Introduction

Porphyra is a red algal genus of laver and includes more than 130 species. Several Porphyra species, including Porphyra yezoensis and Porphyra seriata, have been employed as edible seaweeds and have been cultivated in aquaculture industries in East Asia (Miura 1988; Hwang et al. 2005). Under natural conditions, most Porphyra grow in the intertidal zone and are frequently exposed to a variety of potentially stressful environmental conditions with the turning tides (Sahoo et al. 2002). These changes include changes in humidity, from drying to partial to total immersion; changes in temperature, from heat stress to freezing during hibernal low tides; osmotic shocks; and light intensities (Hwang et al. 1997). It has been demonstrated that plants carry out the appropriate physiological and biochemical changes in order to deal with environmental stresses (Bray et al. 2000). In the acclimation process, the expression of genes involved in abiotic stress tolerance including ion transporters, osmoprotectants, free-radical scavengers, stress proteins such as heat shock protein (HSP), and factors involved in signaling cascades and transcriptional control are induced or upregulated (Bray et al. 2000; Wang et al. 2004).

HSPs play an essential role in protecting cells, and the folding and translocation of nascent proteins, the refolding of denatured proteins, the disassembly of already formed protein aggregates, and so on, under both stress and non-stress conditions (Nelson et al. 1992; Hartl 1996; Feder and Hofmann 1999; Bray et al. 2000; Schroda and Vallon 2009). The presence of HSPs and their induction by environmental stresses have been shown to be a central tolerance mechanism in many organisms (Feder and Hofmann 1999; Ireland et al. 2004). Therefore, it is worthwhile to evaluate the role of HSPs in the adverse stress resistance mechanisms of seaweeds.

HSPs are grouped into five distinct classes named for their approximate molecular masses: HSP100, HSP90, HSP70, HSP60, and small HSP (sHSP) (Bray et al. 2000; Wang et al. 2004; Schroda and Vallon 2009). HSPs are located within both the cytoplasm and organelles, including the nucleus, mitochondria, chloroplasts, and ER (Boston et al. 1996). The HSP100 family chaperones are members of the large ATPase superfamily with a broad spectrum of diverse functional properties (Wang et al. 2004). HSP90 is distinct from other molecular chaperones in that most of its known substrates to date are signal transduction proteins such as steroid hormone receptors and signaling kinases (Wang et al. 2004; Schroda and Vallon 2009). Among the HSP families, HSP70s are a highly conserved and ubiquitous protein family (Karlin and Brocchieri 1998; Feder and Hofmann 1999; Renner and Waters 2007). They perform essential roles in the transport of nascent proteins across membranes into organelles, the folding of newly translated proteins, the repair of misfolded proteins, and helping to target damaged proteins for degradation (Nelson et al. 1992; Hartl 1996; Fink 1999). Under stress conditions, HSP70s are upregulated, participate in the refolding of denatured proteins, maintain cell homeostasis, and protect organisms against damage (Jolly and Morimoto 1999; Hartl and Hayer-Hartl 2002; Mayer and Bukau 2005; Tanaka et al. 2007). HSP70s are a kind of potential biomarker of environmental stresses and can be applied to the monitoring of environmental conditions (Ireland et al. 2004). The HSP60 family is a class of molecular chaperones that is evolutionarily homologous with Escherichia coli GroEL and found in prokaryotes (Hemmingsen et al. 1988; Hartl 1996). The small HSPs (sHSPs) are low molecular mass HSPs (12–40 kDa). In plants, sHSPs form a more diverse family than other HSPs/chaperones with regard to sequence similarity, cellular location, and functions (Vierling 1991). sHSPs function in responses to heat and other stresses, and some sHSPs are expressed during certain developmental stages (Boston et al. 1996).

The Porphyra are an important marine crop and have been proposed as a model plant to study the mechanisms underlying abiotic stress tolerance (Blouin et al. 2011). The recent sequencing of the complete genomes or transcriptome of algae allows us to identify the genes for abiotic stress tolerance in the genome. Herein, we describe the isolation and characterization of the cDNAs encoding for heat shock protein (HSP) from P. seriata. We isolated a total of nine cDNAs encoding for HSP from the P. seriata ESTs and named them PsHSP90, PsHSP70, PsHSP40, and PsHSP20 according to their molecular weight and amino acid sequence homology. We analyze the physiological function of the putative mitochondrial HSP, PsHSP70b gene by using heterologous expression in Chlamydomonas.

Material and Methods

Organisms and Growth Condition

P. seriata was collected and maintained via in vitro cultivation under controlled conditions. Leafy gametophytes of P. seriata were cultured in modified Grund medium (McLachlan 1973) at 10°C with irradiation of 80 μmol photon m−2 s−1 provided by cool-white fluorescent lamps with a photoperiod of 14:10 (light/dark) in a growth room. For heat treatment, growth bottles containing P. seriata were transferred to a 25°C growth chamber with the same light intensity and photoperiod.

Chlamydomonas reinhardtii strain cc-125 (mt+) was grown in Tris–acetate–phosphate (TAP) medium (Harris 1989). Chlamydomonas cells were cultured at 25°C in TAP liquid medium with shaking at 200 rpm under continuous cool fluorescent light (50 μmol photon m−2 s−1).

Isolation and Sequence Analysis of PsHSP cDNA

In a previous study, we generated 3,979 ESTs from two P. seriata cDNA libraries constructed from the gametophyte thalli at 10°C (control) and under high-temperature (25°C) conditions, respectively (Kim et al. 2011). The ESTs were analyzed for Porphyra heat shock protein gene resources. The individual ESTs were searched against the GenBank nr database, using a BLASTX algorithm. The putative HSP cDNAs were identified via keyword searches with BLASTX results.

Colonies containing putative HSP cDNA were selected and cultured, and then plasmid DNAs were purified using a Qiaquick Plasmid Extraction Kit (Qiagen, Hilden, Germany) and sequenced. Sequence editing and amino acid sequence prediction from the selected ESTs were conducted using the Sequencher program (Gene Code Corporation, Ann Arbor, MI, USA). The putative molecular weights and pI values of the deduced polypeptides were predicted with the DNASIS Max program (MiraiBio Inc., San Francisco, CA, USA). The alignments of the deduced amino acid sequences were conducted using the CLUSTAL W program (http://www.ebi.ac.uk/clustalw). The phylogenetic tree was constructed using the program. Conserved motifs or domains are predicted by the Internet program Prosite at http://expasy.org/prosite.

Gene-Specific RT–PCR

Total RNAs were prepared from leafy gametophyte thalli tissues, using a Plant RNeasy kit (Qiagen). The first-strand cDNAs were constructed from 2 μg of total RNA via reverse transcription in 20-μl reaction volumes, using oligo(dT)17 primer and Superscript III reverse transcriptase, in accordance with the manufacturer’s instructions (BRL Life Technologies, Carlsbad, CA, USA). The reactions were conducted for 60 min at 42°C, followed by 5 min of heating at 70°C. The first-strand cDNA reaction was diluted by a factor of 5, and then 2 μl of diluted cDNA were applied to a 50-μl PCR-amplification reaction, containing 5 μl of 10× PCR buffer [200 mM Tris–HCl (pH 8.4), 500 mM KCl], 1 μl of 10 mM dNTPs, 1 μl of each gene-specific primer (10 μM), and 2.5 U of ExTaq DNA polymerase (Takara, Shiga, Japan). PCR reactions were carried out for 35 cycles, each consisting of 30 s at 95°C, 30 s of 58 to 64°C, 90 s of 72°C, and 5 min of termination at 72°C. The annealing temperature and PCR cycle for each of the gene-specific primers were adjusted for optimal PCR reaction. The PCR products were separated on 1% agarose gels and stained with ethidium bromide for photography.

Real-Time Quantitative RT–PCR

Reverse transcription (RT) was carried out using 2 μg of total RNA and 200 U of M-MLV reverse transcriptase (Promega, Madison, WI, USA), 50 μM oligo(dT), 500 μM of each dNTP, and 20 U of ribonuclease inhibitor. For real-time RT–PCR, EvaGreen Real-Time PCR kits (SolGent, Daejeon, Korea) were utilized according to the manufacturer’s instructions with a light cycler, Opticon2 (MJ Research, Ramsey, MN, USA). Amplification was conducted using 40 cycles of 92°C for 20 s, 53°C to 60°C for 20 s, and 72°C for 20 s.

Vector Construction for the PsHSP70b Expression in Chlamydomonas

The pCr102 vector, which harbors regulatory sequences with the psaD promoter and the terminator driven by the Chlamydomonas genome for transgene expression, as well as a hygromycin resistance gene (aph7”) under the control of the tubB2 promoter and the rbcS2 terminator were used for the selection of transformants. The open reading frame of the HSP70 gene was amplified via PCR and subcloned into a pCr102 vector using the NcoI and EcoRV sites, and named pCr102-PsHSP70b.

For the transformation of Chlamydomonas, the pCr102-PsHSP70b plasmid was linearized with XbaI and introduced into Chlamydomonas strain cc-125 via the glass bead method (Kindle 1990). Transformed colonies were selected on TAP agar medium containing 10 μg/ml of hygromycin after 7–14 days of growth.

DNA and RNA Blot Analysis

Chlamydomonas genomic DNA was purified from 100-ml liquid cultures. For Southern blot analysis, 10 μg of genomic DNA was digested with NcoI, separated via 0.8% agarose gel electrophoresis, and then blotted onto Hybond-N nylon membrane (Amersham Biosciences, Uppsala, Sweden). To assess the expression of the introduced PsHSP70b gene, total RNAs were isolated from control and transformed Chlamydomonas cells grown at 25°C and separated on 1.2% agarose gel and subsequently transferred onto nylon membranes. The probe (a 0.9-kb fragment) corresponding to the PsHSP70 cDNA was prepared from XbaI/EcoRV digested transformation vector. For all blot analyses, hybridization with 32P-labeled probes and washing were conducted in accordance with the manufacturer’s instructions. The membrane hybridized with 32P-labeled probe was exposed to a phosphor imaging plate (IP) for 2 days, and signals were detected using a Bio-Imaging Analyzer BAS-1800II (Fuji, Tokyo, Japan).

Heterologous Expression and Analysis of the PsHSP70b Gene in Chlamydomonas

Chlamydomonas cells were grown in TAP medium containing hygromycin at 25°C under a 14-h light/10-h dark cycle. To evaluate high-temperature tolerance, the cells were grown at a concentration of approximately 2–4 × 106 cells/ml and concentrated to 107 cells/ml and diluted to 102–104 in fresh medium. Five microliters of diluted cells was inoculated onto agar plates. For heat stress treatment, the cells were incubated at 37°C for 5 days and subsequently transferred to a 25°C growth chamber under a 14-h light/10-h dark photocycle. C. reinhardtii strain cc-125 and transformed cells with the pCr102 vector, Hyg5414, were used as controls.

Results and Discussion

Identification of the P. seriata HSPs cDNAs

In a previous work, we generated 3,989 ESTs from two cDNA libraries constructed from P. seriata thalli under normal growth conditions and heat stress (Kim et al. 2011). EST analysis via keyword allowed us to identify the cDNAs encoding for putative heat shock protein (HSP). The clustering of the HSP cDNA candidates generated five contigs and four unassembled ESTs, and represented nine unique HSP cDNAs (Table 1). The longest sister cDNA clone in the contig was selected as a representative EST for each HSP contig and was used in the determination of the full sequences.

Table 1 Heat shock proteins isolated P. seriata
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Homology searching using the BLASTX program showed that the nine putative HSP cDNAs encoded for different HSPs. The characteristics of the deduced polypeptides from the nine PsHSP cDNAs are summarized in Table 1. Based on the amino acid sequence homology and molecular weight of the deduced polypeptide, we named the genes PsHSP (Porphyra seriata heat shock protein) 90, PsHSP70, PsHSP40, and PsHSP20. The other three cDNAs, PsHSP_O22, PsHSP_D07, and PsHSP_G17, have 5′-truncated cDNA and sequence homology with putative HSP90, ER HSP70, and DnaJ-like protein, respectively. Figure 1 represents the amino acid sequence of the deduced polypeptide from the nine PsHSP cDNAs. Five major families of HSPs—HSP100, HSP90, HSP70, HSP60, and small HSP (sHSP)—are conservatively recognized in plant and algae genomes (Wang et al. 2004; Schroda and Vallon 2009).

Fig. 1
figure 1figure 1

Deduced amino acid sequence of the PsHSPs (a) and sequence alignment of PsHSP70 (b) from P. seriata. a Deduced amino acid sequences of the PsHSPs, PsHSP90, PsHSP40, PsHSP20, PsHSP_O22, PsHSP_D07, and PsHSP_D17. Amino acid residues are marked with single letters. The putative signal peptide is underlined and the ATPase domain is double-underlined in PsHSP90. The DnaJ domain and zinc finger DNA binding domain are double-underlined in PsHSP40. A putative α-crystallin domain was double-underlined in PsHSP20. The conserved amino acids motif, HDEL for the endoplasmic reticulum (ER) group and EEVD or EEID for the cytosolic group, are marked as bold in PsHSP90, PsHSP_O22, and PsHSP_D07. b Amino acid sequence alignment of the three HSP70s from P. seriata. Amino acid residues are marked with single letters. The putative signal peptide is underlined in PsHSP70b. The three amino acid motifs found in HSP70 are boxed. The conserved amino acids motif, EDID for the cytosolic group, is marked as bold in PsHSP70a. Asterisk (*) and colon (:) indicate identical and similar amino acid residues. Alignments of the deduced amino acid sequences were conducted with the Clustal X program

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Characterization of the PsHSP

Among the identified PsHSPs, the PsHSP90 encodes for a 95.6-kDa polypeptide with 872 amino acids (pI 4.79). Prediction of the signal peptide in the PsHSP90 by the PrediSi program (Prediction of signal peptides, www.pridisi.de) indicated that 25 amino acid residues at the N terminus may be employed as a signal peptide for secretion. PsHSP90 evidences the highest amino acid sequence homology to ER HSP90 from Ostreococcus lucimarinus. HSP90 family members are highly conserved and harbor the N-terminal ATPase domain. PsHSP90 harbors an ATPase domain in the N-terminal region (Fig. 1a). HSP90 family includes seven members in the Arabidopsis genome. Chlamydomonas harbors three genes encoding for the HSP90 family, which is located in the cytoplasm, plastid, and endoplasmic reticulum, respectively (Wang et al. 2004; Schroda and Vallon 2009). The C terminus of each subgroup of HSP contains a highly conserved motif, which is EEVD or EEID for cytosolic, and HDEL for the endoplasmic reticulum (ER) group (Guy and Li 1998; Sung et al. 2001a). PsHSP90 harbors the RDEL sequence in the C terminus, which is similar to the HDEL motif found in the ER HSP. These results indicate that PsHSP90 may be located within the ER. PsHSP_O22 evidences the highest amino acid sequence homology to HSP90 and the EDVD motif in the C-terminal end region. These results indicate that PsHSP_O22 may encode for a cytosolic HSP90. It has been reported that some HSP90 members are upregulated by abiotic stress and perform a function in stress adaptation in plants (Krishna and Gloor 2001), although cytosolic HSP90 has a role in the maturation of signal transduction proteins such as hormone receptors and signaling kinases (Richter and Buchner 2001) in animals.

We identified three cDNAs encoding for an approximate 70-kDa polypeptide and named them PsHSP70a, PsHSP70b, and PsHSP70c (Table 1). PsHSP70a is a 664-amino acid residue polypeptide (71.8 kDa, pI 4.94). PsHSP70b and PsHSP70c are polypeptides comprising 736 amino acids (76.9 kDa, pI 5.28) and 663 amino acids (72.4 kDa, pI 6.29). The PsHSP70 polypeptides harbor three motifs—IDLGTTNS, VYDLGGGTFDVSIL, and VLLVGGMTRVPKVVE—found in the HSP70 family, and also harbor the ATPase domain and substrate binding domain (Fig. 1b). The amino acid sequence alignment of three PsHSP70s showed that there were sequence variations, including deletions and substitutions, in the N terminus and C terminus (Fig. 1b). PsHSP70a harbors an EDID motif at the C-terminal end, which is similar to the EEID motif found in cytosolic HSP. The prediction of signal peptides using the PrediSi program (www.pridisi.de) demonstrates that PsHSP70b harbors a putative signal peptide in its N-terminal region. These results indicate that PsHSP70a may be located in the cytosol and PsHSP70b may be transported into subcellular organelles. PsHSP_D07 evidences the highest amino acid sequence homology to the ER luminal binding protein, Bip, and EEDD motif in the C-terminal region (Table 1, Fig. 1a). These results indicate that PsHSP_D07 may encode for an ER HSP70.

To predict the subfamily of each PsHSP70, different types of HSPs from Arabidopsis, Chlamydomonas, and a few algae were employed to construct a phylogenetic tree using the Clustal X program. In the phylogenetic tree of HSP70s, PsHSP70b and PsHSP70c clustered together with mitochondrial HSP70s, and PsHSP70a and PsHSP_D07 were grouped with the cytosolic and ER HSP family, respectively (Fig. 2). The results of the phylogenetic tree of HSP70s and the motifs or putative signal peptides found in PsHSPs indicate that PsHSP70a is a cytosolic HSP70, and PsHSP70b may be transported to subcellular organelles, specifically the mitochondria. However, the conserved amino acid motif, PEAEYEEAKK, in the mitochondrion (MT) group was not detected in PsHSP70b and PsHSP70c. The signal peptide sequence for the subcellular localization was also not detected in PsHSP70c (Fig. 1b). The localization of PsHSP70 could be confirmed using a reporter gene system such as GFP.

Fig. 2
figure 2

Phylogenetic tree of the PsHSP70s with HSPs from Arabidopsis and Chlamydomonas. The phylogenetic tree of the HSP70 family member was constructed via the unrooted neighbor-joining method using the ClustalX program. Accession numbers of the HSPs from Chlamydomonas and Arabidopsis followed the name of each HSP

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HSP70s have highly specialized functions such as the regulation of the heat stress response and membrane translocation of proteins (Kang et al. 1990; Tomoyasu et al. 1998). In higher plants, HSP70 genes are generally encoded by multigene families. Recently, the completion of genome sequencing in some model plants and unicellular algae demonstrates that Arabidopsis thaliana harbors 14 HSP70 genes consisting of five cytoplasmic, three ER, two mitochondrial, and two chloroplast members, and C. reinhardtii have nine HSP70 genes of two cytoplasmic and four mitochondrial or chloroplast members, and three ER members (Schroda and Vallon 2009), and C. merolae have four HSP70 genes with one each for cytoplasmic, ER, mitochondrial, or chloroplast members, based on cDNA or genomic DNA annotation (Renner and Waters 2007).

The PsHSP40 encodes for 429 amino acids (46.1 kDa, pI 6.61), which are similar to the HSP70 cochaperones, DnaJ(HSP40). PsHSP40 harbors a DnaJ domain at residues 34 to 96 and a zinc finger DNA binding domain at residues 150 to 235 (Fig. 1a). HSP70 cochaperones are usually involved in the regulation of the ATPase activity of their HSP70 partner; they supply it with substrates, and/or connect it with other factors involved in protein folding or degradation (Hennessy et al. 2005). These HSP70 cochaperones were classified into several classes with DnaJ-domain protein regulating ATPase, GrpE-type nucleotide exchange factor, CHIP1 homolog mediate ubiquitinylation of trapped subtract and the HOP-like protein, which mediate the interaction of HSP70 and HSP90. PsHSP_G17 also evidences the highest level of amino acid sequence homology to the DnaJ-like protein and harbors a DnaJ domain (Table 1, Fig. 1a). These results indicate that PsHSP_G17 may encode for a DnaJ/HSP40. The DnaJ/HSP40 interacts, via the DnaJ domain, with its HSP70 partner and stimulates its ATPase activity and supplies it with specific substrates (Liberek et al. 1991; Han and Christen 2003). The DnaJ-domain proteins are multigene family in Chlamydomonas and land plants. Among these, few J-domain proteins harbor the zinc finger domain. PsHSP40 may be one of the DnaJ proteins harboring the zinc finger DNA biding domain in the Porphyra genome.

PsHSP20 encodes for a 25.8-kDa polypeptide with 248 amino acids (pI 6.45) and is similar to the HSP20 family (Table 1). The PsHSP20 polypeptides harbor an α-crystallin domain at residues 139 to 228, which are 90 conserved amino acids at the C-terminal region of the sHSPs (Fig. 1a). The sHSPs are low molecular HSPs with 12–40 kDa and synthesized responses to heat and other stresses. Plants contain a large number of different sHSP genes. In Arabidopsis, 13 different sHSPs are grouped into six classes based on their intracellular localization and sequence relatedness (Scharf et al. 2001). A total of eight sHSP genes are identified from the genome of Chlamydomonas (Schroda and Vallon 2009). The sHSP are not themselves able to refold non-native proteins. They have a high capacity to bind to non-native proteins, probably via hydrophobic interaction, and to stabilize and prevent non-native aggregation. Plant sHSPs respond to a broad range of environmental stresses, including heat, cold, drought, salinity, and oxidative stress (Wang et al. 2004).

Expression of PsHSP Genes

In an effort to evaluate the expression of each PsHSP gene under heat stress conditions, total RNAs were isolated and subsequently subjected to RT–PCR with PsHSP gene-specific primer (Table 2). Figure 3 shows the expression of each PsHSP gene under normal growth condition and heat stress. RT–PCR results show that most of the PsHSP genes are expressed under normal growth conditions, as well as heat stress. However, the transcripts of two PsHSP genes, PsHSP70c and PsHSP_G17, were not observed under the conditions tested. These results indicate that PsHSP70c and PsHSP_G17 are expressed at relatively low levels or at different life stages, either in a tissue-specific manner or under other stimuli. According to the RT–PCR results, it may prove difficult to evaluate the relative expression patterns of the HSP genes, which are expressed abundantly in the cell. However, Fig. 3 shows that the transcript levels of PsHSP70b and PsHSP20 were increased by heat stress. Many plant genes are induced or upregulated in response to heat stress. The genes expressed under heat stress conditions might also be involved in cellular thermotolerance. Among the PsHSP genes, PsHSP70b and PsHSP20 are upregulated by heat stress, and may be involved in the thermotolerance of Porphyra.

Table 2 Lists of primers for the PsHSP and CrHSP genes used in PCR
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Fig. 3
figure 3

Expression pattern of the PsHSP gene under heat stress conditions. Total RNAs were purified from gametophyte thalli and employed for RT–PCR. For heat treatment, gametophyte thalli were transferred into an incubator at 25°C, and then the thallus tissues were harvested at 1-, 3-, 6-, 12-, 24-, and 46-h intervals. The annealing temperatures and PCR cycles were adjusted for some of the primer sets in order to optimize the PCR reactions. The PCR products were separated on 1% agarose gels and stained with ethidium bromide for photography

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In land plants, the sHSPs family of PsHSP20 are the most abundant heat stress-induced proteins, and their transcription levels are increased by various stresses such as cold, drought, and oxidants, in addition to heat stress (Sabehat et al. 1998; Soto et al. 1999; Schroda and Vallon 2009).

HSP70 genes are generally encoded for by multigene families; some members of the HSP70 family are constitutively expressed and other members are expressed in response to heat and other abiotic stresses (Fu et al. 2009; Tominaga et al. 2010). The constitutively expressed HSP70 are frequently involved in assisting in the folding of de novo synthesized polypeptides and the import or translocation of precursor proteins (Frydman 2001; Wang et al. 2004). Other family members that respond to environmental fluctuation may be involved in facilitating refolding and proteolytic degradation of non-native proteins (Hartl 1996; Miernyk 1997). The PsHSP70a that is constitutively expressed may be located in the cytosol and involved in the folding or translocation of newly synthesized polypeptides. The PsHSP70b may be involved in abiotic stress tolerance in Porphyra.

Increase of Thermotolerance by Expression of the PsHSP70b Gene in Chlamydomonas

The PsHSP70b gene was increased at the transcriptional level under heat stress condition. In order to determine the physiological functions of the PsHSP70b gene, we introduced the complete coding sequence of the PsHSP70b into C. reinhardtii strain cc-125 under the control of the psaD promoter (Fig. 4a). The vector alone was also introduced into C. reinhardtii and named Hyg5414. Five PsHSP70b-transformed Chlamydomonas lines were selected on a medium containing hygromycin, and the introduction and expression of the PsHSP70b gene were confirmed via DNA blot and RNA blot analysis (Fig. 4b). These results indicate that the PsHSP70b gene was introduced into the Chlamydomonas genome and expressed. The RNA blot data demonstrate that the transcript level of the PsHSP70b in Chlamydomonas varied in each transformant line (Fig. 4b). The PsHSP70b gene was expressed at much higher levels in transformant line 2 and line 4 than in other lines.

Fig. 4
figure 4

Effect of the PsHSP70b on the growth of Chlamydomonas under heat stress conditions. a Vector map for the expression of PsHSP70b in Chlamydomonas. b Verification of introduction and expression of the PsHSP70b gene in transformed Chlamydomonas. To assess the introduction of PsHSP70b, genomic DNA was digested with NcoI and hybridized with the PsHSP70b gene probe. The expression of the PsHSP70b was evaluated via RNA blot analysis. Total RNA were purified from Chlamydomonas at normal growth condition (25°C). cc-125; wild type, Hyg5414; vector control. c Survival and growth pattern of the wild-type and transformed Chlamydomonas under heat stress conditions. Chlamydomonas were heat treated at 37°C for 5 days and transferred into a 25°C growth chamber, then cultured for 7 days. Then 107 cells/ml were diluted to 102–104 in fresh medium and 5 μl of diluted cells was inoculated onto agar plates

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In order to assess the thermotolerance of the Chlamydomonas harboring the PsHSP70b gene, cc-125 and Hyg5414, and PsHSP70b Chlamydomonas lines were subjected to 37°C (heat treatment) for 5 days or not (control), and then transferred into an incubator at 25°C. One week later, Chlamydomonas cells grew at similar levels on the control culture plates (Fig. 4c). However, when heat stress was applied to the cells, the PsHSP70b Chlamydomonas grew at much higher levels than cc-125 and Hyg5414 (Fig. 4c). These results indicate that the Porphyra PsHSP70b gene contributes to high-temperature tolerance in Chlamydomonas.

The overexpression of HSP70s in plants, which is correlated with enhanced stress tolerance, including thermotolerance, has been previously reported. The overexpression of an ER HSP70 homologue BiP gene in tobacco was shown previously to alleviate water stress and ER stress induced by tunicamycin treatment (Leborgne-Castel et al. 1999; Alvim et al. 2001). Overexpression of DnaK from cyanobacteria (Aphanothece halophytica) enhances heat and salt stress tolerance in transgenic tobacco plants (Sugino et al. 1999; Ono et al. 2001). The constitutive overexpression of cytosolic HSP made Hsc70-1 plants more tolerant to heat shock, but Hsc70-1 also appeared to be deleterious in terms of viability, growth, and development (Sung and Guy, 2003). Transformants overexpressing Hsc70-1 induced reductions in root and shoot meristem activities, thus resulting in dwarfism in those plants (Sung and Guy 2003; Cazale et al. 2009). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes (Sung and Guy 2003). The different phenotypic changes observed in these studies suggest that HSP70s localized to different subcellular compartments participate in different cellular processes. The specific roles of individual HSP70 proteins in organisms are likely to be determined by their location in different subcellular compartments, by differential expression of HSP70 genes in specific cells and at different stages of development or via interaction with specific sets of HSP70-associated proteins (Michaud et al. 1997; May and Soll 2000; Sung et al. 2001b).

PsHSP70b Increases the Transcription of CrHSP Genes

Cellular thermotolerance involves many processes, including alterations of membrane permeability and gene transcription and enzyme function (Vierling 1991; Kampinga et al. 1995; Sangwan et al. 2002). HSP/molecular chaperones are key components contributing to cellular homeostasis in cells under heat stress conditions. Figure 4 suggests that Chlamydomonas cells harboring the PsHSP70b gene evidence much higher thermotolerance than normal cells. Therefore, we hypothesized that the heterologously expressed PsHSP70b may stimulate the expression of the CrHSP genes in the Chlamydomonas genome. In order to address this hypothesis, we assessed the transcription level of five Chlamydomonas HSP (CsHSP) genes including the CrHSP22, CrHSP33, CrHSP70, CrHSP70b, and CrHSP90 genes (Table 2) in the Chlamydomonas harboring the PsHSP70b under normal growth conditions (Fig. 5a). Figure 5a shows that the CrHSP22 and CrHSP70 genes were increased by up to 3- to 5-fold in PsHSP70b transformants under normal growth conditions without heat stress. However, transcripts of the CrHSP33 and CrHSP90 genes were detected at levels similar to those in the control cells (Fig. 5a). It has been shown that the small HSP (sHSP) and HSP70 responded to heat stress in higher plants. These results indicate that heterologously expressed PsHSP70b stimulates the transcription of endogenous CrHSP genes. How, then, is heterologous PsHSP70b involved in the activation of CrHSP gene transcription? It is known that the transcription of the HSP genes is mediated by a specific class of transcription factors, termed heat shock factors (HSFs) (Morimoto 1998). Chlamydomonas HSF1 is weakly expressed under non-stress conditions and is induced rapidly by heat shock (Schulz-Raffelt et al. 2007).

Fig. 5
figure 5

Expression of the CrHSPs (a) and HSF1 gene (b) in transformed Chlamydomonas harboring PsHSP70b. Total RNAs were purified from wild-type Chlamydomonas (cc-125), transformed Chlamydomonas (HSP70-1, HSP70-2, HSP70-3) with PsHSP70b gene, and empty vector control (Hyg5414) under normal growth conditions, and the transcription level of the Chlamydomonas HSP gene (CrHSPs) and HSF1 gene were analyzed via real-time RT–PCR. The TubA1 gene was used as an internal control

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In order to determine whether HSF1 mediates the transcription of the CrHSPs gene in PsHSP70b Chlamydomonas under no heat stress, we assessed the transcription level of the HSF1 gene. Figure 5b shows that transcripts of the HSF1 gene were elevated in the transformed Chlamydomonas, line PsHSP70b-1 and PsHSP70b-2 under normal growth conditions, although transcripts of the HSF1 gene were slightly upregulated in line PsHSP70b-3. These results indicate the possibility that heterologously expressed PsHSP70b may activate the transcription of the HSF1 gene, which may increase the transcript level of the CrHSP22 and CrHSP70 genes. However, previous reports indicate that HSP70 acts as a negative regulator of the HSF in animals and higher plants. HSF undergoes stress-induced trimerization, and binding of the HSP70 to HSF inhibits the trimerization of the HSF (Rabindran et al. 1993; Lee et al. 1995). However, Chlamydomonas HSF1 appears to be constitutively trimeric as in yeast, and its interaction with HSP70 may be involved in the early steps of folding (Sorger and Nelson 1989; Schulz-Raffelt et al. 2007). Therefore, negative regulation of the HSF1 by HSP70 may not apply to Chlamydomonas. Heterologously expressed PsHSP70b may be perceived as a signal molecule and may be involved in activation of the signal pathway for heat tolerance. The substrates for the PsHSP70b involved in those responses remain to be identified.

Genetic approaches with a specific HSP mutant cannot readily detect mutant phenotypes, probably as the result of a high level of functional redundancy. Therefore, overexpression systems have been shown to be a useful tool for assigning the functions of an HSP gene. The constitutively expressed PsHSP70b gene in Chlamydomonas shows that this gene is involved in enhanced heat stress tolerance. This system might be employed for studies of the physiological and molecular functions of the PsHSP70b.