Heat shock transcription factor-mediated thermal tolerance and cell size plasticity in marine diatoms

Domain: earth_science
Published: 2025-04-10
Editorial outcome: major_revision

Abstract
Diatoms are a crucial component of marine ecosystems, recognized for their broad environmental adaptability and wide temperature tolerance. However, the molecular mechanisms underlying their adaptability to diverse temperatures are unknown. In this study, we discover that heat shock transcription factors (HSFs) are potentially important for thermal tolerance in diatoms. Our study focuses on PtHSF2, annotated as HSF2 in Phaeodactylum tricornutum’s genome, which is ubiquitous in diatoms. Overexpression of PtHSF2 markedly enhances thermal tolerance and increases cell size; causes significant differential expression of several genes, including cell division cycle protein 45-like (PtCdc45-like), ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3-related), light-harvesting complex protein 2 (Lhcx2), and fatty acid desaturase. Cleavage Under Targets and Tagmentation (CUT&Tag) and CUT&Tag-qPCR analyses demonstrate that PtHSF2 directly targets and upregulates PtCdc45-like and Lhcx2 while downregulating ATP-binding cassette transporter. Functional validation of PtCdc45-like shows that its overexpression results in larger cell size, enhances antioxidant capacity, and improves cell survival at elevated temperatures. Collectively, our findings elucidate the molecular mechanism by which PtHSF2 mediates high-temperature tolerance in diatoms and validate the functions of its target gene PtCdc45-like. These results highlight the importance of HSFs in diatom temperature adaptation and provide insights into temperature acclimation in microalgae.

Introduction
Global warming, primarily driven by the greenhouse effect, has led to the oceans sequestering over 90% of the excess heat on Earth since 19701. The oceans play a pivotal role in maintaining the planet’s thermal balance, yet the rise in sea temperatures has had detrimental impacts on marine ecosystems. These impacts include substantial coral reef degradation2, declines in marine fauna populations3, and reductions in marine biodiversity4. Marine phytoplankton, critical to oceanic ecosystems, contribute nearly half of global primary productivity5 and are essential in regulating Earth’s climate. Notably, among various phytoplankton groups such as diatoms, dinoflagellates, cyanobacteria, and coccolithophores, diatoms are distinguished by their extensive temperature tolerance6, highlighting their pivotal role in responding to climate change.

Diatoms, which are vital to Earth’s ecosystems and account for approximately one-fifth of global carbon dioxide fixation annually7, are at the forefront of research in the context of global warming. The warming trend has been linked to potential reductions in diatom abundance worldwide by at least 10%, due to increased ocean stratification8. However, there are perspectives suggesting that this stratification may boost diatom productivity and organic carbon output9. A groundbreaking study combining genomic and phenotypic analyses reveals rapid evolutionary adaptation of diatoms to higher temperatures10, underscoring their crucial role as marine phytoplankton in adapting to climate change. Therefore, investigating the molecular mechanisms underlying diatom thermal tolerance is not only of scientific importance but also key to foreseeing and understanding shifts in future marine ecosystems.

Heat shock transcription factors (HSFs) are highly conserved DNA-binding proteins in eukaryotes, playing a crucial role in environmental adaptation by finely tuning gene transcription and enhancing stress resistance11–13. Phaeodactylum tricornutum, a model diatom species widely distributed in coastal and inland waters, exhibits remarkable cell plasticity, including size variation and morphological changes among oval, fusiform, and triradiate forms14. This unique plasticity, combined with its exceptional environmental adaptability, enables P. tricornutum to thrive in highly unstable conditions, such as estuaries and rock pools14. Notably, P. tricornutum exhibits significant temperature adaptability15,16, surviving in natural environments from the tropics to subarctic regions17, and growing in laboratory conditions from 5 to 28°C18,19. However, the molecular mechanisms underlying its thermotolerance remain enigmatic.

The effects of temperature on diatom growth and physiology are well-studied20–22. High-throughput sequencing technologies have paved the way for investigating the molecular mechanisms of diatom responses to temperature stress. Many studies have explored these mechanisms using genomics10, transcriptomics23–25, and proteomics26, providing valuable insights into diatom thermal tolerance. However, these studies primarily focus on omics data, with limited functional analyses of key temperature-responsive genes, especially HSFs. In this study, we investigated the functional mechanism of a conserved diatom HSF in P. tricornutum, which may offer new insights into the molecular mechanisms underlying thermal tolerance in diatoms.

Results
Diatoms, widely distributed in marine ecosystems, are ubiquitous in nearly all aquatic habitats28. Different diatom species exhibit distinct thermal tolerance ranges. For instance, Chaetoceros tenuissimus grows optimally between 25 to 30°C29, while Fragilariopsis cylindrus thrives at −1 to 10°C30. T. pseudonana, Fistulifera solaris, and P. tricornutum have thermal tolerance ranges of 10–35°C10,31, 15–35°C32,33, and 5–28°C18,19, respectively. To elucidate the molecular mechanisms underlying the thermal adaptability of diatoms, an analysis of transcription factor abundance and proportion was conducted in 15 diatom species with sequenced genomes. Results revealed that HSFs are the most abundant transcription factors family in diatoms, ranging from tens to hundreds of genes (Supplementary Table 1). For example, T. weissflogii harbors 393 HSF-encoding genes, constituting 53% of its total transcription factors, while dinoflagellates, coccolithophores, and green algae possess fewer than 10 HSFs, accounting for only 0–7.2% of their transcription factors. Given the global distribution and extensive temperature adaptability of diatoms, it was hypothesized that HSFs play a crucial role in their environmental adaptation. Consistent with previous reports27, we found that P. tricornutum possessed 69 HSF genes, representing the highest proportion (44.2%) among all transcription factors. An analysis of transcriptome data from the MMETSP project34 and the re-assembly data by Johnson et al.35 indicated that 38 HSF genes are actively expressed in P. tricornutum (Supplementary Data 1). Additionally, we observed the expression of HSF orthologues in other species (Supplementary Data 1). We speculate that these HSFs may play significant regulatory roles under specific stress conditions, including high temperature, light stress, and other environmental stresses.

Phylogenetic analysis categorized the 69 HSFs into four major groups: Group I, Group II, Group III, and Group IV (Supplementary Fig. 1A). Except for Group II, the other groups were further subdivided into multiple subclasses. Group IV HSFs displayed closer homology with HSFs from Emiliania huxleyi. Overall, P. tricornutum HSFs were more closely related to each other than to HSFs from Chlamydomonas reinhardtii or Arabidopsis thaliana. Notably, PtHSF2 (Phatr3_J55070) showed the most significant upregulation among the differentially expressed HSFs under elevated temperature conditions (Supplementary Data 2), with its transcript levels varying significantly with temperature changes (15°C to 30°C), peaking at 25°C, followed by 15°C, 30°C, and 20°C (Fig. 1A), suggesting that PtHSF2 may play a crucial role in temperature response.

Furthermore, the analysis of the taxonomic distribution of PtHSF2 homologs in the ocean revealed that 99% of the hits in the marine EUK-SMAGs (Eukaryotes Single-Cell and Metagenome Assembled Genomes) database were from diatoms (Bacillariophyta) (Supplementary Fig. 1B), indicating that diatoms are the primary hosts of this gene. This finding suggests that PtHSF2 is widely present in diatoms and may play a crucial role in their biological functions or adaptation mechanisms. Supplementary Fig. 1C shows that the abundance of PtHSF2 homologs is highest at higher latitudes and relatively low in tropical regions, which could be attributed to differing environmental conditions and ecological niches in these areas. Although diatoms exhibit broad temperature tolerance, they generally thrive better in cooler waters36.

SMART (Simple Modular Architecture Research Tool) domain annotation identified a conserved HSF domain in PtHSF2 between amino acids 89 and 188 (Supplementary Fig. 2A). Annotated as HSF2 in the P. tricornutum genome, it was named PtHSF2. Phylogenetic analysis demonstrated that genes with higher homology to PtHSF2 were derived from other diatom species, such as F. solaris, Pseudo-nitzschia multistriata, and T. pseudonana (Supplementary Fig. 2B). To assess PtHSF2’ regulatory function in microalgae, overexpression and RNA interference (RNAi) plasmids were separately constructed (Fig. 1B). Through genomic polymerase chain reaction (PCR) (Supplementary Fig. 2C), quantitative real-time PCR (qPCR) (Supplementary Fig. 2D), and western blotting (Supplementary Fig. 2E), three overexpression lines (PtHSF2-1, PtHSF2-2, and PtHSF2-3) and three silenced lines (siPtHSF2-1, siPtHSF2-2, and siPtHSF2-3) were successfully established. We measured PtHSF2 protein levels in the PtHSF2 overexpression strain (PtHSF2-proH1) at four temperatures (15°C, 20°C, 25°C, and 30°C) and found a significant increase at elevated temperatures (25°C and 30°C) (Supplementary Fig. 2F).

Alignment analysis of the coding regions of the remaining 68 HSF genes from P. tricornutum with dsPtHSF2 revealed that only 8 HSF genes, including Phatr3_J38294, Phatr3_J48667, Phatr3_J48572, Phatr3_J42824, Phatr3_J45391, Phatr3_EG02624, Phatr3_J50136, and Phatr3_EG00212, contained perfectly matching segments with dsPtHSF2, all of which were shorter than 16bp (Supplementary Fig. 3A). According to Chen et al.37, such short matches are insufficient to cause off-target effects. Furthermore, analysis of the relative transcription levels of these 8 HSF genes showed no significant downregulation (Supplementary Fig. 3B). These results indicate that RNAi targeting PtHSF2 did not cause off-target effects among its paralogues.

To characterize the thermal tolerance of PtHSF2-transformed microalgae, cells were cultured separately at 15°C, 20°C, 25°C, and 30°C. Cell density monitoring revealed that PtHSF2 overexpression cell lines exhibited lower growth rates at 15°C, 20°C, and 25°C compared to WT cells and the RNAi lines (Fig. 1C). While temperatures above 25°C impaired growth, and 30°C exposure for over three days severely inhibited it, PtHSF2 overexpression lines had significantly higher cell density than WT cells at 30°C (Fig. 1C). Confocal microscopy and Nile red staining revealed increased cell size and neutral lipid content in overexpression lines, particularly at 30°C (Fig. 1D–F). Additionally, lipid droplets were observed outside of the PtHSF2-1 cells under 30°C, potentially due to cell rupture or active secretion (Fig. 1D). Previous studies have demonstrated that microalgae actively secrete lipid droplets during periods of high lipid synthesis38. Quantitative analysis showed that cell volume in overexpression lines was 2.1–2.4 times larger than WT cells at 30°C, while knockdown lines were significantly smaller (Fig. 1E).

Thermal stress often impairs photosystem II (PSII), leading to reactive oxygen species (ROS) production and lipid peroxidation39,40. Therefore, ROS and lipid peroxides, such as malondialdehyde (MDA), an abundant byproduct of lipid peroxidation41, serve as indicators of oxidative stress. Analysis of cell survival rates demonstrated that PtHSF2 overexpressing lines exhibited significantly higher survival rates at 30°C compared to WT cells (Fig. 1G). Moreover, overexpression lines exhibited significantly lower ROS (Fig. 1H) and MDA levels (Supplementary Fig. 4A) and higher antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT) (Supplementary Fig. 4B, C). The lipid saturation index was significantly lower in the overexpressing lines and higher in the RNAi lines compared to the WT (Supplementary Fig. 4D). These results suggest that PtHSF2 may mediate the cell’s thermal response by regulating the expression of genes related to cell size, lipid synthesis, and antioxidant defense.

We employed RNA-seq, CUT&Tag, and CUT&Tag-qPCR techniques to unravel the functional mechanisms governed by PtHSF2. The number and quality of sequencing reads from RNA-seq and CUT&Tag libraries were presented in the Supplementary Data 3 and 4, highlighting the sequencing depth and overal data quality across all samples. RNA-seq analysis revealed 1912 significantly differentially expressed genes (DEGs) in the PtHSF2-1 versus WT comparison, with 940 upregulated and 972 downregulated. In the PtHSF2-2 versus WT comparison, 2,404 DEGs were identified, including 1,121 upregulated and 1,283 downregulated. Among these, 1449 DEGs were shared between the two PtHSF2 overexpressing lines, comprising 676 upregulated and 773 downregulated (Supplementary Data 5). Notably, genes encoding Cdc45-like (Phatr3_J9344), light-harvesting complex protein 2 (Lhcx2, Phatr3_EG02404), fatty acid desaturase (FAD, Phatr3_J46275), as well as ataxia telangiectasia mutated (ATM, Phatr3_J51040) and ataxia telangiectasia and Rad3-related (ATR, Phatr3_J44708), were significantly upregulated, while an ATP-binding cassette (ABC) transporter (Phatr3_J42881) containing two ABC_ATPase domains, which shares high homology with F. solaris and Cylindrotheca closterium, was significantly downregulated (Supplementary Data 6–8 and Supplementary Fig. 5A, B). Additionally, the expression levels of 12 PtHSF2 homologs exhibited significant changes, with 5 genes upregulated and 7 downregulated (Supplementary Data 8).

Gene Ontology (GO) annotation and enrichment analysis of the 1449 DEGs revealed that 30.9% participated in cellular processes under the “biological process” category, with 15.0% upregulated and 15.9% downregulated (Fig. 2A and Supplementary Data 9). In the “molecular function” category, 29.4% of DEGs were associated with catalytic activity, with 12.6% upregulated and 16.8% downregulated (Fig. 2B). For the “cellular component” category, DEGs enriched in “cell” and “cell part” terms both accounted for 36.7%, comprising 16.9% upregulated and 19.8% downregulated genes (Fig. 2C). KEGG pathway analysis unveiled significant enrichment of genes involved in cell cycle regulation, antioxidant defense, fatty acids synthesis, AMP-activated protein kinase (AMPK) signaling, and carbohydrate metabolism (Supplementary Data 6). Among these, two mitochondrial inner membrane proteins, MPV17 (Phatr3_J15341 and Phatr3_Jdraft223), associated with antioxidant processes42, were significantly upregulated (Supplementary Data 6). Collectively, these results highlight that PtHSF2 overexpression profoundly influences genes linked to cell cycle regulation and environmental information processing.

CUT&Tag, a potent method for investigating transcription factor-DNA interactions43, identified 295 potential PtHSF2 target genes (Supplementary Data 10), which underwent GO annotation analysis (Fig. 2D–F). These target genes were enriched in catalytic (22.4%) and binding activities (31.2%), participating in intracellular biological processes (32.9%) (Fig. 2D, E). Moreover, a substantial proportion was associated with membrane components (19.3%) and cellular components (38.3%) (Fig. 2F and Supplementary Data 10), suggesting PtHSF2’s involvement in regulating cellular structures and membrane composition.

CUT&Tag, a potent method for investigating transcription factor-DNA interactions43, identified 295 potential PtHSF2 target genes (Supplementary Data 10), which underwent GO annotation analysis (Fig. 2D–F). These target genes were enriched in catalytic (22.4%) and binding activities (31.2%), participating in intracellular biological processes (32.9%) (Fig. 2D, E). Moreover, a substantial proportion was associated with membrane components (19.3%) and cellular components (38.3%) (Fig. 2F and Supplementary Data 10), suggesting PtHSF2’s involvement in regulating cellular structures and membrane composition.

CUT&Tag and CUT&Tag-qPCR analyses were performed. The Venn diagram (Fig. 2G) revealed 35 intersecting genes between potential targets and DEGs (Supplementary Data 11), comprising 18 upregulated and 17 downregulated genes, including Cdc45-like (Phatr3_J9344), Lhcx2 (Phatr3_EG02404), and the ABC transporter (Phatr3_J42881). The limited overlap between DEGs and potential targets likely stems from several factors. First, TFs can regulate gene expression both directly and indirectly44, with some targets showing no detectable expression changes45–47. Second, while RNA-seq offers broad coverage, it lacks specificity, whereas CUT&Tag provides high specificity by targeting proteins of interest with specific antibodies43. Signal intensity analysis around transcription start sites (TSS) showed a significant enrichment peak at 8.25 for PtHSF2, indicating its primary binding sites at the TSS regions, which were absent in IgG controls (Fig. 2H). Using the MEME algorithm48, we identified the top three DNA-binding motifs for PtHSF2 (Fig. 2I), with the most prominent motif being the sequence “GGTTAGGGTTAGGGTTAGGGTTAGGGTTAG.” This sequence represents a putative consensus motif, indicating a conserved recognition pattern across genomic sites. CUT&Tag-qPCR further confirmed PtHSF2’s binding to promoter regions of Cdc45-like (Phatr3_J9344), Lhcx2 (Phatr3_EG02404), and ABC (Phatr3_J42881) transporter genes in both overexpressing lines (Fig. 2J–L).

TF scan positively or negatively regulate the transcription of target genes38,44,49. To elucidate how PtHSF2 modulates its targets, we analyzed transcription levels of potential target genes. In PtHSF2-overexpressing cells, Cdc45-like and Lhcx2 were significantly upregulated, while the ABC transporter was notably downregulated (Fig. 2M–O). The upregulation of FAD (Phatr3_J46275) in overexpressing lines and its downregulation in RNAi lines (Fig. 2P and Supplementary Data 6) corresponded with changes in unsaturated fatty acid content (Supplementary Fig. 4D). However, CUT&Tag did not detect direct targeting of the FAD promoter, suggesting that PtHSF2 regulates FAD indirectly. Interestingly, the regulatory trends for target genes at 30°C (Supplementary Fig. 6A-F) were consistent with those observed at 20°C (Fig. 2M–P; Supplementary Data 5), suggesting that PtHSF2’s regulatory mechanisms remain stable across these temperature conditions.

In the WT background, PtCdc45-like transcription increased from 15°C to 25°C but declined at 30°C compared to 25°C (Fig. 3A), suggesting its critical role in high-temperature responses. To elucidate PtCdc45-like’s biological function, we employed bioinformatics and molecular biology approaches. Bioinformatics analysis revealed that PtCdc45-like coding region spans 2274 bp, encoding a conserved Cdc45-like protein domain between amino acids 29 and 754 (Supplementary Fig. 7A). Phylogenetic analysis showed that PtCdc45-like is evolutionarily conserved among diatoms (Supplementary Fig. 7B).

To characterize its function, overexpression and RNAi plasmids were constructed (Fig. 3B). Putative positive transformants were selected based on bleomycin resistance and validated through genomic PCR, qPCR, and western blot analyses, resulting in six confirmed lines: three overexpression lines (PtCdc45-like-1, −2, −3) and three RNAi lines (siPtCdc45-like-1, −2, −3) (Supplementary Fig. 7C–E). qPCR analysis revealed transcription levels of PtCdc45-like were 2.6-to 3.8-fold higher in the overexpression lines and reduced to 20.2–29.3% of WT levels in RNAi lines (Supplementary Fig. 7D). Western blotting confirmed specific protein expression (~86.9 kDa) in transformants but not in WT cells (Supplementary Fig. 7E). Protein level analysis across varying temperatures demonstrated a temperature-dependent increase in PtCdc45-like protein levels in the PtCdc45-like-proC1 overexpression strain (Supplementary Fig. 7F).

Overexpressing lines exhibited significantly reduced cell densities compared to WT and RNAi lines at 15–25°C, but notably higher densities at 30°C (Fig. 3C). Under normal growth conditions (20°C), overexpression lines displayed significantly larger cell volumes than WT, while RNAi lines had smaller cells, with cell size increasing further at higher temperatures (Fig. 3D, E). These results highlight PtCdc45-like as a key regulator of cell size in P. tricornutum. Notably, the total dry weight of cell pellets, normalized by cell number, revealed that larger cell volumes correlated with higher average cell dry weight (Fig. 3F).

Cell plasticity is influenced by membrane fluidity and cell wall flexibility. The fusiform structure of P. tricornutum, lacking a silicified cell wall50, may facilitate cell size plasticity. Consistent with this, overexpression lines exhibited a lower lipid saturation index, while RNAi lines had higher indices (Fig. 3G). This suggests a positive correlation between cell size and the proportion of unsaturated fatty acids, supported by the upregulation of FAD (Phatr3_J46275) in overexpression lines and its downregulation in RNAi lines (Supplementary Fig. 7G). These findings align with those observed in PtHSF2 transgenic lines (Fig. 2P) and prior studies linking desaturase activity to unsaturated fatty acid synthesis51,52. In microalgae, fatty acids primarily exist as triacylglycerols (TAGs), phospholipids, and glycolipids, with phospholipids and glycolipids serving as the primary components of membranes53. Accordingly, we hypothesize that larger cells had a higher proportion of unsaturated fatty acids in their cell membranes, contributing to enhanced membrane fluidity.

Overexpression lines exhibited lower maximum quantum yield of photosystem II (Fv/Fm) than WT, while RNAi lines showed no difference (Fig. 3H). Interestingly, Lhcx2 (Phatr3_EG02404) transcription decreased in overexpression lines but increased in RNAi lines (Supplementary Fig. 7H), contrasting with its regulation in PtHSF2-transformed lines (Fig. 2N).

Overexpression of both PtCdc45-like and PtHSF2 resulted in comparable phenotypic changes, including increased cell size and altered lipid composition. Furthermore, the ABC transporter (Phatr3_J42881) was significantly downregulated in both PtCdc45-like and PtHSF2 overexpressing lines and upregulated in the RNAi lines (Fig. 2O and Supplementary Fig. 7I). This transporter’s ABC_ATPase domain (Supplementary Fig. 5A), associated with mitochondrial mRNA processing54, suggests that its downregulation may impair mitochondrial function, contributing to slower growth.

Cell cycle progression analysis revealed that overexpression lines (PtHSF2-1 and PtCdc45-like-1) had higher proportions of cells in the S and G2 phases, indicating accumulation or arrest, whereas RNAi lines displayed distributions similar to WT cells (Supplementary Fig. 8A). This is consistent with differential expression of cell cycle-related genes in overexpression lines (Supplementary Data 6; Supplementary Fig. 6E, F; Supplementary Fig. 8B; Supplementary Fig. 9), which may contribute to the observed cell accumulation or arrest during these phases. Among these genes, the key kinases regulating cell cycle and replication checkpoints, ATM and ATR55, were significantly upregulated, suggesting the activation of cell cycle checkpoints. In addition to these cell cycle-related genes, PtCdc45-like significantly altered the transcription levels of FAD, Lhcx2, and ABC (Supplementary Fig. 7G–I). Specifically, its effects on FAD and ABC were consistent with those of PtHSF2, whereas its effect on Lhcx2 was opposite.

To investigate the regulatory relationship between PtCdc45-like and the transcriptional variability of FAD, Lhcx2, ABC, ATM, and ATR we first examined PtHSF2 transcription in PtCdc45-like overexpression and RNAi lines. The results revealed that PtHSF2 was upregulated in the overexpression lines and downregulated in the RNAi lines (Fig. 4A). Combined with our earlier observation that PtHSF2 activates PtCdc45-like transcription, this finding suggests the existence of a positive feedback loop between PtHSF2 and PtCdc45-like.

To further elucidate the regulatory interactions, two types of transgenic cell lines were constructed: Ho-Ci (PtHSF2 overexpression combined with PtCdc45-like knockdown) and Hi-Co (PtHSF2 knockdown combined with PtCdc45-like overexpression). Six lines—Ho-Ci-1, Ho-Ci-2, Ho-Ci-3, Hi-Co-1, Hi-Co-2, and Hi-Co-3—were successfully validated (Supplementary Fig. 10A–F). Next, we assessed FAD and ABC transcription levels in these cell lines. The results indicated that knocking down PtCdc45-like in the PtHSF2 overexpression background did not affect FAD and ABC, and similarly, overexpressing PtCdc45-like in the PtHSF2 knockdown background had no impact on these genes (Fig. 4B, C). These findings suggest that the regulation of FAD and ABC is predominantly mediated by PtHSF2, with the influence of PtCdc45-like being exerted indirectly through its positive feedback on PtHSF2.

In contrast, Lhcx2 transcription was significantly altered in both Ho-Ci and Hi-Co lines, as knocking down PtCdc45-like in the PtHSF2 overexpression background increased Lhcx2 while overexpressing PtCdc45-like in the PtHSF2 knockdown background decreased Lhcx2 (Fig. 4D), indicating that Lhcx2 transcription is influenced by PtCdc45-like. Combined with the data from Fig. 2K, N, these results suggest that Lhcx2 is co-regulated by PtHSF2 and PtCdc45-like, with PtCdc45-like acting as a repressor. Whether this repression is direct or indirect remains to be investigated. ATM and ATR were markedly downregulated in the Ho-Ci lines but upregulated in the Hi-Co lines (Fig. 4E, F). Moreover, both genes were significantly upregulated in PtHSF2 and PtCdc45-like overexpression lines (Supplementary Data 6; Supplementary Fig. 6E, F; Supplementary Fig. 8B; Supplementary Fig. 9), suggesting that PtHSF2 may regulate these genes via PtCdc45-like.

Under normal growth conditions (20°C), knocking down PtCdc45-like in the PtHSF2 overexpression background alleviated the growth retardation (Fig. 4G) and cell enlargement (Fig. 4H) caused by PtHSF2 overexpression. However, under heat stress (30°C), PtCdc45-like knockdown significantly reduced cell density (Fig. 4G). Conversely, overexpressing PtCdc45-like in the PtHSF2 knockdown background yielded the opposite results (Fig. 4G, H). These findings highlight PtCdc45-like as a critical mediator of PtHSF2-regulated cell size and thermal response. Overall, PtHSF2 positively regulates PtCdc45-like and Lhcx2 while negatively regulating ABC and indirectly regulating FAD, with a positive feedback loop between PtHSF2 and PtCdc45-like, and PtCdc45-like acting as a repressor of Lhcx2.

The optimum growth temperature for P. tricornutum (CCMP2561) under laboratory conditions was 20°C. Elevated temperatures negatively impacted cellular growth, often leading to growth inhibition or cell death. Fig. 5A demonstrates that the inhibition rates at 25°C and 30°C were significantly higher in WT cells compared to PtCdc45-like overexpressing cells. Notably, PtCdc45-like overexpressing cells did not show a growth advantage at lower temperatures (15°C and 20°C) (Fig. 3C), suggesting that PtCdc45-like overexpression enhances heat tolerance rather than cold tolerance. To further investigate the thermal tolerance conferred by PtCdc45-like overexpression, we assessed cell survival rates and redox levels under high-temperature conditions (25°C and 30°C). Given the clear growth trend observed at 25°C (Fig. 3C), survival rate analysis was not performed at this temperature. After 13 days of incubation at 30°C, the survival rate of WT cells was only 23.6%, whereas PtCdc45-like overexpression lines (PtCdc45-like-1, PtCdc45-like-2, and PtCdc45-like-3) exhibited significantly higher survival rates of 76.7, 72.5, and 65.0%, respectively (Fig. 5B).

Furthermore, ROS (Fig. 5C) and MDA (Fig. 5D) levels were markedly lower in PtCdc45-like overexpressing cells compared to WT cells, indicating a diminished sensitivity to high-temperature stress. Since oxygenic photosynthesis is a major source of ROS production56, the relatively lower ROS levels observed in PtCdc45-like overexpressing cells could be attributed to the significant suppression of Fv/Fm (Fig. 3H) and the downregulation of Lhcx2 transcription (Supplementary Fig. 7H). Despite no significant increase in SOD levels in overexpressing cells at 30°C compared to WT cells (Fig. 5E), CAT levels were markedly higher in the overexpressing cells (Fig. 5F). Conversely, the PtCdc45-like RNAi lines showed no significant differences in antioxidant enzyme levels compared to WT (Fig. 5D–F). Collectively, these data provided compelling evidence that PtCdc45-like overexpression augmented cellular antioxidant defenses, thereby improving cell tolerance to elevated temperatures.

Both PtHSF2 and PtCdc45-like were identified as crucial contributors to heat tolerance in P. tricornutum. However, it remains unclear whether this mechanism is conserved across other diatom species. To investigate, we subjected T. weissflogii to 20°C, 30°C, and 35°C, followed by transcriptome analysis (quality control details in Supplementary Data 12). Compared to 20°C, the PtHSF2 orthologue in T. weissflogii (snap_masked-ctg119-processed-gene-4.14-mRNA-1, 48.5% similarity) was upregulated 1.21-fold at 30°C and 1.92-fold at 35°C, with the PtCdc45-like orthologue (snap_masked-ctg1929-processed-gene-0.66-mRNA-1, 53.4% similarity) also exhibiting an upregulation trend (Supplementary Data 13). These results suggest that HSF2 may serve as a key regulator of heat tolerance in diatoms and that this regulatory mechanism may be broadly conserved across diatom species.

Methods
Strain and culture conditions
Phaeodactylum tricornutum (CCMP2561), with a fusiform morphotype, was obtained from the National Center for Marine Algae and Microbiota (NCMA, USA), and cultured in sterile f/2 medium (excluding Na2SiO3·9H2O). It was cultivated at 20 ± 1°C in an artificial climate incubator (Ningbo, China), provided with a 12:12 h light-dark cycle at an irradiance of 200 μmol photons m−2 s−1. WT cells were cultured at four different temperatures (15°C, 20°C, 25°C, and 30°C) for 7 days. Subsequently, cells grown at 20°C and 30°C were subjected to transcriptome sequencing, while cells from all four temperatures were used to analyze the transcription levels of PtHSF2 and PtCdc45. Transgenic strains were selected on solid medium with bleomycin. Surviving colonies were subsequently picked and transferred to fresh f/2 liquid medium containing bleomycin (85 μg/mL). Before experimentation, transgenic strains underwent several growth cycles in bleomycin-free medium to negate potential bleomycin antibiotic effects. All experiments were performed according to strict aseptic practices.

Thalassiosira weissflogii (CCMA-189) cultures were maintained under standard growth conditions in f/2 medium at 20°C with a 12:12 h light-dark cycle. This strain was obtained from the Center for Collections of Marine Algae (CCMA), and its cultivation temperature range was between 18 and 25°C. For high-temperature treatment, the cultures were divided into three groups: control (20°C), moderate heat stress (30°C), and severe heat stress (35°C). Each group was incubated at the respective temperature for 5 days, after which cells were collected for transcriptome analysis.

Sequence alignment, phylogenetic analyses, biogeographic and taxonomic distribution
Using the Plant Transcription Factor Database (PlantTFDB 4.0, http://planttfdb.cbi.pku.edu.cn/)99, we identified 69 HSFs from the P. tricornutum protein library downloaded from the National Center for Biotechnology Information database (NCBI, https://www.ncbi.nlm.nih.gov/). Protein libraries for C. reinhardtii and E. huxleyi were downloaded from NCBI, and the A. thaliana protein library was downloaded from UniProt (https://www.uniprot.org/). We predicted HSFs in these libraries using PlantTFDB 4.0 and extracted their gene IDs and amino acid sequences. The 99 obtained HSF protein sequences (Source Data) were aligned using MEGA 6.0 with ClustalW, and a phylogenetic tree was constructed. The results of the alignments were presented in Source Data file. The Neighbor-Joining method inferred evolutionary relationships, with bootstrap values calculated from 1000 replicates to ensure robustness and reliability of the tree topology. Evolutionary distances were computed using the p-distance method.

To construct phylogenetic trees for PtHSF2 and its homologs, as well as for PtCdc45-like and its homologs, we conducted BLASTP searches in the NCBI database to identify the most similar homologs from various species. The sequences were aligned and phylogenetic trees were constructed using MEGA 6.0, following the described procedure. The sequences and alignment results were included in Source Data file.

We utilized the Ocean Gene Atlas (http://tara-oceans.mio.osupytheas.fr/ocean-gene-atlas/) to explore the biogeographic and taxonomic distribution of PtHSF2 and its homologs. The Ocean Gene Atlas is a web service designed for investigating the biogeography of marine planktonic genes100. It includes the marine Eukaryotes Single-Cell and Metagenome Assembled Genomes (EUK-SMAGs) database, which comprises 713 non-redundant and manually curated eukaryotic MAGs and SAGs, covering a total of 10 million genes. The dataset was constructed from 280 billion metagenomic reads obtained from the Tara Oceans project, covering diverse marine environments such as polar, temperate, and tropical sunlit oceans. We searched the protein sequence of HSF (Phatr3_J55070) in the EUK-SMAGs database using BLASTP, and the homologs were obtained at a threshold of 1E−20. The visualization results were directly downloaded from the website.

Gene amplification, vector construction, and transformation
The full-length coding regions (excluding stop codons) of PtHSF2 (GenBank: XM_002184371.1/Phatr3_J55070) and PtCdc45-like (GenBank: OK484425/Phatr3_J9344) were PCR-amplified from P. tricornutum cDNA using primer pairs HSF2-OF/HSF2-OR and Cdc45-like-OF/Cdc45-like-OR, respectively, as detailed in Supplementary Table 2. These amplified products were individually cloned into the pHY21101 vector downstream of the PfcpC (promoter of fucoxanthin chlorophyll a/c binding protein), pHY22 (driven by the HSF2 promoter), and pHY23 (driven by the Cdc45-like promoter) using the ClonExpress II One Step Cloning Kit (Vazyme, China). The 2xFlag tag was appended to the C-terminus of these genes to facilitate protein expression detection.

The resultant recombinant overexpression vectors, namely pHY21-PtHSF2, pHY22-PtHSF2-proH, pHY21-PtCdc45-like, pHY23-PtCdc45-like-proC were introduced into microalgae via electroporation using the Gene Pulser Xcell system (Bio-Rad) as described previously102. Briefly, cells were collected, washed thrice with 0.375 M sorbitol, and resuspended in 0.2 mL of 0.375 M sorbitol. Linearized plasmid (3 μg) and heat-denatured salmon sperm DNA (30 μg) were added to the samples. The samples were then transferred to cuvettes and incubated on ice for 10 min. The electroporation was performed with the following parameters: 500 V field strength, 25 μF capacitance, and 400 Ω resistance. Subsequently, the samples were transferred to 5 mL of liquid medium and incubated in the dark for 24 h. Finally, the cells were harvested and spread onto solid medium supplemented with bleomycin.

PtHSF2 and PtCdc45-like were silenced by using RNA interference (RNAi), and the RNAi vectors were constructed as described previously103 with slight modifications. Specifically, a fragment of the PtHSF2 (spanning 348 to 689 bp) was amplified from P. tricornutum DNA using primers siHSF2-F1 and siHSF2-R1, as listed in Supplementary Table 2. The inverted sequence (from 689 to 348 bp) was amplified using primers siHSF2-F2 and siHSF2-R2. Given the absence of intron in the PtHSF2, the first intron of the PtHSF (GenBank: XM_002177210.1) was amplified using primers siHSF-F3 and siHSF-R3 to facilitate hairpin loop formation. These fragments were subsequently cloned into the pHY21 vector using the ClonExpress Ultra One Step Cloning Kit (Vazyme, China). Similarly, fragments of the PtCdc45-like (from 1 to 465 bp and its inverted sequence from 368 to 1 bp) were amplified using primer pairs siCdc45-like-F1/R1 and siCdc45-like-F2/R2, respectively, as listed in Supplementary Table 2. These fragments were then cloned into the pHY21 vector in both sense and antisense orientations. The resulting RNAi vectors (pHY21-siPtHSF2 and pHY21-siPtCdc45-like) were introduced into microalgae cells via electroporation102.

Following previous studies104, we introduced two plasmids simultaneously into P. tricornutum cells to generate cell lines with the desired genetic modifications. Specifically, plasmid pHY21-PtHSF2 and plasmid pHY21-siPtCdc45-like were co-electroporated into WT cells to create the PtHSF2 overexpression and PtCdc45-like knockdown cell line (Ho-Ci). Conversely, plasmid pHY21-siPtHSF2 and plasmid pHY21-PtCdc45-like were co-transformed to generate the PtHSF2 knockdown and PtCdc45-like overexpression cell line (Hi-Co). The electroporation conditions, as well as the selection and cultivation of the transformed microalgae, were conducted as previously described.

Evaluation of transformants by molecular approaches
Screening on solid media containing bleomycin yielded hundreds of transformants for each plasmid. Ten transformants for each plasmid were then randomly selected for further screening and validation. Transformants that failed to grow in the antibiotic-containing liquid media were excluded. False positives were identified and eliminated through genomic PCR, and qPCR was subsequently employed to select transformants with stable high or low expression of the target gene. These selection processes ensured that the observed effects were attributable to the introduced genes rather than random variation among the transgenic lines. Specifically, to verify the integration of the transgene into the host cells, genomic PCR was conducted using primers Pt89a and Pt91r (Supplementary Table 2) that were designed to amplify the backbone of the expression cassette. The PCR products were separated by gel electrophoresis and purified using a Gel Extraction Kit (Omega Bio-tek, USA). Subsequently, the purified products were validated by sequencing analysis. Genomic PCR was performed to confirm the integration of the silencing expression cassettes into the genome using the forward primer Pt89a, which targets the vector backbone, and the reverse primers siHSF2R4/siCdc45-like-R3, which target introns. The relative transcript levels of target genes were assessed by quantitative real-time PCR (qPCR). Total RNA was isolated from WT and transgenic lines using a RNAiso Plus Kit (Takara, Japan). The isolated RNA was reverse-transcribed using a HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, China) to generate first-strand complementary DNA. The qPCR assay was conducted in 96-well plates with a 20 μL reaction volume using the AceQ qPCR SYBR Green Master Mix Kit (Vazyme, China) on the Bio-Rad CFX96 real-time PCR system. Each sample was tested in triplicate, and expression levels were referenced against the endogenous gene β-actin and TBP (TATA box binding protein), which were selected based on their stable expression across different conditions in P. tricornutum, as verified in previous studies105,106. To ensure consistent comparisons among different strains, cells used for molecular validation were cultured under standard conditions (20°C) until they reached the exponential growth phase. All primers and their sequences utilized in this study are listed in Supplementary Table 2. According to the MIQE guidelines107, the specificity of the primers was confirmed by a single peak in the melt curve of the amplification products (Source Data). The amplification efficiency of the primers, determined from standard curves, ranged between 90 and 110% (Source Data). Ct values for each qPCR reaction were provided in Source Data file. Relative transcription levels were calculated using the comparative Ct method108 and were normalized to the reference genes β-actin and TBP. Total protein was extracted and denatured. Protein expression in transgenic cells was probed by western blot using an anti-Flag antibody (1:3000, Rabbit monoclonal, ab205606, Abcam, UK) targeting the Flag-tagged PtHSF2 and PtCdc45-like.

Analysis of off-target effects
Previous work on dsRNA specificity37 has shown that dsRNAs triggering RNAi require one of the following criteria: (i) >80% sequence similarity with target genes; (ii) ≥16 bp contiguous fragments that perfectly match the target gene or >26 bp fragments that almost perfectly match the target gene with one or two mismatches (single mismatches inserted between matching fragments of ≥5 bp or mismatched couplets inserted between matching fragments of ≥8 bp). Therefore, these parameters can be used to predict the off-target effects of dsRNAs. The dsRNA of PtHSF2 (designated as dsPtHSF2) was aligned with the coding regions of the remaining 68 HSF genes from P. tricornutum (Source Data) using MEGA 6.0 with ClustalW. Based on the alignment results (Source Data), 8 genes with perfectly matching segments to dsPtHSF2 were selected for further validation of their relative transcription levels using qPCR. Since no homologous gene of PtCdc45-like was found in P. tricornutum and no segments with continuous perfect matches of ≥5 bp in length were found with the dsRNA targeting PtCdc45-like (designated as dsPtCdc45-like), further analysis of the off-target effects of PtCdc45-like RNAi was not conducted.

Cell density, growth rate, inhibition rate, and survival rate analysis
We synchronized exponentially growing cells by subjecting them to an extended dark period (20 h) and then transferred them to fresh medium for normal culture (12 h light: 12 h dark). Referring to previous studies10,19, each strain was acclimated to the experimental temperature. After a 7-day acclimation period, a second transfer was performed, and measurements of various parameters commenced. To ensure sufficient cell quantities for subsequent measurements and to avoid growth differences due to initial density variations, the initial cell density was set to 1×106 cells/mL. The cultures were then maintained at 15°C, 20°C, 25°C, and 30°C, respectively. Cell density was monitored using a light microscope and a Bright-Line hemocytometer, and the data were plotted as growth curves. The growth inhibition rate at 15°C, 25°C, and 30°C was calculated by comparing cell density with that at 20°C on day 13. Cell viability was assayed using 1.0% Trypan blue109. Briefly, cell culture (1 mL) was collected and centrifuged to separate the supernatant and cells. The supernatant was discarded and the cells were resuspended with 100 μL of 1.0% Trypan blue. The samples were incubated at room temperature for 3 h, centrifuged to remove the supernatant, and washed twice with PBS (Phosphate buffered saline) to remove the unbound dye. Finally, the cells were resuspended with PBS and observed and counted under a microscope.

Laserscanning confocal microscopic observation and morphology analysis
Cell morphology and oil bodies were visualized using a confocal laser scanning microscope (LSM880 with AiryScan, Carl Zeiss, Germany)110. Specifically, cells from day 12, collected at consistent culture times, were stained with Nile red (0.1 mg/mL in acetone; Sigma-Aldrich, USA) and incubated in the dark for 10 min at room temperature. Observations were made using a 100×/1.4-NA oil objective lens, with excitation and emission wavelengths set at 530 nm and 592 nm, respectively. Images of each sample were captured randomly. The cell size parameters were quantified by Fiji ImageJ tool111. Briefly, images with embedded scale bar were imported into the ImageJ program. The length of the scale bar was measured by line tool. The unit of length (in pixels) was set to micron, based on the actual length of the scale bar. Subsequently, cell length and width were measured using line tool. The geometry of P. tricornutum is half parallelepiped and its volume was calculated according to the following equation112: V=l*w*h/2, where V is volume, l is cell length, w is cell width, and h is height (equal to the width).

Neutral lipid content analysis
To detect the neutral lipid content, cells were stained with Nile red (0.1 mg/mL in acetone) and the relative fluorescence intensity was analyzed using a Multifunctional Microplate Reader (BioTek Synergy H1, USA)110. Briefly, 10 μL Nile red was added to 1 mL of cell culture, and the samples were mixed well and incubated in the dark for 20 min at room temperature. The samples were transferred to a 96-well plate and the fluorescence intensity was detected at excitation and emission wavelengths of 530 and 580 nm, respectively. The unstained cell cultures were used as blank controls. Finally, the neutral lipid content was analyzed by the relative fluorescence intensity of Nile red.

Fatty acid composition analysis
To analyze the composition of the fatty acids, they were extracted and methylated, and detected using a gas chromatography-mass spectrometry (GC-MS)113 with slight modifications. 50 mL of cell culture was harvested and the supernatant was discarded. Samples were transferred to 15 mL tubes and 5 mL 2 M KOH (in methanol) was added. Cells were broken by sonication and incubated at 75°C for 10 min. Samples were stratified at room temperature and the supernatant was transferred to 50 mL tubes. 5 mL 2 M KOH was added to samples and the above steps were repeated twice. The supernatants were combined and an equal volume of 2 M HCl (in methanol) was added. Samples were incubated at 75°C for 10 min, stratified at room temperature, and the supernatant was transferred to new 50 ml tubes. Subsequently, hexane was added for extraction of methylated fatty acids. 6 μL 10 mg/mL methyl nonadecanoate (in hexane) was added to samples, and the samples were concentrated to 1.5 mL using a Termovap Sample Concentrator. The methylated fatty acids were analyzed by GC-MS and identified leveraging the National Bureau of Standards spectrum library. Lipid saturation index was calculated using the following equation114: Saturation index = Total fatty acid / Unsaturated fatty acid × 100. Amount of total fatty acid

Analysis of photosynthetic efficiency
The maximum photochemical efficiency of PSII (Fv/Fm) was measured as described previously with slight modifications113. Briefly, 2 mL cell culture was harvested and incubated in the dark for 10 min. Samples were then transferred into a measuring cuvette, and chlorophyll fluorescence parameters were determined with a PhytoPAM Phytoplankton Analyzer (Walz, Germany). The Fv/Fm was calculated based on the following formula: Fv/Fm = Fm − Fo / Fm, where Fv is variable fluorescence, Fm is maximum fluorescence, and Fo is initial fluorescence.

Transcriptome sequencing and analyses
Research has indicated that while many transcription factors are activated only under specific conditions, such as light stress and high temperature115, and analyzing transcription profiles and DNA binding sites in organisms exhibiting visible phenotypes due to transcription factor overexpression is a common approach to studying transcription factor mechanisms116. This method can be applied to organisms cultured under normal culture conditions without requiring specific condition induction116. In this study, we observed that overexpression of PtHSF2 under normal culture conditions (20°C) led to phenotypic changes such as increased cell volume and slowed growth. Therefore, we conducted RNA-seq and CUT&Tag experiments on cells cultured at 20°C. After incubation at 20°C to stationary phase, total RNA was extracted from PtHSF2 overexpressing cells and WT cells using RNAiso reagent (Takara, Japan), with three parallel experiments each cell line. Then, mRNA was enriched with Oligo(dT) beads and fragmented by fragmentation buffer. The fragmented mRNAs were reverse transcribed into cDNA using a random hexamer primer, and subsequently, the second-strand cDNA was synthesized. The double-stranded cDNA was purified with QiaQuick PCR extraction kit, and the purified cDNA fragments were subjected to end repair, poly(A) addition and Illumina sequencing adapters ligation. Next, the ligated samples were separated by size via agarose gel electrophoresis and then enriched by PCR amplification. Finally, the resultant products were sequenced using the NovaSeq 6000 platform (Illumina, San Diego, CA, USA) at the Novogene company (Peking, China). RNA extraction and transcriptome sequencing for T. weissflogii were carried out following the same protocol.

Raw sequenced data were assessed for quality using SeqKit tool. Trimmomatic was used to trim low-quality bases and remove adapter sequences, producing high-quality reads for subsequent analysis. The cleaned reads were aligned to the corresponding reference genomes of P. tricornutum and T. weissflogii using Bowtie 2. RPKM (Fragments Per Kilobase per Million bases) values were calculated and log2-transformed to assess gene expression levels. DEGs analysis was performed using DESeq2. Genes with an adjusted |log2(FC)| ≥ 1 and p-value < 0.05 were considered significantly differentially expressed. Significant DEGs were further verified by qPCR, and relative gene expression was determined by comparative Ct method and normalized by reference genes β-actin and TBP.

For GO term enrichment analysis, the SwissProt database downloaded from NCBI was used as the reference for annotation. The diamond tool was utilized with an e-value cutoff of 1e–5 to map the DEGs to the SwissProt database. After mapping, UniProt IDs were obtained, and corresponding GO IDs were retrieved from the idmapping.tb.gz file, downloaded from UniProt. WEGO (Web Gene Ontology Annotation Plot, http://wego.genomics.cn) was then used to analyze and obtain Level 2 GO terms, enabling the identification of enriched biological processes, molecular functions, and cellular components in our datasets. Additionally, KEGG pathway enrichment analysis was conducted using the Assign KO Tool (https://www.kegg.jp/kegg/mapper/assign_ko.html).

CUT&Tag combined with electroporation
CUT&Tag was performed as previously described43 with modifications, and Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme, China) was used. In addition, since P. tricornutum has a cell wall, proteins were delivered into the nucleus using electroporation117,118. In brief, PtHSF2-1, PtHSF2-2, and WT cells were used in this experiment, with three biological replicates and one technical replicate for each line. A 1:1 mixture of cell samples from these two overexpression lines was employed as a negative control. Cells were collected after incubation at 20°C to stationary phase. Cell pellets were washed thrice with wash buffer and resuspended in 200 μL antibody buffer containing a 1:50 dilution of the primary antibody (Rabbit Anti-Flag, ab205606, Abcam, UK). Non-specific IgG (1:50, Rabbit IgG, ab172730, Abcam, UK) was added to the negative control. After gentle vortexing, samples were transferred into cuvettes and electroporated as described previously102. The electroporation parameters were set as follows: 500 V field strength, 25 μF capacitance, and 400 Ω resistance. The samples were transferred into 1.5 mL tubes and incubated at 4°C overnight. Centrifugation was performed to remove the supernatant, and cells were resuspended in 200 μL Dig-wash buffer pre-mixed with secondary antibody (1:50, Goat Anti-Rabbit IgG H&L, ab6702, Abcam, UK). The mixture was transferred into cuvettes, and the electroporation was performed as described previously102. Samples were then transferred into 1.5 mL tubes and incubated at room temperature for 1 h. Cells were washed thrice in Dig-wash buffer and resuspended in 0.04 μM Hyperactive pA -Transposon adapter complex (in Dig-300 buffer) with gentle vortexing. Subsequently, samples were transferred into cuvettes for electroporation102. Samples were transferred into 1.5 mL tubes, and the tubes were placed on a rotator at room temperature for 1 h. The supernatant was removed, and cells were washed thrice with Dig-300 buffer and resuspended in 50 μL Trueprep Tagment Buffer L. Samples were incubated at 37°C for 1 h to fragment the DNA. 5 μL 20 mg/mL Proteinase K, 6 μL 10% SDS and 10 μL 0.5 M EDTA were added to samples. The samples were thoroughly mixed and incubated for 10 min at 55°C. DNA was extracted by phenol-chloroform-isoamyl alcohol method.

A TruePrep Index Kit V2 for Illumina (Vazyme, China) was used for PCR amplification. DNA fragments were used as templates, and universal i5 primer, uniquely barcoded i7 primers, and CUT&Tag Amplification Mix were added. PCR was performed with the conditions below: 72°C for 3 min, 95°C for 3 min, 12 cycles of 98°C for 10 s and 60°C for 5 s, 72°C for 1 min, and 4°C hold. Amplification products were purified using VAHTS DNA Clean Beads (Vazyme, China). For library pooling, 10 μL of sample from each of the three libraries of the WT line were homogenously mixed to form the sequencing library for WT. Similarly, 5 μL of sample from each of the six libraries of the two overexpression lines were homogenously mixed to form the sequencing library for PtHSF2. Additionally, 10 μL of sample from each of the three libraries of the negative control were homogenously mixed to form the sequencing library. These three libraries were sequenced on an Illumina NovaSeq platform (BGI, Shenzhen, China).

Data processing and analysis
Paired-end reads were aligned using Bowtie 2 version 2.2.5 (https://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Peak calling was executed with Model-based Analysis of ChIP-Seq 2 (MACS2)119. Notably, we constructed a promoter database for P. tricornutum using the hiPromoter software (Patent No. ZL202110822106.X) and used BLAST to pinpoint the reads in peaks to promoters. GO and KEGG pathway enrichment analysis of potential target genes were conducted as previously mentioned. Binding motifs of PtHSF2 were analyzed using Multiple EM for Motif Elicitation (MEME)48.

CUT&Tag-qPCR for profiling the binding of PtHSF2
To further validate the results of CUT&Tag high-throughput sequencing, CUT&Tag-qPCR analysis was performed on three potential target genes (Cdc45-like, Lhcx2, and ABC) using unmerged libraries. Promoter-specific primer pairs were designed according to the principles of ChIP-qPCR assays. The primer pairs are listed in Supplementary Table 2, with specificity confirmed by a single peaks in melt curve analysis and amplification efficiencies of 90–110% (Source Data). The Ct values obtained from qPCR using these primers are shown in the Source Data file. The promoters of β-actin and TBP, which are located far from the promoters of Cdc45-like, Lhcx2, and ABC, were used as internal controls.

Comprehensive analysis of the expression of 69 P. tricornutum HSFs across MMETSP datasets
To investigate the expression of 69 HSFs across various species, we utilized RNA-seq datasets from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP)34. We downloaded the reassembled data, including transcriptome assemblies and expression quantification datasets (https://zenodo.org/records/3247846), provided by Johnson et al.35. To assess the expression levels of HSFs in P. tricornutum, we performed a BLASTn search using 69 HSF sequences with an e-value threshold of 1e–5. Additionally, HSF orthologues in other species were identified using tBLASTn with the same threshold. Expression levels were determined by integrating the BLAST results with the expression quantification datasets from the MMETSP reassembled transcriptome.

Redox levels
The content of reactive oxygen species (ROS) was determined by a ROS Assay Kit (Beyotime, China). Briefly, 1 mL cell culture were harvested and washed twice with PBS. Cells were resuspended in 1 mL 10 μM fluorescent probe DCFH-DA and incubated at 37°C for 20 min. Cells were washed thrice in PBS to remove probes did not enter cells. 1 mL PBS was added to resuspend cells, and fluorescence intensity was measured using a microplate reader.

Lipid peroxidation was assessed using a Lipid Peroxidation MDA Assay Kit (Beyotime, China). Firstly, total protein was prepared using a Cell Lysis Buffer (Beyotime, China) and protein concentration was determined by a BCA Protein Assay Kit (Beyotime, China). 0.2 mL protein sample was mixed with 0.4 mL MDA detection solution and heated at 100 °C for 15 min. Subsequently, 0.2 mL supernatant was transferred into a 96-well plate, and the absorbance was measured at 532 nm.

Catalase (CAT) activity was assayed using ammonium molybdate-chromogenic method120 with a Catalase Assay Kit (Solarbio, China). In short, 30 mL cell culture were harvested and washed twice with PBS. Cells were resuspended in 1 mL extraction solvent and sonicated. Centrifugation was performed to separate the cells and supernatant. 20 μL supernatant was mixed with 100 μL H2O2 substrate and incubated at 25°C for 10 min. 180 μL CAT assay buffer was added to samples, and the mixture was left at room temperature for 10 min. Samples were transferred into 96-well plates, and the absorbance was obtained at 405 nm.

Superoxide dismutase (SOD) activity was determined using a Total Superoxide Dismutase Assay Kit with WST-8 (Beyotime, China). 50 mL cell culture were collected and washed twice with PBS. 200 μL SOD sample preparation solution was added, and samples were homogenized using a tissue grinder. Subsequently, centrifugation was performed and the supernatant was collected. 20 μL supernatant, 160 μL WST-8 working solution, and 20 μL enzyme working solution were added to 96-well plates. The samples were thoroughly mixed and incubated at 37°C for 30 min. Absorbance was measured at 450 nm.

Flow cytometry
Flow cytometry analysis was performed to examine the DNA content and determine the cell cycle distribution. Five microalgal strains (WT, PtHSF2-1, siPtHSF2-1, PtCdc45-like-1, siPtCdc45-like-1) were used in this experiment. Following a previously established method121 with minor modifications, we monitored the DNA content changes in exponentially growing cells over 12 consecutive time points. Starting at the beginning of the light period (0 h), we sampled 5 mL of culture every 2 h. Cells were collected, washed thrice with 1× PBS, and then fixed overnight at 4°C in 1 mL of 70% ethanol. Post-fixation, we added 500 μL of SYBR Green I working solution, diluted in PBS from a 20× stock solution in DMSO, and incubated the samples at room temperature for 15 min in the dark. Fluorescence was detected using a flow cytometer with an excitation wavelength of 488 nm, an emission wavelength of 520 nm, and a detection filter range of 510–530 nm.

Statistical analysis
Statistical analyses and visualization were performed using GraphPad Prism (version 8.0). Data from at least three independent experimental replicates were given and presented as means ± SD. Two-tailed unpaired Student’s t test was used to assess the differences between two experimental groups. Significant difference was indicated at P < 0.05 (*) or P < 0.01 (**) level.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript “Thermal tolerance and cellular plasticity in marine phytoplankton diatoms modulated by epigenetic mechanisms for environmental adaptation” authors investigated the role of the heat shock transcription factor 2 (HSF2) in the thermal tolerance in the diatom Phaeodactylum tricornutum and uncovered PtCdc45-like gene as one of the major targets of PtHSF2. The work explores a little understood mechanism in thermo tolerance of an important group of phytoplankton.\nI am fully aware that functional studies in diatoms are not easy to carry out and I appreciate the use of state-of-the-art techniques such as CUT&Tag. However, I am convinced that several open questions need to be addressed (in a way that is feasible to do in diatoms) to draw conclusions about the mechanism of HSF2 action.\nMajor comments:\n1. The most important issue is for me the missing analysis of the impact of HSF2 and Cdc45-like gene on the cell cycle progression. First of all, the authors report that both HSF2 and Cdc45-like over-expressing strains have wider cells than the respective control strains, resulting in higher cell volume. Authors themselves speculate that this could be due to a cell cycle arrest (line 312). The presence of two chloroplasts per cell in Figure 1F for PtHSF2 over-expression strains and Figure 3B for PtCdc45-like over-expression strains further supports the idea of cell accumulation/cell cycle arrest in the S or G2 phase, which could explain the higher cell volume. Moreover, the Cdc45 protein is a conserved member of eukaryotic replisomes. Therefore, the analysis of the cell cycle progression of PtHSF2 and PtCdc45-like is essential to understand the phenotypes induced by over-expression and knock-down of these genes.\nVerifying the presence or absence of cell cycle arrest is important also for the interpretation of the RNAseq results, as it was shown that many genes in diatoms show cell cycle stage-dependent expression.\n2. To further test the hypothesis that PtCdc45-like is a direct target of PtHSF2 and part of PtHSF2 overexpression phenotype is mediated by PtCdc45 overexpression, I suggest testing whether PtCdc45-like downregulation can alleviate part of the PtHSF2 overexpression phenotype. The authors have all the constructs/strains to test this genetic interaction.\nI recommend to address the above questions and adjust the discussion and conclusions according to the results that will be obtained.\nMinor comments:\n3. Title of the article - I find the title very general. It’s perhaps only my personal preference, but I recommend using a title that better describes the findings of the presented research, including the names of the studied genes.\n4. Methods explaining how were obtained results presented in Supplementary Figure 1 are missing.\n5. Supplementary Figure 1 C shows that the abundance of PtHSF2 homologs is highest at higher latitudes and relatively low in tropical parts of the oceans. How can it be explained?\n6. Please give a better explanation for Supplementary Figure 1B.\n7. Taking into account the focus of this study on heat stress tolerance, I am missing information about the expression of the expression of the PtHSF2 gene at 20C and 30C.\n8. Authors show that they generated PtHSF2 RNAi lines, but there is no analysis done with these lines. If there is no phenotypic difference from wild-type, it is necessary to mention it at least in the text. Also, the thermotolerance of PtCdc45-like RNAi lines should be shown.", "reviewer_2": "This manuscript by Huang et al. reveals the role of epigenetic mechanisms in facilitating the adaptation of marine phytoplankton to environmental stress, with a focus on the thermal tolerance and cellular plasticity. The insights gleaned are pivotal in understanding and projecting marine ecosystem responses to global warming. Notably, it marks the application of a novel CUT&Tag technique to decode the operational mechanism of transcription factor PtHSF2 within microalgae.\nOverall, this manuscript appears to be well-positioned for acceptance, given its novelty and relevance to a broad readership interested in marine biology, climate change, and epigenetics. However, I have several other comments on this manuscript, and they should be considered before the paper can be published.\nMajor points:\n1. The authors mention in the manuscript that P. tricornutum has 69 heat shock transcription factors (HSFs), but have not explained why PtHSF2 was chosen for this study. The authors should explain it.\n2. CUT&Tag technology is a highly sensitive and streamlined method for studying protein-DNA interactions on chromatin, offering advantages such as low background noise and suitability for single-cell analysis. However, there is few report of its application in algae. To promote the application of this technology in the field of algal research, I suggest specifying the relevant experimental details, such as electroporation parameters, including voltage and pulse duration in Line 525.\nMinor points:\n3. Substitute \"heat shock transcription factors\" with \"HSFs\" in Lines 39 and 79.\n4. Add citation for the statement in Line 99, \"Diatoms are widely distributed in marine ecosystems and are ubiquitous in almost all aquatic habitats\".\n5. Lines 102 to103, omit \"heat shock transcription factors\".\n6. The \"is\" in Line 109 should be changed to \"was\".\n7. Citations are needed for the statement made in Lines 196-197.\n8. Change \"led to the increase\" in Lines 237-238 to \"led to the enlargement\".\n9. Switch \"PtCdc45-like-transformed algae\" in Line 243 to \"PtCdc45-like-transformed lines\".\n10. Replace \"strains\" in Line 247 with \"lines\".\n11. Line 261, modify \"Cell size plasticity\" to \"Cell plasticity\".\n12. Line 269, correct the typo \"thnt\" to \"that\".\n13. Line 273, replace \"speculate\" with \"speculated\".\n14. Analyze and attach a relevant citation to the statement \"Heightened temperatures stimulate the generation of reactive oxygen species (ROS) within cells, thereby triggering lipid peroxidation\" in Line 286.\n15. Provide reference in Lines 346-347 \"Accumulating evidence shows that P. tricornutum exhibits extensive temperature adaptability.\"\n16. Line 551, \"19 ggnog 5.0\" appears to be a potential spelling error, and the correct expression is likely \"EggNOG 5.0.\" If that's the case, please correct the spelling error.", "reviewer_3": "In this manuscript, the authors conducted a systematic study to elaborate on the regulation mechanism of thermal tolerance mediated by heat shock transcription factor and target gene CDC-45 like in the model diatom Phaeodactylum tricornutum. Gene overexpression and silence methodologies were employed to address the issues , along with monitoring cell physiological response. Overall, the work flow of this manuscript is clearly written, though some results are not included. The introduction and discussion are not comprehensive and adequate yet. This work provides further understanding into the thermal adaptivity of diatom. The key finding of this study is the identification of several HSF-target genes, CDC-45 like etc. Except for this, the other findings described in this manuscript are not as new and appealing, which makes it not qualified for publication on NCOMM.\nThere are some concerns about the results and conclusion. The first concern is that gene transcript regulation via transcription factor is not epigenetic regulation. And the author argues that CDC45 is an important control gene for cell size. In a unicellular phytoplankton physiological study, the algae cell size is sensitive to ambient stress and cell growth status. It is often to find that cells grown under unfavorable conditions have a relatively larger cell size. Thus, the phenomenon observed in this study might also be likely due to the overall cell physiological response caused by gene interference, instead of a direct effect from CDC45. As well as regarding lipid accumulation, which has been widely examined in the development of microalgae biofuel. Comparison between this work and the regulation mechanism reported in those studies were not fully discussed.\n1. Line 23-24. As a matter of fact, many studies have been conducted to address the cellular mechanism in terms of temperature adaptation. e.g. The ISME Journal (2019) 13:2415–2425, and the quote from the book “The Molecular Life of Diatom”:\n2. It is not surprised that HSF play important role in temperature response. I would say, it is well known that HSF is the key player in this mechanism.\n3. The growth curve of RNAi strains should be examined and presented as well.\n4. Line179, besides Cdc45-like, Lhcx2 and ABC transporter, do not see a summary of other genes in the assemble of 35 genes.\n5. Line 188, what does this nt sequence represent? In Figure 2 J-L, the abundance of which promoter binding motif was examined? Please make a clear description.\n6. Line 206, Wrong number in supplementary tables. Why refer to a primer table? The description of Phatr3_J46275 is abrupt and lacks contextual connection. Should be “FAD” in the legend of Figure 2P?\n7. Line 285, Given the Fv/Fm was significantly inhibited in the PtCdc45-like-overexpressing strains, and lower transcription level of Lhcx2, it is not surprised to see the relative lower cellular ROS content.", "reviewer_4": "Report on the manuscript NCOMMS-24-06983\nThe manuscript “Thermal tolerance and cellular plasticity in marine phytoplankton diatoms modulated by epigenetic mechanisms for environmental adaptation” by Huang et al relates to the molecular mechanisms involved in the acclimatory response of diatom to increased sea water temperature condition. Available evidences indicate that diatoms, a prominent group of marine primary producers, present a larger thermal tolerance range than other phytoplankton functional types. Knowledge on the mechanisms involved in diatom thermal tolerance range, however, are limited. Huang et al report an expansion of the transcription factor Heat Shock Factors (HSFs) in diatom genomes. In addition, reverse genetic approach carried out in the model diatom Phaeodactylum tricornutum suggests that the HSF orthologue HSF2 may play a positive role in the response to increased temperature via transcriptional activation of the target cyclin dependent kinase Cdc45-like with concurrent effects on cell size and growth, lipid and oxidative stress metabolisms.\nDocumentation on the molecular mechanisms involved in the thermal tolerance of marine microalgae is central to better understand their ecophysiology and predict their response to ongoing ocean warming. This topic, therefore, is of particular interest for a large and broad audience. The introduction is concise and focused, presenting well the background and needed research. The result section follows a logical sequence and the choice of the methods and approaches are sound and complementary. However, while the manuscript presents valuable novel information their significance remains relatively limited. Specifically, while the manuscript suggests that HSF2 and Cdc45-like may play a positive role in the tolerance of P. tricornutum to increased temperature it remains to be addressed if this mechanism is conserved in diatoms eg by providing evidences on the role of HSF2 in different diatom species of contrasting thermal tolerance ranges and/or in another model diatom species of contrasting ecophysiology such as Thalassiosira pseudonana. In addition, it remains unknown whether HSF2 and Cdc45-like directly contribute to the apparent larger thermal tolerance range observed in diatoms compared to that in other phytoplankton functional types eg are HSF2 and Cdc45-like orthologues absent or regulated differently in coccolithophores and green algae? Also, the experimental approach focuses on the response to increased temperature but does not address the acclimatory response to decreased temperature thus limiting our understanding on the importance of HSF2 and Cdc45-like for the diatom thermal tolerance range per se. In addition, and importantly, I think that in some instances the designs of the experiments have shortcomings, the experiments are incomplete and the data are not presented in a clear way - altogether this reduces the solidity of the results and conclusions. In consequence I believe that the manuscript in its current version is not suited for publication in Nature Communications. This is a very interesting topic and the work carried out by Huang et al is substantial. I have provided below my detailed comments and suggestions, which I hope, may contribute to improve the manuscript.\nLine 3\nI do not think that the term “epigenetic” fits with the study. Please see Deans and Maggert 2015 (doi.org/10.1534/genetics.114.173492) for a recent discussion on the signification of this term.\nLine 102\nMore than four diatom species have fully sequenced genomes (see Grigoriev et al 2021 doi.org/10.1093/nar/gkaa898). Please consider to include them in the analysis.\nLine 103\nAre all these HSFs expressed? Please consider to exploit the RNAseq data set from the MMETSP project (Keeling et al 2014 doi.org/10.1371/journal.pbio.1001889) to investigate this point.\nLine 103\nIf known, please indicate whether these four diatom species have different thermal tolerance ranges.\nLine 113\nWhy “two branches”? Many (sub)branches are shown.\nLine 116\nIs “this HSF” or orthologues present in microalgae species from phytoplankton functional types other than diatoms?\nLine 125\nIt is well established that gene knock-down by RNAi can present off-target effect (Chen et al 2021 10.1080/15476286.2020.1868680). This is particularly relevant when the target is a member of a multigene family such as HSF here. The authors should provide supports and if possible experimental evidences that only HSF2, and no other paralogues, was downregulated in the siRNA lines eg by providing a sequence alignment supporting that the targeted sequence is specific to HSF2 and by analyzing the expression levels of HSF2 paralogues in the RNAi lines (eg by RT-qPCR).\nLine 130\nThree instead of two independent transgenic lines per construct (overexpressing and RNAi lines) would be preferable.\nLine 134\nPlease consider to assess the phenotype of the transgenic lines in response to a milder increase in temperature than from 20C to 30C; as shown in Fig.1B and C both WT and HSF2 overexpressing lines do not grow at 30C. The WT line is just dying faster.\nLine 149\n- It is reported that both HSF2 overexpressing and RNAi lines have been generated but the phenotypic characterization is shown only for the formers. This is a shortcoming; phenotypic characterization of the HSF2 siRNA lines is needed in this paragraph.\n- Please consider to investigate if HSF2 (and Cdc45-like) expression is upregulated in the WT background when grown at 30C versus 20C. This will provide support for their intrinsic role in the response to increased temperature.\n- It is not supported at this stage that HSF2 may regulate the expression of genes involved in the cell cycle per se.\nLine 151\n- Why the RNAseq and CUT&Tag experiments were conducted in cultures grown at 20C (and not at 30C) while the increased tolerance of the HSF2 overexpressing lines versus WT is evidenced at 30C?\nLine 154\n- Please state how many genes were found differentially expressed (up and down), using which fold change threshold and p-value.\n- Please indicate and analyze separately the genes found up and down regulated.\n- Please indicate here how the RNAseq results overlap between the two HSF2 overexpressing lines in term of the genes found up and down regulated in both lines.\n- Add in SI the list of genes found differential expressed with their fold change and p-values.\n- It would be interesting to report how the expression of the HSF orthologues are regulated in the HSF2 overexpressing lines versus WT line.\nLine 159\n- Please analyze the up and down regulated genes separately.\n- It is not clear how the GO term enrichment analysis was conducted. Please describe better this information in the material and method (reference gene list used, fold change, p-value?).\nLine 170\n- Please add in supplementary metrics relative to the CUT&Tag NGS experiment eg number of sequenced reads and their quality and indicate how many were mapped to promotor regions.\n- Please indicate here that the CUT&Tag experiment was carried out from mixed samples of the two overexpressing lines and state how many mixed samples were analyzed.\n- The design above is a shortcoming – the two overexpressing lines should have been analyzed separately to support the replicability of the results across the lines.\n- Also, as for the RNAseq experiment, why the experiment was carried out in cultures grown at 20C while the increased heat stress tolerance was evidenced at 30C?\nLine 173\n“Predominantly”; please provide numbers and statistics.\nLine 179\n- Only 35 out of the 295 gene targets found in CUT&Tag were among the 1449 genes found differentially expressed in RNAseq. How the authors explain this limited overlap?\n- Please consider the genes found up and down regulated in RNAseq separately when considering their overlap with the gene targets found in CUT&Tag.\nLine 181\nPlease provide here the accession number of the Lhcx2 and ABC genes.\nLine 184\n“Predominantly”; Please provide number and statistics to support this claim.\nLine 188\n“Previous…”; this information is already stated in line 167. The qPCR is simply a confirmation of the CUT&Tag NGS library.\nLine 196\nPlease provide references supporting that a same transcription factor can activate and repress the transcription of different gene targets.\nLine 208\nFAD is not the only gene found differentially expressed in RNAseq but not found targeted by HSF2 in CUT&Tag – in fact the bulk of the RNAseq result does not overlap with the CUT&Tag result (see comment line 179). Please discuss this observation eg difference in sensitivity and coverage between the two techniques? Could the regulatory action of HSF2 be mostly indirect?\nLine 217\n- I understand the rational in analyzing Cdc45-like knock-down and overexpressing lines in order to assess if they phenocopy their HSF2 counterparts. Still, the possible genetic interaction between these 2 genes could be better assessed by (also) characterizing the phenotype of HSF2 know-down lines overexpressing Cdc45-like and reciprocally.\n- Also, given the possible off-target effect of the RNAi construct together with the use of only two independent Cdc45-like knock-down lines it is warranted to generate and characterize the phenotype of Cdc45-like knock-down lines complemented by a RNAi resistant version of Cdc45-like expressed under its own promoter.\nLine 243\nWhy “single cell dry weight”; is it not total dry weight cell pellet normalized by the cell number instead?\nLine 248\n- Please define “material” – ABC transporters fall under many (sub)families.\n- Is this ABC transporter (J42881) the same one as the one mentioned in line 181 (targeted in qPCR)? If so, please indicate its regulation in the HSF2 overexpressing lines as found by RNAseq in the paragraph starting line 151.\n- Both Cdc45-like and the ABC transporter J42881 were identified as target of HSF2 in CUT&Tag (line 193). It is also stated that HSF2 activates the transcription of Cdc45-like but represses the one of ABC transporter as determined in qPCR (line 202). How the authors explain that the overexpression of Cdc45-like alone (ie in the absence of HSF2 overexpression) downregulates ABC transporter? Is it possible that ABC is targeted by both HSF2 and Cdc45-like in parallel, or could there be a positive feedback loop between Cdc45-like and HSF2? I would be curious to know the expression level of HSF2 in the Cdc45-like knock-down and overexpressing lines.\nLine 269\n“thnt”. Typing error.\nLine 273\nIt is difficult to speculate on the possible increase in lipid content in the Cdc45-like overexpressing lines specifically in the cell membrane as the HSF2 overexpressing lines present an increased accumulation of intracellular lipid content (line 142 and Fig1E and F).\nLine 276\n- In this paragraph the phenotypic characterization (sensitivity to elevated temperature, ROS, MDA/SOD/CAT contents) of the Cdc45-like knock-down lines must be reported.\n- Also, the phenotype of the Cdc45-like lines (both overexpressing and knock-down) when grown under 20C must be shown.\nLine 279\n“The optimum growth…”.\nThis sentence should be stated at the beginning of the Results section.\nLine 287\nPlease introduce what is malonaldehyde ant its known cellular role.\nLine 288\nWhy “discrepancy”? The Cdc45-like overexpressing lines present an increased tolerance to heat stress congruent with a lower content of ROS.\nLine 294\nPlease indicate which genes involved in antioxidant defense were eventually found upregulated and/or targeted by HSF2 in the HSF2 overexpressing lines.\nLine 299\n“…emerging studies revealed that HSFs also target genes beyond heat shock proteins”. Please indicate which genes specifically and in which organisms. Are those genes also found up regulated/targeted by HSF2 in the HSF2 overexpressing lines?\nLine 300\n“Our novel finding demonstrated that PtHSF2 regulated the transcription of…”.\nThis is only based on the RNAseq and CUT&Tag experiments conducted in the HSF2 overexpressing lines grown at 20C. It would be necessary to conduct comparative RNAseq or targeted RT-qPCR experiments in the HSF2 knock-down lines versus WT line grown under 20C and 30C to better support this claim. The HSF2 knock-down lines are available.\nLine 303\n“…PhHSF2 was involved in regulating thermal tolerance…”.\nAgain, phenotypic characterization of the HSF2 knock-down lines is needed (increased sensitivity to elevated temperature?).\nLine 307\n“Mcm2-7 and GINS”.\nPlease explain abbreviations.\nLine 309\n“Fig.5”.\nPlease cite Fig.3 instead (experimental results) as Fig.5 is a model.\nLine 312\n“Hence we speculated that…”.\nThis speculation is unclear to me – do P. tricornutum WT cells increase in size during G1, or has it been reported before that cells with an extended G1 phase (as a consequence of S phase impairment) become larger?\nLine 323\n“Larger cells had lower growth rate than smaller cells in diatoms”.\nMaybe because large cells need more energy and nutrients than small cells to grow irrespectively of the thermal tolerance range.\nLine 324\n“..several ABC transporters were found to be significantly decreased in…”.\nPlease detail which ABC transporters and their putative functions.\nLine 333\n“…larger P. tricornutum cells, while growing slower at 20C than smaller cells exhibited superior antioxidant abilities and higher survival rates at elevated temperature”.\n- From this rational it would be intuitive to think that P. tricornutum WT cell (and in other diatom species) increases in size in response to increased temperature – has such observation been reported before?\n- Also, how an abrupt transition from 20C to 30C is relevant with what P. tricornutum and diatoms in general experience in their environment?\nLine 343\n“..correlate the cell size of P. tricornutum with its broad temperature adaptation and to provide insights into the molecular mechanisms by which diatoms tolerate high temperature”\n- Providing experimental evidences in different diatom species of contrasting thermal tolerance ranges and sizes is needed to support this claim.\n- Please distinguish adaptation (such as in the study cited line 63 reference 10) and short term acclimatory response (this study).\nLine 349\n“..whose shape plasticity is crucial for environmental adaptation.”\n- Do different morphotypes of P. tricornutum present different thermal tolerance ranges? And, do P. tricornutum switch morphotype under increased temperature?\n- Please distinguish cell size and morphology in the text.\nLine 401\nPlease indicate how many transgenic lines were obtained and how the ones shown in the study were selected.\nLine 420\n- RT-qPCR experiments should include at least 2 validated references genes.\n- Please provide information of the RT-qPCR experiments following the MIQE guidelines (Bustin 2009 10.1373/clinchem.2008.112797).\nLine 426\n- The initial cell density of the cultures is indicated as 1 x106 cells/mL. To my experience this is very high even for P. tricornutum. In consequence the cultures present a very short (day 1 to 3) exponential growth phase over the course of the 13 days culture experiment (Fig.1B). Please justify the choice of the starting cell density.\n- Also, please clarify the design of the culture experiment regarding how the cultures were exposed to the different temperature regimes eg were the cultures maintained at 20C and then transferred (how quickly?) to the 20C and 30C growth conditions?\nLine 427\nPlease clarify what is “15ggnog15ine”.\nLine 509\nPlease follow and indicate the MIQE guidelines or similar for the qPCR experiments (eg provide specificity, efficiency and Ct values of the qPCR reaction for each primer pairs, demonstrate that a sufficient number of reference genes were used for relative quantification).\nLine 516\nPlease clearly indicate the number of independent lines, and biological and technical replicates used.\nLine 545\nPlease provide a table presenting the statistics of the NGS reads including their number and quality for each library.\nLine 557\nSame comment as in line 509.\nLine 550\nPlease detail the procedure used for the GO term enrichment analysis (fold-change and number of gene used as threshold, statistics, reference gene list).\nFig 1C\nIs does not seem right to plot daily growth rate. The growth rate should be calculated within the exponential growth phase which is very short here (around day 1 to 3) due the very high starting cell density (see comment line 426).\nFig 1D and E\n“D” seems to correspond to “E” and vice versa.\nFigure 1F\nHow the authors explain the presence of red dots outside the cells in the picture of the PtHSF2-1 line under 30C?\nFigure 2A-F\n- Please analyze separately the up and down regulated genes found in RNAseq.\n- What means in the x-axis “Genes involved (%)”?\nFigure 2G\nPlease analyze separately the up and down regulated genes found in RNAseq.\nFigure 2H\nPlease label the y-axis.\nFigure 2I\nPlease better show what the y-axis corresponds to.\nFigure 3\nPlease state at which temperature the lines were exposed.\nTables\nThe two tables have formatting problems:\nIn the first table the name of the genes in the first left column are truncated.\nIn the second table the name of the entries in the top raw are truncated\nFor both tables, please provide a short description note to understand what the tables correspond to.\nSupplementary Fig.1A\n- Please provide sequences and sequence alignments, and indicate the algorithms used for the phylogenetic analysis.\n- Please provide branch lengths and bootstrap values.\n- It could be worth adding some green algae or coccolithophores species in the phylogenetic analysis to infer the conservation/speciation of the HSF orthologues across different phytoplankton functional types.\nSupplementary Fig.1B\nPlease explain what is the “EUK_SMAGs database”.\nSupplementary Fig.2B\n- Please provide sequences and sequence alignments, and indicate the algorithms used for the phylogenetic analysis.\n- Please provide branch lengths and bootstrap values.\n- To what correspond the mauve circles at the nodes?\nSupplementary Fig.2C\nPlease provide a positive control for PCR amplification in WT gDNA.\nSupplementary Fig.2D\nPlease provide information pertained to the qPCR experiment following the MIQE guidelines or similar.\nSupplementary Fig.2F\n- Is “E” and not “F”.\n- Please provide pictures of the full gels and films. This is particularly important to ascertain the specificity of the FLAG antibody.\nSupplementary Fig.4B, C and E\n- Same comments as for Supplementary Fig.2B, C and E.\n- Please indicate to what “n” corresponds to: technical or biological replicates?\nSupplementary Table 1\nPlease provide gene sequences.\nSupplementary Table 3\n- Is Supplementary Table “2” and not “3”.\n- Please add information of the specific use of the primers in the study (eg “validation of….”).\n- Provide here information on the qPCR primer pairs efficiency, Ct values etc."}, "author_response": "Response to Editor’s and Reviewers’ Comment\nWe thank the editor and reviewers for their suggestions and constructive comments.\nThe offered comments were taken carefully and we have responded to the queries and\ncomments raised by the referees and accordingly revised the manuscript. As per the\nreviewer’s suggestions, the carried-out amendments and revisions are differentiated\nby using red color font in the revised manuscript.\nReviewer #1 (Remarks to the Author):\nIn the manuscript “Thermal tolerance and cellular plasticity in marine phytoplankton\ndiatoms modulated by epigenetic mechanisms for environmental adaptation” authors\ninvestigated the role of the heat shock transcription factor 2 (HSF2) in the thermal\ntolerance in the diatom Phaeodactylum tricornutum and uncovered PtCdc45-like gene\nas one of the major targets of PtHSF2. The work explores a little understood\nmechanism in thermo tolerance of an important group of phytoplankton.\nI am fully aware that functional studies in diatoms are not easy to carry out and I\nappreciate the use of state-of-the-art techniques such as CUT&Tag. However, I am\nconvinced that several open questions need to be addressed (in a way that is feasible\nto do in diatoms) to draw conclusions about the mechanism of HSF2 action.\nResponse: We appreciate the time and effort that you have dedicated to providing\nyour valuable feedback on our manuscript.The provided comments are considered\ncarefully and carried out the experiments.\nMajor comments:\n1. The most important issue is for me the missing analysis of the impact of HSF2 and\nCdc45-like gene on the cell cycle progression. First of all, the authors report that both\nHSF2 and Cdc45-like over-expressing strains have wider cells than the respective\ncontrol strains, resulting in higher cell volume. Authors themselves speculate that this\ncould be due to a cell cycle arrest (line 312). The presence of two chloroplasts per cell\nin Figure 1F for PtHSF2 over-expression strains and Figure 3B for PtCdc45-like over-\nexpression strains further supports the idea of cell accumulation/cell cycle arrest in the\nS or G2 phase, which could explain the higher cell volume. Moreover, the Cdc45\nprotein is a conserved member of eukaryotic replisomes. Therefore, the analysis of the\ncell cycle progression of PtHSF2 and PtCdc45-like is essential to understand the\nphenotypes induced by over-expression and knock-down of these genes.\nVerifying the presence or absence of cell cycle arrest is important also for the\ninterpretation of the RNAseq results, as it was shown that many genes in diatoms\nshow cell cycle stage-dependent expression.\nResponse:Thank you for your insightful and detailed comments. We agree that\nanalyzing the impact of HSF2 and Cdc45-like genes on cell cycle progression is\ncrucial to understanding the observed phenotypes. We haveaddressed your concerns\nas follows:\n(1)Analysis of Cell Cycle Progression: As you mentioned, both HSF2 and\nCdc45-like over-expressing strains exhibit wider cells and higher cell volume, which\nwe speculated might be due to cell cycle arrest.Research has indicated that\nchloroplast division in Phaeodactylum tricornutum occurs during the S phase 1. We\nsynchronized exponentially growing cells by subjecting them to an extended dark\nperiod (20 h) and then transferred them to fresh medium for normal culture (12 h light:\n12 h dark). Samples were collected at consistent culture times for confocal analysis,\nrevealing that the enlarged cells contained two chloroplasts.This finding further\nsupports the idea of cell accumulation or cell cycle arrest in the S or G2 phase. (L. No.\n537-544, 773-775, 793, 1291-1293, Fig. 1D and Fig. 3E)\n(2) Flow Cytometry Analysis: To address this issue, we have performed flow\ncytometry analysis to examine the DNA content and determine the cell cycle\ndistribution of PtHSF2 and PtCdc45-like transgenic strains. It was found that PtHSF2\nand PtCdc45-like overexpression cells exhibited cell accumulation or cell cycle arrest\nin the S or G2 phase. (L. No. 375-387, 543-544, 983-995, Supplementary Fig. 8)\n(3) RNAseq Data Reanalysis:We recognize that cell cycle arrest could influence\ngene expression profiles. Therefore, verifying the cell cycle status of our strains is\nessential for accurately interpreting the RNAseq data. Given the cell cycle-dependent\nexpression of many genes in diatoms, we reanalyzed the RNA sequencing results and\nidentified several cell cycle-related genes that were significantly differentially\nexpressed. (L. No. 387-390, Supplementary Table 6)\nWe have included the results of these additional experiments and analyses in the\nrevised manuscript to provide a comprehensive understanding of the impact of HSF2\nand Cdc45-like genes on cell cycle progression and the associated phenotypes.\n2. To further test the hypothesis that PtCdc45-like is a direct target of PtHSF2 and part\nof PtHSF2 overexpression phenotype is mediated by PtCdc45 overexpression, I\nsuggest testing whether PtCdc45-like downregulation can alleviate part of the PtHSF2\noverexpression phenotype. The authors have all the constructs/strains to test this\ngenetic interaction.\nResponse:Thank you for your valuable suggestion. We agree that investigating the\nphenotypic differences between PtCdc45-like RNAi lines and PtHSF2 overexpressing\nlines could further elucidate the phenotypic changes caused by the upregulation of\nPtCdc45-like expression in PtHSF2 overexpressing strains. This will provide crucial\ninsights into the genetic interaction between PtHSF2 and PtCdc45-like.\nWe have knocked down PtCdc45-like in the PtHSF2 overexpression backgroundto\nobserve phenotypic changes.These experiments included measuring cell size and\nmonitoring cell density to determine if the PtHSF2-induced phenotypes are mediated\nby PtCdc45-like.These results have been included in our revised manuscript. (L. No.\n401-405, 420-424, 710-718, 1372-1373, Supplementary Fig. 9A-C, Fig. 4E and F)\nI recommend to address the above questions and adjust the discussion and conclusions\naccording to the results that will be obtained.\nResponse:Thank you again for your constructive comments on our manuscript, and\nwe have revised the discussion and conclusions.\nMinor comments:\n3. Title of the article - I find the title very general. It’s perhaps only my personal\npreference, but I recommend using a title that better describes the findings of the\npresented research, including the names of the studied genes.\nResponse:Thank you for your valuable feedback. We agree that the title could be\nmore specific to better reflect the findings of our research. We have revised the title to\nmore accurately describe the studied genes and their roles in thermal tolerance and\ncellular plasticity. (L. No. 2-4)\n4. Methods explaining how were obtained results presented in Supplementary Figure\n1 are missing.\nResponse: We apologize for omitting this part of the methodand appreciate your\nmeticulous scrutiny.We have added the methods for obtaining the results shown in\nSupplementary Figure 1 in the Methods section. (L. No. 643-657, 663-674)\n5. Supplementary Figure 1 C shows that the abundance of PtHSF2 homologs is\nhighest at higher latitudes and relatively low in tropical parts of the oceans. How can\nit be explained?\nResponse: Supplementary Fig. 1 Cpresents the geographic distribution and\nabundance of PtHSF2 homologous genes, and in combination with Supplementary\nFig. 1 B we find that these homologous genes are mainly derived from diatoms\n(Bacillariophyta). These results suggested that diatoms were particularly abundant at\nhigh latitudes, which were consistent with previous reports 2, 3.In terms of why\ndiatoms are more abundant at higher latitudes, there are mainly the following reasons:\n(i) the availability of nutrients such as nitrate, phosphate and silicate is higher at\nhigher latitudes 4 (ii) although diatoms have extensive temperature tolerance,most\ndiatoms grow better in cooler water 5, 6. (L. No. 149-153)\n6. Please give a better explanation for Supplementary Figure 1B.\nResponse: We apologize for the lack of a detailed explanation of this part of the\nresults, which led to an inconvenience in understanding.We have provided a detailed\nexplanation in the manuscript. (L. No. 143-147)\n7. Taking into account the focus of this study on heat stress tolerance, I am missing\ninformation about the expression of the expression of the PtHSF2 gene at 20C and\n30C.\nResponse: Thank you for your valuable feedback. We acknowledge the importance of\nincluding data on PtHSF2 gene expression at different temperatures, given the focus\nof this study on heat stress tolerance. Through qPCR analysis, we observed a\nsignificant increase in PtHSF2 expression at 30 °C compared to its expression level at\n20 °C in the wild-type background. (L. No. 138-142, 627-629, 1437-1440, Fig. 1A)\n8. Authors show that they generated PtHSF2 RNAi lines, but there is no analysis done\nwith these lines. If there is no phenotypic difference from wild-type, it is necessary to\nmention it at least in the text. Also, the thermotolerance of PtCdc45-like RNAi lines\nshould be shown.\nResponse: Thank you for your professional comments, we agree with this. We have\nanalyzed the characterization of PtHSF2 RNAi lines and added the results to the text\n(L. No. 180-181, 192-194, 211-212, Fig.1C-H). We have also tested and\nsupplemented the thermotolerance of PtCdc45-like RNAi lines. (L. No. 455-456, Fig.\n5)\nReviewer #2 (Remarks to the Author):\nThis manuscript by Huang et al. reveals the role of epigenetic mechanisms in\nfacilitating the adaptation of marine phytoplankton to environmental stress, with a\nfocus on the thermal tolerance and cellular plasticity. The insights gleaned are\npivotal in understanding and projecting marine ecosystem responses to global warming.\nNotably, it marks the application of a novel CUT&Tag technique to decode the\noperational mechanism of transcription factor PtHSF2 within microalgae. Overall,\nthis manuscript appears to be well-positioned for acceptance, given its novelty and\nrelevance to a broad readership interested in marine biology, climate change, and\nepigenetics. However, I have several other comments on this manuscript, and they\nshould be considered before the paper can be published.\nResponse: We thank you for reviewing our manuscript and giving us the opportunity\nto revise it.The provided comments and concerns were considered carefully, and we\nhave modified the manuscript as per the suggestions.\nMajor points:\n1. The authors mention in the manuscript that P. tricornutum has 69 heat shock\ntranscription factors (HSFs), but have not explained why PtHSF2 was chosen for this\nstudy. The authors should explain it.\nResponse:We appreciate your insightful comment. Our decision to focus on PtHSF2\nwas driven by preliminary analyses of relative transcript levels, which indicated that\nits expression levels showed significant variation with temperature changes. This\nobservation suggested that PtHSF2 might play a crucial role in the temperature\nresponse mechanism of P. tricornutum.We have incorporated this important result\ninto the revised manuscript as Fig. 1A and made corresponding modifications to the\ncontent of the manuscript. (L. No. 138-142, Fig. 1A)\n2. CUT&Tag technology is a highly sensitive and streamlined method for studying\nprotein-DNA interactions on chromatin, offering advantages such as low background\nnoise and suitability for single-cell analysis. However, there is few report of its\napplication in algae. To promote the application of this technology in the field of algal\nresearch, I suggest specifying the relevant experimental details, such as\nelectroporation parameters, including voltage and pulse duration in Line 525.\nResponse: We are grateful for this suggestion. We have added a detailed description\naccording to the comment. (L. No. 894-895)\nMinor points:\n3. Substitute \"heat shock transcription factors\" with \"HSFs\" in Lines 39 and 79.\nResponse: Thank you for pointing this out.This phrase was modified according to the\ncomment. (L. No. 39, 89)\n4. Add citation for the statement in Line 99, \"Diatoms are widely distributed in marine\necosystems and are ubiquitous in almost all aquatic habitats\".\nResponse: Thank you for pointing this out, and the reference has been added. (L. No.\n111, 1125-1126)\n5. Lines 102 to 103, omit \"heat shock transcription factors\".\nResponse: The phrase “heat shock transcription factors” has been deleted. (L. No. 118)\n6. The \"is\" in Line 109 should be changed to \"was\".\nResponse: Thank you for your careful scrutiny, we have corrected this tense. (L. No. 123)\n7. Citations are needed for the statement made in Lines 196-197.\nResponse: Thank you for your valuable feedback. References have been added. (L.\nNo. 288, 1162-1163, 1181-1182, 1198-1199)\n8. Change \"led to the increase\" in Lines 237-238 to \"led to the enlargement\".\nResponse: Thank you for your suggestion. We have made the changes as per the\ncomment. (L. No. 333)\n9. Switch \"PtCdc45-like-transformed algae\" in Line 243 to \"PtCdc45-like-\ntransformed lines\".\nResponse: Thank you for pointing this out. Changes have been made as per the\ncomment. (L. No. 338)\n10. Replace \"strains\" in Line 247 with \"lines\".\nResponse: Thank you for your suggestion. The word “strains” has been replaced with\n“lines”. (L. No. 328)\n11. Line 261, modify \"Cell size plasticity\" to \"Cell plasticity\".\nResponse: Thank you for your valuable feedback. Changes have been made as per the\ncomment. (L. No. 341)\n12. Line 269, correct the typo \"thnt\" to \"that\".\nResponse:We apologize for the spelling error and thank you for pointing it out.The\nerror has been corrected. (L. No. 349)\n13. Line 273, replace \"speculate\" with \"speculated\".\nResponse: Thank you for pointing this out. The tense has been corrected. (L. No. 356)\n14. Analyze and attach a relevant citation to the statement \"Heightened temperatures\nstimulate the generation of reactive oxygen species (ROS) within cells, thereby\ntriggering lipid peroxidation\" in Line 286.\nResponse: Thank you for your constructive feedback. According to the comment, we\nhave analyzed this section and have attached relevant citations to support this\nstatement in the revised manuscript. (L. No.197-204, 1165-1173)\n15. Provide reference in Lines 346-347 \"Accumulating evidence shows that P.\ntricornutum exhibits extensive temperature adaptability.\"\nResponse: Thank you for your valuable feedback. Relevant references have been\nadded. (L. No. 609)\n16. Line 551, \"19 ggnog 5.0\" appears to be a potential spelling error, and the correct\nexpression is likely \"EggNOG 5.0.\" If that's the case, please correct the spelling error.\nResponse:Thank you for bringing this to our attention. We appreciate your careful\nreview.We have reanalyzed the GO data and reflected these updates in the revised\nmanuscript. (L. No. 872-879)\nReviewer #3 (Remarks to the Author):\nIn this manuscript, the authors conducted a systematic study to elaborate on the\nregulation mechanism of thermal tolerance mediated by heat shock transcription\nfactor and target gene CDC-45 like in the model diatom Phaeodactylum tricornutum.\nGene overexpression and silence methodologies were employed to address the issues,\nalong with monitoring cell physiological response. Overall, the work flow of this\nmanuscript is clearly written, though some results are not included. The introduction\nand discussion are not comprehensive and adequate yet. This work provides further\nunderstanding into the thermal adaptivity of diatom. The key finding of this study is\nthe identification of several HSF-target genes, CDC-45 like etc. Except for this, the\nother findings described in this manuscript are not as new and appealing, which\nmakes it not qualified for publication on NCOMM.\nThere are some concerns about the results and conclusion. The first concern is that\ngene transcript regulation via transcription factor is not epigenetic regulation. And the\nauthor argues that CDC45 is an important control gene for cell size. In a unicellular\nphytoplankton physiological study, the algae cell size is sensitive to ambient stress\nand cell growth status. It is often to find that cells grown under unfavorable conditions\nhave a relatively larger cell size. Thus, the phenomenon observed in this study might\nalso be likely due to the overall cell physiological response caused by gene\ninterference, instead of a direct effect from CDC45. As well as regarding lipid\naccumulation, which has been widely examined in the development of microalgae\nbiofuel. Comparison between this work and the regulation mechanism reported in\nthose studies were not fully discussed.\nResponse: We appreciate the time and effort that you have dedicated to providing\nyour constructive comments on our manuscript.We have carefully considered your\nfeedback and would like to address each point you raised, as well as outline the\nimprovements we have made to the manuscript.\n(1) Regarding the missing results: To enhance the robustness of our results, we\nconducted the following additional experiments:\n- Investigated the interaction between the PtHSF2 and PtCdc45-like genes. (Fig. 4)\n- Monitored cell growth curves and cell volume at four temperatures (15 ℃, 20 ℃,\n25 ℃, and 30 ℃). (Fig. 1C and E, Fig. 3C and D)\n- Assessed the redox levels of PtHSF2 transformants at 30 ℃. (Fig. 1H,\nSupplementary Fig. 4A-C)\n- Characterized all RNAi knockdown lines. (Fig. 1C-H, Fig. 3C-H, Fig. 5)\n- Conducted transcriptome analyses of Thalassiosira weissflogii at 20 ℃, 30 ℃, and\n35 ℃. (Supplementary Table 13)\n(2) Regarding the introduction and discussion:We have expanded the\nintroduction and discussion sections to include more background information and\nliterature review, thoroughly introducing the role of heat shock transcription factors in\ndiatoms. Additionally, we have provided a more comprehensive discussion on how\nour findings relate to existing literature. (L. No. 77-88, 474-486, 509-518)\n(3) Distinction between gene transcript regulation and epigenetic regulation:\nThank you for pointing this out. We apologize for not understanding “epigenetics”\ncorrectly, which has led to its misuse. We have modified this in the revised manuscript.\n(L. No. 2-4)\n(4) Role of PtCdc45-like gene and consideration of overall physiological\nresponses: As you mentioned, the cell size of unicellular microalgae is influenced by\nboth environmental factors and growth status.We have added experimental data to\nfurther validate the direct impact of the PtCdc45-like gene on cell size.Additionally,\nwe have provided a more detailed discussion in the revised manuscript (L. No. 330-\n332, 394-430, 509-518, Fig. 3D, E, and Fig. 4).\n(5) Comparing our findings on lipid accumulation with previous studies: As is\nwell known, P. tricornutum is an oil-rich microalgae that has been widely studied for\nmicroalgal biofuels 7.Reports indicate that the lipid content and composition of P.\ntricornutum are influenced not only by environmental factors such as nitrate\ncontent/source, salinity, and temperature 8, 9, 10 but also by enzymes involved in lipid\nsynthesis, such as acetyl-CoA carboxylase, fatty acid synthase, fatty acid desaturase,\nand diacylglycerol acyltransferase 7, 11, 12.Our study demonstrated that the proportion\nof unsaturated fatty acids in PtHSF2 and PtCdc45-like overexpressing lines was\nsignificantly higher than in wild-type cells (Supplementary. Fig. 4D and Fig. 3G).\nThese overexpressing cells showed a notable upregulation of a fatty acid desaturase\n(FAD), whereas knockdown lines exhibited the opposite pattern (Fig. 2P and\nSupplementary. Fig. 7F), suggesting the pivotal role of FAD in converting saturated to\nunsaturated fatty acids. These findings align with previous reports 13, 14, which\nindicated that increased desaturase expression enhances unsaturated fatty acid\nsynthesis.(L. No. 351-354, 1207-1213)\n1. Line 23-24. As a matter of fact, many studies have been conducted to address the\ncellular mechanism in terms of temperature adaptation. e.g. The ISME Journal (2019)\n13:2415–2425, and the quote from the book “The Molecular Life of Diatom”.\nResponse: Thank you for highlighting the existing studies on the cellular mechanism\nof temperature adaptation. Indeed, numerous research efforts have explored this area.\nFor instance, the ISME Journal (2019) 13:2415–2425 elucidated the molecular\nmechanisms underlying temperature adaptation in two diatom species with different\noptimal growth temperatures using transcriptomics, significantly enhancing our\nunderstanding of this subject. Additionally, we appreciate your recommendation of\n“The Molecular Life of Diatoms”, which gathers the latest data and developments on\ndiatoms from genes to ecosystems. Its insights contribute to our understanding of\ntemperature adaptation mechanisms at the molecular level. We have cited the above\ntwo important references in the revised manuscript. (L. No. 77-88, 1098-1120)\n2. It is not surprised that HSF play important role in temperature response. I would\nsay, it is well known that HSF is the key player in this mechanism.\nResponse:Thank you for your feedback. We acknowledge that the role of HSFs in\ntemperature response is well-established, as HSFs are critical regulators in this\nmechanism.The biological functions of HSFs have been extensively studied in model\norganisms such as Saccharomyces cerevisiae15, 16, Arabidopsis thaliana 17,\nDrosophila18, 19, and humans 20, and they have traditionally been recognized as\nhaving the function of regulating the expression of heat shock gene in response to\nvarious stimuli 21. However, there is limited research on the function of diatoms HSFs,\nespecially the connection between the abundant HSFs in diatoms and their extensive\nenvironmental adaptability.Our study aims to build upon existing knowledge to\nspecifically investigate how PtHSF2 regulates the transcriptional expression of target\ngenes, thereby influencing cell size and heat tolerance in P. tricornutum.We have\nemphasized the novel aspects of our findings to contribute to a deeper understanding\nof the biological functional mechanisms of HSFs. We have revised the manuscript to\nhighlight the novelty of our results more clearly. (L. No. 474-486, 613-615, 1232-\n1266)\n3. The growth curve of RNAi strains should be examined and presented as well.\nResponse: Thank you for your valuable suggestion. We agree with this comment. In\nresponse, we have conducted the required experiments and included the growth\ncurves of the RNAi strains in the revised manuscript. (L. No. 180-184, 327-330, Fig.\n1C and Fig. 3C)\n4. Line179, besides Cdc45-like, Lhcx2 and ABC transporter, do not see a summary of\nother genes in the assemble of 35 genes.\nResponse: Thank you for pointing this out. We apologize for the oversight. We\nappreciate your thorough review. These 35 potential target genes have been listed in\nSupplementary Table 11, and we have revised the manuscript. (L. No. 263-264 )\n5. Line 188, what does this nt sequence represent? In Figure 2 J-L, the abundance of\nwhich promoter binding motif was examined? Please make a clear description.\nResponse: Thank you for your insightful comments.We apologize for not providing a\nsufficient explanation, which may have caused inconvenience in understanding.\nThe nucleotide sequence “GGTTAGGGTTAGGGTTAGGGTTAGGGTTAG” shown\nin Fig. 2I represents the highest affinity DNA binding motif identified for PtHSF2 in\nour study.To obtain these binding motifs, we performed CUT&Tag-seq (Cleavage\nUnder Targets and Tagmentation followed by sequencing) analysis. This method\nallows us to identify the specific DNA sequences that PtHSF2 binds to in the genome.\nThe sequenced data were then analyzed using MEME 22, a motif discovery algorithm,\nwhich scans the sequences for recurrent patterns. These patterns, known as motifs,\nindicate the preferred binding sites of PtHSF2. The sequence\n“GGTTAGGGTTAGGGTTAGGGTTAGGGTTAG” is a consensus motif, which\nmeans it represents a common DNA binding pattern recognized by PtHSF2 across\nmultiple genomic locations. The presence of this motif suggests that PtHSF2 has a\nstrong affinity for binding to these specific nucleotide arrangements. We have\nclarified this in the revised manuscript. (L. No. 277-282)\nIn Fig. 2J-L, we sequentially examined the abundance of promoter sequences for\nCdc45-like (Phatr3_J9344), Lhcx2 (Phatr3_EG02404), and ABC (Phatr3_J42881).\nWe have added this information to the revised manuscript.(L. No. 1469-1470, Fig.\n2J-L)\n6. Line 206, Wrong number in supplementary tables. Why refer to a primer table? The\ndescription of Phatr3_J46275 is abrupt and lacks contextual connection. Should be\n“FAD” in the legend of Figure 2P?\nResponse:Thank you for your thorough review and insightful comments. We\napologize for the errors and any confusion caused. Regarding the wrong number in\nsupplementary tables, we have corrected the numbering and verified all references to\nsupplementary materials in the manuscript to ensure accuracy. The reference to a\nprimer table was indeed misplaced. We have revised the text (L. No. 298,\nSupplementary Table 6). We acknowledge that the description of Phatr3_J46275 was\nabrupt and lacked context. We have revised this section to provide a more\ncomprehensive introduction and connection to the surrounding text (L. No. 294-296,\nSupplementary Fig. 4D). Thank you for pointing out this incorrect legend, we have\ncorrected it. (L. No. 1474)\n7. Line 285, Given the Fv/Fm was significantly inhibited in the PtCdc45-like-\noverexpressing strains, and lower transcription level of Lhcx2, it is not surprised to\nsee the relative lower cellular ROS content.\nResponse: Thank you for your comment. We agree with your observation.Indeed, in\nphotosynthetic organisms, the process of oxygenic photosynthesis generates a\nsignificant amount of ROS 23.Therefore, the significant inhibition of Fv/Fm in the\nPtCdc45-like overexpressing strains, along with the lower transcription level of Lhcx2,\nindeed correlates with the relatively lower cellular ROS content.We have elaborated\non this point in the revised manuscript to provide a clearer connection between these\nobservations. (L. No. 449-452, 1223-1224)\nReviewer #4 (Remarks to the Author):\nReport on the manuscript NCOMMS-24-06983\nThe manuscript “Thermal tolerance and cellular plasticity in marine phytoplankton\ndiatoms modulated by epigenetic mechanisms for environmental adaptation” by\nHuang et al relates to the molecular mechanisms involved in the acclimatory response\nof diatom to increased sea water temperature condition. Available evidences indicate\nthat diatoms, a prominent group of marine primary producers, present a larger thermal\ntolerance range than other phytoplankton functional types. Knowledge on the\nmechanisms involved in diatom thermal tolerance range, however, are limited. Huang\net al report an expansion of the transcription factor Heat Shock Factors (HSFs) in\ndiatom genomes. In addition, reverse genetic approach carried out in the model\ndiatom Phaeodactylum tricornutum suggests that the HSF orthologue HSF2 may play\na positive role in the response to increased temperature via transcriptional activation\nof the target cyclin dependent kinase Cdc45-like with concurrent effects on cell size\nand growth, lipid and oxidative stress metabolisms.\nDocumentation on the molecular mechanisms involved in the thermal tolerance of\nmarine microalgae is central to better understand their ecophysiology and predict their\nresponse to ongoing ocean warming. This topic, therefore, is of particular interest for\na large and broad audience. The introduction is concise and focused, presenting well\nthe background and needed research. The result section follows a logical sequence\nand the choice of the methods and approaches are sound and complementary.\nHowever, while the manuscript presents valuable novel information their significance\nremains relatively limited.Specifically, while the manuscript suggests that HSF2 and\nCdc45-like may play a positive role in the tolerance of P. tricornutum to increased\ntemperature it remains to be addressed if this mechanism is conserved in diatoms eg\nby providing evidences on the role of HSF2 in different diatom species of contrasting\nthermal tolerance ranges and/or in another model diatom species of contrasting\necophysiology such as Thalassiosira pseudonana. In addition, it remains unknown\nwhether HSF2 and Cdc45-like directly contribute to the apparent larger thermal\ntolerance range observed in diatoms compared to that in other phytoplankton\nfunctional types eg are HSF2 and Cdc45-like orthologues absent or regulated\ndifferently in coccolithophores and green algae? Also, the experimental approach\nfocuses on the response to increased temperature but does not address the acclimatory\nresponse to decreased temperature thus limiting our understanding on the importance\nof HSF2 and Cdc45-like for the diatom thermal tolerance range per se. In addition,\nand importantly, I think that in some instances the designs of the experiments have\nshortcomings, the experiments are incomplete and the data are not presented in a clear\nway - altogether this reduces the solidity of the results and conclusions. In\nconsequence I believe that the manuscript in its current version is not suited for\npublication in Nature Communications. This is a very interesting topic and the work\ncarried out by Huang et al is substantial. I have provided below my detailed\ncomments and suggestions, which I hope, may contribute to improve the manuscript.\nResponse: Thank you for your thorough review and insightful comments on our\nmanuscript. We greatly appreciate your feedback and constructive suggestions, which\nhave helped us enhance the quality and robustness of our study.Below, we have\naddressed each of your concerns in detail:\n(1) Conservation of the Mechanism in Other Diatom Species: We understand the\nimportance of exploring whether the mechanisms we identified are conserved across\ndifferent diatom species, especially those with varying thermal tolerance ranges. In\nour study, we conducted high-temperature treatments on Thalassiosira weissflogii at\n30°C and 35°C, followed by transcriptome sequencing analysis. The results showed\nthat, compared to 20°C, the expression levels of the HSF2 orthologue (snap_masked-\nctg119-processed-gene-4.14-mRNA-1, 48.5% similarity) in Thalassiosira weissflogii\nsignificantly increased with rising temperatures, showing 1.21-fold and 1.92-fold\nupregulation at 30°C and 35°C, respectively. As shown in the Supplementary Table 13,\nthe Cdc45-like orthologue (snap_masked-ctg1929-processed-gene-0.66-mRNA-1,\n53.4% similarity) also exhibited an upregulation trend. Based on these findings, we\nhypothesize that the HSF2 transcription factor is a key regulator of heat tolerance in\ndiatoms, and this regulatory role may be widespread among diatoms.\n(2)Comparison with Other Phytoplankton Functional Types: Furthermore, to\ninvestigate whether HSF2 and Cdc45-like contribute to the broader temperature\ntolerance range observed in diatoms compared to other phytoplanktons, We conducted\northologue comparison analyses across the genomes of several sequenced microalgae,\nincluding Emiliania huxleyi (a coccolithophore) and Chlamydomonas reinhardtii (a\ngreen alga). The results showed that the HSF2 orthologue (XP_005770267.1) was\npresent in E. huxleyi with a sequence similarity of 39.7%, while no orthologue for\nCdc45-like was detected. In C. reinhardtii, both HSF2 orthologue (XP_001702071.2)\nand Cdc45-like orthologue (XP_001696501.1) were identified, with sequence\nsimilarities of 37.9% and 26.3%, respectively (Supplementary Table 13). These\nfindings indicate that the HSF2 orthologs and Cdc45-like genes in E. huxleyi and C.\nreinhardtii exhibit lower similarity, or even absence, compared to their diatom\ncounterparts. These results suggest that these microalgae might rely on distinct\ngenetic mechanisms to regulate their response to temperature fluctuations. Given\nthese findings, the absence or low similarity of HSF2 and Cdc45-like orthologs in\ncoccolithophores and green algae might contribute to their narrower thermal tolerance\nrange compared to diatoms. Together, these analyses imply that HSF2 and Cdc45-like\ngenes might play a more central role in the thermal tolerance mechanisms of diatoms,\npotentially explaining their broader adaptability to temperature fluctuations.These\nupdates have been reflected in the revised manuscript. (L. No. 460-469, 543-555)\n(3)Acclimatory Response to Decreased Temperature: We acknowledge that our\ninitial manuscript exclusively focused on investigating the response to elevated\ntemperatures.We have conducted additional experiments to assess the role of HSF2\nand Cdc45-like in the acclimatory response to decreased temperature (15 ℃).The\nresults indicated that overexpressing cells did not demonstrate a growth advantage at\nlow temperatures (15 ℃), and no significant differences were observed between the\nRNAi strains and the wild-type (Fig. 1C and Fig. 3C).This provides a more\ncomprehensive understanding of their roles in the thermal tolerance range of diatoms.\nWe have added these results to the revised manuscript. (L. No. 179-184, 327-330)\n(4)Experimental Design and Data Presentation: We appreciate your feedback\nregarding the experimental design and data presentation.To improve the reliability of\nour results and conclusions, we have made every effort to address the shortcomings in\nthe experimental design and the areas where the experiments were incomplete.\nHere is a point-by-point response to your comments and concerns.Your feedback\nis greatly appreciated if there are any areas that need improvement.\nLine 3\nI do not think that the term “epigenetic” fits with the study. Please see Deans and\nMaggert 2015 (doi.org/10.1534/genetics.114.173492) for a recent discussion on the\nsignification of this term.\nResponse: Thank you for providing valuable comments and recommending the\nreference Deans and Maggert 2015 (doi.org/10.1534/genetics.114.173492) to us.We\nhave carefully read this literature, which greatly assisted us in our understanding of\n\"epigenetic\". We agree with your comments and have modified the title accordingly.\n(L. No. 2-4)\nLine 102\nMore than four diatom species have fully sequenced genomes (see Grigoriev et al\n2021 doi.org/10.1093/nar/gkaa898). Please consider to include them in the analysis.\nResponse: Thank you for your reminding. According to your suggestion, we have\nretrieved 16 species of diatoms with sequenced genomes (two of which are duplicated)\nin the PhycoCosm (https://phycocosm.jgi.doe.gov) database. We have counted the\nnumber and proportion of their HSFs and supplemented Supplementary Table 1.\nBased on these results, we have also revised the manuscript. (L. No. 117,\nSupplementaryTable1)\nLine 103\nAre all these HSFs expressed? Please consider to exploit the RNAseq data set from\nthe MMETSP project (Keeling et al 2014 doi.org/10.1371/journal.pbio.1001889) to\ninvestigate this point.\nResponse: Thank you for your valuable suggestion. In this regard, We conducted a\nstudy based on the MMETSP project24 and the re-assembly data by Johnson et al.\n(2019) (https://github.com/dib-lab/dib-MMETSP) 25. Our results showed that 38 HSF\ngenes of P. tricornutum are expressed (Supplementary Table 2). Additionally, we\nobserved the expression of HSF orthologues in other species (Supplementary Table 2).\nWe speculate that these HSFs may play significant regulatory roles under specific\nstress conditions, including high temperature, light stress, and other environmental\nstresses. (L. No. 126-132)\nLine 103\nIf known, please indicate whether these four diatom species have different thermal\ntolerance ranges.\nResponse: Thank you for your comment. We appreciate your suggestion to include\ninformation on the thermal tolerance ranges of these four diatom species. It has been\nreported that the thermal tolerance ranges of Thalassiosira pseudonana CCMP 1335,\nFistulifera solaris JPCC DA0580 and Phaeodactylum tricornutum CCMP 2561 are\n10-35 ℃ 26, 27, 15-35 ℃ 28, 29 and 5-28 ℃ 30, 31, respectively.Unfortunately, the\nspecific thermal tolerance range of Thalassiosira weissflogii is not well-documented\nin the current literature. However, studies have shown that T. weissflogii does not\ngrow at temperatures below 8 ℃ 32, while it can grow well at 30 ℃ 33. These reports\nshowed that these four diatoms had different thermal tolerance ranges. We have added\nthe known thermal tolerance ranges of diatoms to the manuscript. (L. No. 111-115,\n1128-1146)\nLine 113\nWhy “two branches”? Many (sub)branches are shown.\nResponse: We apologize for not explaining Supplementary Figure 1A in detail, and\nthank you for pointing this out.Indeed there are many (sub)branches here. We have\nrevised the manuscript. (L. No. 133-135, Supplementary Fig. 1A)\nLine 116\nIs “this HSF” or orthologues present in microalgae species from phytoplankton\nfunctional types other than diatoms?\nResponse: Thank you for your insightful question.By performing a BLASTP search\nin the NCBI database to retrieve homologous sequences of this HSF(Phatr3 J55070),\nthe results shown in the figure below indicate that the top 119 sequences with the\nhighest similarity are all derived from diatoms (Rebuttal Fig. 1).Consistent results\nwere obtained from the marine EUK-SMAGs (Eukaryotes Single-Cell and\nMetagenome Assembled Genomes) database (Supplementary Fig. 1B), indicating that\ndiatoms are the primary source of this HSF.While other phytoplankton species, such\nas Chlamydomonas reinhardtii, Prorocentrum lima, and Cyanidioschyzon merolae,\nalso possess homologs, their homology is comparatively lower (Supplementary Fig.\n2B).These results suggest that this HSF is conserved in diatoms but not in other\nphytoplankton.We have incorporated this information into the revised manuscript. (L.\nNo. 143-148)\nRebuttal Fig. 1 Analysis of the species distribution of PtHSF2 homologs using\nBLASTP in the NCBI database\nLine 125\nIt is well established that gene knock-down by RNAi can present off-target effect\n(Chen et al 2021 10.1080/15476286.2020.1868680). This is particularly relevant\nwhen the target is a member of a multigene family such as HSF here. The authors\nshould provide supports and if possible experimental evidences that only HSF2, and\nno other paralogues, was downregulated in the siRNA lines eg by providing a\nsequence alignment supporting that the targeted sequence is specific to HSF2 and by\nanalyzing the expression levels of HSF2 paralogues in the RNAi lines (eg by RT-\nqPCR).\nResponse:Thank you very much for your professional comments and providing us\nwith a meaningful reference.Previous work on dsRNA specificity 34 has shown that\ndsRNAs triggering RNAi require one of the following criteria:(i) >80% sequence\nsimilarity with target genes; (ii) ≥16 bp contiguous fragments that perfectly match the\ntarget gene or >26 bp fragments that almost perfectly match the target gene with one\nor two mismatches (single mismatches inserted between matching fragments of ≥5 bp\nor mismatched couplets inserted between matching fragments of ≥8 bp). Therefore,\nthese parameters can be used to predict the off-target effects of dsRNAs.\nAligning the coding regions of the remaining 68 HSF genes from P. tricornutum with\nthe dsRNA of PtHSF2 (designated as dsPtHSF2), it was found that only 8 genes,\nincluding Phatr3_J38294, Phatr3_J48667, Phatr3_J48572, Phatr3_J42824,\nPhatr3_J45391, Phatr3_EG02624, Phatr3_J50136, and Phatr3_EG00212, contained\nperfectly matching segments with dsPtHSF2 (Supplementary Fig. 3A).Moreover,\nthese perfectly matched consecutive fragments were all less than 16 bp. To further\nconfirm whether dsPtHSF2 induces RNAi of these eight genes, qPCR analysis was\nconducted to assess their transcription levels.Supplementary Fig. 3B, the relative\ntranscription levels of these 8 genes did not significantly decrease. These results\nindicate that dsPtHSF2 did not trigger RNAi of PtHSF2paralogues. We have added\nthese results in the revised manuscript. (Supplementary Fig. 3, L. No. 169-177, 756-\n771, 1159-1160)\nLine 130\nThree instead of two independent transgenic lines per construct (overexpressing and\nRNAi lines) would be preferable.\nResponse: Thank you for your valuable feedback. We concur with this point and have\nconsidered it. As per your recommendation, we have successfully developed a third\ntransgenic microalgae strain (PtHSF2-3, siPtHSF2-3, PtCdc45-like-3, siPtCdc45-like-\n3) and included additional experimental data in the revised manuscript.\n(Supplementary Fig. 2C-E, Supplementary Fig. 7C-E, L. No. 166-168, 318-323)\nLine 134\nPlease consider to assess the phenotype of the transgenic lines in response to a milder\nincrease in temperature than from 20C to 30C; as shown in Fig.1B and C both WT\nand HSF2 overexpressing lines do not grow at 30C. The WT line is just dying faster.\nRsponse:Thank you for your constructive suggestion. As you mentioned, cell growth\nis severely inhibited at 30 °C, with the WT cells dying more rapidly (Fig. 1C). We\nbelieve that evaluating the characterization of transgenic microalgae at a milder\ntemperature is both interesting and necessary. Therefore, we have assessed the\nphenotype of the cells at 25 °C and included relevant data in the revised\nmanuscript.(Fig. 1C-E, Fig. 3C and D, L. No. 179-184, 327-330)\nLine 149\n- It is reported that both HSF2 overexpressing and RNAi lines have been generated\nbut the phenotypic characterization is shown only for the formers. This is a\nshortcoming; phenotypic characterization of the HSF2 siRNA lines is needed in this\nparagraph.\nResponse:Thank you for pointing this out, and we agree with this. We have\ncharacterized the RNAi strains and supplemented the relevant data in the revised\nmanuscript. (Fig. 1C-H, Supplementary Fig.4, L. No. 180-181, 192-194, 211-212)\n- Please consider to investigate if HSF2 (and Cdc45-like) expression is upregulated in\nthe WT background when grown at 30C versus 20C. This will provide support for\ntheir intrinsic role in the response to increased temperature.\nResponse:Thank you for providing constructive feedback. We wholeheartedly agree\nwith this point and have assessed the changes in the relative transcription levels of\nPtHSF2 and Cdc45-like in WT cells as the temperature increases (15 °C, 20 °C, 25 °C,\n30 °C). In the wild-type background, the relative transcript levels of PtHSF2 (Phatr3_\nJ55070) exhibited significant variation with temperature changes (15 °C to 30 °C),\nwith the highest expression observed at 25 °C, followed by 15 °C, 30 °C, and 20 °C\n(Fig. 1A). In contrast, the relative transcription level of Cdc45-like increased with\nrising temperatures between 15 °C and 25 °C, but was lower at 30 °C than at 25 °C\n(Fig. 3A). Our results indicated that PtHSF2 targeted and upregulated PtCdc45-like\n(Fig. 2J, M). However, an anomaly was observed: the transcription level of PtHSF2 at\n15 °C was higher than at 20 °C, while the transcription level of PtCdc45-like at 15 °C\nwas not higher than at 20 °C.These data suggest that in WT cells, the transcription\nlevel of PtCdc45-like is regulated by PtHSF2 at temperatures above 20 °C. However,\nat lower temperatures (15 °C), PtCdc45-like transcription may also be influenced by\ncold.This could explain why PtHSF2 transcription increased at 15 °C in WT cells\nwithout a corresponding increase in cell size (Fig. 1A and E).We have added these\nresults to the revised manuscript.(L. No. 138-142, 306-308, 519-529)\n- It is not supported at this stage that HSF2 may regulate the expression of genes\ninvolved in the cell cycle per se.\nResponse: Thank you for carefully reviewing our manuscript and pointing out this\noversight. We have made the necessary revisions in the manuscript. (L. No. 214)\nLine 151\n- Why the RNAseq and CUT&Tag experiments were conducted in cultures grown at\n20C (and not at 30C) while the increased tolerance of the HSF2 overexpressing lines\nversus WT is evidenced at 30C?\nResponse:Thank you for your insightful question. RNA-seq and CUT&Tag\nexperiments were conducted to elucidate the regulatory mechanisms of PtHSF2 on its\ntarget genes. Research has indicated that while many transcription factors are\nactivated only under specific conditions, analyzing transcription profiles and DNA\nbinding sites in organisms with visible phenotypes resulting from overexpression of\ntranscription factors is a common approachto studying transcription factor\nmechanisms 35. This method can be applied to organisms cultured under standard\nculture conditions without requiring specific condition induction 35.\nIn this study, we observed that overexpression of PtHSF2 under normal culture\nconditions (20 °C) led to phenotypic changes such as increased cell volume (Fig. 1E)\nand slowed growth (Fig. 1C).Therefore, we conducted RNA-seq and CUT&Tag\nexperiments on cells cultured at this temperature. These methods allowed us to\nelucidate the influence of PtHSF2 overexpression on the overall transcriptional\nlandscape and analyze its DNA-binding targets independently of environmental\nfactors such as temperature.\nTo reinforce our findings, we performed qPCR to assess the relative transcription\nlevels of several key differentially expressed genes (identified at 20 °C) in PtHSF2-\ntransformed lines cultured at 30 °C.These complementary experiments confirmed\nthat the transcription levels of the target genes at 30 °C (Supplementary Fig. 6) were\nconsistent with those at 20 °C (Fig. 2M-P),thereby supporting our conclusion\nregarding PtHSF2’s role in increased thermal tolerance. The relevant results have\nbeen included in the revised manuscript. (L. No. 301-304, 840-849, 1410-1415)\nLine 154\n- Please state how many genes were found differentially expressed (up and down),\nusing which fold change threshold and p-value.\n- Please indicate and analyze separately the genes found up and down regulated.\n- Please indicate here how the RNAseq results overlap between the two HSF2\noverexpressing lines in term of the genes found up and down regulated in both lines.\n- Add in SI the list of genes found differential expressed with their fold change and p-\nvalues.\n- It would be interesting to report how the expression of the HSF orthologues are\nregulated in the HSF2 overexpressing lines versus WT line.\nResponse: Thank you for your insightful comments. Here are the details:\n- In response, we would like to clarify that using a |log₂(FC)| threshold of ≥ 1 and a p-\nvalue of < 0.05, our RNA-seq analysis revealed that in the comparison of ptHSF2-1\nversus WT, 1,912 significantly differentially expressed genes (DEGs) were identified,\nwith 940 genes upregulated and 972 downregulated. In the comparison of ptHSF2-2\nversus WT, 2,404 significantly differentially expressed genes were identified,\nincluding 1,121 upregulated and 1,283 downregulated genes (as shown in\nSupplementary Table 5). (L. No. 220-224, 867-869)\n- We have separately analyzed the genes found upregulated and downregulated and\nincluded this analysis in the revised manuscript (as shown in Supplementary Table 5).\n( L. No. 220-224)\n- The overlap between the two PtHSF2 overexpressing lines showed that 1,449 DEGs\nwere common to both lines, with 676 genes upregulated and 773 downregulated\n(Supplementary Table 5). (L. No. 224-226)\n-We have added a list of the differentially expressed genes with their fold change and\np-values in (Supplementary Table 5).\n- We have also provided a detailed account of the regulation of HSF orthologue\nexpression in the HSF2 overexpressing lines in comparison to the WT line.\nSpecifically, the expression levels of 12 PtHSF2 homologs exhibited significant\n exhibited significant\nchanges, with 5 genes upregulated and 7 downregulated (Supplementary Table 5). (L.\nNo. 232-234)\nThese updates have been reflected in the revised manuscript.\nLine 159\n- Please analyze the up and down regulated genes separately.\n- It is not clear how the GO term enrichment analysis was conducted. Please describe\nbetter this information in the material and method (reference gene list used, fold\nchange, p-value?).\nResponse:Thank you for your questions. We have provided a separate analysis of the\nupregulated and downregulated genes in the Supplementary Table 5 of the revised\nmanuscript. Additionally, we have clarified the GO term enrichment analysis in the\nMaterials and Methods section, including the reference gene list used, fold change,\nand p-value. (L. No. 220-224, 872-879)\nLine 170\n- Please add in supplementary metrics relative to the CUT&Tag NGS experiment eg\nnumber of sequenced reads and their quality and indicate how many were mapped to\npromotor regions.\nResponse:Thank you for your suggestion. We have added supplementary metrics\nrelated to the CUT&Tag NGS experiment, including the number of sequenced reads,\ntheir quality, and the proportion mapped to promoter regions (Supplementary Table 4).\n(L. No. 218-220)\n- Please indicate here that the CUT&Tag experiment was carried out from mixed\nsamples of the two overexpressing lines and state how many mixed samples were\nanalyzed.\nResponse: Thank you for pointing this out. We apologize for not stating the details of\nthe samples associated with the CUT&Tag experiment in the manuscript. In the\nCUT&Tag assay, we performed experiments on the WT line and two lines\noverexpressing PtHSF2 (PtHSF2-1 and PtHSF2-2), with three biological replicates\nand one technical replicate for each line. A 1:1 mixture of cell samples from two\noverexpression lines was employed as a negative control. However, due to fund\nconstraints, we consolidated the libraries and conducted high-throughput sequencing\non the pooled library. Specifically, 10 μL of sample from each of the three libraries of\nthe WT line was mixed homogenously as the sequencing library for WT; 5 μL of\nsample from each of the six libraries of the two overexpression lines was mixed\nhomogenously as the sequencing library for PtHSF2;and 10 μL of sample from each\nof the three libraries of the negative control was mixed homogenously as the\nsequencing library. As per your comments, we have made changes to the manuscript.\n(L. No. 915-921)\n- The design above is a shortcoming – the two overexpressing lines should have been\nanalyzed separately to support the replicability of the results across the lines.\nResponse: Thank you for your comments and professional suggestions. We\nacknowledge the validity of your point. To address this limitation, we validated the\nlibraries of the two overexpressing lines separately by using CUT&Tag-qPCR, and\nthe results were consistent in both transformed strains (Fig. 2J-L). We have revised\nthe manuscript. (L. No. 285, 933-935)\n- Also, as for the RNAseq experiment, why the experiment was carried out in cultures\ngrown at 20C while the increased heat stress tolerance was evidenced at 30C?\nResponse: Thank you for your insightful comment.The optimal growth temperature\nfor P. tricornutum (CCMP2561) is 20 °C 36.The RNA-seq experiment was conducted\non cultures grown at 20 °C to establish a baseline gene expression profile under\nnormal, non-stress conditions.This baseline is essential for understanding the\nfundamental regulatory mechanisms of PtHSF2 in the absence of heat stress.It has\nbeen reported that in many cases, overexpression of natural proteinsis sufficientfor\nphenotypic activation and induction of physiologically relevant responses 35.\nTherefore, transcriptional profiling of TFs overexpressing cells grown under normal\nculture conditions can more accurately reflect the direct target genes regulated by TFs\ncompared to transcriptome analysis under induced conditions 35. Additionally, our\nprimary objective was to elucidate the potential function of PtHSF2 in the thermal\nresponse of P. tricornutum.To achieve this, we cultured PtHSF2-overexpressing lines\nat 30 °C and conducted analyses to characterize their heat tolerance.Considering that\nthese results are not sufficient to demonstrate comparable transcription levels of key\npotential target genes at 20 °C and 30 °C,we used qPCR to assess the relative\ntranscription levels of several key potential target genes in cultures maintained at\n30 °C.The results indicated that the relative transcript levels of these target genes at\n30 °C (Supplementary Fig.6) are comparable to those at 20 °C (Fig. 2M-P). We have\nincorporated the results into the manuscript and revised it. (L. No. 301-304, 840-849,\n1410-1415)\nLine 173\n“Predominantly”; please provide numbers and statistics.\nResponse: Thank you for your feedback. We have revised the manuscript to include\nspecific numbers and statistics where 'predominantly' was previously used. (L. No.\n256-260)\nLine 179\n- Only 35 out of the 295 gene targets found in CUT&Tag were among the 1449 genes\nfound differentially expressed in RNAseq. How the authors explain this limited\noverlap?\nResponse: Thank you for raising the question regarding the small overlap between\ndifferentially expressed genes and target genes, which has facilitated a more in-depth\nexploration of these findings. Several factors may account for this limited overlap.\nFrom a functional standpoint of transcription factors, in addition to directly regulating\ngene transcription, they can also indirectly influence gene expression through cascade\nregulation 37, suggesting that differentially expressed genes may encompass both\ndirect and indirect target genes.Moreover, transcription factor targets can be\ncategorized as either functional or non-functional 38.This indicates that transcription\nfactors may bind to certain promoters without impacting gene expression 39, 40,\nrequiring the presence of other co-factors, such as coactivators or corepressors, to\nactivate or inhibit transcription collaboratively 37, 41. From a technical standpoint, for\nthe same cell type in a similar state, RNA-seq offers a more extensive coverage but\nlacks specificity, including all differentially expressed genes in the final results.In\ncontrast, CUT&Tag leverages a protein A/G-Tn5 transposase fusion protein to\naccurately target proteins of interest under the steerage of specific antibodies and then\nfragments the DNA around the target site, thereby offering high specificity and\nreduced background noise 42.We have provided this additional commentary on the\nlimited overlap to the revised manuscript. (L. No. 267-271, 492-508)\n- Please consider the genes found up and down regulated in RNAseq separately when\nconsidering their overlap with the gene targets found in CUT&Tag.\nResponse:Thank you for your suggestion. We have separately considered the genes\nfound upregulated and downregulated in the RNAseq analysis when examining their\noverlap with the gene targets identified in the CUT&Tag experiment in the revised\nmanuscript (Supplementary Table 11). (L. No. 264-265)\nLine 181\nPlease provide here the accession number of the Lhcx2 and ABC genes.\nResponse: We appreciate your feedback regarding this matter. The accession numbers\nfor the Lhcx2 and ABC genes are as follows: Lhcx2 (Phatr3_EG02404), ABC\n(Phatr3_J42881). We have included this information in the revised manuscript.(L. No.\n228-230)\nLine 184\n“Predominantly”; Please provide number and statistics to support this claim.\nReponse: Thank you for your feedback. We have revised the manuscript to include\nspecific numbers and statistics to support the claim where “predominantly” was\npreviously used. Specifically, we have provided data showing that the distribution of\nsignal intensity around transcription start sites (TSS) in PtHSF2 showed a peak\nintensity of 8.25, indicating a significant enrichment of binding at these sites,\nunderscoring PtHSF2’s primary binding location at the TSS, whereas the IgG control\ndid not exhibit such enrichment(Fig. 2H). (L. No. 273-276)\nLine 188\n“Previous…”; this information is already stated in line 167. The qPCR is simply a\nconfirmation of the CUT&Tag NGS library.\nResponse: Thank you for your feedback. We appreciate your attention to detail.As\nyou correctly noted, the qPCR functions primarily as a validation step for the\nCUT&Tag NGS library. We have revised the manuscript to eliminate redundancy and\nensure clarity in the presentation of our findings. (L. No.282)\nLine 196\nPlease provide references supporting that a same transcription factor can activate and\nrepress the transcription of different gene targets.\nResponse: Thank you for your feedback and request for references.The ability of a\nsingle transcription factor to both activate and repress the transcription of different\ngene targets is well-documented.Several studies support this dual regulatory function:\n-Hu et al. (2007) “Genetic reconstruction of a functional transcriptional regulatory\nnetwork.” Nature genetics, 39(5), 683-687.\n- Wei et al. (2011) “Genome-wide Analyses of Transcription Factor GATA3-\nMediated Gene Regulation in Distinct T Cell Types.” Immunity, 35(2): 299-311.\n- Li et al. (2019) “Transcriptional regulation of microalgae for concurrent lipid\noverproduction and secretion.” Science advances, 5(1), eaau3795.\nThese references provide comprehensive evidence supporting the dual functionality of\na transcription factor in regulating the transcription of different gene targets.We have\nincorporated these references into the manuscript. (L. No. 288, 1162-1163, 1181-1182,\n1198-1199)\nLine 208\nFAD is not the only gene found differentially expressed in RNAseq but not found\ntargeted by HSF2 in CUT&Tag – in fact the bulk of the RNAseq result does not\noverlap with the CUT&Tag result (see comment line 179). Please discuss this\nobservation eg difference in sensitivity and coverage between the two techniques?\nCould the regulatory action of HSF2 be mostly indirect?\nResponse: Thank you for your insightful observation. It is indeed noteworthy that\nFAD is not the sole gene found to be differentially expressed in RNA-seq\n(Supplementary Table 5 and 6) but is not targeted by HSF2 in CUT&Tag\n(Supplementary Table 10).The limited overlap between the results of the two\ntechniques can be attributed to various factors.RNA-seq provides comprehensive\ncoverage of all differentially expressed genes, offering a broad view of gene\nexpression changes 43. However, it lacks specificity in identifying direct transcription\nfactor interactions. In contrast, CUT&Tag is designed to specifically identify direct\nbinding sites of transcription factors 42, thus offering high specificity but potentially\nlimited coverage of all differentially expressed genes. This discrepancy in coverage\nand sensitivity explains why many RNA-seq identified DEGs might not overlap with\nthe CUT&Tag targets. Moreover, the observation that many differentially expressed\ngenes are not directly targeted by PtHSF2 in CUT&Tag suggests that PtHSF2’s\nregulatory actions may indeed be predominantly indirect.Transcription factors often\ninfluence gene expression through complex regulatory networks involving various co-\nfactors and secondary effects 37, 40. Additionally, it is possible that a large proportion\nof binding sites are non-functional, indicating that the binding of transcription factors\ndoes not affect gene transcription 38, 40.In summary, differentially expressed genes\nmay encompass direct and indirect targets, while binding sites may include both\nfunctional and non-functional targets. These factors may account for the limited\nintersection between RNA-seq and CUT&Tag. We have discussed these points in the\nrevised manuscript.(L. No. 492-508)\nLine 217\n- I understand the rational in analyzing Cdc45-like knock-down and overexpressing\nlines in order to assess if they phenocopy their HSF2 counterparts. Still, the possible\ngenetic interaction between these 2 genes could be better assessed by (also)\ncharacterizing the phenotype of HSF2 know-down lines overexpressing Cdc45-like\nand reciprocally.\nResponse: We greatly appreciate your comments and professional advice.We fully\nagree with your suggestions.We did not directly knock down or overexpress the target\ngenes in the transformed microalgae background. Instead, following a previous study\n44, we simultaneously transformed two plasmids into the WT cells and obtained\ntransformants with PtHSF2 overexpression and PtCdc45-like knockdown (named Ho-\nCi), as well as PtHSF2 knockdown and PtCdc45-like overexpression (named Hi-Co).\nWe then performed phenotypic analyses on the Ho-Ci and Hi-Co lines.\nThe results showed that under normal growth conditions (20 °C), knocking down\nPtCdc45-like in the PtHSF2 overexpression background alleviated the growth\nretardation (Fig. 4E) and cell enlargement (Fig. 4F) caused by PtHSF2 overexpression.\nAt 30 °C, cell density monitoring showed that knocking down PtCdc45-like in the\nPtHSF2 overexpression background significantly reduced cell density (Fig. 4E).\nConversely, overexpressing PtCdc45-like in the PtHSF2 knockdown background\ncompensated for the phenotypes associated with PtHSF2 overexpression (Fig. 4E, F).\nThis demonstrates that PtCdc45-like is a key gene mediating PtHSF2’s regulation of\ncell size and heat tolerance. We have included these findings in the manuscript. (L. No.\n401-405, 420-430, 710-718, Fig. 4E and F, Supplementary Fig. 9A-F)\n- Also, given the possible off-target effect of the RNAi construct together with the use\nof only two independent Cdc45-like knock-down lines it is warranted to generate and\ncharacterize the phenotype of Cdc45-like knock-down lines complemented by a RNAi\nresistant version of Cdc45-like expressed under its own promoter.\nResponse: We are deeply grateful for your professional and rigorous suggestions.\nBased on the previous comments(Line 130), we have successfully constructed the\nthird transformed microalgae strain siPtCdc45-like-3.Phenotypic analysis indicates\nthat the three siPtCdc45-like strains exhibit consistent characteristics (Fig. 3C-H).\nAdditionally, to determine whether the dsRNA targeting PtCdc45-like (designated as\ndsPtCdc45-like) has off-target effects,we initially utilized Sequenceserver 45 to align\ndsPtCdc45-like with the Coding DNA Sequences (CDS) of the genome.The\nalignment results revealed that, apart from PtCdc45-like, there were no other hits with\ncontinuous perfect matches of ≥5 bp in length (results not shown), implying that this\ndsRNA will not induce off-target effects 34.We believe that it would be significant to\ngenerate PtCdc45-like knock-down lines expressed under its own promoter. However,\nin light of the results presented above, we have not yet generated PtCdc45-like knock-\ndown lines under the control of its own promoter. We have clarified this situation in\nthe revised manuscript (L. No. 767-771). We kindly ask for your consideration of this\nproposed approach, and welcome any feedback or suggestions you may have.\nLine 243\nWhy “single cell dry weight”; is it not total dry weight cell pellet normalized by the\ncell number instead?\nResponse: We sincerely apologize for any confusion caused by the inaccurate\ndescription provided here.In reality, this result corresponds to the analysis of total dry\nweight cell pellet normalized by the cell number. We have made adjustments to the\n“single cell dry weight”. (L. No. 338-339)\nLine 248\n- Please define “material” – ABC transporters fall under many (sub)families.\nResponse: Thank you for your comment. BLAST analysis of the complete proteomes\nfrom several common model organisms, including Arabidopsis thaliana, Oryza sativa,\nTriticum aestivum, and Chlamydomonas reinhardtii, revealed no homologous proteins\nfor this ABC transporter (Phatr3_J42881). However, in various diatoms, the protein\nshows high homology, particularly in non-model species such as Fistulifera solaris\nand Cylindrotheca closterium (Supplementary Fig. 5B). As a result, assigning this\nprotein to a specific family or subfamily based on known species remains challenging.\nNotably, the ABC_ATPase domain of this transporter (Phatr3_J42881)\n(Supplementary Fig. 5A) is implicated in the processing of mitochondrial mRNA\ntranscripts. It is therefore hypothesized that downregulation of this ABC transporter\nmay impair mitochondrial mRNA transcript processing 46, potentially compromising\nmitochondrial function and contributing to slower growth. These updates have already\nbeen incorporated into the revised manuscript. (L. No. 226-232, 370-374)\n- Is this ABC transporter (J42881) the same one as the one mentioned in line 181\n(targeted in qPCR)? If so, please indicate its regulation in the HSF2 overexpressing\nlines as found by RNAseq in the paragraph starting line 151.\nResponse: Yes, this ABC transporter (J42881) is the same as the one mentioned in line\n181 (targeted in qPCR).Following your suggestion, we have indicated in the initial\nparagraph the regulation of the ABC transporter (J42881) in the HSF2 overexpressing\nlines. (L. No. 226-232)\n- Both Cdc45-like and the ABC transporter J42881 were identified as target of HSF2\nin CUT&Tag (line 193). It is also stated that HSF2 activates the transcription of\nCdc45-like but represses the one of ABC transporter as determined in qPCR (line\n202). How the authors explain that the overexpression of Cdc45-like alone (ie in the\nabsence of HSF2 overexpression) downregulates ABC transporter? Is it possible that\nABC is targeted by both HSF2 and Cdc45-like in parallel, or could there be a positive\nfeedback loop between Cdc45-like and HSF2? I would be curious to know the\nexpression level of HSF2 in the Cdc45-like knock-down and overexpressing lines.\nResponse:Thank you for highlighting this interesting and critical phenomenon and\nproposing a solution for us.We conducted two experiments to elucidate why\noverexpression of PtCdc45-like alone leads to the transcriptional downregulation of\nABC transporter (Phatr3_J42881).Firstly, we examined the transcription levels of\nPtHSF2 in the PtCdc45-like overexpression and RNAi lines separately.The results\ndemonstrated an upregulation of PtHSF2 transcription in the overexpression line and\na downregulation in the RNAi line (Fig. 4A).This, combined with the previous\nfinding that PtHSF2 activates the transcription of PtCdc45-like, suggests the existence\nof a positive feedback loop between PtHSF2 and PtCdc45-like.Secondly, to elucidate\nwhether PtCdc45-like alone influences the transcription of ABC transporter\n(Phatr3_J42881),we assessed the transcription levels of ABC transporter in the Ho-Ci\nlines. The results indicated that in the PtHSF2 overexpression background, knocking\ndown PtCdc45-like did not significantly alter the transcription levels of ABC (Fig.\n4C). These results indicate that ABC is primarily regulated directly by PtHSF2, while\nthe changes in ABC observed in PtCdc45-like transgenic lines (Supplementary Fig.\n7H) are likely due to PtCdc45-like’s positive feedback regulation of PtHSF2.We have\nincorporated these results into the manuscript and made revisions accordingly. (L. No.\n394-400, 406-411)\nLine 269\n“thnt”. Typing error.\nResponse: Thank you for pointing this out, we have corrected it. (L. No. 349)\nLine 273\nIt is difficult to speculate on the possible increase in lipid content in the Cdc45-like\noverexpressing lines specifically in the cell membrane as the HSF2 overexpressing\nlines present an increased accumulation of intracellular lipid content (line 142 and\nFig1E and F).\nResponse: We are grateful for your insightful comment regarding the potential\nincrease in lipid content specifically in the cell membrane of the Cdc45-like\noverexpressing lines.We completely concur with your statement.As explicitly\nindicated in line 187 and supported by Fig. 1D and F, the HSF2 overexpressing lines\nalready demonstrate an increased accumulation of intracellular lipid content.\nConsequently,speculating on the possible increase in lipid content specifically in the\ncell membrane of the Cdc45-like overexpressing lines may be inappropriate.\nMeanwhile, we must apologize for any confusion resulting from our inappropriate\ndescription. In light of the increased proportion of unsaturated fatty acids\n(Supplementary Fig. 4D),our intended message was to highlight the rise in the\nproportion of unsaturated fatty acids rather than their overall content.We have revised\nthe term “content” in the manuscript. (L. No. 357)\nLine 276\n- In this paragraph the phenotypic characterization (sensitivity to elevated temperature,\nROS, MDA/SOD/CAT contents) of the Cdc45-like knock-down lines must be\nreported.\nResponse: Thank you for your valuable feedback. We have evaluated the phenotypic\ncharacteristics of PtCdc45-like knock-down lines under high temperature conditions,\nincluding sensitivity to temperature increases, levels of ROS, MDA, SOD, and CAT.\nThese results have been incorporated into the manuscript. (Fig. 5C-F, L. No. 446-456)\n- Also, the phenotype of the Cdc45-like lines (both overexpressing and knock-down)\nwhen grown under 20C must be shown.\nResponse: Thank you for your feedback. We have included the phenotype of both the\nCdc45-like overexpressing and knock-down lines when grown under 20 °C as\nrequested. (Fig. 3C-H, L. No. 327-332, 365-367)\nLine 279\n“The optimum growth…”.\nThis sentence should be stated at the beginning of the Results section.\nResponse: Thank you for your valuable suggestion. As per your advice, we have\nrepositioned the sentence “The optimum growth...” to the beginning of this paragraph.\n(L. No. 432-433)\nLine 287\nPlease introduce what is malonaldehyde ant its known cellular role.\nResponse: Thank you for your comment. Malondialdehyde (MDA)is a compound\nthat is produced as a byproduct of lipid peroxidation 47. It is recognized as a reliable\nmarker for oxidative stress and lipid peroxidation in cellular systems 48.We have\ndescribed malondialdehyde and its cellular functions in the revised manuscript. (L. No.\n203-204)\nLine 288\nWhy “discrepancy”? The Cdc45-like overexpressing lines present an increased\ntolerance to heat stress congruent with a lower content of ROS.\nResponse: Thank you for your feedback.We apologize for any misunderstanding\ncaused by our wording selection, and have made revisions to the manuscript\naccordingly. (L. No. 448)\nLine 294\nPlease indicate which genes involved in antioxidant defense were eventually found\nupregulated and/or targeted by HSF2 in the HSF2 overexpressing lines.\nResponse: Thank you for your thoughtful comment.In accordance with your request,\nwe have conducted additional analysis of differentially expressed genes and target\ngene\ntargets related to antioxidant defense in the RNA-seq and CUT&Tag results,\nrespectively.Two mitochondrial inner membrane proteins, MPV17 (Phatr3_J15341\nand Phatr3_Jdraft223), which are involved in antioxidant processes 49, were\nsignificantly upregulated (Supplementary Table 6). Additionally, a gene encoding\nglutathione peroxidase (Phatr3_J13005) was identified as a PtHSF2 target\n(Supplementary Table 10), although no differential expression was observed for this\ngene.These findings demonstrate that PtHSF2 plays a pivotal role in regulating genes\ninvolved in antioxidant defense. We have incorporated these results into the revised\nmanuscript. (L. No. 246-248)\nLine 299\n“…emerging studies revealed that HSFs also target genes beyond heat shock proteins”.\nPlease indicate which genes specifically and in which organisms. Are those genes also\nfound up regulated/targeted by HSF2 in the HSF2 overexpressing lines?\nResponse: Thank you for your feedback.Several studies have emerged elucidating the\nrole of heat shock factors (HSFs) in the regulation of genes beyond heat shock\nproteins (HSPs).For instance, in mammals, the transcription factor HSF1 has been\nidentified to target the promoter of tumor necrosis factor-α (TNFα) 50 and interleukin\n(IL)-6 51, thereby regulating the transcription of these non-heat shock protein\nencoding genes. Furthermore, in Caenorhabditis elegans, research has shown that an\nacyl-CoA dehydrogenase 1 (acdh-1) and four vitellogenin lipid transporters (vit-1, -3,\n-4, and -5) are upregulated by HSF-1 under non-stress conditions 52. The only HSF in\nSaccharomyces cerevisiae has been discovered to activate the expression of RPN4\n(regulatory particle non-ATPase subunit 4) and PDR3 (pleiotropic drug resistance 3)\n53. Additionally, research on HSF2 has revealed that in mammals, HSF2 regulates\nnon-heat shock proteins such as proto-oncogene c-fos 54, HIF-1α (Hypoxia-inducible\nfactor-1α) 55, EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1),\nand PROM2 (Prominin 2) 56.In Arabidopsis thaliana, HSF2 regulates non-heat shock\ngenes including APX2 (ascorbate peroxidase 2) 17, 57, GolS1/S2 (galactinol synthase 1\nand 2), and IPS2 (inositol 3-phosphate synthase 2) 17.Among these previously\nidentified HSF2 targets, none were regulated by PtHSF2 in our study.We have\nenriched the discussion section of the revised manuscript with insights from these\nreferences. (L. No. 474-486, 1232-1266)\nLine 300\n“Our novel finding demonstrated that PtHSF2 regulated the transcription of…”.\nThis is only based on the RNAseq and CUT&Tag experiments conducted in the HSF2\noverexpressing lines grown at 20C. It would be necessary to conduct comparative\nRNAseq or targeted RT-qPCR experiments in the HSF2 knock-down lines versus WT\nline grown under 20C and 30C to better support this claim. The HSF2 knock-down\nlines are available.\nResponse:Thank you for pointing out the need for additional validation to support our\nclaim.We agree that conducting comparative RNAseq or targeted RT-qPCR\nexperiments in HSF2 knock-down lines versus WT lines grown under both 20 °C and\n30 °C would provide stronger evidence for the regulatory role of PtHSF2. Following\nyour suggestions, we conducted targeted RT-qPCR experiments on cells cultured at\n20 °C and 30 °C, including WT lines, PtHSF2 overexpressing lines, and PtHSF2\nknock-down lines.The genes validated in these experiments include Cdc45-like,\nLhcx2, ABC, and FAD. Fig. 2M-Pshow the relative transcription levels of the 4 genes\nin PtHSF2 transgenic lines at 20 °C,indicating that Cdc45-like, Lhcx2, and FAD were\nsignificantly upregulated in overexpression lines and significantly downregulated in\nknockdown lines. Conversely, ABC was downregulated in overexpression lines and\nupregulated in knockdown lines. The relative transcription levels of these genes at\n30 °C (Supplementary Fig. 6) were consistent with those at 20 °C. We have\nincorporated these results into the revised manuscript and made corresponding\nmodifications to the text (L. No. 301-304, Supplementary Fig. 6)\nLine 303\n“…PtHSF2 was involved in regulating thermal tolerance…”.\nAgain, phenotypic characterization of the HSF2 knock-down lines is needed\n(increased sensitivity to elevated temperature?).\nResponse: We appreciate your feedback and agree that phenotypic characterization of\nthe HSF2 knock-down lines is essential to support our claim that “PtHSF2 was\ninvolved in regulating thermal tolerance”.To address your suggestion, we have\nperformed phenotypic characterization of the PtHSF2 knock-down lines,including an\nanalysis of their sensitivity to elevated temperatures (Fig.1G and H, Supplementary\nFig. 4A-C). The results show that PtHSF2 knockdown has a minimal effect on\nsensitivity to high temperatures. We have incorporated the results into the revised\nmanuscript.\nLine 307\n“Mcm2-7 and GINS”.\nPlease explain abbreviations.\nResponse: Thank you for your insightful comments and for pointing out the need to\nclarify abbreviations in our manuscript.Mcm2-7 refers to the Minichromosome\nMaintenance proteins 2-7,which form a hexameric complex essential for the initiation\nand elongation phases of DNA replication.GINS stands for Go-Ichi-Ni-San, a protein\ncomplex named after the Japanese words for the numbers 5, 1, 2, and 3, reflecting the\nfour subunits of the complex (Sld5, Psf1, Psf2, and Psf3).We have provided\nexplanations for Mcm2-7 and GINS in the revised manuscript. (L. No. 531-532)\nLine 309\n“Fig.5”.\nPlease cite Fig.3 instead (experimental results) as Fig.5 is a model.\nResponse:Thank you for your feedback and suggestion regarding the figure citation.\nWe appreciate your attention to detail.We have revised the manuscript as per your\nsuggestion. (L. No. 534)\nLine 312\n“Hence we speculated that…”.\nThis speculation is unclear to me – do P. tricornutum WT cells increase in size during\nG1, or has it been reported before that cells with an extended G1 phase (as a\nconsequence of S phase impairment) become larger?\nResponse: Thank you for your insightful comments and questions regarding our\nspeculation. We appreciate the opportunity to clarify the following points:\n(1) P. tricornutum WT cells increase in size during G1 phase:Under natural\nconditions, diatoms require several years of asexual reproduction to reach sexual\nmaturity before they can undergo sexual reproduction 58.Therefore, the life cycle of\ndiatoms is predominantly characterized by a long vegetative phase of mitotic cell\ndivisions, with only a brief meiotic phase 59.The cell cycle of eukaryotic organisms,\nincluding diatoms, is primarily divided into interphase and the mitotic phase, with\ninterphase further subdivided into gap1 phase (G1), DNA synthesis phase (S), and\ngap2 phase (G2) 59.Throughout the cell cycle, cells sequentially undergo a series of\nevents. The G1 phase is the first stage of interphase,during which the cell experiences\nsignificant growth, including an increase in volume, extensive synthesis of proteins\nand RNA, and the accumulation of sufficient nutrients and energy to prepare for\nsubsequent DNA replication and cell division 60, 61.In cell biology, it has been\nobserved that microalgae 60, 62, 63, 64, yeast 65, 66, plant cells 67, 68, and animal cells 69, 70\nall exhibit significant growth during the G1 phase. Therefore, we hypothesize that P.\ntricornutum WT cells also follow this pattern.\n(2) Cells with an extended G1 phase (due to S phase impairment) become larger:\nThere are studies reporting that when the S phase is impaired (inhibition and/or delay),\nleading to an extended G1 phase, cells tend to become larger. For instance, In the\ngreen alga Chlamydomonas, cell cycle inhibitors, which block the transition from the\nG1 phase to the S phase, delay the onset of the S phase at high concentrations,\nresulting in an extended G1 phase during which the cells continue to grow, leading to\nan increase in cell size 64.The same mechanism is observed in both budding yeast and\nmammalian cells. In yeast, high concentrations of Whi5 G1/S transcriptional repressor\nextend the G1 phase 71, delaying the S phase and resulting in increased cell size. In\nmammals, Whi5’s functional orthologis the retinoblastoma protein (Rb) 72, which\nregulates the G1/S transition by inhibiting the early 2 factor (E2F) activity 73.\nResearch indicates that Rb overexpression prolongs the G1 phase, thereby delaying\nentry into the S phase and increasing cell size 74.\nOverall, these studies indicate that cells typically increase in size during the G1\nphase, and an extended G1 phase resulting from S phase abnormalities (inhibition\nand/or delay) leads to even larger cell size.However, our analysis of cell cycle\nprogression in WT, PtHSF2-1, siPtHSF2-1, PtCdc45-like-1, and siPtCdc45-like-1\ncells showed that the enlarged cells (PtHSF2-1 and PtCdc45-like-1) did not arrest in\nG1 phasebut rather accumulated or arrested in the S and G2 phase (Supplementary\nFig. 8).Therefore, this finding is inconsistent with our previous assumption.We have\nrevised the manuscript to incorporate this result from the cell cycle progression\nanalysis. (L. No. 375-387, 541-544)\nLine 323\n“Larger cells had lower growth rate than smaller cells in diatoms”.\nMaybe because large cells need more energy and nutrients than small cells to grow\nirrespectively of the thermal tolerance range.\nResponse: Thank you for your insightful comment.You raised an important point\nregarding the lower growth rate of larger cells in diatoms compared to smaller cells.\nIndeed, one possible explanation for this phenomenon is that larger cells require more\nenergy and nutrients to sustain their growth.This increased demand can be attributed\nto the higher metabolic activity needed to maintain cellular functions in a larger\nvolume.For instance, research on cancer cells indicates that cell size and metabolic\ndemand are closely linked.Larger cells have higher protein synthesis rates, which\ncorrelates with increased energy requirements and nutrient uptake, such as essential\namino acids and glucose, to support their growth and maintenance 75.A study 76\nindicates that larger diatoms exhibit lower growth rates, likely due to the higher\nenergetic and nutrient demands required for their maintenance and growth.\nAdditionally, larger phytoplankton species generally have slower growth rates, partly\ndue to their greater resource requirements 77. We have revised the discussion section\nbased on these literature. (L. No. 564-566, 1304-1310)\nLine 324\n“..several ABC transporters were found to be significantly decreased in…”.\nPlease detail which ABC transporters and their putative functions.\nResponse:Thank you for your insightful comment regarding the ABC transporters.\nWe have provided a detailed description of the specific ABC transporters identified\nand their putative functions in the revised manuscript. Below is the revised text\naddressing your concern at line 566-575 of the manuscript:\nMoreover, several ABC transporters, including Phatr3_J42881, Phatr3_J44752, and\nPhatr3_Jdraft1613, were found to be significantly decreased in PtHSF2\noverexpressing cells, with Phatr3_J42881 also reduced in PtCdc45-like\noverexpressing cells (Fig. 2O, Supplementary Fig. 7H and Supplementary Table 7).\nBased on the STRING analysis (https://string-db.org/), Phatr3_Jdraft1613 is predicted\nto interact with Phatr3_J44752, acetyl-CoA carboxylases (1 and 2), and the K+\nchannel inward rectifier domain-containing protein (KCNJ) (Supplementary Fig. 5C).\nThese findings suggest a potential role for these ABC transporters in lipid and\npotassium ion transport, with their decreased expression likely impairing material\ntransport and consequently reducing growth rates 78.\nLine 333\n“…larger P. tricornutum cells, while growing slower at 20C than smaller cells\nexhibited superior antioxidant abilities and higher survival rates at elevated\ntemperature”.\n- From this rational it would be intuitive to think that P. tricornutum WT cell (and in\nother diatom species) increases in size in response to increased temperature – has such\nobservation been reported before?\n- Also, how an abrupt transition from 20C to 30C is relevant with what P. tricornutum\nand diatoms in general experience in their environment?\nResponse:Thank you for your insightful comments. Yes, there is evidence to support\nthis observation.A study indicated that in P. tricornutum, cell size increases\nsignificantly with rising temperatures. Specifically, the research demonstrated that cell\nvolume increased by approximately 10% when the temperature was raised from 18 °C\nto 25 °C 79.An earlier study also indicated that P. tricornutum cells grown at 23 °C to\n25 °C were larger than those grown at lower temperatures 80.Another study,\nemploying flow cytometry analysis, demonstrated that under nitrogen limitation\nconditions, the cell size gradually increased as the temperature rose from 20 °C to\n30 °C 81. In addition to P. tricornutum, it has been observed in other diatoms that cell\nsize increases with rising temperatures. Specifically, in the diatom Thalassiosira\npseudonana, it was found that temperatures above the thermal optimum reduce cell\ngrowth and silica production while increasing cell volume and protein content 82. A\nstudy on Skeletonema marinoi revealed that the cell volume of this diatom increased\nby 220% from low to high temperatures 83.Specifically, the research compared the\nresponses of S. marinoi strains from the 1960s, 1990s, and 2010s across a temperature\nrange of 6 °C to 22 °C. The results indicated that modern (2010s) strains exhibited a\nsignificant increase in cell volume at higher temperatures. While our findings align\nwith previously reported observations that elevated temperatures can lead to an\nincrease in cell size in some diatoms, including P. tricornutum,not all diatoms exhibit\nthis behavior.Some diatoms show a decrease in cell size with rising temperatures 84.\nFor example,high temperatures resulted in decreased cell sizes in the benthic diatoms\nAmphora coffeaeformis and Nitzschia ovalis 85.Therefore, in diatoms, whether cell\nsize increases with rising temperatures cannot be generalized; specific analyses are\nrequired for different species.We have revised the discussion section based on these\nliterature. (L. No. 584-596, 1107-1112, 1328-1343)\nThe growth temperature range of diatoms is species-specific. Generally, diatoms\nexhibit the ability to thrive across a broad spectrum of temperatures, typically ranging\nfrom approximately 2 °C to 28 °C 86.P. tricornutumgrows optimally at temperatures\nbetween 15 °C and 25 °C but can survive in a broader range from 5 °C to 28 °C,but is\nunable to sustain growth at temperatures exceeding 30 °C 30, 36, 81.Consequently, we\naimed to investigate the tolerance of transgenic microalgae (PtHSF2 and PtCdc45-like)\nat 30 °C. However, as P. tricornutum is primarily found in coastal regions, literature\nindicates that coastal seawater temperatures can fluctuate significantly, but typically\ndo not exceed a 10 °C variation.For instance, studies have shown that coastal waters\nin Washington state experience temperature variations of 4 °C to 6 °C 87. In Chinese\ncoastal areas, seawater temperature changes during marine heatwaves (MHWs) range\nfrom 0.4 °C to 3.7 °C 88.Therefore, the abrupt transition from 20 °C to 30 °C may not\nbe appropriate.Given this, wefurther evaluated the characteristics of the transgenic\nmicroalgae at a milder high temperature of 25 °C, and subsequently modified the\nmanuscript based on the results. (L. No. 179-184, 327-328, 434-436, 441-442, 779-\n780, Fig. 1C-E, Fig. 3C and D, Fig.5)\nLine 343\n“..correlate the cell size of P. tricornutum with its broad temperature adaptation and to\nprovide insights into the molecular mechanisms by which diatoms tolerate high\ntemperature”\n- Providing experimental evidences in different diatom species of contrasting thermal\ntolerance ranges and sizes is needed to support this claim.\n- Please distinguish adaptation (such as in the study cited line 63 reference 10) and\nshort term acclimatory response (this study).\nResponse: Thank you for your valuable feedback. We appreciate your insights and\nhave addressed your comments as follows:\n(1) Experimental evidence in different diatom species:To support the claim of\ncorrelating cell size with temperature adaptation, we have included additional\nexperimental evidence from studies on various diatom species exhibiting different\nthermal tolerance ranges and sizes. We refer specifically to a 300-generation thermal\nacclimation study on Thalassiosira pseudonana, conducted under four temperature\nconditions: control (22 °C), moderate (26 °C), severe (32 °C), and fluctuating\n(22 °C/32 °C), which assessed the thermal tolerance of the evolved lineages compared\nto the ancestral lineage 27.The study found that the evolved lineages acclimated to\nhigh temperatures exhibited a higher optimal growth temperature and an increase in\ncell size.Notably, cells acclimated to 32 °C were twice the size of control cells at\n22 °C, indicating a correlation between cell size and high-temperature tolerance.\nAnother investigation into the thermal adaptation of Skeletonema marinoi from the\nBaltic Sea revealed a relationship between cell size and heat tolerance 83.Over the\npast 60 years, the thermal tolerance of S. marinoi has progressively increased,with\nhigh-temperature-tolerant strains from the 2010s exhibiting cell volumes 220% larger\nthan those of low-temperature-tolerant strains from the 1960s and 1990s.These studies\nprovide a comparative framework to understand how P. tricornutum fits into the\nbroader context of diatom temperature adaptation.We have added these references to\nthe revised manuscript. (L. No. 603-605, 1107-1108)\n(2) Distinguishing adaptation and short-term acclimatory response:We\nacknowledge that adaptation and short-term acclimation response are indeed distinct\nprocesses, and we appreciate the opportunity to clarify these terms as they relate to\nour study and the cited literature.Adaptation involves genetic changes over multiple\ngenerations, allowing a species to survive and reproduce in a particular environment.\nThis process is driven by natural selection and results in heritable traits that are passed\ndown to future generations. Thalassiosira pseudonana was acclimated to four\ndifferent temperature conditions 27. After approximately 300 generations of selection,\nit was observed that T. pseudonana exhibited enhanced adaptation to warming.\nFurther genome resequencing revealed evolutionary changes in genes related to\ntranscriptional regulation, cellular oxidative stress, and redox processes, indicating\ntrue adaptive evolution.\nIn contrast,short-term acclimatory responses, such as those observed in our study,\ninvolve physiological, biochemical, or behavioral changes that occur within an\nindividual's lifespan in response to immediate environmental changes.These changes\ndo not involve genetic alterations. Our research focuses on the immediate response of\nPtHSF2 and PtCdc45-like transgenic microalgae strains to sudden temperature\nchanges, investigating how these transgenic strains adjust their cellular functions and\nmorphology in the short term to cope with thermal stress.\nBy distinguishing these processes, we underscore that our study examines the\nimmediate, reversible physiological adjustments of our transgenic microalgae strains\nto thermal stress, whereas the cited literature in line 61 (reference 10) addresses\ngenetic adaptations that occur over much longer timescales.\nWe have revised the manuscript to emphasize that our study focused on the\nshort-term temperature response capacity of P. tricornutum.(L. No. 606)\nLine 349\n“..whose shape plasticity is crucial for environmental adaptation.”\n- Do different morphotypes of P. tricornutum present different thermal tolerance\nranges? And, do P. tricornutum switch morphotype under increased temperature?\n- Please distinguish cell size and morphology in the text.\nResponse: Thank you for your insightful comments. We appreciate the opportunity to\nclarify these points.\n(1) Thermal tolerance of different morphotypes and morphotype switching:\nCurrent research indicates that P. tricornutum can exhibit different morphotypes,\nincluding fusiform, oval, and triradiate forms 89. However, there is limited specific\ndata on the thermal tolerance ranges of each morphotype.There is evidence indicating\nthat temperature changes can induce morphological changes in different strains of P.\ntricornutum.For instance,previous studies 31, 89 have shown thatincreasing the\ntemperature from 15 °C to 28 °C caused strains Pt3 (CCAP1052⁄1B) and Pt9\n(CCMP633) to transition from oval to fusiform shape,with this process being\nreversible in strain Pt9; additionally, a small portion of oval cells in strain Pt1 8.6\n(CCMP2561) also transitioned to fusiform cells. Despite evidence indicating that\ntemperature changes can induce morphological transitions in P. tricornutum, the\nunderlying molecular mechanisms remain insufficiently validated. Future studies\nfocusing on temperature-induced morphotype changes would be valuable to address\nthis knowledge gap. (L. No. 612-613, 1328-1334)\n(2) Distinguishing cell size and morphology: We acknowledge the importance of\nclearly distinguishing between cell size and morphology in our manuscript.Cell size\nrefers to the physical dimensions of the cells, typically measured in micrometers\n(μm). In contrast, morphology encompasses the overall shape and structural features\nof the cells, including attributes such as form (e.g., fusiform, oval) and structural\nadaptations. To address this, we have revised the text to explicitly differentiate\nbetween these terms. (L. No. 611)\nLine 401\nPlease indicate how many transgenic lines were obtained and how the ones shown in\nthe study were selected.\nResponse: Thank you for your feedback, and we apologize for overlooking these\ndetails in the manuscript.By screening on solid media containing bleomycin, we\nobtained hundreds of transformants for each plasmid, although a complete count was\nnot performed. We then randomly selected 10 transformants for each plasmid for\nscreening and validation. Transformants that failed to grow in the antibiotic-\ncontaining liquid media were excluded. False positives were identified and eliminated\nthrough genomic PCR, and qPCR was subsequently used to select transformants with\nstable high or low expression of the target gene.These selection processes ensured\nthat the observed effects were due to the introduced genes rather than random\nvariation among the transgenic lines. We have incorporated these details into the\nrevised manuscript. (L. No. 720-727)\nLine 420\n- RT-qPCR experiments should include at least 2 validated references genes.\n- Please provide information of the RT-qPCR experiments following the MIQE\nguidelines (Bustin 2009 10.1373/clinchem.2008.112797).\nResponse: Thank you for your professional insights.We appreciate your\nrecommendations and have addressed the concerns as follows:\n(1)Reference Genes for RT-qPCR: We acknowledge the importance of using two\nvalidated reference genes for RT-qPCR experiments. In the revised manuscript,we\nincluded two reference genes, β-actin and TBP (TATA box binding protein), which\nwere selected based on their stable expression across different conditions in P.\ntricornutum, as verified in previous studies 90, 91.These reference genes were used to\nnormalize the expression levels of our target genes to ensure accurate and reliable\nquantification. (L. No. 743-745, 1375-1380)\n(2) MIQE Guidelines:We have revised the manuscript to include detailed\ninformation about our RT-qPCR experiments following the MIQE guidelines 92.\nSpecifically, we have provided the following information:\n- Primer specificity:We provided the melting curves of each primer pair’s\namplification products, showing single peaks that indicate the specificity of the\namplification products.\n- Primer amplification efficiency: We diluted WT cDNA in a gradient to create six\nconcentrations. qPCR was performed using these different concentrations of cDNA as\ntemplates. After amplification, standard curves were constructed with the log values\nof the template dilution series on the X-axis and the corresponding Ct values on the Y-\naxis. The amplification efficiency of each primer pair, as determined from the\nstandard curve, ranged from 90% to 110%.\n- Ct values: We have included the Ct values of all qPCR reactions in the raw data.\n- Normalization: The relative transcription levels were calculated using the\ncomparative Ct method 93 and normalized to the reference genes β-actin and TBP.\nWe have included this information in the revised manuscript under the Methods\nsection. We hope these revisions address your concerns adequately and enhance the\nrigor and clarity of our study. Thank you again for your valuable feedback. (L. No.\n746-752, 1382-1386, Source Data)\nLine 426\n- The initial cell density of the cultures is indicated as 1 x106 cells/mL. To my\nexperience this is very high even for P. tricornutum. In consequence the cultures\npresent a very short (day 1 to 3) exponential growth phase over the course of the 13\ndays culture experiment (Fig.1B). Please justify the choice of the starting cell density.\n- Also, please clarify the design of the culture experiment regarding how the cultures\nwere exposed to the different temperature regimes eg were the cultures maintained at\n20C and then transferred (how quickly?) to the 20C and 30C growth conditions?\nResponse: Thank you for your insightful comments.We have addressed the concerns\nas follows:\n(1) Initial Cell Density Justification:We acknowledge that an initial cell density\nof 1 x106 cells/mL might seem high for P. tricornutum.We chose this initial density\nbased on the results of preliminary experiments,aiming to ensure sufficient cell\nnumbers for subsequent measurements of growth, physiological parameters, and\nredox levels.High temperatures (30 °C) lead to rapid death in wild-type cells.If the\ninitial density is too low, very few cells will remain after a complete culture cycle of\napproximately 13 days.Therefore, we selected a higher initial density.Additionally, to\navoid growth differences due to varying initial densities, we set the initial density for\nother temperature conditions at 1 x106 cells/mL and ensured the culture volume was\nconsistent across all flasks. We observed that differences in culture volume can also\naffect growth outcomes. We have provided an explanation for the establishment of\nthis density in the revised manuscript. (L. No. 777-779)\n(2) Experimental Design Clarification: We appreciate your request for\nclarification on the experimental design.Referring to previous studies on\nPhaeodactylum tricornutum31 and Thalassiosira pseudonana 27 exposed to different\ntemperatures, we initially transferred each strain to acclimate them to the designated\nexperimental temperature.After a 7-day acclimation period, we performed a second\ntransfer and commenced measurements of various parameters.\nWe have updated the manuscript to include these details. (L. No. 775-777)\nLine 427\nPlease clarify what is “15ggnog15ine”.\nResponse: Thank you for your feedback. We apologize for any confusion and\ninconvenience caused by the spelling errors. We have corrected it to “Bright-Line”.(L.\nNo. 781)\nLine 509\nPlease follow and indicate the MIQE guidelines or similar for the qPCR experiments\n(eg provide specificity, efficiency and Ct values of the qPCR reaction for each primer\npairs, demonstrate that a sufficient number of reference genes were used for relative\nquantification).\nResponse: Thank you again for your professional suggestions.We have redone all\nqPCR experiments in the manuscript according to MIQE guidelines.Primer\nspecificity was confirmed by the presence of a single peak in the melt curve of the\namplified products (Source Data), and amplification efficiency was calculated using\nstandard curves (Source Data). All Ct values are provided in the Source Data file.\nRelative quantification was performed using two reference genes (β-actin and TBP).\nThe manuscript has been revised in accordance with these guidelines. (L. No. 870-871)\nLine 516\nPlease clearly indicate the number of independent lines, and biological and technical\nreplicates used.\nResponse:Thank you for your feedback. We have clearly indicated the number of\nindependent lines, and biological and technical replicates used in the revised\nmanuscript.Specifically, PtHSF2-1, PtHSF2-2, and WT cells were used in this\nexperiment, with three biological replicates and one technical replicate for each line.\nA 1:1 mixture of cell samples from these two overexpression lines was employed as a\nnegative control. (L. No. 886-889)\nLine 545\nPlease provide a table presenting the statistics of the NGS reads including their\nnumber and quality for each library.\nResponse: Thank you for your suggestion. We have included a table in the\nsupplementary materials presenting the statistics of the NGS reads, including their\nnumber and quality for each library (Supplementary Table 3 and 4). (L. No. 218-220)\nLine 557\nSame comment as in line 509.\nResponse: Thank you for your feedback and the recommended reference.We have re-\nperformed the CUT&Tag-qPCR experiments according to the MIQE guidelines, and\nhave included the details of the process and the results in the revised manuscript. (L.\nNo. 936-941)\nLine 550\nPlease detail the procedure used for the GO term enrichment analysis (fold-change\nand number of gene used as threshold, statistics, reference gene list).\nResponse:Thank you for your thorough review of our manuscript. We appreciate your\nrequest for further details on the GO term enrichment analysis. Per your request, the\nMethods section has been revised to include a detailed description of the procedure.\n(L. No. 872-879)\nFig 1C\nIs does not seem right to plot daily growth rate. The growth rate should be calculated\nwithin the exponential growth phase which is very short here (around day 1 to 3) due\nthe very high starting cell density (see comment line 426).\nResponse: Thank you for your professional comment.We agree that plotting the daily\ngrowth rate may not be appropriate given the very short exponential growth phase due\nto the high starting cell density.In light of this, we have removed the growth rate data\nfrom the revised manuscript and adjusted the content accordingly.Thank you for\nbringing this to our attention.\nFig 1D and E\n“D” seems to correspond to “E” and vice versa.\nResponse: Thank you for your feedback.We appreciate your attention to detail and\napologize for any confusion caused. We have reviewed the figures and agree that they\nwere mislabeled. We have corrected the labeling in the revised manuscript.\nFigure 1F\nHow the authors explain the presence of red dots outside the cells in the picture of the\nPtHSF2-1 line under 30C?\nResponse: Thank you for your attention to detail. The red dots observed outside the\ncells are lipid droplets stained with Nile Red.By monitoring Nile Red fluorescence,\nwe found an increase in fluorescence in PtHSF2 overexpressing cells under high\ntemperature, indicating a higher neutral lipid content. Neutral lipids are primarily\nstored in lipid droplets. There are two possible explanations for the presence of lipid\ndroplets outside the cells:(i) exposure to high temperature may have compromised\ncell integrity, causing cell rupture and the subsequent release of lipid droplets into the\nextracellular space; (ii) the cells synthesized a large number of lipid droplets and\nactively secreted them outside the cells, which is consistent with our previous findings\n94.We have provided a description and explanation of this detail in the revised\nmanuscript. (L. No. 187-192, 1162-1163)\nFigure 2A-F\n- Please analyze separately the up and down regulated genes found in RNAseq.\n- What means in the x-axis “Genes involved (%)”?\nResponse:We appreciate the your insightful suggestion regarding the separate\nanalysis of upregulated and downregulated genes from the RNA-seq data. We have\nconducted a separate analysis of the upregulated and downregulated genes, and this\nanalysis has been included in the revised manuscript (Supplementary Table 5). (L. No.\n220-226)\nWe have clarified the x-axis label “Genes involved (%)” in Figure 2A-C to specify\nthat it represents the percentage of differentially expressed genes (DEGs) associated\nwith a specific GO term (Level 2) within the overlap between the two HSF2\noverexpressing lines (with an adjusted |log₂(FC)| ≥ 1 and p-value < 0.05), relative to\nthe total of 1449 DEGs identified in this overlap during the RNA-seq analysis. (L. No.\n867-869, 1459-1462)\nThe x-axis label “Genes involved (%)” in Figure 2D-F indicates the percentage of\npotential target genes associated with a specific GO term (Level 2) among the 295\npotential target genes identified by CUT&Tag. (L. No. 1462-1465)\nWe have already clarified this in the revised manuscript's figure 2A-F legend.\nFigure 2G\nPlease analyze separately the up and down regulated genes found in RNAseq.\nResponse:Thank you for your suggestion. In Figure 2G, we have separately analyzed\nthe upregulated and downregulated genes found in the RNAseq data (Supplementary\nTable 5). The results are now presented in the updated Figure 2G.\nFigure 2H\nPlease label the y-axis.\nResponse: Thank you for your suggestion. We have labeled the y-axis in Figure 2H in\nthe revised manuscript\nFigure 2I\nPlease better show what the y-axis corresponds to.\nResponse:Thank you for your suggestion. We have clarified what the y-axis\ncorresponds to in Figure 2I in the revised manuscript.\nFigure 3\nPlease state at which temperature the lines were exposed.\nResponse: Thank you for your comment. We have updated Fig. 3 to include the\nspecific temperatures at which the lines were exposed. This information is now\nclearly stated in the figure legend and annotated within the figure itself.(L. No. 1485,\nFig. 3)\nTables\nThe two tables have formatting problems:\nIn the first table the name of the genes in the first left column are truncated.\nIn the second table the name of the entries in the top raw are truncated\nFor both tables, please provide a short description note to understand what the tables\ncorrespond to.\nResponse: Thank you for your valuable feedback. In response to your comments, we\nhave revised the formatting of these tables and added brief description notes to help\nreaders understand their content.\nSupplementary Fig.1A\n- Please provide sequences and sequence alignments, and indicate the algorithms used\nfor the phylogenetic analysis.\n- Please provide branch lengths and bootstrap values.\n- It could be worth adding some green algae or coccolithophores species in the\nphylogenetic analysis to infer the conservation/speciation of the HSF orthologues\nacross different phytoplankton functional types.\nResponse: Thank you for your insightful comments and suggestions regarding\nSupplementary Fig.1A. We have addressed your points as follows:\n(1) Sequences and Sequence Alignments: We have included the sequences and\nsequence alignments used for the phylogenetic analysis in the Source Data file.The\nalignments were performed using the ClustalW.\n(2)Algorithms Used for Phylogenetic Analysis: The phylogenetic tree was\nconstructed using the Neighbor-Joining method implemented in MEGA6.\n(3) Branch Lengths and Bootstrap Values: We have added the bootstrap values to\nthe phylogenetic tree. The bootstrap values were calculated with 1000 replicates to\nensure robustness and reliability of the tree topology.Additionally, we included atree\nscale to indicate branch lengths.\n(4) Addition of Green Algae and Coccolithophores Species:To enhance the\nanalysis and infer the conservation/speciation of HSF orthologues across different\nphytoplankton functional types, we have included sequences from additional species,\nincluding green algae (Chlamydomonas reinhardtii), coccolithophores (Emiliania\nhuxleyi), and Arabidopsis thaliana.The revised phylogenetic tree now reflects these\nadditions and provides a more comprehensive view of HSF2 orthologue distribution\nand evolution.\nWe have updated the manuscript and supplementary materials accordingly.\nThank you for your valuable feedback, which has significantly improved the quality\nand depth of our analysis. (L. No. 645-657)\nSupplementary Fig.1B\nPlease explain what is the “EUK_SMAGs database”.\nResponse: Thank you for your question regarding the “EUK_SMAGs database” in\nSupplementary Fig.1B.The “EUK_SMAGs database” refers to the database of the\nmarine Eukaryotic MAGs and SAGs (Metagenome and Single-Cell Assembled\nGenomes), which are integral to the Ocean Gene Atlas v2.0 95.This database\ncomprises 713 non-redundant and manually curated eukaryotic MAGs and SAGs,\nencompassing a total of 10 million genes. The dataset was constructed from 280\nbillion metagenomic reads obtained from the Tara Oceans project, covering diverse\nmarine environments such as polar, temperate, and tropical sunlit oceans. The genome\nsizes within the EUK_SMAGs database range from 10 Mb to 1.3 Gb. The primary\npurpose of the EUK_SMAGs database is to offer a comprehensive catalog of\neukaryotic genes, enabling the exploration of the biogeography, phylogeny, and\nfunctional diversity of marine plankton.\nWe have included this explanation in the revised manuscript to provide clearer context\nfor the use of the EUK_SMAGs database in our analysis. (L. No. 666-671)\nSupplementary Fig.2B\n- Please provide sequences and sequence alignments, and indicate the algorithms used\nfor the phylogenetic analysis.\n- Please provide branch lengths and bootstrap values.\n- To what correspond the mauve circles at the nodes?\nResponse: Thank you for your valuable feedback. We have addressed your concerns\nas follows:\n(1) Sequences and Sequence Alignments: We have provided the sequences and\nsequence alignments used for the phylogenetic analysis in the Source Data file. The\nalignments were performed using the ClustalW algorithm.\n(2) Algorithms Used for Phylogenetic Analysis: The phylogenetic tree was\nconstructed using the Neighbor-Joining method implemented in the MEGA6 software.\n(3)Branch Lengths and Bootstrap Values: We have included branch lengths and\nbootstrap values on the phylogenetic tree. Bootstrap values were calculated with 1000\nreplicates to ensure the robustness of the tree topology.\n(4)Mauve Circles at the Nodes: The mauve circles at the nodes represent\nbootstrap support values. Larger circles indicate higher bootstrap values, reflecting\ngreater confidence in the corresponding nodes.\nAs per your request, we have revised Supplementary Fig. 2B and its legend to\ninclude these details. (L. No. 660-662, Supplementary Fig. 2B)\nSupplementary Fig.2C\nPlease provide a positive control for PCR amplification in WT gDNA.\nResponse: Thank you for your professional comments.We have repeated the genomic\nPCR validation experiments and updated the results in Supplementary Fig. 2C.\nSpecifically, we used wild-type (WT) genomic DNA (gDNA) as a template and\nperformed amplification with primers Pt89a and Pt91r, which produced a non-specific\nband at 750 bp. In contrast, using the same primers to amplify the gDNA of PtHSF2\noverexpression transformants yielded a specific band at 1338 bp, which matches the\nexpected size. For the RNAi silenced strains, due to the difficulty in amplifying the\ncomplete hairpin structure, we used the forward primer Pt89a targeting the expression\ncassette and the reverse primer siHSF2R4 targeting the intron for amplification. The\namplification product size was consistent with the expected 603 bp. (Supplementary\nFig. 2C)\nSupplementary Fig.2D\nPlease provide information pertained to the qPCR experiment following the MIQE\nguidelines or similar.\nResponse: Thank you for your insightful comments.We have undertaken the qPCR\nexperiments again in accordance with the MIQE guidelines and have incorporated the\nnecessary details into the revised manuscript. (Supplementary Fig. 2D, Source Data)\nSupplementary Fig.2F\n- Is “E” and not “F”.\n- Please provide pictures of the full gels and films. This is particularly important to\nascertain the specificity of the FLAG antibody.\nResponse: Thank you for your insightful comments regarding Supplementary Fig.2E.\n(1) Correction of Labeling:We apologize for the labeling error in the figure\nlegend.The figure should indeed be labeled as “E” and not “F” We have corrected the\nfigure legend.\n(2)Providing Full Gels and Films:We understand the importance of providing\nfull films to verify the specificity of the FLAG antibody. We have included images of\nfull films in the supplementary materials. (Source Data)\nWe appreciate your feedback, which has allowed us to enhance the transparency\nand reliability of our Western blot analysis.\nSupplementary Fig.4B, C and E\n- Same comments as for Supplementary Fig.2B, C and E.\n- Please indicate to what “n” corresponds to: technical or biological replicates?\nResponse: Thank you for your feedback. We have addressed your concerns as follows:\n(1) Supplementary Fig.7B: We have provided the sequences and sequence\nalignments used for the phylogenetic analysis of PtCdc45-like in the Source Data file.\nWe have updated the Methods section (L. No. 658-662) to include the algorithms and\nsoftware used for the phylogenetic analysis.The Supplementary Fig.4B have been\nrevised to clearly display the branch lengths and bootstrap values. Additionally, the\nfigure legend now clarifies that the mauve circles at the nodes represent bootstrap\nsupport values.\n(2) Supplementary Fig.7C: Similar to the modifications in Supplementary Fig.2C,\nwe used gDNA from WT and PtCdc45-like overexpression strains as templates for\nPCR amplification with the Pt89a and Pt91r primers. The amplification product of the\nPtCdc45-like overexpression strains exhibited a specific band at 2451 bp, which\ncorresponds to the expected size, while the WT showed a nonspecific band at 750 bp.\nFurthermore, using Pt89a and siCdc45-like-R3 primers to amplify the interference\nstrains, we observed a specific band at the expected size of 545 bp. (Supplementary\nFig.7C)\n(3) Supplementary Fig.7E: We have corrected the label for Supplementary\nFig.7E.Additionally, we have included pictures of the full films from the Western blot\nanalysis in the Source Data file.\n(4) Clarification of “n”: We have clarified that the “n” represents 3 biological\nreplicates.\nSupplementary Table 1\nPlease provide gene sequences.\nResponse: Thank you for your valuable comment.We have included the gene IDs and\ntheir corresponding protein sequences for HSF transcription factors from various\nspecies in the Source Data file.\nSupplementary Table 3\n- Is Supplementary Table “2” and not “3”.\n- Please add information of the specific use of the primers in the study (eg “validation\nof….”).\n- Provide here information on the qPCR primer pairs efficiency, Ct values etc.\nResponse: Thank you for your professional comments.We appreciate your\nrecommendations and have addressed the concerns as follows:\n(1) Correction of Table Number: We apologize for the error in labeling. The table\nshould be labeled as Supplementary Table 14 in the revised manuscript.\n(2) Information on Primer Use: We have added detailed information regarding\nthe specific use of the primers in the study (Supplementary Table 14).\n(3) qPCR Primer Pairs Efficiency and Ct Values etc: We have provided the\nnecessary information related to the qPCR primers in the Source Data file, including\nefficiency and Ct values, in accordance with MIQE guidelines.\nReferences\n1. Huysman MJ, et al. Genome-wide analysis of the diatom cell cycle unveils a\nnovel type of cyclins involved in environmental signaling. Genome Biol. 11,\n1-19 (2010).\n2. Malviya S, et al. Insights into global diatom distribution and diversity in the\nworld’s ocean. PNAS 113, E1516-E1525 (2016).\n3. Benoiston A-S, et al. The evolution of diatoms and their biogeochemical\nfunctions. Philos Trans R Soc Lond B Biol Sci. 372, 20160397 (2017).\n4. Sigman DM, Hain MP. The biological productivity of the ocean. Nat. Edu.\nKnowled. 3, 21 (2012).\n5. Anderson NJ. Diatoms, temperature and climatic change. Eur. J. Phycol. 35,\n307-314 (2000).\n6. Lürling M, Eshetu F, Faassen EJ, Kosten S, Huszar VL. Comparison of\ncyanobacterial and green algal growth rates at different temperatures.\nFreshwater Biol. 58, 552-559 (2013).\n7. Butler T, Kapoore RV, Vaidyanathan S. Phaeodactylum tricornutum: a diatom\ncell factory. Trends Biotechnol. 38, 606-622 (2020).\n8. Yodsuwan N, Sawayama S, Sirisansaneeyakul S. Effect of nitrogen\nconcentration on growth, lipid production and fatty acid profiles of the marine\ndiatom Phaeodactylum tricornutum. Agric. Nat. Resour. 51, 190-197 (2017).\n9. Garcıa MC, Mirón AS, Sevilla JF, Grima EM, Camacho FG. Mixotrophic\ngrowth of the microalga Phaeodactylum tricornutum: influence of different\nnitrogen and organic carbon sources on productivity and biomass composition.\nProcess Biochem. 40, 297-305 (2005).\n10. Qiao H, Cong C, Sun C, Li B, Wang J, Zhang L. Effect of culture conditions\non growth, fatty acid composition and DHA/EPA ratio of Phaeodactylum\ntricornutum. Aquaculture 452, 311-317 (2016).\n11. Dolch L-J, Maréchal E. Inventory of fatty acid desaturases in the pennate\ndiatom Phaeodactylum tricornutum. Mar. Drugs 13, 1317-1339 (2015).\n12. Dinamarca J, Levitan O, Kumaraswamy GK, Lun DS, Falkowski PG.\nOverexpression of a diacylglycerol acyltransferase gene in Phaeodactylum\ntricornutum directs carbon towards lipid biosynthesis. J. Phycol. 53, 405-414\n(2017)\n13. Peng K-T, et al. Delta 5 fatty acid desaturase upregulates the synthesis of\npolyunsaturated fatty acids in the marine diatom Phaeodactylum tricornutum.\nJ. Agric. Food. Chem. 62, 8773-8776 (2014).\n14. Hamilton ML, Haslam RP, Napier JA, Sayanova O. Metabolic engineering of\nPhaeodactylum tricornutum for the enhanced accumulation of omega-3 long\nchain polyunsaturated fatty acids. Metab Eng. 22, 3-9 (2014).\n15. Wiederrecht G, Seto D, Parker CS. Isolation of the gene encoding the S.\ncerevisiae heat shock transcription factor. Cell 54, 841-853 (1988).\n16. Hahn J-S, Thiele DJ. Activation of the Saccharomyces cerevisiae heat shock\ntranscription factor under glucose starvation conditions by Snf1 protein kinase.\nBiol. Chem. 279, 5169-5176 (2004).\n17. Nishizawa A, Yabuta Y, Yoshida E, Maruta T, Yoshimura K, Shigeoka S.\nArabidopsis heat shock transcription factor A2 as a key regulator in response\nto several types of environmental stress. Plant J. 48, 535-547 (2006).\n18. Jedlicka P, Mortin MA, Wu C. Multiple functions of Drosophila heat shock\ntranscription factor in vivo. The EMBO J. 16, 2452-2462 (1997).\n19. Zhong M, Orosz A, Wu C. Direct sensing of heat and oxidation by Drosophila\nheat shock transcription factor. Mol. Cell 2, 101-108 (1998).\n20. Gomez-Pastor R, Burchfiel ET, Thiele DJ. Regulation of heat shock\ntranscription factors and their roles in physiology and disease. Nat. Rev. Mol.\nCell Biol. 19, 4-19 (2018).\n21. Pirkkala L, Nykänen P, Sistonen L. Roles of the heat shock transcription\nfactors in regulation of the heat shock response and beyond. The FASEB J. 15,\n1118-1131 (2001).\n22. Bailey TL, Williams N, Misleh C, Li WW. MEME: discovering and analyzing\nDNA and protein sequence motifs. Nucleic Acids Res. 34, W369-W373 (2006).\n23. Foyer CH, Hanke G. ROS production and signalling in chloroplasts:\ncornerstones and evolving concepts. Plant J. 111, 642-661 (2022).\n24. Keeling PJ, et al. The Marine Microbial Eukaryote Transcriptome Sequencing\nProject (MMETSP): illuminating the functional diversity of eukaryotic life in\nthe oceans through transcriptome sequencing. PLoS Biol. 12, e1001889 (2014).\n25. Johnson LK, Alexander H, Brown CT. Re-assembly, quality evaluation, and\nannotation of 678 microbial eukaryotic reference transcriptomes. GigaScience\n8, giy158 (2019).\n26. Boyd PW, et al. Marine phytoplankton temperature versus growth responses\nfrom polar to tropical waters–outcome of a scientific community-wide study.\nPloS one 8, e63091 (2013).\n27. Schaum C-E, Buckling A, Smirnoff N, Studholme D, Yvon-Durocher G.\nEnvironmental fluctuations accelerate molecular evolution of thermal\ntolerance in a marine diatom. Nat. Commun. 9, 1719 (2018).\n28. Sato R, Maeda Y, Yoshino T, Tanaka T, Matsumoto M. Seasonal variation of\nbiomass and oil production of the oleaginous diatom Fistulifera sp. in outdoor\nvertical bubble column and raceway-type bioreactors. J. Biosci. Bioeng. 117,\n720-724 (2014).\n29. Matsumoto M, et al. Outdoor cultivation of marine diatoms for year-round\nproduction of biofuels. Mar. Drugs 15, 94 (2017).\n30. Kudo I, Miyamoto M, Noiri Y, Maita Y. Combined effects of temperature and\niron on the growth and physiology of the marine diatom Phaeodactylum\ntricornutum (Bacillariophyceae). J. Phycol. 36, 1096-1102 (2000).\n31. De Martino A, et al. Physiological and molecular evidence that environmental\nchanges elicit morphological interconversion in the model diatom\nPhaeodactylum tricornutum. Protist 162, 462-481 (2011).\n32. Walter B, Peters J, Van Beusekom J, St. John M. Interactive effects of\ntemperature and light during deep convection: a case study on growth and\ncondition of the diatom Thalassiosira weissflogii. ICES J. Mar. Sci. 72, 2061-\n2071 (2015).\n33. Sas AAA, Arshad A, Das SK, Pau SSN, Cob ZC. Optimum Temperature and\nSalinity Conditions for Growth, Lipid Contents, and Fatty Acids Composition\nof Centric Diatoms Chaetoceros Calcitrans and Thalassiosira Weissflogii.\nPertanika Journal of Science & Technology31, 2 (2023).\n34. Chen J, et al. Off-target effects of RNAi correlate with the mismatch rate\nbetween dsRNA and non-target mRNA. RNA Biol. 18, 1747-1759 (2021).\n35. Chua G, et al. Identifying transcription factor functions and targets by\nphenotypic activation. PNAS 103, 12045-12050 (2006).\n36. Baker KG, Geider RJ. Phytoplankton mortality in a changing thermal seascape.\nGlobal Change Biol. 27, 5253-5261 (2021).\n37. Hu Z, Killion PJ, Iyer VR. Genetic reconstruction of a functional\ntranscriptional regulatory network. Nat. Genet. 39, 683-687 (2007).\n38. Gao F, Foat BC, Bussemaker HJ. Defining transcriptional networks through\nintegrative modeling of mRNA expression and transcription factor binding\ndata. BMC Bioinf. 5, 1-10 (2004).\n39. Monteiro PT, Pedreira T, Galocha M, Teixeira MC, Chaouiya C. Assessing\nregulatory features of the current transcriptional network of Saccharomyces\ncerevisiae. Sci. Rep. 10, 17744 (2020).\n40. Shlyueva D, Stampfel G, Stark A. Transcriptional enhancers: from properties\nto genome-wide predictions. Nat. Rev. Genet. 15, 272-286 (2014).\n41. Lee TI, Young RA. Transcriptional regulation and its misregulation in disease.\nCell 152, 1237-1251 (2013).\n42. Kaya-Okur HS, et al. CUT&Tag for efficient epigenomic profiling of small\nsamples and single cells. Nat. Commun. 10, 1930 (2019).\n43. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for\ntranscriptomics. Nat. Rev. Genet. 10, 57-63 (2009).\n44. Daboussi F, et al. Genome engineering empowers the diatom Phaeodactylum\ntricornutum for biotechnology. Nat. Commun. 5, 3831 (2014).\n45. Priyam A, et al. Sequenceserver: a modern graphical user interface for custom\nBLAST databases. Mol. Biol. Evol. 36, 2922-2924 (2019).\n46. Shaw PL, McAdams NM, Hast MA, Ammerman ML, Read LK, Schumacher\nMA. Structures of the T. brucei kRNA editing factor MRB1590 reveal unique\nRNA-binding pore motif contained within an ABC-ATPase fold. Nucleic\nAcids Res. 43, 7096-7109 (2015).\n47. Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: production, metabolism,\nand signaling mechanisms of malondialdehyde and 4‐hydroxy‐2‐nonenal.\nOxid. Med. Cell. Longevity 2014, 360-438 (2014).\n48. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation\nproducts: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 186, 407-\n421 (1990).\n49. Krick S, et al. Mpv17l protects against mitochondrial oxidative stress and\napoptosis by activation of Omi/HtrA2 protease. PNAS 105, 14106-14111\n(2008).\n50. Singh IS, He J-R, Calderwood S, Hasday JD. A high affinity HSF-1 binding\nsite in the 5′-untranslated region of the murine tumor necrosis factor-α gene is\na transcriptional repressor. J. Biol. Chem. 277, 4981-4988 (2002).\n51. Inouye S, et al. Heat shock transcription factor 1 opens chromatin structure of\ninterleukin-6 promoter to facilitate binding of an activator or a repressor. J.\nBiol. Chem. 282, 33210-33217 (2007).\n52. Brunquell J, Morris S, Lu Y, Cheng F, Westerheide SD. The genome-wide role\nof HSF-1 in the regulation of gene expression in Caenorhabditis elegans.\nBMC Genomics 17, 1-18 (2016).\n53. Hahn JS, Neef DW, Thiele DJ. A stress regulatory network for co‐ordinated\nactivation of proteasome expression mediated by yeast heat shock\ntranscription factor. Mol. Microbiol. 60, 240-251 (2006).\n54. Wilkerson DC, Skaggs HS, Sarge KD. HSF2 binds to the Hsp90, Hsp27, and\nc-Fos promoters constitutively and modulates their expression. Cell Stress\nChaperones 12, 283 (2007).\n55. Chen R, Liliental J, Kowalski P, Lu Q, Cohen S. Regulation of transcription of\nhypoxia-inducible factor-1α (HIF-1α) by heat shock factors HSF2 and HSF4.\nOncogene 30, 2570-2580 (2011).\n56. Smith RS, et al. HSF2 cooperates with HSF1 to drive a transcriptional\nprogram critical for the malignant state. Sci. Adv. 8, eabj6526 (2022).\n57. Schramm F, Ganguli A, Kiehlmann E, Englich G, Walch D, von Koskull-\nDöring P. The heat stress transcription factor HsfA2 serves as a regulatory\namplifier of a subset of genes in the heat stress response in Arabidopsis. Plant\nMol. Biol. 60, 759-772 (2006).\n58. Seckbach J, Kociolek P. The Diatom World, vol. 19. Springer Science &\nBusiness Media, (2011).\n59. Bulankova P, Bilcke G, Vyverman W, De Veylder L. Cellular hallmarks and\nregulation of the diatom cell cycle. The Molecular Life of Diatoms, Springer\nInternational Publishing, 229-263 (2022).\n60. Zachleder V, Bišová K, Vítová M. The cell cycle of microalgae. The\nphysiology of microalgae,Springer International Publishing, 3-46 (2016).\n61. Goranov AI, et al. The rate of cell growth is governed by cell cycle stage.\nGenes Dev. 23, 1408-1422 (2009).\n62. Fattorini N, Maier UG. Targeting of proteins to the cell wall of the diatom\nThalassiosira pseudonana. Discover Mater. 1, 1-10 (2021).\n63. Bišová K, Zachleder V. Cell-cycle regulation in green algae dividing by\nmultiple fission. J. Exp. Bot. 65, 2585-2602 (2014).\n64. Heldt FS, Tyson JJ, Cross FR, Novák B. A single light-responsive sizer can\ncontrol multiple-fission cycles in Chlamydomonas. Curr. Biol. 30, 634-644.\ne637 (2020).\n65. Talia SD, Skotheim JM, Bean JM, Siggia ED, Cross FR. The effects of\nmolecular noise and size control on variability in the budding yeast cell cycle.\nNature 448, 947-951 (2007).\n66. Garmendia-Torres C, Tassy O, Matifas A, Molina N, Charvin G. Multiple\ninputs ensure yeast cell size homeostasis during cell cycle progression. Elife 7,\ne34025 (2018).\n67. D’Ario M, et al. Cell size controlled in plants using DNA content as an\ninternal scale. Science 372, 1176-1181 (2021).\n68. R. Jones A, et al. Cell-size dependent progression of the cell cycle creates\nhomeostasis and flexibility of plant cell size. Nat. Commun. 8, 15060 (2017).\n69. Dolznig H, Grebien F, Sauer T, Beug H, Müllner EW. Evidence for a size-\nsensing mechanism in animal cells. Nat. Cell Biol. 6, 899-905 (2004).\n70. Tzur A, Kafri R, LeBleu VS, Lahav G, Kirschner MW. Cell growth and size\nhomeostasis in proliferating animal cells. Science 325, 167-171 (2009).\n71. Schmoller KM, Turner J, Kõivomägi M, Skotheim JM. Dilution of the cell\ncycle inhibitor Whi5 controls budding-yeast cell size. Nature 526, 268-272\n(2015).\n72. Medina EM, Turner JJ, Gordan R, Skotheim JM, Buchler NE. Punctuated\nevolution and transitional hybrid network in an ancestral cell cycle of fungi.\nElife 5, e09492 (2016).\n73. Dyson N. The regulation of E2F by pRB-family proteins. Genes Dev. 12,\n2245-2262 (1998).\n74. Zatulovskiy E, Zhang S, Berenson DF, Topacio BR, Skotheim JM. Cell growth\ndilutes the cell cycle inhibitor Rb to trigger cell division. Science 369, 466-471\n(2020).\n75. Dolfi SC, et al. The metabolic demands of cancer cells are coupled to their\nsize and protein synthesis rates. Cancer Metab. 1, 1-13 (2013).\n76. Finkel ZV, Beardall J, Flynn KJ, Quigg A, Rees TAV, Raven JA.\nPhytoplankton in a changing world: cell size and elemental stoichiometry. J.\nPlankton Res. 32, 119-137 (2010).\n77. Litchman E, Klausmeier CA, Schofield OM, Falkowski PG. The role of\nfunctional traits and trade‐offs in structuring phytoplankton communities:\nscaling from cellular to ecosystem level. Ecol. Lett. 10, 1170-1181 (2007).\n78. Wu LF, Chen PC, Lee CM. The effects of nitrogen sources and temperature on\ncell growth and lipid accumulation of microalgae. Int. Biodeterior. Biodegrad.\n85, 506-510 (2013).\n79. Li M, Young JN. Temperature sensitivity of carbon concentrating mechanisms\nin the diatom Phaeodactylum tricornutum. Photosynth. Res. 156, 205-215\n(2023).\n80. Fawley MW. Effects of light intensity and temperature interactions on growth\ncharacteristics of Phaeodactylum tricornutum (bacillariophyceae) 1. J. Phycol.\n20, 67-72 (1984).\n81. Siegel P, Baker KG, Low‐Décarie E, Geider RJ. High predictability of direct\ncompetition between marine diatoms under different temperatures and nutrient\nstates. Ecol. Evol. 10, 7276-7290 (2020).\n82. Sheehan CE, Baker KG, Nielsen DA, Petrou K. Temperatures above thermal\noptimum reduce cell growth and silica production while increasing cell\nvolume and protein content in the diatom Thalassiosira pseudonana.\nHydrobiologia 847, 4233-4248 (2020).\n83. Hattich G, et al. Temperature optima of a natural diatom population increases\nas global warming proceeds. Nat. Clim. Change, 14, 518-525 (2024).\n84. Liang Y, Koester JA, Liefer JD, Irwin AJ, Finkel ZV. Molecular mechanisms\nof temperature acclimation and adaptation in marine diatoms. ISME J. 13,\n2415-2425 (2019).\n85. Tew KS, et al. Effects of elevated CO2 and temperature on the growth,\nelemental composition, and cell size of two marine diatoms: potential\nimplications of global climate change. Hydrobiologia 741, 79 - 87 (2014).\n86. Suzuki Y, Takahashi M. Growth responses of several diatom species isolated\nfrom various environments to temperature. J. Phycol. 31, 880-888 (1995).\n87. Koehlinger JA, Newton J, Mickett J, Thompson L, Klinger T. Large and\ntransient positive temperature anomalies in Washington’s coastal nearshore\nwaters during the 2013–2015 northeast Pacific marine heatwave. Plos one 18,\ne0280646 (2023).\n88. Li Z, Wan L, Liu Y, Wang Z, Wu L. Analysis of Marine Heatwaves in China’s\ncoastal seas and adjacent offshore waters. Atmosphere 14, 1738 (2023).\n89. Martino AD, Meichenin A, Shi J, Pan K, Bowler C. Genetic and phenotypic\ncharacterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions\n1. J. Phycol. 43, 992-1009 (2007).\n90. Siaut M, et al. Molecular toolbox for studying diatom biology in\nPhaeodactylum tricornutum. Gene 406, 23-35 (2007).\n91. Ma Y-H, et al. Antisense knockdown of pyruvate dehydrogenase kinase\npromotes the neutral lipid accumulation in the diatom Phaeodactylum\ntricornutum. Microb. Cell Fact. 13, 1-9 (2014).\n92. Bustin SA, et al. The MIQE Guidelines: Minimum information for publication\nof quantitative real-time PCR experiments. Clin. Chem. 55, 611-622 (2009).\n93. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative\nCT method. Nat. Protoc. 3, 1101-1108 (2008).\n94. Li D-W, et al. Transcriptional regulation of microalgae for concurrent lipid\noverproduction and secretion. Sci. Adv. 5, eaau3795 (2019).\n95. Vernette C, et al. The Ocean Gene Atlas v2. 0: online exploration of the\nbiogeography and phylogeny of plankton genes. Nucleic Acids Res. 50, W516-\nW526 (2022).\nResponse to Editor and Reviewers\nWe would like to thank the editor and reviewers for their time and valuable feedback,\nwhich have been very helpful in improving the clarity and quality of our manuscript.\nBelow, we provide detailed responses to each comment and indicate the changes made\nin the manuscript.\nReviewer #1 (Remarks to the Author):\nOverall, this manuscript is much improved following the authors’ response to the\ncomments from all reviewers. The additional experiments helped to clarify the\nrelationships between the investigated genes (PtHSF2 and PtCDC45) and their\ninfluence on the thermal tolerance in the diatom Phaedactylum tricornutum. I have no\nmajor comments but just two minor comments below.\nLine 138- 140 — I am still missing a better explanation of why you focused solely on\nthe PtHSF2. It is mentioned that this the transcripts of this HSF varies significantly with\nthe temperature. But is this different from the other HSFs? Could you show the\nunderlying data for other HSF to illustrate better your choice of HSF2?\nResponse: Thank you for your valuable feedback. Based on RNA-seq and differential\ngene expression analysis from our preliminary experiments, we found that PtHSF2\n(Phatr3_J55070) is the most significantly upregulated HSF under elevated temperature\nconditions (Supplementary Table 3). Specifically, P. tricornutum was cultured at 20°C\nand 30°C for 7 days, followed by RNA-seq analysis. Differential expression analysis\nwas conducted using the criteria of |log₂(fold change)| > 1 to identify significantly\ndifferentially expressed genes. The results revealed a significant upregulation of\nPtHSF2 expression with the temperature shift from 20°C to 30°C, making it the top-\nupregulated HSF among all detected heat shock factors. Based on these findings, we\nhypothesize that PtHSF2 plays a key role in the heat stress response, which led us to\nfocus on its functional characterization in this manuscript. (L. No. 135-137)\nLine 278-282 - I recommend calling the identified sequence “putative consensus motif”.\nConfirmation that it is the main target sequence of HSF2 binding needs additional\nexperiments.\nResponse:Thank you for your insightful suggestion. We agree with your viewpoint and\nhand\nhave revised the manuscript to describe the sequence as a “putative consensus motif,”\nappropriately reflecting the preliminary nature of our findings. (L. No. 262)\nReviewer #2 (Remarks to the Author):\nMy concerns have been addressed in the revisions.\nResponse: We sincerely appreciate your positive feedback and are delighted that our\nrevisions have effectively addressed your concerns.\nReviewer #3 (Remarks to the Author):\nThe manuscript has been substantially improved. Here are some minor comments.\n1. The author address needs to be checked.\nResponse: Thank you for bringing this to our attention. We have thoroughly reviewed\nand revised the author address to ensure its consistency and accuracy across the\nmanuscript. (L. No. 16)\n2. Line 139, how about the transcriptional expression of other HSFs? Average value\nsignificantly lower than HSF2?\nResponse: Thank you for your valuable comment. In our preliminary experiments, P.\ntricornutum was cultured at 20°C and 30°C for 7 days,followed by RNA-seq analysis.\nUsing a threshold of |log₂(fold change)| > 1 to identify significantly differentially\nexpressed genes, we found that PtHSF2 (Phatr3_J55070) exhibited the most significant\nupregulation among all differentially expressed heat shock factors (Supplementary\nTable 3). Based on these findings, we hypothesize that PtHSF2 is actively involved in\nthe regulation of the heat stress response, which is why we focused on investigating its\nrole in the heat stress response mechanism in this study. (L. No. 135-137)\n3. Line 168, please state at what growth condition and stage, the expression level was\ndetermined for comparison among different strains.\nResponse: Thank you for your insightful comment.Cells used for molecular validation\nwere cultured under standard conditions (20 °C) until they reached the exponential\ngrowth phase to ensure consistent comparisons among different strains. We have\nclarified these growth conditions and the stage in the Methods section. (L. No. 725-727)\n4. Supplementary Fig 5B, according to Supple Table 1, should be Fugacium kawagutii.\nResponse: We thank the reviewer for carefully pointing out the discrepancy. We agree\nwith the reviewer’s observation and have corrected the Supplementary Fig. 5B to\naccurately reflect Fugacium kawagutii. (Supplementary Fig. 5B)\n5. Better visualize the expression level (in supplementary figure) of key genes involved\nwith cell-cycle arrest as listed in Supplementary Table 6, i.e. CDC families should be\nanalyzed generally.\nResponse: We appreciate this constructive suggestion. To address your concerns\ncomprehensively, we have improved the visualization of the significantly differentially\nexpressed genes (DEGs) involved in cell cycle regulation, as listed in Supplementary\nTable 7.Among these genes, only Cdc45-like belongs to the CDC family.Furthermore,\nwe have incorporated a discussion based on current literature, hypothesizing the\nmechanisms by which the altered expression of these genes may contribute to cell cycle\narrest. (L. No. 519-534, Supplementary Fig. 8B)\nReviewer #4 (Remarks to the Author):\nI thank the authors for having addressed my comments points by points. The additional\nresults and information provided are substantial. To my understanding, however, I\nreckon that some of these results are not consistent with a natural role (i.e. in WT) of\nPtHSF2 in P. tricornutum thermal tolerance. This is based on the following three points:\n(1) Cell volume and tolerance to increased temperature – Fig.1E and 3D.\nIt is correct that the volume of P. tricornutum WT cell increases (by only ca. 10-20%)\nin response to increased temperature (references 79 and 80 provided in the response\nand this work). However, these references and the present work do not establish a causal\nrelationship between the increase in cell volume and the tolerance to elevated\ntemperature. In addition, the PtHSF2 and PtCdc45-like overexpressing and knock-\ndown lines, as the WT line, do present such increase in cell volume in response to\nincreased temperature suggesting that this phenomenon, if of any role in the acclimation\nto elevated temperature, is independent of PtHSF2 and PtCdc45-like.\nResponse: We thank the reviewer for this valuable comment.We fully acknowledge the\npoint raised. While both the literature and our current findings indicate a modest\nincrease in cell volume in response to elevated temperature, we recognize that this\nchange is not directly correlated with enhanced thermal tolerance. The observed\nincrease in cell volume seems to be a general physiological response of P. tricornutum\nto temperature stress, rather than a specific adaptive mechanism contributing to thermal\ntolerance.In light of this, we have revised the manuscript to clarify that the increase in\ncell volume is a non-specific acclimation response, and we have reframed our\nconclusions to avoid any direct linkage between cell volume expansion and increased\nthermal tolerance. (L. No. 456-462)\nIn addition, we greatly appreciate the reviewer’s perspective that cell volume changes\nmay be independent of PtHSF2 and PtCdc45-like regulation. However, based on our\nfindings, we propose that these genes play a critical role in modulating cell volume.\nSpecifically, while the RNAi lines exhibit a slight increase in cell volume under high-\ntemperature stress, their volumes remain significantly smaller than those of the WT.\nConversely, the overexpression lines show a pronounced increase in cell volume\ncompared to the WT. These results are consistent with observations under non-stress\nconditions (20°C), where PtHSF2 and PtCdc45-like overexpression led to increased\ncell volume, while knockdown resulted in reduced cell volume (Fig. 1E and Fig. 3D).\nTherefore, while the increase in cell volume under high-temperature stress may indeed\nrepresent an acclimation response, the distinct differences observed among WT,\noverexpression, and knockdown lines suggest that PtHSF2 and PtCdc45-like contribute\nto cell volume regulation. We acknowledge the complexity of this phenomenon and\nhave carefully revised the manuscript to highlight that other regulatory mechanisms\nmay also be involved. (L. No. 216-217, 339-342, 348, 372-376, 519-534,\nSupplementary Table 7; Supplementary Fig. 6E, F; Supplementary Fig. 8B;\nSupplementary Fig. 9)\nWe hope that this clarification and the revisions made in the manuscript address the\nreviewer’s concerns.\n(2) Transcriptional regulation of PtHSF2 and Cdc45-like – Fig.1A and 3A.\nThere is no clear positive correlation in the WT line between the expression levels of\nPtHSF2 and PtCdc45-like and the temperature increase. Especially, the transcription\nlevels of PtHSF2 and Cdc45-like are lower at 30C than at 25C. This does not agree with\nthe observation that the PtHSF2 and PtCdc45-like overexpressing lines present an\nincreased tolerance to 30C compared to the WT line.\nResponse: We appreciate the reviewer’s detailed comment and provide the following\nexplanations to address the concerns raised:\n1. Nonlinear regulation of gene transcription in response to environmental\nstimuli: Environmental stimuli often induce gene expression changes that do\nnot necessarily exhibit a linear correlation with the intensity of the stimulus.Our\nresults indicate that while the transcription levels of PtHSF2 and PtCdc45-like\nat 30°C were lower than those at 25°C, their levels at 30°C were significantly\nhigher than at the optimal growth temperature of 20°C (Fig. 1A and Fig. 3A).\nThis demonstrates a clear responsiveness of these genes to elevated\ntemperatures, although the relationship between transcription levels and\ntemperature does not follow a simple linear trend. Similar nonlinear\ntranscriptional responses have been widely documented in other systems. For\ninstance: In yeast species exposed to temperatures ranging from 10°C to 46°C,\nHsp70 expression peaked 6–8°C above the optimal growth temperature, with\nexpression levels declining at higher temperatures 1; In Heterosigma akashiwo,\nCO₂-responsive genes, such as SLC4 (SLC4 family bicarbonate transporter),\nALAT_GGAT (alanine aminotransferase), and CA (carbonic anhydrase),\nexhibited peak transcription levels at 400–600 ppm CO₂ but decreased at higher\nconcentrations 2; In human embryonic kidney (HEK) cells and cochlear tissues\nexposed to cisplatin-induced stress, the transcription levels of stress-related\ngenes such as Chop (C/EBP homologous protein) and DR5 (death receptor 5)\ninitially rose with increasing cisplatin concentrations but decreased once a\nthreshold concentration was surpassed 3; Similarly, in human intervertebral disc\nNP (nucleus pulposus) cells exposed to varying pH levels (7.4, 7.1, 6.8, and 6.5),\nthe expression of TIMP-1 (tissue inhibitor of metalloproteinases 1) initially\ndecreased with increasing acidity (pH 7.4–7.1) but then increased as the pH\ndecreased further (pH 7.1–6.5) 4.\n2. The dual role of HSFs in cold and heat stress responses: HSFs are well-\nestablished mediators of abiotic stress responses 5, with individual HSFs often\nresponding to multiple stressors 6. For example, in wheat seedlings, TaHSF3 is\nupregulated under both low and high temperatures, and its overexpression in\nArabidopsis enhances tolerance to extreme temperatures 7. Similarly, in animals,\nthe relative expression levels of HSF1 and HSP70 have been observed to\nincrease under both heat and cold stress conditions 8. In alignment with these,\nour findings demonstrate that PtHSF2 was significantly upregulated at both low\n(15°C) and high temperatures (25°C and 30°C) (Fig. 1A), highlighting its dual\nrole in mediating responses to both cold and heat stress.\n3. The incomplete correlation between gene transcription levels and protein\nabundance: In both bacteria and eukaryotes, intracellular protein\nconcentrations are generally correlated with the abundances of their\ncorresponding mRNAs, but the correlation is not particularly strong, with the\nsquared Pearson correlation coefficient (R²) typically around 0.40 9. This\nsuggests that a decrease in transcription levels does not necessarily result in a\nproportional decrease in protein abundance. Additionally, during stress\nresponses, post-transcriptional and post-translational regulatory mechanisms\nmay play critical roles in modulating the levels and functions of mRNAs and\nproteins 10. Therefore, it is insufficient to evaluate gene function based solely\non transcriptional levels. Moreover, a decline in transcription levels may\nrepresent an energy-saving strategy under extreme conditions.\nIn conclusion, although the transcription levels of PtHSF2 and PtCdc45-like at 30°C\nwere lower than those at 25°C, they remained significantly elevated at both 25°C and\n30°C compared to those at the optimal growth temperature (20°C). These findings\nsupport the roles of these genes in temperature stress responses.\nTo address this apparent inconsistency, we further assessed the protein expression\nlevels of PtHSF2 and PtCdc45-like under different temperature conditions. As shown\nin Supplementary Fig. 2F and Supplementary Fig. 7F, PtHSF2 levels in the PtHSF2-\nproH1 strain, driven by the HSF2 promoter, and PtCdc45-like levels in the PtCdc45-\nlike-proC1 strain, driven by the Cdc45-like promoter, exhibit a clear linear increase\nwith rising temperatures. These results suggest that post-transcriptional or translational\nregulation could contribute to the observed increased protein abundance at 30°C,\naligning with the enhanced heat tolerance observed in the overexpression lines. (L. No.\n163-166, 295-298, 467-497)\n(3) Thermal tolerance of siPtHSF2 and siPtCdc45-like lines – Fig.1C and 3C.\nThe siPtHSF2 and siPtCdc45-like lines present a tolerance to increased temperature\nsimilar to the WT line suggesting that PtHSF2 and Cdc45-like do not play a crucial role\nin thermal tolerance.\nResponse: We appreciate the reviewer’s thoughtful observation regarding the thermal\ntolerance of siPtHSF2 and siPtCdc45-like lines presented in Fig. 1C and Fig. 3C. The\ncomment that these lines exhibit a thermal tolerance similar to the WT raises important\npoints regarding the roles of PtHSF2 and PtCdc45-like in thermal stress responses.\nBelow, we provide a detailed response and clarification:\n1. Residual expression of target genes: While RNAi-mediated knockdown\neffectively reduced PtHSF2 and PtCdc45-like transcription levels, it did not\nachieve complete suppression. This incomplete silencing likely retains a level\nof gene functionality sufficient to sustain the baseline thermal tolerance\nobserved in these lines. As shown in Supplementary Fig. 2D and Supplementary\nFig. 7D, the gene expression levels of PtHSF2 and PtCdc45-like were reduced\nby approximately 77.9% and 73.5%, respectively, leaving residual activity that\nmay obscure phenotypic differences under the tested conditions.\n2. Potential compensatory mechanisms: Thermal tolerance is likely governed\nby a network of genes and pathways with overlapping or compensatory\nfunctions. While PtHSF2 and PtCdc45-like are involved in thermal stress\nresponses, other heat shock factors or regulatory proteins may compensate for\ntheir partial loss of function. Notably, P. tricornutum possesses 69 heat shock\nfactors (HSFs), suggesting potential redundancy or functional overlap. We\nhypothesize that homologous genes may compensate for the loss of PtHSF2 in\nsiPtHSF2 lines, preserving thermal tolerance.\n3. The potential indirect regulatory role of PtCdc45-like: Additionally, we have\nreconsidered the function of PtCdc45-like in thermal responses. Given its\nestablished role in DNA replication during the S phase, the enhanced thermal\ntolerance observed in PtCdc45-like overexpression lines may result indirectly\nfrom its effects on DNA replication and cell cycle regulation. Our findings\nreveal that PtCdc45-like overexpression induced slower cell growth (Fig. 3C)\nand cell cycle arrest (Supplementary Fig. 8A) under non-stress conditions\n(20°C). These findings align with previous studies indicating that Cdc45\noverexpression causes severe replication stress, leading to cell cycle arrest and\nproliferation inhibition 11, 12. Mechanistically, replication stress caused by\nCdc45 overexpression results in replication fork stalling and activates the\nATM/Chk2 (ataxia telangiectasia mutated/checkpoint kinase 2) signaling\npathway 11. Our findings reveal that under non-temperature stress conditions,\nthe overexpression of PtHSF2 and PtCdc45-like markedly increased the\ntranscriptional levels of ATM and ATR (ataxia telangiectasia and Rad3-related)\nlevels (Supplementary Table 7; Supplementary Fig. 8B; Supplementary Fig. 9\nA, B).\nATM and ATR are critical kinases that govern cell cycle and replication\ncheckpoints by phosphorylating the checkpoint effector kinases Chk2 and Chk1\nin response to DNA damage 13. This activation initiates DNA repair mechanisms,\nensuring that damaged DNA is adequately repaired before progression into the\nnext cell cycle phase 13. Our results indicate that at 30°C, the transcriptional\nlevels of ATM and ATR were significantly elevated in both PtHSF2 and\nPtCdc45-like overexpression lines (Supplementary Fig. 6E, F; Supplementary\nFig. 9C, D). This upregulation likely promotes the repair of heat-induced DNA\ndamage, thereby enhancing cellular heat stress responses and improving\nsurvival under elevated temperatures.\nWe have revised the Discussion section to address this comment and provide further\nclarification. We hope this explanation comprehensively addresses the reviewer’s\nconcerns. (L. No. 519-546)\nIt is a consequent and important work, but as it stands, I think that the results are not\nsupporting and congruent enough for the manuscript to be published in Nature\nCommunications.\nReferences\n1. Keyport Kik S, et al. An adaptive biomolecular condensation response is\nconserved across environmentally divergent species. Nat. Commun. 15, 3127\n(2024).\n2. Hennon GM, Williamson OM, Limón MDH, Haley ST, Dyhrman ST. Non-\nlinear physiology and gene expression responses of harmful alga Heterosigma\nakashiwo to rising CO2. Protist 170, 38-51 (2019).\n3. Li J, et al. Modulating the unfolded protein response with ISRIB mitigates\ncisplatin ototoxicity. Sci. Rep.14, 22382 (2024).\n4. Gilbert HT, Hodson N, Baird P, Richardson SM, Hoyland JA. Acidic pH\npromotes intervertebral disc degeneration: Acid-sensing ion channel-3 as a\npotential therapeutic target. Sci. Rep. 6, 37360 (2016).\n5. Guo M, Liu J-H, Ma X, Luo D-X, Gong Z-H, Lu M-H. The plant heat stress\ntranscription factors (HSFs): structure, regulation, and function in response to\nabiotic stresses. Front. Plant Sci. 7, 114 (2016).\n6. Liu HC, Liao HT, Charng YY. The role of class A1 heat shock factors (HSFA1s)\nin response to heat and other stresses in Arabidopsis. Plant Cell Environ. 34,\n738-751 (2011).\n7. Zhang S, et al. Overexpression of TaHSF3 in transgenic Arabidopsis enhances\ntolerance to extreme temperatures. Plant Mol. Biol. Rep.31, 688-697 (2013).\n8. Pawar HN, Kumar GR, Narang R, Agrawal RK. Heat and cold stress enhances\nthe expression of heat shock protein 70, heat shock transcription factor 1 and\ncytokines (IL-12, TNF-and GMCSF) in buffaloes. Int. J. Curr. Microbiol. App.\nSci. 3, 307-317 (2014).\n9. Vogel C, Marcotte EM. Insights into the regulation of protein abundance from\nproteomic and transcriptomic analyses. Nat. Rev. Genet. 13, 227-232 (2012).\n10. Guerra D, Crosatti C, Khoshro HH, Mastrangelo AM, Mica E, Mazzucotelli E.\nPost-transcriptional and post-translational regulations of drought and heat\nresponse in plants: a spider’s web of mechanisms. Front. Plant Sci.6, 57 (2015).\n11. Köhler C, et al. Cdc45 is limiting for replication initiation in humans. Cell Cycle\n15, 974-985 (2016).\n12. Fu Y, Lv Z, Kong D, Fan Y, Dong B. High abundance of CDC45 inhibits cell\nproliferation through elevation of HSPA6. Cell Proliferation55, e13257 (2022).\n13. Smith J, Tho LM, Xu N, Gillespie DA. The ATM–Chk2 and ATR–Chk1\npathways in DNA damage signaling and cancer. Adv Cancer Res. 108, 73-112\n(2010)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Overall, this manuscript is much improved following the authors’ response to the comments from all reviewers. The additional experiments helped to clarify the relationships between the investigated genes (PtHSF2 and PtCDC45) and their influence on the thermal tolerance in the diatom Phaedactylum tricornutum. I have no major comments but just two minor comments below. \nLine 138- 140 — I am still missing a better explanation of why you focused solely on the PtHSF2. It is mentioned that this the transcripts of this HSF varies significantly with the temperature. But is this different from the other HSFs? Could you show the underlying data for other HSF to illustrate better your choice of HSF2?\nLine 278-282 - I recommend calling the identified sequence “ putative consensus motif”. Confirmation that it is the main target sequence of HSF2 binding needs additional experiments.", "reviewer_2": "My concerns have been addressed in the revisions.", "reviewer_3": "The manuscript has been substantially improved. Here are some minor comments.\n1.The author address needs to be checked.\n2.Line 139, how about the transcriptional expression of other HSFs? Average value significantly lower than HSF2?\n3.Line 168, please state at what growth condition and stage, the expression level was determined for comparison among different strains.\n4.Supplementary Fig 5B, according to Supple Table 1, should be Fugacium kawagutii.\n5.Better visualize the expression level (in supplementary figure) of key genes involved with cell-cycle arrest as listed in Supplementary Table 6, i.e. CDC families should be analyzed generally.", "reviewer_4": "I thank the authors for having addressed my comments points by points. The additional results and information provided are substantial. To my understanding, however, I reckon that some of these results are not consistent with a natural role (i.e. in WT) of PtHSF2 in P. tricornutum thermal tolerance. This is based on the following three points:\n(1) Cell volume and tolerance to increased temperature – Fig.1E and 3D.\nIt is correct that the volume of P. tricornutum WT cell increases (by only ca. 10-20%) in response to increased temperature (references 79 and 80 provided in the response and this work). However, these references and the present work do not establish a causal relationship between the increase in cell volume and the tolerance to elevated temperature. In addition, the PtHSF2 and PtCdc45-like overexpressing and knock-down lines, as the WT line, do present such increase in cell volume in response to increased temperature suggesting that this phenomenon, if of any role in the acclimation to elevated temperature, is independent of PtHSF2 and PtCdc45-like.\n(2) Transcriptional regulation of PtHSF2 and Cdc45-like – Fig.1A and 3A.\nThere is no clear positive correlation in the WT line between the expression levels of PtHSF2 and PtCdc45-like and the temperature increase. Especially, the transcription levels of PtHSF2 and Cdc45-like are lower at 30C than at 25C. This does not agree with the observation that the PtHSF2 and PtCdc45-like overexpressing lines present an increased tolerance to 30C compared to the WT line.\n(3) Thermal tolerance of siPtHSF2 and siPtCdc45-like lines – Fig.1C and 3C.\nThe siPtHSF2 and siPtCdc45-like lines present a tolerance to increased temperature similar to the WT line suggesting that PtHSF2 and Cdc45-like do not play a crucial role in thermal tolerance.\nIt is a consequent and important work, but as it stands, I think that the results are not supporting and congruent enough for the manuscript to be published in Nature Communications."}, "author_response": "Response to Editor and Reviewers\nWe would like to thank the editor and reviewers for their time and valuable feedback, which have been very helpful in improving the clarity and quality of our manuscript.\nBelow, we provide detailed responses to each comment and indicate the changes made in the manuscript.\nReviewer #1 (Remarks to the Author):\nOverall, this manuscript is much improved following the authors’ response to the comments from all reviewers. The additional experiments helped to clarify the relationships between the investigated genes (PtHSF2 and PtCDC45) and their influence on the thermal tolerance in the diatom Phaedactylum tricornutum. I have no major comments but just two minor comments below.\nLine 138- 140 — I am still missing a better explanation of why you focused solely on the PtHSF2. It is mentioned that this the transcripts of this HSF varies significantly with the temperature. But is this different from the other HSFs? Could you show the underlying data for other HSF to illustrate better your choice of HSF2?\nResponse: Thank you for your valuable feedback. Based on RNA-seq and differential gene expression analysis from our preliminary experiments, we found that PtHSF2 (Phatr3_J55070) is the most significantly upregulated HSF under elevated temperature conditions (Supplementary Table 3). Specifically, P. tricornutum was cultured at 20°C and 30°C for 7 days, followed by RNA-seq analysis. Differential expression analysis was conducted using the criteria of |log₂(fold change)| > 1 to identify significantly differentially expressed genes. The results revealed a significant upregulation of PtHSF2 expression with the temperature shift from 20°C to 30°C, making it the top-upregulated HSF among all detected heat shock factors. Based on these findings, we hypothesize that PtHSF2 plays a key role in the heat stress response, which led us to focus on its functional characterization in this manuscript. (L. No. 135-137)\nLine 278-282 - I recommend calling the identified sequence “putative consensus motif”. Confirmation that it is the main target sequence of HSF2 binding needs additional experiments.\nResponse:Thank you for your insightful suggestion. We agree with your viewpoint and have revised the manuscript to describe the sequence as a “putative consensus motif,” appropriately reflecting the preliminary nature of our findings. (L. No. 262)\nReviewer #2 (Remarks to the Author):\nMy concerns have been addressed in the revisions.\nResponse: We sincerely appreciate your positive feedback and are delighted that our revisions have effectively addressed your concerns.\nReviewer #3 (Remarks to the Author):\nThe manuscript has been substantially improved. Here are some minor comments.\n1. The author address needs to be checked.\nResponse: Thank you for bringing this to our attention. We have thoroughly reviewed and revised the author address to ensure its consistency and accuracy across the manuscript. (L. No. 16)\n2. Line 139, how about the transcriptional expression of other HSFs? Average value significantly lower than HSF2?\nResponse: Thank you for your valuable comment. In our preliminary experiments, P. tricornutum was cultured at 20°C and 30°C for 7 days,followed by RNA-seq analysis.\nUsing a threshold of |log₂(fold change)| > 1 to identify significantly differentially expressed genes, we found that PtHSF2 (Phatr3_J55070) exhibited the most significant upregulation among all differentially expressed heat shock factors (Supplementary Table 3). Based on these findings, we hypothesize that PtHSF2 is actively involved in the regulation of the heat stress response, which is why we focused on investigating its role in the heat stress response mechanism in this study. (L. No. 135-137)\n3. Line 168, please state at what growth condition and stage, the expression level was determined for comparison among different strains.\nResponse: Thank you for your insightful comment.Cells used for molecular validation were cultured under standard conditions (20 °C) until they reached the exponential growth phase to ensure consistent comparisons among different strains. We have clarified these growth conditions and the stage in the Methods section. (L. No. 725-727)\n4. Supplementary Fig 5B, according to Supple Table 1, should be Fugacium kawagutii.\nResponse: We thank the reviewer for carefully pointing out the discrepancy. We agree with the reviewer’s observation and have corrected the Supplementary Fig. 5B to accurately reflect Fugacium kawagutii. (Supplementary Fig. 5B)\n5. Better visualize the expression level (in supplementary figure) of key genes involved with cell-cycle arrest as listed in Supplementary Table 6, i.e. CDC families should be analyzed generally.\nResponse: We appreciate this constructive suggestion. To address your concerns comprehensively, we have improved the visualization of the significantly differentially expressed genes (DEGs) involved in cell cycle regulation, as listed in Supplementary Table 7.Among these genes, only Cdc45-like belongs to the CDC family.Furthermore, we have incorporated a discussion based on current literature, hypothesizing the mechanisms by which the altered expression of these genes may contribute to cell cycle arrest. (L. No. 519-534, Supplementary Fig. 8B)\nReviewer #4 (Remarks to the Author):\nI thank the authors for having addressed my comments points by points. The additional results and information provided are substantial. To my understanding, however, I reckon that some of these results are not consistent with a natural role (i.e. in WT) of PtHSF2 in P. tricornutum thermal tolerance. This is based on the following three points:\n(1) Cell volume and tolerance to increased temperature – Fig.1E and 3D.\nIt is correct that the volume of P. tricornutum WT cell increases (by only ca. 10-20%) in response to increased temperature (references 79 and 80 provided in the response and this work). However, these references and the present work do not establish a causal relationship between the increase in cell volume and the tolerance to elevated temperature. In addition, the PtHSF2 and PtCdc45-like overexpressing and knock-down lines, as the WT line, do present such increase in cell volume in response to increased temperature suggesting that this phenomenon, if of any role in the acclimation to elevated temperature, is independent of PtHSF2 and PtCdc45-like.\nResponse: We thank the reviewer for this valuable comment.We fully acknowledge the point raised. While both the literature and our current findings indicate a modest increase in cell volume in response to elevated temperature, we recognize that this change is not directly correlated with enhanced thermal tolerance. The observed increase in cell volume seems to be a general physiological response of P. tricornutum to temperature stress, rather than a specific adaptive mechanism contributing to thermal tolerance.In light of this, we have revised the manuscript to clarify that the increase in cell volume is a non-specific acclimation response, and we have reframed our conclusions to avoid any direct linkage between cell volume expansion and increased thermal tolerance. (L. No. 456-462)\nIn addition, we greatly appreciate the reviewer’s perspective that cell volume changes may be independent of PtHSF2 and PtCdc45-like regulation. However, based on our findings, we propose that these genes play a critical role in modulating cell volume. Specifically, while the RNAi lines exhibit a slight increase in cell volume under high-temperature stress, their volumes remain significantly smaller than those of the WT. Conversely, the overexpression lines show a pronounced increase in cell volume compared to the WT. These results are consistent with observations under non-stress conditions (20°C), where PtHSF2 and PtCdc45-like overexpression led to increased cell volume, while knockdown resulted in reduced cell volume (Fig. 1E and Fig. 3D). Therefore, while the increase in cell volume under high-temperature stress may indeed represent an acclimation response, the distinct differences observed among WT, overexpression, and knockdown lines suggest that PtHSF2 and PtCdc45-like contribute to cell volume regulation. We acknowledge the complexity of this phenomenon and have carefully revised the manuscript to highlight that other regulatory mechanisms may also be involved. (L. No. 216-217, 339-342, 348, 372-376, 519-534, Supplementary Table 7; Supplementary Fig. 6E, F; Supplementary Fig. 8B; Supplementary Fig. 9)\nWe hope that this clarification and the revisions made in the manuscript address the reviewer’s concerns.\n(2) Transcriptional regulation of PtHSF2 and Cdc45-like – Fig.1A and 3A.\nThere is no clear positive correlation in the WT line between the expression levels of PtHSF2 and Cdc45-like and the temperature increase. Especially, the transcription levels of PtHSF2 and Cdc45-like are lower at 30C than at 25C. This does not agree with the observation that the PtHSF2 and PtCdc45-like overexpressing lines present an increased tolerance to 30C compared to the WT line.\nResponse: We appreciate the reviewer’s detailed comment and provide the following explanations to address the concerns raised:\n1. Nonlinear regulation of gene transcription in response to environmental stimuli: Environmental stimuli often induce gene expression changes that do not necessarily exhibit a linear correlation with the intensity of the stimulus.Our results indicate that while the transcription levels of PtHSF2 and PtCdc45-like at 30°C were lower than those at 25°C, their levels at 30°C were significantly higher than at the optimal growth temperature of 20°C (Fig. 1A and Fig. 3A). This demonstrates a clear responsiveness of these genes to elevated temperatures, although the relationship between transcription levels and temperature does not follow a simple linear trend. Similar nonlinear transcriptional responses have been widely documented in other systems. For instance: In yeast species exposed to temperatures ranging from 10°C to 46°C, Hsp70 expression peaked 6–8°C above the optimal growth temperature, with expression levels declining at higher temperatures 1; In Heterosigma akashiwo, CO₂-responsive genes, such as SLC4 (SLC4 family bicarbonate transporter), ALAT_GGAT (alanine aminotransferase), and CA (carbonic anhydrase), exhibited peak transcription levels at 400–600 ppm CO₂ but decreased at higher concentrations 2; In human embryonic kidney (HEK) cells and cochlear tissues exposed to cisplatin-induced stress, the transcription levels of stress-related genes such as Chop (C/EBP homologous protein) and DR5 (death receptor 5) initially rose with increasing cisplatin concentrations but decreased once a threshold concentration was surpassed 3; Similarly, in human intervertebral disc NP (nucleus pulposus) cells exposed to varying pH levels (7.4, 7.1, 6.8, and 6.5), the expression of TIMP-1 (tissue inhibitor of metalloproteinases 1) initially decreased with increasing acidity (pH 7.4–7.1) but then increased as the pH decreased further (pH 7.1–6.5) 4.\n2. The dual role of HSFs in cold and heat stress responses: HSFs are well-established mediators of abiotic stress responses 5, with individual HSFs often responding to multiple stressors 6. For example, in wheat seedlings, TaHSF3 is upregulated under both low and high temperatures, and its overexpression in Arabidopsis enhances tolerance to extreme temperatures 7. Similarly, in animals, the relative expression levels of HSF1 and HSP70 have been observed to increase under both heat and cold stress conditions 8. In alignment with these, our findings demonstrate that PtHSF2 was significantly upregulated at both low (15°C) and high temperatures (25°C and 30°C) (Fig. 1A), highlighting its dual role in mediating responses to both cold and heat stress.\n3. The incomplete correlation between gene transcription levels and protein abundance: In both bacteria and eukaryotes, intracellular protein concentrations are generally correlated with the abundances of their corresponding mRNAs, but the correlation is not particularly strong, with the squared Pearson correlation coefficient (R²) typically around 0.40 9. This suggests that a decrease in transcription levels does not necessarily result in a proportional decrease in protein abundance. Additionally, during stress responses, post-transcriptional and post-translational regulatory mechanisms may play critical roles in modulating the levels and functions of mRNAs and proteins 10. Therefore, it is insufficient to evaluate gene function based solely on transcriptional levels. Moreover, a decline in transcription levels may represent an energy-saving strategy under extreme conditions.\nIn conclusion, although the transcription levels of PtHSF2 and PtCdc45-like at 30°C were lower than those at 25°C, they remained significantly elevated at both 25°C and 30°C compared to those at the optimal growth temperature (20°C). These findings support the roles of these genes in temperature stress responses.\nTo address this apparent inconsistency, we further assessed the protein expression levels of PtHSF2 and PtCdc45-like under different temperature conditions. As shown in Supplementary Fig. 2F and Supplementary Fig. 7F, PtHSF2 levels in the PtHSF2-proH1 strain, driven by the HSF2 promoter, and PtCdc45-like levels in the PtCdc45-like-proC1 strain, driven by the Cdc45-like promoter, exhibit a clear linear increase with rising temperatures. These results suggest that post-transcriptional or translational regulation could contribute to the observed increased protein abundance at 30°C, aligning with the enhanced heat tolerance observed in the overexpression lines. (L. No. 163-166, 295-298, 467-497)\n(3) Thermal tolerance of siPtHSF2 and siPtCdc45-like lines – Fig.1C and 3C.\nThe siPtHSF2 and siPtCdc45-like lines present a tolerance to increased temperature similar to the WT line suggesting that PtHSF2 and Cdc45-like do not play a crucial role in thermal tolerance.\nResponse: We appreciate the reviewer’s thoughtful observation regarding the thermal tolerance of siPtHSF2 and siPtCdc45-like lines presented in Fig. 1C and Fig. 3C. The comment that these lines exhibit a thermal tolerance similar to the WT raises important points regarding the roles of PtHSF2 and PtCdc45-like in thermal stress responses. Below, we provide a detailed response and clarification:\n1. Residual expression of target genes: While RNAi-mediated knockdown effectively reduced PtHSF2 and PtCdc45-like transcription levels, it did not achieve complete suppression. This incomplete silencing likely retains a level of gene functionality sufficient to sustain the baseline thermal tolerance observed in these lines. As shown in Supplementary Fig. 2D and Supplementary Fig. 7D, the gene expression levels of PtHSF2 and PtCdc45-like were reduced by approximately 77.9% and 73.5%, respectively, leaving residual activity that may obscure phenotypic differences under the tested conditions.\n2. Potential compensatory mechanisms: Thermal tolerance is likely governed by a network of genes and pathways with overlapping or compensatory functions. While PtHSF2 and PtCdc45-like are involved in thermal stress responses, other heat shock factors or regulatory proteins may compensate for their partial loss of function. Notably, P. tricornutum possesses 69 heat shock factors (HSFs), suggesting potential redundancy or functional overlap. We hypothesize that homologous genes may compensate for the loss of PtHSF2 in siPtHSF2 lines, preserving thermal tolerance.\n3. The potential indirect regulatory role of PtCdc45-like: Additionally, we have reconsidered the function of PtCdc45-like in thermal responses. Given its established role in DNA replication during the S phase, the enhanced thermal tolerance observed in PtCdc45-like overexpression lines may result indirectly from its effects on DNA replication and cell cycle regulation. Our findings reveal that PtCdc45-like overexpression induced slower cell growth (Fig. 3C) and cell cycle arrest (Supplementary Fig. 8A) under non-stress conditions (20°C). These findings align with previous studies indicating that Cdc45 overexpression causes severe replication stress, leading to cell cycle arrest and proliferation inhibition 11, 12. Mechanistically, replication stress caused by Cdc45 overexpression results in replication fork stalling and activates the ATM/Chk2 (ataxia telangiectasia mutated/checkpoint kinase 2) signaling pathway 11. Our findings reveal that under non-temperature stress conditions, the overexpression of PtHSF2 and PtCdc45-like markedly increased the transcriptional levels of ATM and ATR (ataxia telangiectasia and Rad3-related) levels (Supplementary Table 7; Supplementary Fig. 8B; Supplementary Fig. 9 A, B).\nATM and ATR are critical kinases that govern cell cycle and replication checkpoints by phosphorylating the checkpoint effector kinases Chk2 and Chk1 in response to DNA damage 13. This activation initiates DNA repair mechanisms, ensuring that damaged DNA is adequately repaired before progression into the next cell cycle phase 13. Our results indicate that at 30°C, the transcriptional levels of ATM and ATR were significantly elevated in both PtHSF2 and PtCdc45-like overexpression lines (Supplementary Fig. 6E, F; Supplementary Fig. 9C, D). This upregulation likely promotes the repair of heat-induced DNA damage, thereby enhancing cellular heat stress responses and improving survival under elevated temperatures.\nWe have revised the Discussion section to address this comment and provide further clarification. We hope this explanation comprehensively addresses the reviewer’s concerns. (L. No. 519-546)"}, {"round": 3, "reviewer_comments": {"reviewer_3": "No more comments.", "reviewer_4": "The author's responses are sound and strong. The manuscript is well improved and suitable for publication in Nature Communications."}, "author_response": ""}]

Decision letter