Unique high-temperature tolerance mechanisms of zoochlorellae Symbiochlorum hainanensis derived from scleractinian coral Porites lutea

ABSTRACT Global warming is a key issue that causes coral bleaching mainly because of the thermosensitivity of zooxanthellae. Compared with the well-studied zooxanthellae Symbiodiniaceae in coral holobionts, we rarely know about other coral symbiotic algae, let alone their thermal tolerance. In this study, a zoochlorellae, Symbiochlorum hainanensis, isolated from the coral Porites lutea, was proven to have a threshold temperature of 38°C. Meanwhile, unique high-temperature tolerance mechanisms were suggested by integrated transcriptomics and real-time quantitative PCR, physiological and biochemical analyses, and electron microscopy observation. Under heat stress, S. hainanensis shared some similar response strategies with zooxanthellae Effrenium sp., such as increased ascorbate peroxidase, glutathione peroxidase, superoxide dismutase activities and chlorophyll a, thiamine, and thiamine phosphate contents. In particular, more chloroplast internal layered structure, increased CAT activity, enhanced selenate reduction, and thylakoid assembly pathways were highlighted for S. hainanensis’s high-temperature tolerance. Notably, it is the first time to reveal a whole selenate reduction pathway from SeO42− to Se2− and its contribution to the high-temperature tolerance of S. hainanensis. These unique mechanisms, including antioxidation and maintaining photosynthesis homeostasis, efficiently ensure the high-temperature tolerance of S. hainanensis than Effrenium sp. Compared with the thermosensitivity of coral symbiotic zooxanthellae Symbiodiniaceae, this study provides novel insights into the high-temperature tolerance mechanisms of coral symbiotic zoochlorellae S. hainanensis, which will contribute to corals’ survival in the warming oceans caused by global climate change. IMPORTANCE The increasing ocean temperature above 31°C–32°C might trigger a breakdown of the coral-Symbiodiniaceae symbioses or coral bleaching because of the thermosensitivity of Symbiodiniaceae; therefore, the exploration of alternative coral symbiotic algae with high-temperature tolerance is important for the corals’ protection under warming oceans. This study proves that zoochlorellae Symbiochlorum hainanensis can tolerate 38°C, which is the highest temperature tolerance known for coral symbiotic algae to date, with unique high-temperature tolerance mechanisms. Particularly, for the first time, an internal selenium antioxidant mechanism of coral symbiotic S. hainanensis to high temperature was suggested.

The thermal tolerance of coral symbiotic algae is very important for the health and survival of coral holobionts in the warming oceans.Studies have found that some Symbiodiniaceae types have relatively higher thermal tolerance, e.g., Symbiodi nium thermophilum (11) and Durusdinium trenchii (10,12), but totally, coral symbiotic zooxanthellae Symbiodiniaceae is sensitive to thermal stress with lower threshold temperature, i.e., 31°C-32°C.The increasing ocean temperature above 32°C might trigger a breakdown of the coral-Symbiodiniaceae symbioses (13) or coral bleaching caused by thermosensitive Symbiodiniaceae's escape or hypopigmentation (1,2,10,14); therefore, the exploration of other kinds of coral symbiotic algae with high-temperature tolerance is important for the corals' protection under warming oceans.
Besides zooxanthellae Symbiodiniaceae, corals host other kinds of symbiotic photosynthetic algal symbionts, e.g., Ostreobium (15).In 2018, a zoochlorellae, Symbio chlorum hainanensis (Chlorophyta, Ulvophyceae), was first isolated from the bleached scleractinian coral Porites lutea in the South China Sea and named by us (16).Meanwhile, we found that S. hainanensis was wildly distributed in scleractinian corals Platygyra lamellina, Porites lutea, and Favia speciosa.Particularly, the abundance of S. hainanensis became higher when these corals were bleached under thermal stress, accompanied by an abundant decrease in coral symbiotic Symbiodiniaceae (17).This phenomenon indicates the possible roles of S. hainanensis in maintaining the coral holobionts' health under warming oceans by replacing zooxanthellae Symbiodiniaceae.Zoochlorellae has been found to be able to enhance the acclimation capacity of green hydra under heat stress (18), but we rarely know about the response of coral zoochlorellae to thermal stress compared to coral zooxanthellae.In 2020, we proved that S. hainanensis could maintain growth at 32°C, which is generally a lethal temperature to most Symbiodinia ceae (19).Thus, the high-temperature tolerance of zoochlorellae S. hainanensis arouses our great interest, and it is hypothesized that it probably has unique high-temperature tolerant mechanisms that are different from zooxanthellae Symbiodiniaceae.In this study, unique high-temperature tolerance mechanisms of coral symbiotic Symbiochlo rum hainanensis to a high temperature of 38°C were predicted by transcriptomics first and then verified by real-time quantitative PCR (RT-qPCR) and physiological and biochemical analysis along with electron microscopy observation using thermosensitive zooxanthellae Effrenium sp. as a control.

Physiological and biochemical changes in S. hainanensis under thermal stress
Zoochlorellae S. hainanensis grew well under 26°C and survived under 38°C but died when it was exposed to 39°C (Fig. 1A), demonstrating that 38°C is an extreme thermal stress to this alga.Contrary to S. hainanensis, zooxanthellae Effrenium sp.died quickly when it was exposed to 34°C (Fig. 1B), showing a lower heat tolerance than S. hainanen sis.
The content of chlorophyll a in S. hainanensis cells increased significantly under higher temperatures of 32°C and 38°C, compared with the 26°C control (Fig. 1C).This phenomenon also occurred in Effrenium sp. at 29°C and 32°C compared with the control at 26°C (Fig. 1D).The diameter of S. hainanensis cells was about 5-10 µm (Fig. 2A, C, and  E).Interestingly, the internal layers of chloroplasts in S. hainanensis cells under higher temperatures (Fig. 2D and F) become more abundant compared with the control (Fig. 2B).In the case of Effrenium sp. (Fig. 2G through L), the diameter of cells was about 4-7 µm.However, there was no significant change in the internal layers in Effrenium sp.chloroplasts under heat stress, which was different from S. hainanensis.
Based on this study, the O 2 •− content in S. hainanensis did not change significantly under higher temperatures compared with the control (Fig. 3A).H 2 O 2 content decreased significantly under the extreme high temperature of 38°C (Fig. 3B), whereas malondialde hyde (MDA) content increased significantly under the extreme high temperature of 38°C (Fig. 3C).In contrast, the O 2 •− , H 2 O 2 , and MDA contents of Effrenium sp.increased under higher temperatures (Fig. 3D through F).
Compared with the control at 26°C, the activities of antioxidases APX, CAT, GPX, and SOD in S. hainanensis increased particularly under the extremely high temperature of 38°C (Fig. 3G through I), whereas the Prdx activity remained the same as the con trol (Fig. 3J).In the case of Effrenium sp., the activities of antioxidases APX, GPX, and SOD displayed similar change trends as S. hainanensis (Fig. 3L through N).It is worth mentioning that, different from S. hainanensis, no significant CAT activity was detected in Effrenium sp., and the Prdx activity decreased in heat stress groups (Fig. 3O).

Transcriptome profiles of S. hainanensis and Effrenium sp. under thermal stress
The RNA sequencing (RNA-Seq) data and de novo-assembled unigenes of S. hainanensis and Effrenium sp. are summarized in Tables S1 and S2, respectively, and differentially expressed genes (DEGs) of both algae are listed in Tables S3 and S4, respectively.The principal component analysis (PCA) showed that the gene transcriptions of S. hainanensis were obviously different between the control and thermal stress groups (Fig. 4A).The top five terms of three GO ontologies were related to photosynthesis and antioxidation (Fig. 4B through C).In particular, selenocompound metabolism (ko00450) and thiamine metabolism (ko00730) were significantly changed under higher temperatures (Fig. 4D  and E), indicating their possible relationship with thermal tolerance.The expression of two key genes (MET3 and TRR1) in selenocompound metabolism was significantly upregulated (Fig. 5A).Five key genes (adk, dxs, iscS, TH2, and thiN) related to thiamine metabolism and two genes (MET17 and sir) in sulfur metabolism (ko00920) exhibited significantly upregulated expression (Fig. 5A).
PCA, GO enrichment, and KEGG enrichment analyses were also performed for Effrenium sp. (Fig. 4F through J).In order to compare the heat resistance mechanisms of these two different algae, only the genes in Effrenium sp.corresponding to the DEGs in S. hainanensis were compared, regardless of the level of change in gene expression.Much less DEGs were detected in Effrenium sp.(8,543 DEGs, Table S4) than in S. hainanensis (29,393 DEGs, Table S3).The top GO terms were related to the cell cycle, and only one KEGG pathway (i.e., cholesterol metabolism) exhibited significant changes in both thermal stress groups.In Effrenium sp., the expression of nine genes in chlorophyll a biosynthesis was upregulated (Fig. 5B), which is similar to S. hainanensis.Similar to S. hainanensis, most genes related to thiamine metabolism in Effrenium sp. were upregu lated under heat stress (Fig. 5B).But totally, Effrenium sp.showed different transcrip tome profiles from S. hainanensis; for example, only a part of HSP transcripts (mainly HSP70) was upregulated in Effrenium sp.under heat stress (Fig. 5B), and no CAT-encod ing transcript change was detected in Effrenium sp.Meanwhile, the upregulated TRR1 transcripts were not significant under higher temperatures; some MET3 transcripts were upregulated, while others were downregulated.In sulfur metabolism, one sir transcript was downregulated and MET17 was not detected.In the case of genes associated with thylakoid formation, only ALB 3.2 and TatA transcripts and one of the five thylakoid quadruplicate in each group.For both KEGG enrichment and GO enrichment analysis, DEG-eliminated sequences from the X1C1 group or the S1C1 group were used for each alga, respectively.For panels B, C, G, and H, the x axis represents the ratio between the gene number of significantly upregulated genes and the gene number of genes annotated in each pathway; size of the burbles represents the number of genes that showed significantly different expression; color of the burbles represents log 10 (P-value).For panels D, E, I, and J, terms were sorted by the ratio of upregulated DEGs in total DEGs in reverse order; x axis represents the ratio between the gene number of significantly upregulated genes and the gene number of genes annotated in each term; size of the burbles represents the number of genes that showed significant upregulation; color of the burbles represents different ontology.
membrane protein TERC-encoding transcripts were upregulated, while others (such as TatC, THFIID, and THF1) displayed a decreasing trend.

DISCUSSION
Based on the thermal response of coral Symbiodiniaceae (3,(5)(6)(7)(8)(9) and Effrenium sp., in this study, S. hainanensis has the highest temperature tolerance known for coral symbiotic algae, i.e., 38°C.The expression of HSPs is commonly considered to be associated with stress (20), HSP-related DEGs in zoochlorellae S. hainanensis and zooxanthellae Effrenium sp.under thermal stress indicate that algal cells were indeed in a stress response state (Fig. 5).HSPs are known to have function in protein processing, such as protein folding, protein translocation, and maintaining the conformation of unstable/wrong-fol ded proteins, as well as their signaling functions (20).In S. hainanensis, the upregulation of genes encoding HSPs, especially small HSPs, HSP70 and HSP90, indicated that the heat response system was triggered, and the alga was conserving its protein homeostasis.In contrast to S. hainanensis, a few HSPs were upregulated in Effrenium sp., indicating that some of its protein homeostasis maintaining mechanism might be damaged or dysfunctional under thermal stress, which would result in the destruction of protein homeostasis inside algal cells.However, the unchanged O 2 •− content and decreased H 2 O 2 content in S. hainanensis indicated its higher ability to remove reactive oxygen species (ROS) than Effrenium sp. (Fig. 3A through F); this is probably one of the reasons why S. hainanensis can survive under the extreme high temperature of 38°C.Compared with Effrenium sp., the presence of CAT, the remaining activity of Prdx, and the reduc ing product of the selenate reduction pathway (Se 2− ) could be the mechanisms that contribute to the enhanced ROS removal capacity in S. hainanensis.The upregulated DEGs related to antioxidases, selenate reduction, thiamine biosynthesis, chlorophyll a synthesis, and thylakoid assembly in S. hainanensis X1 are summarized in Fig. 7, showing the unique thermal resistance mechanisms of S. hainanensis.Considering the wide distribution and increased abundance of S. hainanensis in bleaching corals (16,17,19,21), S. hainanensis might play important roles in corals' resistance to thermal stress, particularly when thermosensitive zooxanthellae escape in the warming oceans caused by global climate change.Meanwhile, the transplanting of S. hainanensis might be a strategy to help corals survive in warming oceans caused by global climate change because of its high thermal tolerance.

The specific contribution of antioxidant enzymes, particularly CAT, to the high-temperature tolerance of S. hainanensis
Elevated temperature not only affects temperature-dependent biochemical reactions but also increases intracellular oxidative pressure (22).In this study, the MDA content change may reflect the oxidative pressure particularly under the ultimate temperatures, i.e., 38°C and 32°C, for S. hainanensis and Effrenium sp., respectively (Fig. 3C and F).The increase in ROS such as O 2 •− and H 2 O 2 under heat stress will cause algal oxidative damage (22).Hence, the scavenging of ROS will contribute to heat resistance.The O 2 •− content in S. hainanensis under different experiment temperatures remained low, and the H 2 O 2 content under the extreme temperature significantly decreased (Fig. 3A and  B).On the contrary, O 2 •− and H 2 O 2 contents in Effrenium sp.increased (Fig. 3D and E), indicating that its ROS removal capacity was weakened under thermal stress.The lower O 2 •− and H 2 O 2 contents in S. hainanensis indicate its higher ability to relieve oxidative pressure than Effrenium sp.The expression of genes encoding antioxidant enzymes SOD, APX, CAT, and GPX in S. hainanensis was upregulated under thermal stress (Fig. 5A); consequently, the activities of SOD, APX, CAT, and GPX increased (Fig. 3G through K).It is known that antioxidases are able to transfer O 2 •− to H 2 O 2 and finally to H 2 O (22); therefore, ROS inside the algal cells is maintained at a low level.However, the different activity changes of these four enzymes indicated their different contributions to the heat resistance of S. hainanensis.Specifically, the GPX and SOD activities were higher when the algal cells were heated (32°C and 38°C), but the APX and CAT activities were only higher under an extremely high temperature (38°C).It can be speculated that GPX and SOD are the core antioxidant enzymes in the heat resistance of S. hainanensis, and APX and CAT are reserves, which can only be called under the extremely high temperature.The increased intercellular SOD activity in Chlorella ellopsoidea (23), Breviolum (clade B1), and Cladocopium (clade C1) (8) indicates SOD's role in the algal response to heat stress.In Chlamydomonas reinhardtii, APX activity was found to be increased under elevated temperatures (24).Although transcripts encoding antioxidant enzymes like APX and SOD displayed a decreasing trend in Effrenium sp.under thermal stress, their expression changes did not reach a significant level.Although catalase peroxidase (KatG) has been found in Breviolum (clade B1) (25), the capacity of H 2 O 2 degradation in Breviolum (clade B1) and Effrenium (clade E1) displayed no significant change under thermal stress (25,26).Accordingly, it is speculated that different coral symbiotic algae have different response patterns of antioxidant enzymes to thermal stress.In particular, our results suggested the importance of antioxidase CAT in the high-temperature tolerance of S. hainanensis because no CAT activity was detected in Effrenium sp.Based on the result from Bayer et al. (27), Symbiodinium sp.CassKB8 and Breviolum sp.Mf1.05b appear to lack CAT (23), and no CAT activity change was detected in dinoflagellate Cladocopium goreaui during the thermal exposure period (28).Thus, CAT might lead to a much more effective antioxidant system in S. hainanensis and aid its higher tolerance than Effrenium sp.

Selenate reduction and thiamine biosynthesis related to the high-tempera ture tolerance of S. hainanensis
In S. hainanensis, besides the roles of multiple antioxidant enzymes, the enhancement of selenate reduction and thiamine biosynthesis pathways probably contributes to the tolerance of S. hainanensis to high temperatures (Fig. 7).It is worth mentioning that a whole pathway of selenate reduction was detected in S. hainanensis (Fig. 6E), which was correlated with this algal high-temperature tolerance.In this pathway, SeO 4 2− is successively catalyzed to Se 2− by sulfate adenylyltransferase (EC 2.7.7.4, encoded by MET3) and thioredoxin reductase (EC 1.8.1.9,encoded by TRR1).The upregulated expression of two genes MET3 and TRR1 (Fig. 5A and 6A) and the increased content of Se 2− in S. hainanensis cells (Fig. 6B) were detected under heat stress in this study.As a result of this upregulation, theoretically, the content of substrate (SeO ) should be reduced, which was supported by the decreased contents of SeO 4 2− and SeO 3 2− and the increase of Se 2− in the algal cells.In contrast to S. haina nensis, there is no significant content change in SeO 3 2− or Se 2− in Effrenium sp. cells (Fig. 6H), as well as no significant change in the related genes' expression (Fig. 5B), indicating this selenate reduction pathway does not contribute to the heat resistance of Effrenium sp.Selenium has been reported to play a key role in the cellular antioxidant defense mechanism (29).For example, Se at low concentration positively promoted the antioxidative effect of Chlorella pyrenoidosa by increasing the levels of glutathione peroxidase, catalase, linolenic acid, and photosynthetic pigments (30) and increased the activity of antioxidant enzymes (SOD and CAT) and the amount of antioxidant metabolites (phenols, flavonoids, and carotenoids) in Ulva sp.(31).Maronić et al. (32) also highlighted the importance of the algal Se detoxification strategy, especially the role of selenoenzymes and other selenoproteins with antioxidant function.Similarly, based on the upregulation of specific genes and the increased Se 2− yield concentration under heat stress, this study suggests an internal selenium antioxidant mechanism of S. hainanensis to high temperature.Taken together the present knowledge of the thermal response mechanisms of well-studied Chlamydomonas (33), coral symbiotic Symbiodiniaceae (3)(4)(5)(6)(7)(8)(9), and the thermal response of Effrenium sp. in this study, it is the first time to find a correlation between the upregulated selenate reduction pathway and high-temperature tolerance of coral symbiotic algae, which could be one of the reasons why its antioxidant system is more effective than Effrenium sp.
Tunc-Ozdemir et al. (34) found the role of thiamine in the protection of cells against oxidative damage in Arabidopsis thaliana and found that thiamine-induced tolerance to oxidative stress was accompanied by decreased production of reactive oxygen species, as evidenced from decreased protein carbonylation and hydrogen peroxide accumula tion.In this study, the expression of six genes (MET17, iscS, dxs, TH2, thiN, and adk) responsible for synthesizing thiamine and its three phosphates was upregulated in S. hainanensis under heat stress (Fig. 5A, 6C, D, and F), which was proved by the increased contents of thiamine, TMP, TDP, and TTP under heat stress.Prior studies have noted that the antioxidant/anti-heat function of thiamine and TDP is common in algae and plants, such as the cyanobacterium Nodularia spumigena, dinoflagellate Prorocentrum minimum (35), Zea mays, and Arabidopsis thaliana (36).Thus, thiamine biosynthesis could contribute to thermal tolerance in S. hainanensis by increasing the content of its antioxidant products thiamine and TDP.Combined with the similar increase of thiamine and TDP in thermally stressed Effrenium sp., it could be proposed that this is a universal mechanism in the thermal stress response of coral symbiotic algae.The increased TTP of algae under thermal stress (Fig. 6D) suggests TTP's possible correlation with thermal tolerance.However, to date, we rarely know about the function of TTP in stress response, except that it was suggested to serve as "alarmones" when cells are under starvation (37).Thus, TTP's roles in the antioxidation of S. hainanensis need further study.

Maintenance of photosynthesis homeostasis by enhancing thylakoid assembly for the high-temperature tolerance of S. hainanensis
Based on the KEGG and GO enrichment analyses, in addition to antioxidation, photo synthesis homeostasis maintenance might be another contributor to the high-temper ature tolerance of S. hainanensis.Under heat stress, the expression of seven genes (ChlD, ChlH, ChlI, CHLM, CTH1, HEMC, and PPOX) involved in chlorophyll a biosynthesis was upregulated (Fig. 5A).Consistent with this result, the chlorophyll a content in S. hainanensis cells increased (Fig. 1C).The enhanced chlorophyll a biosynthesis indicated that S. hainanensis was compensating for heat-induced chlorophyll loss or increasing the energy inflow under heat stress.In addition, the increased thylakoid formation was found to be involved in the response of S. hainanensis to heat stress (Fig. 7).Five genes (ALB3.2,SQD1, TatA, Thf1, and VIPP1) associated with thylakoid formation were upregulated in S. hainanensis under heat stress (Fig. 5A).These genes are involved in thylakoid membrane lipid synthesis (SQD1), thylakoid membrane protein synthesis (TatA), integration of light-harvesting complex into thylakoid membrane (ALB3.2),as well as thylakoid assembly and stacking (Thf1 and VIPP1).In contrast, the increased thylakoid assembly was not observed in Effrenium sp. (Fig. 2G through L, Fig. 5B), indicating the possible damage to photosynthesis homeostasis of this alga under heat stress.The internal layers of chloroplasts in S. hainanensis cells under higher temperatures (Fig. 2D  and F) became more abundant compared with the control (Fig. 2B), whereas there was no significant change in internal layers in Effrenium sp.chloroplasts under heat stress.Thus, it can be speculated that S. hainanensis probably increase the assembly or the formation of thylakoids under thermal stress (Fig. 7).Presumably, it might be compensat ing for the thylakoid losses caused by heat or forming new thylakoid de novo to maintain photosynthesis energy inflow in S. hainanensis.Similar to our results, the formation of aberrant prolamellar body-like structures was observed in the chloroplast of heat-tol erating Chlamydomonas reinhardtii under elevated temperature, which is considered to be associated with photosynthesis maintenance (38).Similarly, the enlargement of chloroplasts along with the increase in chlorophyll fluorescence and pigment content of S. hainanensis were detected in our previous study (19).Coupled with the morphologic change of chloroplasts in both the 32°C and 38°C groups (Fig. 2), it is presumably suggested that S. hainanensis probably try to maintain the photosynthesis homeostasis by increasing the assembly of thylakoid and more chloroplast internal layered structure.

Conclusions
Compared with the thermosensitive zooxanthellae Effrenium sp.(threshold tempera ture: 32°C), zoochlorellae S. hainanensis has a heat-resistant temperature of 38°C, which is the highest thermal tolerance of coral symbiotic algae.Besides the similar heat response strategies as Effrenium sp., e.g., increased APX, GPX, and SOD activities and chlorophyll a, thiamine, and thiamine phosphates' contents, S. hainanensis has unique high-temperature tolerance mechanisms, including more chloroplast internal layered structure, increased CAT activity, and enhanced selenate reduction and thylakoid assembly pathways.Particularly, for the first time, an internal selenium antioxidant mechanism of coral symbiotic S. hainanensis to high temperature was suggested.The revealed unique high-temperature tolerance mechanisms of zoochlorellae S. hainanensis efficiently remove ROS to maintain the low-level inner cellular superoxide (O 2 •− ) content and ensure photosynthesis homeostasis.The revealed 38°C high-temperature tolerance and the related molecular mechanisms of S. hainanensis greatly expand our understand ing of the heat resistance of coral symbiotic algae.
To determine the extreme thermal stress temperature, the cultures of S. hainanen sis under 26°C were changed to 32°C, 38°C, and 39°C, respectively, on the 10th day (mid-exponential phase), while the cultures of Effrenium sp.under 26°C were changed to 29°C, 32°C, and 34°C, respectively, on the 25th day (mid-exponential phase).The initial cell density was 1 × 10 5 cell/mL, and five replicate cultures were used.From the fourth day, algal cells were sampled by sterile dropper every 3 days and then stained to distinguish the viable cells as described by Malerba et al. (41).The number of viable cells was counted using a light microscope, with five replicate counts performed.
To reveal the thermal tolerance mechanisms of S. hainanensis and Effrenium sp., both algae were exposed to 32°C and 38°C, 29°C and 32°C, respectively, using 26°C as the control.The controls before and after the heat treatment (named C0 and C1) were used to rule out the effects of time (Table S7).The thermal treatment started on the 10th day for S. hainanensis and on the 25th day for Effrenium sp., i.e., their mid-exponential phase, and lasted for 3 days.Then, the algal cells (heated for 3 days and the control) were collected by centrifugation (6,500 × g, 15 min) using a high-speed refrigerated centrifuge (H1650R, Cence, Hunan, China), after being washed three times with sterile artificial seawater (NaCl, 453.80 mM; MgCl

Pigments' content analysis and transmission electron microscope observa tion
Pigments' content was determined using the acetone-based method (42).Specimen sections with a thickness of about 70 nm were sliced using a cryo-ultramicrotome (UC6FC7, Leica, Wetzlar, Germany) and observed by a 120-kV transmission electron microscope (Tecnai G2 Spirit Bio twin, FEI Corp., Hillsboro, OR, USA) (17).

Antioxidation biochemical analyses
Algal cells were ground in liquid nitrogen.The contents of superoxide (O 2 •− ) and hydrogen peroxide (H 2 O 2 ) were determined as described by Malerba and Cerana (43) and Gao et al. (44), respectively, to evaluate the intercellular ROS level.The level of membrane lipid peroxidation and the content of MDA were measured according to Malerba and Cerana (43).Five replicates were used for each test.
APX activity was detected using the kit D799461-0050 (Sangong, Shanghai, China).One unit of APX activity was defined as the amount of enzyme that oxidizes 1 µmol of ascorbate per minute in the reaction system.Catalase activity was detected by the kit D799597-0050 (Sangong, Shanghai, China).One unit of CAT activity was defined as the degradation of 1 µmol H 2 O 2 per minute in the reaction system.GPX activity was detected by the kit D799617-0050 (Sangong, Shanghai, China).One unit of GPX activity was defined as the oxidation of 1 µmol NADPH per minute in the reaction system.SOD activity was detected by the kit D799593-0050 (Sangong, Shanghai, China).One unit of SOD activity was defined as the amount of enzyme that inhibited the rate of ferricyto chrome c reduction by 50%.Prdx activity was determined by the kit D799592-0100 (Sangong, Shanghai, China).One unit of Prdx activity was defined as every 0.005 change of A 470 per minute per milliliter in the reaction system.

Transcriptome analysis
Total RNA was extracted as described in our previous study (19).The extracted RNA was divided into two parts, one for RNA sequencing and another for real-time quantitative PCR confirmatory analysis.In the case of RNA-Seq, 16 libraries (four replicates × four groups [C0, C1, W1, and W2] for each alga) were constructed and sequenced.
The quality control and short read assembly of RNA-Seq data were performed as described in our previous study (19).Transcripts with read counts ≤ 1 were dis carded to reduce interference.The annotation of de novo-assembled unigenes was performed according to our previous study (19).Differentially expressed gene analysis was performed using DESeq2 R package version 1.26.0 (45), the thresholds to evaluate the significance for contigs were set as P value = 0.01 and log 2 (fold change) = ±1.For differently expressed gene analysis, after eliminating DEGs from the C1 control group, GO enrichment and KEGG enrichment were performed using clusterProfiler v.3.14.3 R package (46).In GO enrichment, the default arguments were used; for each term, the proportion of upregulated DEGs in total DEGs was calculated and sorted in reverse order.In KEGG enrichment analysis, the default arguments were used, and the thresholds to evaluate the significance of change for each pathway were set as P value = 0.05.

Real-time quantitative PCR analysis
A Tiangen FastKing RT Kit KR116 (Tiangen, Beijing, China) was used for the first-strand cDNA synthesis.RT-qPCR was conducted using Tiangen Talent qPCR PreMix (SYBR Green, FP209).The selected unigenes and primers (designed based on their sequence) are listed in Tables S5 and S6 for S. hainanensis and Effrenium sp., respectively.A BioRad C1000 Thermal Cycler (BioRad, Hercules, CA, USA) was used for PCR: 95°C for 5 min; 55°C for 20 s, and 72°C for 20 s, 40 cycles.The melting curve procedure was as follows: 95°C for 30 s; 55°C for 65 s and then rose to 95°C.The 2 −∆∆Ct method was used to calculate relative gene expression values (47).

FIG 1
FIG 1 Growth curve and pigments' content of S. hainanensis X1 (A and C) and Effrenium sp.S1 (B and D) under different temperatures.In panels A and B, error bars represent ±standard deviations (SD), and some error bars are obscured by data point markers; the batch experiments were conducted in triplicates.One of the representative data sets is presented here.In panels C and D, whiskers represent the minimum and maximum of at least five samples in three independent experiments.X1C1: 26°C; X1W1: 32°C; X1W2: 38°C; S1C1: 26°C; S1W1: 29°C; and S1W2: 32°C.

FIG 6
FIG 6 RT-qPCR verification and selenate reduction, thiamine, and thiamine phosphate compounds' analyses.Bar plot of RT-qPCR results (A) and selenium compounds' content (B) in the selenate reduction pathway in S. hainanensis X1.Bar plot of RT-qPCR results (C) and thiamine and thiamine phosphates' content (D) in the thiamine biosynthesis pathway in S. hainanensis X1.Scheme of the detected upregulated selenate reduction pathway (E) and upregulated thiamine biosynthesis pathway (F) in S. hainanensis X1.Bar plot of RT-qPCR results (G), selenium compounds' content (H), and thiamine and thiamine phosphates' content (I) in Effrenium sp.S1.For RT-qPCR, the data represent mean ± SD of quadruplicates; β-actin (X1_44387) and 18S rRNA (S1_42608) were used as internal controls, respectively.For compound contents' results, the data represent mean ± SD of at least six samples in two independent experiments (at least three each).The statistical difference (one-way ANOVA) between treatment and control is indicated as *P < 0.05, **P < 0.01, or ***P < 0.001.For the pathway, solid and dotted arrows represent one-step or multi-step reactions, respectively; italics represent genes; the color indicates the relative expression change (red represents upregulated).X1C1: 26°C; X1W1: 32°C; X1W2: 38°C; S1C1: 26°C; S1W1: 29°C; and S1W2: 32°C.

FIG 7
FIG 7 Schematic summary of the upregulated DEGs in S. hainanensis X1 response to thermal stress.DEGs were selected as adjusted P value < 0.01 and log2 (fold change) ≥ ±1 in thermal stressed groups (X1W1 group, 32°C and X1W2 group, 38°C) compared to the control (X1C1 group, 26°C) on the third day.Genes related to antioxidases, selenate reduction, thiamine biosynthesis, chlorophyll a synthesis, and thylakoid formation are shown.Solid and dotted arrows represent one-step or multi-step reactions, respectively.Italics represent genes.The semicircle indicates the change of gene relative expression or substance content in algae cells: left semicircle represents S. hainanensis X1, and right semicircle represents Effrenium sp.S1; color in the semicircle indicates the change trend: red represents upregulated or increased, blue represents downregulated or decreased, and white represents no significant chang.The dotted black border represents that this gene or substance was not detected.