3.3 Cross-temperature differential gene expression
We first examined the expression data for temperature-dependent responses in each species by a two-dimensional plot of the first two principal components of a PCA. This analysis showed temperature-dependent separation of replicates for the heat-tolerant,B. calyciflorus s.s. This separation was less clear for the heat-sensitive species, in which replicates from 20 oC and 23 oC clustered together, while all samples treated at 26 °C formed a separate cluster (Figure S2). Overall, we found a greater number of differentially expressed genes (DEGs) in the heat-tolerant species than the heat-sensitive. In both species we captured a greater number of DEGs in pairwise comparison controlvs. high heat (Figure 3A, 3B). However, in the heat-tolerant species, we captured a greater number of genes differentially expressed between control and mild heat (1268) than between mild and high heat (64). In contrast, in the heat-sensitive we found the opposite pattern (Figure 3A, 3B).
We focused on pairwise comparison mild vs. high heat in both species as we had evidence from population growth rate data that there is a strong effect of temperature in this transition. In B. calyciflorus s.s., among the 64 genes differentially expressed between mild (26 oC) and high (32 oC) heat, were genes encoding for RNA polymerases, histone proteins and transferases. The above genes showed different temperature induction with genes encoding for RNA polymerases and N-acetyltranferases being up-regulated at high heat. Genes encoding for histones such as H2B family were either up- or down- regulated at high heat (Figure S3A). In heat-sensitive B. fernandoi , among the 310 differentially expressed between mild (23 oC) and high (26oC) heat, we found genes encoding for several histone methyltranferase proteins such as H3K4, H3K79, H4K20. All above genes were up-regulated with heat (Figure S3B).
We further focused on genes that are differentially expressed in all the three pairwise comparisons. These genes are of high importance as they are the most responsive to temperature presenting either up- or down-regulation along with temperature increase. In the heat tolerantB calyciflorus s.s., there were 23 genes differentially expressed in all pairwise comparisons. Among these, genes encoding for ribosomal proteins and glutathione S-transferase were up-regulated at lower temperatures, while proteases related genes were up-regulated at higher temperatures (Figure S4A). In the heat-sensitive B. fernandoi,there were two genes that were differentially expressed in all pairwise comparisons. These genes encode for E3 ubiquitin-ligase and a mediator of RNA polymerase were up-regulated at higher temperatures (Figure S4B).