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.