabolites with positively identified chemical signatures (Supplemental Dataset S1) and identified 135 metabolites (83.6%) significantly different between at least two of the samples (p<0.01, ANOVA, Tukey’s post hoc). We chose the metabolome of C. reinhardtii grown at 28°C as the control and compared all other samples to it (Figure 3B). Not all metabolites belonging to the same class contributed equally to the observed differences, and we present the 20 metabolites that have the largest differences in abundance between the treatments (Table 2).
A subset of metabolites showed comparable accumulation patterns in both species. Carbohydrates and glycerol are well-known cryoprotectants in cold-adapted organisms (Roser, Melick, Ling & Seppelt 1992; Tulha, Lima, Lucas & Ferreira 2010; Su et al. 2016), including algae (Leya 2013). Here, we observed high accumulation of several carbohydrates (e.g. 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 acid accumulation showed a strong dependence on growth temperature and is increased at the lowest temperature for both algae. Notably, α-ketoglutarate (α-KG), 3-phosphoglycerate (3-PGA), and phosphoenolpuruvate (PEP) showed the highest increases in abundance, particularly in UWO241 grown at 4°C (FC 48.1, 39.8, and 18.0 respectively). Lactic acid is the exception, and 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). Fatty acid and lipid metabolism are important for algal cold adaptation (Lyon & Mock 2014; Jung et al. 2016; Suzuki, Hulatt, Wijffels & Kiron 2019). Ergosterol and linoleic acid exhibited a decreased accumulation in both species grown in the cold (Table 2), although overall lipid levels were not significantly affected by low temperatures (Figure 3B). Finally, we detected high accumulation of the antioxidant dehydroascorbic acid in both C. reinhardtii (FC 19.7) and UWO241 (FC 20.1) grown at their respective lowest temperatures (Table 2). Threonic acid, a product of ascorbate catabolism (Debolt et al ., 2007), also showed increased levels in both species, suggesting an important role for the ascorbate pathway during cold adaptation and acclimation.
We observed species-specific differences in the primary metabolomes. Glucose, a carbohydrate with known roles in osmotolerance, cold stress and freezing tolerance (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). This implies that there is a metabolic switch in the primary carbon metabolism of UWO241. Sugar alcohols are important molecules in cold-stress tolerance in plants and algae (Roser et al . 1992; Leya, 2013); the accumulation of metabolites from this compound class was increased in C. reinhardtii but not in UWO241 (Figure 3B), although both species showed a significant decrease in several sugar alcohols (e.g., mannitol and galactinol; Table 2) at low temperatures. Amino acid metabolism was significantly affected by low temperature inC. reinhardtii and nearly all detected amino acids increased in abundance (Figure 3A). Again, we did not observe this in UWO241, and amino acid abundance was largely unchanged or decreased (e.g., cysteine and aspartic acid; Table 2). 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 its accumulation was constitutively high at all temperatures (FC 13.5 – 34.7; Table 2). We also observed a strong species-specific pattern in the accumulation of N-containing compounds, including those involved in purine and pyrimidine metabolism. These compounds 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 (e.g., thymidine, Figure 3A, Table 2). Altogether, we suggest that these data reflect metabolic adaptations in UWO241 to life in a perennially cold environment.