Heat shock induced global metabolomic and transcriptomic changes in UWO241.
Next, we examined global metabolomic heat-induced changes in UWO241 cultures grown at 4°C, 10°C and 15°C and subsequently exposed to heat stress at 24°C for 6 hours. 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 5). 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 15°C before the 6h heat stress (Figure 5). HCA (Figure 6a) revealed the strongest response 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.
Next, we compared the accumulation of all 163 positively identified metabolites (Figure 6b, Supplemental Dataset S2) and report the 20 metabolites that showed the largest abundance difference between steady-state and heat stress (Table 2). Most major 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, except for carboxylic acids and sugar phosphates which increased significantly in the 10°C cultures (but not in those grown at 15°C). These metabolites are already present in high amounts in UWO241 acclimated to 4°C (Figure 3b). 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 conditions (Table 2). These increases follow a temperature dependent pattern, with the highest FC seen in the cultures acclimated to 4°C prior to heat shock.
Using the same experimental design we examined transcriptomic responses with RNA-Seq analysis based on the 16,325 gene models in the functionally annotated UWO241 genome (Zhang et al. 2021a). Differentially expressed genes (DEGs) were defined as those having an absolute FC >4 and a p-value <0.05. Similar to the metabolomic analyses (Figure 2; Figure 3), we found that steady-state culturing temperature had only a very minor effect on the overall transcriptome status in UWO241, with <3% of the total number of genes encoded in the UWO241 genome being identified as DEGs in the 10°C and 15°C-grown cultures, when compared to those at 4°C (Figure 7a, 7b; Supplementary Dataset S3). Exposure to 24°C induced a significant heat-induced response at the level of the transcriptome. The cultures acclimated to 10°C exhibited the strongest response with 1909 DEGs (11.7%; 939 up- and 970 down-regulated), followed by the cultures grown at 4°C with 1610 DEGs (9.8%; 826 up- and 784 down-regulated). The cultures grown at 15°C were the least responsive to heat stress with 837 DEGs (5.1%; 456 up- and 381 down-regulated) (Figure 7c, 7d; Supplementary Dataset S4). Regardless of the initial culturing temperature, pathways enriched for up-regulated DEGs included those in protein processing and endocytosis (Table 3; Supplementary Dataset S5). Pathways involved in energy metabolism, nucleic acid metabolism, amino acid metabolism, ribosome biogenesis, fatty acid biogenesis and cofactor synthesis were enriched in down-regulated DEGs (Table 3; Supplementary Dataset S5).