HSP gene family and expression profiling in UWO241 under heat stress
HSPs are highly conserved among evolutionary distant species and are often used as molecular heat-stress markers. We identified a total of 55 full-length, transcript-supported HSP genes in the UWO241 genome (Table 4, Supplementary Dataset S6). This number was quite large compared to that reported for the C. reinhardtii genome (41 full-length HSP genes) (Schroda & Vallon 2009) (Table 4). This expansion in the HSP gene family could be related to the extremophilic lifestyle of UWO241 or simply due to the more distant evolutionary relationship between UWO241 and C. reinhardtii (Possmayer et al. 2016). To distinguish between these possibilities, we screened the genomes of other green algae with publicly available draft nuclear genome sequences. We detected the highest number of HSP genes in the two psychrophiles: UWO241 (55) and ICE-L (51). The subfamilies containing HSP100s, HSP60s and small HSPs were significantly expanded in both species (Table 4; Supplementary Dataset S6); however, UWO241 had more HSP70 genes and ICE-L had more HSP90 genes.
Previous work identified widespread duplication events in the UWO241 genome, including hundreds of highly similar full-length duplicate genes (HSDs; ≥90% pairwise identity) (Zhang et al. 2021a; Zhang, Hu & Smith 2021b). Indeed, our analysis revealed that certain C. reinhardtii HSP genes had multiple homologs in the UWO241 genome (ClpB3, HSP70A, HSP70D, HSP70G, CPN60A, HSP22A). Not all of these qualify as duplicates according to the strict cut-off used by Zhanget al . (2021), but nevertheless they share a high % identity at the amino acid level (55-94%) and contain highly conserved domains important for HSP function (Supplementary Dataset S6; Supplementary Figure S4). The HSP70 gene subfamily was the most expanded one in UWO241, primarily due to a total of 6 cytosolic HSP70A isoforms. The other psychrophilic alga ICE-L encoded 3 cytosolic HSP70A genes, while the genomes of the other explored species encoded for only one or two cytosolic HSP70A genes (Supplementary Figure S5).
To examine whether HSP gene expression is affected by temperature in UWO241, all full-length HSP genes were profiled using the RNA-Seq dataset described above. HSPs are induced by cold stress in C. reinhardtii (Maikova et al, 2016) and we asked whether the steady-state culturing temperature (4°C, 10°C, 15°C) affects HSP expression in UWO241. Most HSP genes were not significantly regulated in the cultures grown at different steady-state temperatures, except for ClpB1, HSP70B-2 and all HSP22A homologs that were either down-regulated or below the detection threshold in the 10°C cultures compared to those at 4°C (Figure 8). This suggests that steady-state temperature has only a minor effect on HSP regulation in UWO241. Some HSPs had high expression under steady-state conditions, in this case defined as FPKM>100 (Supplementary Dataset S3). These include all members of the HSP90 and CPN60 families, one HSP100 gene (ClpB3-4) and some (but not all) members of the HSP70 family (HSP70A-1 and -2, HSP70B-1, HSP70C, HSP70E and BIP1).
Exposure to heat stress for 6 hours induced the expression of some, but not all, HSP genes. (Figure 8, Supplementary Table 5). Most highly expressed transcripts at steady-state conditions were not upregulated under heat stress, apart from HSP90A, which was up-regulated. The genes with largest expression differences between steady-state and heat stress were the HSP22A homologs (HSP22A-2, -3, -6 to -9; FC≈7-32,000), followed by several HSP70 (HSP70A-3 to A-5, HSP70B-2) and ClpB (ClpB3-1, ClpB1) homologs (Figure 8; Supplementary Dataset S6). These data imply that some HSPs may have homeostatic roles and are constitutively expressed at high levels under steady-state conditions while others may play stress-related roles.