Molecular mechanism underlying Pyropia haitanensis PhHsp22-mediated increase in the high-temperature tolerance of Chlamydomonas reinhardtii

Global warming is one of the key limiting factors affecting the cultivation of Pyropia haitanensis which is an economically important macroalgae species grown in southern China. However, the mechanism underlying the high-temperature tolerance of P. haitanensis remains largely unknown. In a previous study, we showed that the expression of the small heat shock protein 22 gene (Hsp22) is upregulated in P. haitanensis in response to high-temperature stress, but the associated regulatory mechanism was not fully elucidated. In this study, a transgenic Chlamydomonas reinhardtii expression system was used to functionally characterize P. haitanensis Hsp22. Our analyses indicated that the C-terminal of PhHsp22 is highly conserved and contains an A-crystal structure domain. A phylogenetic analysis revealed PhHsp22 is not closely related to small heat shock protein genes in other species. Additionally, PhHsp22 expression significantly increased at 3 and 6 h after initiating 33 °C treatment, which improved the survival rate of transgenic C. reinhardtii during the early stage of high-temperature treatment. The further transcriptome analysis revealed that PhHsp22 expression can promote pathways related to energy metabolism, metabolites metabolism, and protein homeostasis in transgenic C. reinhardtii cells exposed to high temperatures. Therefore, PhHsp22 may be crucial for the response of Pyropia species to high-temperature stress. Furthermore, this gene may be useful for breeding new high-temperature algal strains.


Introduction
Pyropia species are economically important marine red algae used to produce food, fertilizer, and medicine (Blouin et al. 2011;Brawley et al. 2017;Cao et al. 2020). With the expansion of artificial seeding and the development of a floating culture method, the farming and processing of Pyropia species is now a major part of the largest seaweed industries in East Asian countries, including China, Japan, and South Korea (Sahoo et al. 2002;Hwang et al. 2019). However, because of global warming, seawater temperatures have gradually increased in recent years which has adversely affected the cultivation and yield of Pyropia species (Shi et al. 2017;Wang et al. 2018a;Hwang et al. 2019). To adapt to global climate change and to continue to develop improved methods for cultivating Pyropia species, several high-temperature (HT) tolerant Pyropia haitanensis strains (Z-26, Z-61, and ZS-1) have been selected and are now widely cultivated in southern China (Chen et al. 2008). Although the mechanisms underlying this high-temperature tolerance have been investigated (Shi et al. 2017;Wang et al. 2018a, b), they remain largely uncharacterized. Therefore, deciphering the molecular basis for the heat stress tolerance of P. haitanensis is important for increasing the commercial value and productivity of red algal species.
The development and application of high-throughput sequencing technology and molecular biology research tools Jing Chang and Jianzhi Shi contributed equally to this work. have resulted in some recent progress regarding the elucidation of the molecular mechanisms behind P. haitanensis heat stress tolerance. For example, Wang et al. (2018b) compared the transcriptomes between Z-61 and wild-type P. haitanensis strains, revealing that Z-61 is more thermotolerant than the wild type because of an increase in the transcriptional regulation of genes mainly related to photosynthesis, antioxidant systems, and energy metabolism. In another investigation, 151 heat stress-responsive P. haitanensis proteins associated with various processes, mainly including photosynthesis, protein synthesis and degradation, defense response, and energy and carbohydrate metabolism, were identified based on the iTRAQ technique (Shi et al. 2017). On the basis of a lipidomics analysis, Chen et al. (2016b) detected a number of lipid biomarkers for high-temperature stress. Additionally, some candidate genes related to high-temperature responses have been cloned, including genes encoding manganese superoxide dismutase (Yang et al. 2013) and carbonic anhydrases (Chen et al. 2016a). In order to cope with HTs and other abiotic stresses, Pyropia species initiate various defense mechanisms to carry out the appropriate physiological and biochemical changes (Qian et al. 2015;Im et al. 2015;Wang et al. 2018aWang et al. , 2019Wang et al. , 2020Zheng et al. 2020). Notably, regardless of the pathway that is activated by HT stress, non-native proteins aggregate and partially unfolded proteins are produced and accumulate in Pyropia species, with detrimental effects on the thalli cells.
Heat shock proteins (HSPs) are molecular chaperones that can prevent irreversible protein aggregations (Liberek et al. 2008;Tyedmers et al. 2010). Based on their molecular weight, homology and functions, HSPs can be classified into several families. Small heat shock proteins (sHSPs) are the most ubiquitous HSPs subgroup, with molecular weights ranging from 12 to 42 kDa (Zhang et al. 2016). They are usually undetectable in vegetative tissues under normal growth conditions but can be induced by environmental stresses and developmental stimulation. In cells exposed to abiotic stresses, sHSPs function as the first line of defense by binding and stabilizing proteins at intermediate stages of folding, assembly, degradation, and translocation across membranes (Waters 2012). Compared to sHSP family in plants, those in red macroalgae seem to be less diverse. To date, only a few sHSPs have been identified in Chondrus crispus (Collén et al. 2013), Porphyra umbilicalis (Brawley et al. 2017), Pyropia yezoensis (Uji et al. 2019), and P. haitanensis . Identifying and clarifying the physiological functions of sHSPs in response to HT will provide a basis for molecular analyses and the selective breeding of Pyropia species. We previously cloned the P. haitanensis Hsp22 gene and found its expression was upregulated more than 1000-folds under HT stress . The stable genetic transformation system of hygromycin resistance gene by codon optimization has only been reported in P. yezoensis (Uji et al. 2014). Although successful genetic transformation system in P. yezoensis has been reported, transgenic strains of P. yezoensis can only be propagated through monospore germination, which is a very convenient and fast way of propagation. However, P. haitanensis has no such asexual reproduction, resulting in it being difficult to obtain stable transformed cells for P. haitanensis. Therefore, sHSP functions remain largely unknown in these species.
Unlike Pyropia species, Chlamydomonas reinhardtii is a unicellular green alga broadly used for elucidating fundamental biological processes. There are several genetic resources and analytical tools available for C. reinhardtii, including nuclear, chloroplast, mitochondrial transformation systems, RNAi knockdown methods, a sequenced genome, and hundreds of genetically characterized mutants (Kindle 1990;Shimogawara et al. 1998;Neupert et al. 2012). Moreover, many heterologous genes have been expressed in C. reinhardtii, including genes from microalgae (Cordero et al. 2011), macroalgae (Kim et al. 2011), higher plants (Siripornadulsil et al. 2002), and humans (Rasala and Mayfield 2011). Specifically, Jin et al. (2017) inserted the Pyropia tenera HSP19.3 gene into C. reinhardtii and confirmed that the physiological function of the encoded protein may influence HT tolerance. Accordingly, C. reinhardtii may be suitable for expressing P. haitanensis genes. However, the molecular mechanism underlying the HSPs function related to C. reinhardtii HT tolerance is unclear. The objectives of the present study were to establish a stable genetic transformation system of P. haitanensis gene (PhHsp22) in C. reinhardtii and investigate the PhHsp22expression pattern. Additionally, the regulation and expression of genes in response to HT stress were examined via the genome-wide high-throughput transcriptomic analysis of C. reinhardtii overexpressing PhHsp22.

Algae and growth conditions
The Pyropia haitanensis Z-61 strain used in this study is tolerant to HT and produces a high yield. It was selected and purified by researchers in the Laboratory of Germplasm Improvements and Applications of Pyropia in Jimei University, Fujian Province, China. Five randomly selected blades (15 ± 2 cm long) were cultured at 21°C with a 10-h light:14-h dark cycle (50 μmol photons m -2 s -1 irradiance). Additionally, Chlamydomonas reinhardtii strain "CC-400 cw15 mt+" was used to examine the physiological function of PhHsp22. The C. reinhardtii cells were grown in Trisacetate-phosphate (TAP) medium at 25°C with shaking (100 rpm) and a 14:10 (light/dark) cycle with cool fluorescent light (50 μmol photons m −2 s −1 ).

Sequence and phylogenetic tree analyses of PhHsp22
The PhHsp22 nucleotide sequence, deduced amino acid sequence, and open reading frame were analyzed with tools available on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) . Conserved domains were identified with SMART (http://smart.embl-heidelberg.de/). MEGA6 program was used to construct a phylogenetic tree according to the maximum likelihood method.

RNA extraction and cDNA synthesis
Total RNA was extracted from P. haitanensis and C. reinhardtii and purified with the E.Z.N.A. Plant RNA Kit (Omega, Germany). The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm/280 nm (A 260 /A 280 ) and 260 nm/230 nm (A 260 /A 230 ) with a NaroDrop ND-1000 spectrophotometer (LabTech, USA). The integrity of the purified RNA was assessed by 1.2% agarose gel electrophoresis. The total RNA (1 μg) was used as the template for the first-strand cDNA synthesis with the PrimeScript RT reagent kit (TaKaRa, Japan) and random primers. The cDNA samples were diluted with nuclease-free water to 5 ng μL −1 and then used as the templates for the quantitative real-time (qRT)-PCR analysis.

Isolation of PhHsp22 and construction of a transformation vector
The complete PhHsp22 cDNA was cloned with PhHsp22 specific primers (Table S1). The pMD19-T (TaKaRa, Japan) and pChlamy_3 (Invitrogen, USA) vectors were used for constructing the transformation vector. The amplified PhHsp22 sequence was first inserted into the pMD19-T vector. The resulting recombinant plasmid and pChlamy_3 were digested with KpnI and PstI (TaKaRa, Japan), after which the PhHsp22 and linearized pChlamy_3 vectors were ligated to form the transformation vector.

Chlamydomonas reinhardtii nuclear transformation
The nuclear transformation of C. reinhardtii was completed according to a modified version of the electroporation method described by Yamano et al. (2013). Briefly, C. reinhardtii cells were cultured until the cell density reached 1-2 × 10 6 cells mL −1 , after which a 10 mL aliquot of the cultured cells was centrifuged at 600×g for 5 min. The pelleted cells were re-suspended in 200 μL TAP medium containing 40 mM sucrose for a final density of 1 × 10 8 cells mL −1 . The pChlamy-PhHsp22 plasmids (2 μg) were linearized with PstI and added to the cell suspension. The cell suspension was added to an electroporation cuvette (ECM830, USA). Parameters were optimized as three Pps of 300 V with 6 ms pulse length, 50 ms pulse interval. After electroporation, the cell suspension from the cuvette was transferred into 10 mL TAP medium containing 40 mM sucrose and incubated at dim light (1~2 μmol photons m −2 s −1 ) for 24 h. Transformed colonies were selected on TAP agar medium containing 10 μg mL −1 hygromycin after 7-14 days of growth.

Stress treatment and quantitative real-time PCR analysis
Chlamydomonas was treated at 33°C for 3, 6, and 12 h to observe the biomass. Cell growth was monitored based on the cell counts determined by measuring the optical density at 750 nm (Kumar et al. 2013). To assess the effects of HT on gene expression, cDNA was prepared for HT treatment samples for a qRT-PCR analysis with PhHsp22-specific primers (Hsp22QF and Hsp22QF). A β-tubulin gene was used as an internal control. The primers for qRT-PCR are listed in Table S1. The qRT-PCR was performed with 96-well plates and the ABI 7300 real-time PCR Detection system. The 20 μL reaction volumes comprised 10 μL 2 × SYBR Green Master Mix (Takara, China), 0.4 μL sense and antisense primers (20 mM), 2 μL diluted template, 0.4 μL ROX Reference Dye (50×), and 6.8 μL RNA-free water. The qRT-PCR program was as follows: 95°C for 30 s and 40 cycles of 95°C for 5 s and 60°C for 31 s. A dissociation curve analysis of the amplification was completed after the PCR cycles to confirm that only one PCR product was amplified and detected.
Transcriptome assembly, annotation, and differential gene expression analysis Only RNA samples with an A 260 /A 280 ratio of 1.9-2.1 and an A 260 /A 230 > 2.0 were used in the subsequent analyses. The de novo transcriptome assembly and annotation were completed by GENE DENOVO Biotech, Ltd. (Guangzhou, China). Gene expression levels were calculated based on the RPKM (reads per kilobase per million reads) method (Mortazavi et al. 2008). The gene expression data were then used to analyze differentially expressed genes (DEGs) with the DESeq2 software (Love et al. 2014).

Statistical analysis
All experiments were repeated three times. The standard deviation of the mean was used to determine the precision of the mean. The significance of any differences between the treated and control samples was assessed by a one-way ANOVA and post hoc testing with the LSD test of SPSS 12.0 (SPSS Inc, USA). The threshold for significance was set at p < 0.05.

Identification of PhHsp22 cDNA
In a previous work, we cloned P. haitanensis Hsp22 from a cDNA library based on high-throughput sequencing. PhHsp22 encodes a 19.1 kDa polypeptide with 172 amino acids (pI of 5.24) . The prediction of domain structures in PhHsp22 by SMART revealed the presence of PhHsp22 contained A-crystal structure domain from amino acids 52 to 149, as well as the HSP20 domain at amino acids 60-164 (Fig. 1a). A phylogenetic analysis supported the existence of a sister-group relationship between P. haitanensis and Chondrus crispus but implied that Pyropia species diverged from Cyanophyta, Chlorophyta, Phaeophyta, and land plant species (Fig. 1b).

Confirmation of C. reinhardtii transformation and
PhHsp22 expression under high-temperature stress To investigate whether the constitutive expression of PhHsp22 can improve the HT tolerance of transgenic C. reinhardtii, we transformed C. reinhardtii with the PhHsp22 coding sequence under the control of a tandem promoter comprising the Hsp70A and Rbc S2 promoters (Fig.  S1). Many independently regenerated hygromycin-resistant lines were obtained. The transformation was then confirmed by PCR analyses using PhHsp22-specific primers (Table S1). The expected amplicons were produced for all randomly selected hygromycin-resistant lines, but not for the wild-type control, indicating PhHsp22 was integrated into the genome of the hygromycin-resistant lines (Fig. 2a). The 5th transgenic lines (T5) were selected for the subsequent HT tolerance analyses at the transcriptional and morphological levels. The PhHSP22 expression levels in T5 were determined in a qRT-PCR assay. As shown in Fig. 2c, at least a fifteenfold increase of PhHsp22 expression in transgenic C. reinhardtii was induced by HT stress, with peak expression levels at 33°C (Fig. 2b). Thus, we evaluated the effects of this temperature on the transgenic C. reinhardtii over time (Fig. 2c). As shown in Fig. 2c, the maximal expression of the PhHsp22 gene is detected after 3 h of induction at 33°C. The PhHsp22 expression level decreased gradually after 6 h and would gradually decline to the normal level after 12 h.
Tolerance of transgenic C. reinhardtii to hightemperature stress The transgenic C. reinhardtii was clearly more tolerant to HT treatment (33°C) than the WT control (Fig. 3). The cultures for the T5 were obviously greener than WT cultures at 33°C. The differences between the WT and transgenic cultures exhibited a time-dependent profile.

Verification of differentially expressed genes
To further verify the reliability of the RNA-seq data, 8 unigene sequences were randomly selected from the DEGs of the transcriptome for qPCR analysis. There was a good correlation between the transcript abundances based on the qRT-PCR analysis and the expression profiles determined by RNA-Seq ( Figure S2).

Functional analyses of differentially expressed genes
Based on the abovementioned results, we selected the PhHsp22-overexpressing transgenic C. reinhardtii incubated at 33°C for 3 h for a transcriptome analysis, with the WT C. reinhardtii used as the control. The mRNA samples, including different strains of C. reinhardtii, were subjected to paired-end sequencing using the Illumina HiSeq2500 platform. Details regarding the transcriptome assembly are provided in supplementary table S2. The high-temperature stress affected the WT control more than the transgenic C. reinhardtii. Specifically, compared with the WTunstressed (CK) expression levels, the 3-h heat treatment of the WT C. reinhardtii (WT-3 h) upregulated and downregulated the expression of 3666 and 3487 genes, respectively. In contrast, the 3-h heat treatment of the transgenic C. reinhardtii (HSP-3 h) upregulated and downregulated the expression of 363 and 556 genes, respectively, relative to the HSP-CK expression levels. Additionally, there were 6958 DEGs between WT-CK and HSP-CK, whereas only 89 DEGs were detected between WT-3 h and HSP-3 h (Fig. 4a). Furthermore, the top 20 enriched KEGG pathways among the DEGs between WT-CK and HSP-CK included proteasome, photosynthesisantenna proteins, citrate cycle, metabolic pathway, and ABC transporters (Fig. 4b). The expression levels of most of the DEGs related to these pathways were significantly upregulated in HSP-CK (Figs. 4c and 5).
To reveal the functional relationships among the DEGs involved in proteasome, ubiquitin-mediated proteolysis, protein processing in the endoplasmic reticulum, and citrate cycle, a protein-protein interaction network was generated with the STRING program. This network showed that the subgenes encoded diverse proteins, including protein disulfide isomerase (prtp), heat shock protein 70 (HSP70), 20S proteasome alpha subunit E (PAE1), and malate dehydrogenase (MMDH1) (Fig. 6). Moreover, the expression levels of the DEGs associated with the top 20 enriched KEGG pathways were mainly downregulated and upregulated in HSP-3 h and CK-3 h samples, respectively (Fig. 7).

Discussion
In the present study we determined that PhHsp22 contained the theoretical domain of ACD and HSP20, which is consistent with the domains of Arabidopsis thaliana sHSPs . The N-terminal amino acid sequence of sHSPs is poorly conserved in other species, indicating that the Nterminal of PhHsp22 has lower conservation . In contrast, the C-terminal is highly conserved and contains ACD . A previous study confirmed that the C-terminal region of PsHsp20 in Porphyra species is highly conserved . Our findings suggest that the PhHsp22 may play a similar function of response to stress at the C-terminal. sHSPs have a high capacity to bind to non-native proteins, but not able to refold, which maybe interact through hydrophobic. Phylogenetic analysis demonstrated that PhHsp22 was poorly clustered with the sHSPs genes from other species (Fig. 1b), which reflect the diversity in the evolution of these genes.
HT stress has a complex effect on cell functions and can completely inactivate enzymes, resulting in protein denaturation (Kampinga et al. 1995). Accordingly, HT may disrupt the synthesis of native proteins in plants, while accelerating the production of HSPs and heat shock factors (Nover et al. 2001;Wang et al. 2004). Our earlier research involving transcriptome analyses and cloning revealed that the synthesis of sHSPs in Pyropia species might increase to mitigate the harmful effects of environmental stresses, including high temperatures , drought (Shi et al. 2019), and salinity . Because only P. yezoensis has a stable Fig. 4 Functional analyses of differentially expressed genes. a Global overview of the differentially expressed genes (DEGs) among samples (WT-CK, wild-type unstressed control; WT-3 h, wild type exposed to high-temperature stress for 3 h; HSP-CK, transgenic C. reinhardtii unstressed control; HSP-3 h, transgenic C. reinhardtii exposed to hightemperature stress for 3 h). b Top 20 enriched KEGG pathways among the DEGs between WT-CK and HSP-CK. c Number of up-and downregulation genes related to the top 20 enriched KEGG pathways transformation system, there is a lack of relevant research on P. haitanensis (Uji et al. 2014). We introduced PhHsp22 into the C. reinhardtii genome and analyzed its heat-induced expression in the current study. Driven by the Hsp70A and RbcS2 tandem promoter, PhHsp22 in transgenic lines could respond positively to heat stress (Fig. 2b). The selection of an appropriate promoter is crucial for gene transformations and transgenes expression in plants (Potenza et al. 2004). Unfortunately, only a few promoters have been confirmed as suitable for red algae (Huddy et al. 2012;Son et al. 2012). There are some promoters in P. yezoensis that can be used normally, but they have not been proven to be suitable for P. haitanensis (Fukuda et al. 2008;Uji et al. 2010). The observed upregulated expression of PhHsp22 suggests that Hsp70A and RbcS2 promoters may be effective for driving transgene expression in C. reinhardtii. In addition, the present result findings imply that PhHsp22 contributes to HT stress response in C. reinhardtii. During the high-temperature (33°C ) treatment, the promoter of hsp70a-rbcs2 induced high expression of phhsp22 in transgenic C. reinhardtii from 3 to 6 h (Fig. 2c). Meanwhile, overexpression of PhHsp22 contributes to heat stress tolerance in Chlamydomonas (Fig. 3). These results are similar to those of an earlier transgenic study (Jin et al. 2017). The expression of the Pyropia tenera HSP19.3 gene inserted into the C. reinhardtii genome is reportedly upregulated soon after initiating a HT treatment and confers Fig. 5 Heat map of the differentially expressed genes involved in the proteasome, ubiquitin-mediated proteolysis, protein processing in the endoplasmic reticulum (ER), and citrate cycle (TCA cycle) between WT-CK (wild-type unstressed control) and HSP-CK (transgenic C. reinhardtii unstressed control) the tolerance to heat (Jin et al. 2017). Our data suggest that PhHsp22 from P. haitanensis might play a significant role in resisting HT stress.
As chaperones, sHSPs can bind to denatured proteins in an ATP-independent pattern to prevent them from aggregating thereby maintaining cellular homeostasis. PhHsp22 is the most heat stress-responsive P. haitanensis sHSPs gene. Moreover, its positive effects on the HT tolerance of transformed C. reinhardtii have been confirmed. However, it remains unclear how much of this HT tolerance is due to this gene. Therefore, we compared the transcriptional profiles of WT and PhHsp22-overexpressing C. reinhardtii exposed to the same HT stress. The results suggest that PhHsp22 mainly affects the proteasome, photosynthesis-antenna proteins, citrate cycle, oxidative phosphorylation, biosynthesis of amino acid, protein processing in endoplasmic reticulum, ubiquitinmediated proteolysis, and other pathways (Figs. 4 and 5). Thus, the transgenic C. reinhardtii had fewer DEGs than WT control following the same HT treatment (Fig. 4a), indicating that PhHsp22 can maintain the cellular homeostasis of C. reinhardtii in response to HT stress.
Misfolded and denatured proteins inevitably accumulate in Pyropia thalli under HT stress conditions. The misfolded and unfolded proteins are perceived as endoplasmic reticulum Fig. 6 Protein-protein interaction network for differentially expressed genes related to proteasome, protein processing in endoplasmic reticulum, ubiquitin-mediated proteolysis, and citrate cycle (TCA cycle) in transgenic C. reinhardtii based on a STRING analysis. The proteins encoded by the sub-genes in the network are presented in different colors.
Red color represents a degree between 634 and 425, whereas purple color represents a degree between 425 and 200. The green line indicates a combined score greater than 543, whereas the gray line indicates a combined score of less than 543. The circle size corresponds to the number of protein interactions. Protein annotations are listed in Table S2 stress by specific sensor proteins in the endoplasmicreticulum membrane, ultimately resulting in the expression of genes encoding chaperones. In addition, misfolded and unfolded proteins are ubiquitinized by 26S proteasome for endoplasmic reticulum-associated degradation (ERAD) or inhibit protein translation to reduce the abundance of synthetic proteins and avoid endoplasmic reticulum stress (Helenius and Aebi 2004;Zhu 2016). The quality control system recognizes proteins in the endoplasmic reticulum based on interactions with calnexin and calreticulin (Helenius and Aebi 2004). Correctly folded proteins are transported to the Golgi apparatus through COP II, whereas misfolded and denaturation proteins are degraded by a proteasome or refolded via HSPs and other molecular chaperones (Wang et al. 2018a;Shi et al. 2019;Wen et al. 2020). In a recent study, all of the genes annotated by ubiquitin-mediated proteolysis, including those encoding the ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, ubiquitin ligase, and most of the genes related to 26S proteasome pathway exhibited upregulated in P. haitanensis under severe HT conditions (Wang et al. 2018a). This HT-induced expression may be due to the fact that the ubiquitin-proteasome system mediates the removal of damaged and/or misfolded proteins and modulates regulatory protein contents. Additionally, Wen et al. (2020) pointed out that increases in the abundance of HSPs and proteins involved in ubiquitinmediated proteasome activity stabilize the dynamic balance between protein folding and removal in P. haitanensis in response to hypersalinity. Here, the expression levels of 26 of 35 DEGs related to ubiquitin-mediated proteolysis as well as 25 of 38 DEGs related to protein processing in endoplasmic reticulum were upregulated in the PhHsp22-overexpressing transgenic C. reinhardtii (Fig. 5). Furthermore, PhHsp22 upregulated the expression of all but one of the 36 DEGs related to proteasomes which are large multi-subunit proteases in the cytosol (Bochtler et al. 1999). Proteasomes can recognize, unfold, and digest protein substrates that have been marked for degradation with an ubiquitin moiety (Bochtler et al. 1999). Thus, the C. reinhardtii cells expressing PhHsp22 may be protected from fluctuations in environmental conditions via buffering proteasome and HSPs pools (Hanna et al. 2007). HSP expansions mediating protein homeostasis might reflect the adaptation of Pyropia species to the intertidal Fig. 7 Numbers of up-and downregulated genes among the top 20 enriched KEGG pathways between HSP-CK and HSP-3 h (a) as well as between WT-CK and WT-3 h. b WT-CK, wildtype unstressed control; WT-3 h, wild type exposed to heat stress for 3 h; HSP-CK, transgenic C. reinhardtii unstressed control; HSP-3 h, transgenic C. reinhardtii exposed to heat stress for 3 h environment . The protein-protein interaction network analysis also indicated that HSPs influence key regulatory activities (Fig. 6). Therefore, our results imply that PhHsp22 contributes to the HT tolerance of C. reinhardtii by maintaining cellular homeostasis.
Additionally, we previously determined that severe HT stress substantially upregulates the expression of genes related to encoding energy metabolism in P. haitanensis, thereby enhancing carbohydrate metabolism to provide energy and the related carbon skeletons for key metabolic processes (Wang et al. 2018a) such as the citrate cycle is responsible for the oxidation of respiratory substrates that drive ATP synthesis (Sweetlove et al. 2010) and considerably influences the ability of plants to resist the effects of abiotic stresses (Mailloux et al. 2007). First, pyruvate dehydrogenase converts pyruvic acid to acetyl coenzyme A (acetyl-CoA). The formation of citric acid from acetyl-CoA and oxaloacetic acid via a reaction catalyzed by citric acid synthetase represents the beginning of the citrate cycle. Citric acid synthetase is the rate-limiting enzyme of the citrate cycle. We observed that compared with the WT levels, the expression levels of 28 of 29 DEGs related to pyruvate dehydrogenase, malate dehydrogenase, succinate dehydrogenase, citrate synthase, and other key enzymes were obviously upregulated in the transgenic C. reinhardtii cells (Fig. 5). This is consistent with previous studies, C. reinhardtii switching from regular metabolism to a specialized metabolism for stress protection (Schroda et al. 2015). Within the first 3 h of HS stress, several of TCA/ glycolytic enzymes and the glyoxylate cycle were significantly downregulated, while the main metabolism metabolites are rapidly depleted (Hemme et al. 2014). At the same time, the quantitative results also verify this point ( Figure S2). Therefore, PhHsp22 may mediate energy metabolism in P. haitanensis exposed to high-temperature stress. The enhancement of the citrate cycle and other energy metabolism-related processes not only increase energy production but also provide sufficient amounts of carbon skeletons for amino acid biosynthesis, DNA replication, arginine biosynthesis, and protein processing (Fig. 4c) to protect thalli from HT stress.

Conclusion
In this study, the function of the PhHsp22 gene from P. haitanensis was verified by transformed into C. reinhardtii. The early upregulation of PhHsp22 expression following a HT treatment suggests this gene contributes to the early response to HT stress. Moreover, its HT-induced expression increases the survival of transgenic C. reinhardtii via its effects on cellular energy, secondary metabolites, HSPs, and proteasome pools. The results of this investigation may clarify the regulatory effects of HSPs on the HT tolerance of P. haitanensis. Furthermore, PhHsp22 may be useful for the breeding of new macroalgal strains adapted to global warming.