The primary metabolome of UWO241 grown at 4°C responds strongly to heat stress
To understand the heat-induced changes to psychrophilic metabolism, we analyzed the primary metabolome of UWO241 exposed to the non-permissive temperature of 24°C for 6h. PCA analysis of all 771 detected metabolites demonstrated a separation along both principal components between the metabolome of the UWO241 cultures grown at 4°C and the metabolome of the same cultures exposed to 24°C (Figure 6). We also observed a separation between the metabolomes of UWO241 grown at 10°C and those exposed to heat, albeit only along PC2. There was minimal separation along either component between the cultures acclimated to the highest growth temperature of 15°C before and after 6h heat stress (Figure 6). HCA (Figure 7A) and volcano plot analysis (Table 3) revealed a strong response at the level of the metabolome when cultures grown at 4°C were exposed to 24°C for 6 hrs (222 DAMs, 29%). This heat stress response was attenuated in cultures acclimated to 10°C, and even more so in the cultures acclimated to 15°C, with 71 (9%) and 26 (3%) DAMs after heat exposure, respectively.
Detailed analysis on the 161 positively identified metabolites revealed that 98 metabolites (54.8%) were significantly different between control and heat stress samples (ANOVA, p<0.01, Tukey’s post hoc). We organized the positively identified metabolites by chemical categories and normalized all control samples to an arbitrary value of 1. We compared the metabolomes from cultures exposed to 24°C to the appropriate steady-state growth temperature (i.e., 4°C metabolome was compared to 4°C + 6h heat stress metabolome). We reported the differences between the metabolite classes (Figure 7B, Supplemental Dataset S2) and the 20 metabolites that showed the largest abundance difference between steady-state and heat stress treatments (Table 4). Most metabolite classes, including carbohydrates, sugar alcohols, amino acids, lipids and antioxidants, increased in abundance in the cultures grown at 4°C and exposed to 24°C, with the exception of carboxylic acids and sugar phosphates, which increased significantly in the 10°C cultures (but not in those grown at 15°C). Notably, these metabolites are already present in high amounts in UWO241 acclimated to 4°C (Figure 3B). We also analyzed the 20 metabolites that showed the most significant change in accumulation in heat stressed UWO241 (Table 4). Glucose-6-phosphate was the only compound that showed a small but significant decrease regardless of the initial culturing temperature (FC 1.7-2.6). Ergosterol (FC 439.1) and α-tocopherol (FC 308.1) increased at very high amounts in all cultures exposed to heat, regardless of the initial growth conditions (Table 5). These increases followed a temperature dependent pattern, with the highest FC seen in the cultures initially grown at 4°C.