Isolation and characterization of a stress-responsive gene encoding a CHRD domain-containing protein from a halotolerant green alga

Accepted Manuscript Isolation and characterization of a stress-responsive gene encoding a CHRD domain-containing protein from a halotolerant green alga

Ryo Ishinishi, Hideyuki Matsuura, Satoshi Tanaka, Saaya Nozawa, Keisuke Tanada, Norihito Kawashita, Kazuhito Fujiyama, Hitoshi Miyasaka, Kazumasa Hirata PII: DOI: Reference:

S0378-1119(17)30825-9 doi:10.1016/j.gene.2017.10.012 GENE 42231

To appear in:

Gene

Received date: Revised date: Accepted date:

7 June 2017 12 September 2017 6 October 2017

Please cite this article as: Ryo Ishinishi, Hideyuki Matsuura, Satoshi Tanaka, Saaya Nozawa, Keisuke Tanada, Norihito Kawashita, Kazuhito Fujiyama, Hitoshi Miyasaka, Kazumasa Hirata , Isolation and characterization of a stress-responsive gene encoding a CHRD domain-containing protein from a halotolerant green alga. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.012

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Isolation and characterization of a stress-responsive gene encoding a CHRD domain-containing protein from a halotolerant green alga Ryo Ishinishia, Hideyuki Matsuuraa,*, Satoshi Tanakac, Saaya Nozawaa, Keisuke Tanadaa, Norihito Kawashitab,1, Kazuhito Fujiyamae, Hitoshi Miyasakad, and Kazumasa Hirataa a

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Applied Environmental Biology Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan b Pharmainformatics and Pharmacometrics Laboratory, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan c The Kansai Electric Power Co., Inc., Advanced Technology Laboratory, Keihanna Engineering Center, 1-7 Seika-cho, Souraku-gun, Kyoto 619-0237, Japan d Department of Applied Life Science, Sojo University, 4-22-1 Ikeda, Nishiku, Kumamoto 860-0082, Japan e International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

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*Corresponding author Hideyuki Matsuura E-mail address: [email protected]

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Present address: Computational Life Science Laboratory, Faculty of Sciences and Engineering, Kindai

University, 3-4-1 Kowakae, Higashiosaka City, Osaka 577-8502, Japan

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ABSTRACT The genetic basis of stress resistance in extremophilic microalgae is not well studied. In this study, a gene of unknown function, the cluster58 or CL58 gene, was identified from the halotolerant green alga Chlamydomonas W80 and characterized. The CL58 gene encodes a protein containing a domain of unknown function, the CHRD domain, and a putative secretory signaling sequence at its N-terminus. The levels of CL58 mRNA increased in response to high copper levels and low temperatures. When the CL58 gene was heterologously expressed as a fusion gene with the NanoLuc luciferase gene in Chlamydomonas reinhardtii, a majority of the NanoLuc activity was detected in the culture medium compared with that in the intracellular fraction. A mutagenic analysis revealed that the putative secretory signaling sequence was sufficient for the secretion of the CL58-NanoLuc fusion protein. In addition, we expressed the protein encoded by the CL58 gene in Escherichia coli; the recombinant, soluble protein was then purified. In summary, we identified a novel gene from C. W80 that appears to encode a stress-responsive, CHRD domain-containing secreted protein.

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Keywords: stress response, Chlamydomonas reinhardtii, secretory protein, recombinant protein, unknown function

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1. Introduction Abiotic stresses, such as drought, high/low temperatures, and high salinity, are factors that critically restrain plant growth and reproduction (Atkinson and Urwin, 2012; Bray et al., 2000). The average yields of most major crop plants are considered to be suppressed by >50% by abiotic stresses (Boyer, 1982). In addition, climate change is expected the lead to environmental conditions that are less conducive to plant growth. Therefore, the development of stress-tolerant plants is a prerequisite for meeting the increasing pressure on global food productivity caused by growing human populations. Extremophilic microalgae have the ability to thrive under extreme conditions, such as high/low temperatures, extreme pH, and high salinity, or in the presence of xenobiotics, such as heavy metals (Varshney et al., 2015). Such microalgae are recognized as an underexploited source of genes that may prove to be useful for plant biotechnology, particularly for the molecular breeding of abiotic stresstolerant plants; that is, these microalgae may serve as sources of anti-stress genes (Hirooka et al., 2009; Waditee et al., 2005; Yoshimura et al., 2004). For example, the gene encoding ascorbate peroxidase from Cyanidioschyzon merolae, an extremophile that tolerates acidic conditions, high temperatures, and reactive oxygen species (ROS) (Misumi et al., 2007), was shown to confer tolerance to oxidative or hightemperature stresses when overexpressed in Arabidopsis thaliana plants (Hirooka et al., 2009). In addition, A. thaliana plants expressing N-methyltransferase enzymes, which are derived from the halotolerant cyanobacterium Aphanothece halophytica, improved plant tolerance to various abiotic stresses (Waditee et al., 2005). Moreover, microalgae are promising as potential sustainable sources of energy, commercial products, and human and animal food (Varshney et al., 2015); extremophilic microalgae are now being considered as candidates for biotechnological exploitation as sources of genes that could allow non-extremophiles to cope with various extreme environmental conditions (Varshney et al., 2015). In this context, it is important to reveal the genetic basis of tolerance to extreme conditions in extremophilic microalgae, as well as to identify the anti-stress genes involved. The marine green alga Chlamydomonas W80, which was isolated off the coast of Wakayama in Japan (Miura et al., 1986; Miyasaka et al., 1998), is an extremophile. C. W80 is highly tolerant to oxidative stress by methyl viologen (MV), high NaCl concentrations, and cadmium stress (Miyasaka et al., 2000; Tanaka et al., 2011); it is known to have much higher activities of ROS-degrading enzymes than the freshwater alga C. reinhardtii (Tanaka et al., 2011). Importantly, the gene that encodes glutathione peroxidase (GPX)-like protein in C. W80 successfully confers oxidative stress tolerance caused by MV or chilling after its expression in tobacco plants (Yoshimura et al., 2004). In addition, a number of unique anti-stress genes isolated from C. W80 have been characterized, including genes encoding the CFo ATP synthase subunit II homolog (Suda et al., 2009), cysteine protease (Usui et al., 2007), group 3 late embryogenesis abundant protein gene (Tanaka et al., 2004), glutathione peroxidaselike protein gene (Takeda et al., 2003), ascorbate peroxidase gene homolog (Takeda et al., 2000), NADP+-glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose-1,7-bisphosphatase (Tamoi et al., 2001). In this study, we focused on the novel gene cluster58 (CL58), which was isolated from C. W80, based on the data of expressed sequence tag (EST). This gene is predicted to encode a protein with a domain of unknown function, termed CHRD (Hyvönen, 2003), and a secretory signaling sequence. The transcript levels of the gene were found to increase in response to high concentrations of copper (Cu) and low temperatures. We created transgenic C. reinhardtii strains that express fusion proteins with luciferase (NanoLuc) and experimentally confirmed that the CHRD domain-containing protein has a secretory signaling sequence. In addition, CL58 was successfully expressed in Escherichia coli as a maltosebinding protein (MBP) fusion protein and was subsequently purified.

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2. Materials and Methods 2.1. Chlamydomonas strains and culture conditions The marine green alga C. W80 was cultured in modified Okamoto medium (MOM; pH 8.0), as described previously (Tanaka et al., 2011). For quantitative real-time PCR analysis, C. W80 was cultured in MOM under high salt (1.5 M NaCl), MV (150 µM), hydrogen peroxide (1 mM H2O2), high Cu (750 µM CuSO4), and low temperature (4°C) conditions for 8 h. The cell-walled C. reinhardtii strain C-9 was obtained from the National Institute for Environmental Studies in Japan (NIES-2235) and cultured in Tris-acetate-phosphate (TAP) medium with shaking at 25°C under continuous illumination at 50 μmol photons/m2/s.

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2.2. Cloning of CL58 cDNA Total RNA was isolated from 50 mL of C. W80 cell suspension cultures, for which the OD680 was 1.3 (approximately 70 mg fresh weight), using Isogen (Nippon Gene) and the RNeasy Plant Mini Kit (Qiagen), in accordance with the manufacturers’ instructions. Total RNA (1.5 μg) was subjected to 5′- and 3′-RACE with a GeneRacer kit (Invitrogen), in accordance with the manufacturer’s instructions. In brief, total RNA was dephosphorylated with calf intestinal alkaline phosphatase and then treated with tobacco acid pyrophosphatase to remove the 5′ cap structure from the intact, full-length mRNAs. Next, the GeneRacer RNA Oligo was ligated onto the 5′ end of the RNA using T4 RNA ligase. The ligated RNA was then converted into first-strand cDNA using the GeneRacer Oligo dT Primer and used as a template for PCR amplification. For 5′-RACE, the GeneRacer 5′ Primer and a CL58-specific primer_1 were used for the first round of PCR, and the GeneRacer 5′ Nested Primer and a CL58-specific primer_2 were used for nested PCR. For 3′-RACE, the GeneRacer 3′ Primer and a CL58-specific primer_3 were used for the first round of PCR, and the GeneRacer 3′ Nested Primer and a CL58-specific primer_4 were used for nested PCR. Gel-purified RACE PCR products were subcloned into the pUC118 plasmid using the Mighty Cloning Reagent Set (Takara) and sequenced. A full-length cDNA clone of the CL58 gene was obtained by PCR, in which the first-strand cDNA constructed using the GeneRacer kit, as described above, was used as a template. The primers used were the GeneRacer 5′ Primer and a CL58-specific primer_5 for the first round of PCR and the GeneRacer 5′ Nested Primer and a CL58-specific primer_6 for the second round of PCR. The gel-purified PCR products were sequenced, as described above. The primers used for these analyses are shown in Table S1. The nucleotide sequence of the CL58 gene was deposited as the “CHRD gene” in DDBJ/EMBL/GenBank under accession number LC257982.

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2.3. Computer analysis of DNA and amino acid sequences The subcellular localization of proteins and the location of a putative secretory signaling peptide were predicted using the WoLF PSORT (Horton et al., 2007), PredAlgo (Tardif et al., 2012), and TargetP (Emanuelsson et al., 2000) programs. Additional computer analyses of DNA and amino acid sequences were performed using Geneious Pro R10 (http://www.geneious.com) (Kearse et al., 2012). A BLASTP search was performed in Geneious against the NCBI non-redundant protein database using the default settings. Multiple sequence alignments were performed using the default settings in the ClustalW tool in Geneious Pro.

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2.4. Quantitative RT-PCR Three milliliters of C. W80 cell suspension cultures, for which the OD680 was adjusted to 2.3 (approximately 6.5 × 107 cells/mL), was grown in a 12-well plate under continuous illumination at 150 μmol photon/m2/s. For the quantitative RT-PCR analysis, the phosphorus source (5 mM total phosphorus from both K2HPO4 and KH2PO4) in MOM was replaced with 5 mM disodium glycerophosphate and 5 mM Tricine (pH 8.0) to avoid precipitation when high concentrations of CuSO4 were added to MOM. After 8 h of culture, total RNA was isolated and purified, as described above. Approximately 5 μg of total RNA and a random hexamer were added to the cDNA synthesis reaction, and synthesis using the Transcriptor First-Strand cDNA Synthesis Kit (Roche) was performed. Real-time PCR was then performed with LightCycler TaqMan Master and LightCycler 2.0 (Roche) using cDNA derived from approximately 50 ng of RNA. Standard curves were prepared using plasmid DNA, and the mRNA levels of the CL58 gene were estimated by comparison with the relative values of the glyceraldehyde 3phosphate dehydrogenase (GAPDH) gene, as described previously (Miyasaka et al., 2016). The primers and TaqMan probes specific for the CL58 gene are provided in Table S1; the primers and TaqMan probes specific for the GAPDH gene were described previously (Miyasaka et al., 2016).

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2.5. Plasmid construction for transformation of C. reinhardtii The coding region of the CL58 gene was synthesized by GenScript (Piscataway, NJ), subcloned into the pUC57 plasmid at the NdeI/EcoRI sites, and amplified by PCR using the CL58-G primer set. The PCR product was cloned into the NdeI/EcoRI sites of the pNYAN vector (PsaDp::YFP vector) (Kobayashi et al., 2016) using a Gibson assembly kit [New England Biolabs (NEB)] to create the PsaDp::CL58-YFP vector. NanoLuc sequence (Hall et al., 2012) was synthesized by Eurofins Genomics as the codon-optimized sequence for C. reinhardtii, cloned into pTAC-2 (BioDynamics Laboratory), and amplified by PCR using the delta-YFP primer set. The PCR product was assembled with the inverse-PCR product, which was amplified using the NanoLuc-G-F and -R primer set and either the PsaDp::CL58-YFP or the PsaDp::YFP vector as a template, to create the PsaDp::CL58NLuc or PsaDp::NLuc vector, respectively. For the construction of PsaDp::CL5826-163-NLuc, nucleotide sequences encoding the putative secretory signaling peptide (amino acids 1–25) were amplified by PCR using the signal-G primer set and the PsaDp::CL58-NLuc vector as a template. The resulting PCR product was cloned into the NotI/EcoRI sites of the PsaDp::NLuc vector. For the construction of PsaDp::ars1-NLuc, the signaling peptide region of ars1 (Rasala et al., 2012) was amplified by PCR using the ars1-G primer set and the genomic DNA of the C-9 strain, which was isolated using the DNeasy Plant Mini Kit (Qiagen), as a template. The PCR product was inserted into the PsaDp::NLuc vector that had been digested with NdeI and EcoRI. The sequences for all of the constructed plasmids were verified by DNA sequencing. The oligonucleotides used for these procedures are listed in Table S1.

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2.6. Transformation of C. reinhardtii Transformation of the C. reinhardtii strain C-9 was performed using a high-efficiency electroporation method and the square electric pulse-generating electroporator NEPA21 (Nepa Gene), as described previously (Yamano et al., 2013), with the following exceptions: 2 μL of 200–800 μg/mL plasmids was mixed with 38 μL of the cell suspension, the parameter of Pp was 300 V with an 8-ms pulse length, and 10 μg/mL paromomycin was used for selection instead of 30 μg/mL hygromycin B.

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2.7. Screening for the identification of positive transformants using luminescence Each paromomycin-resistant colony was transferred to a master plate (1.5% agar TAP plate containing 10 μg/mL paromomycin). Aliquots of transformants grown on the master plate for more than 4 days were transferred to 10 μL of the 1× Cell Culture Lysis Reagent (CCLR; Promega), 2 μL of the resulting cell lysate was mixed with 2 μL of the Nano-Glo Luciferase Assay Reagent (Promega), and luminescent intensity was measured using a luminometer (MiniLumat LB9506; Berthold Technologies) configured to measure light emission over a 5-s period.

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2.8. NanoLuc activity assay of total soluble protein in the culture medium Five hundred microliters of stationary-phase cultures were mixed with 2.5 mL of TAP medium and cultured for 24 h. The cell suspension, corresponding to 5.0 × 107 cells, was placed into a 1.5-mL tube, and the cells were collected by centrifugation (1,500 × g for 10 min) and washed twice with 500 μL of TAP medium. The cells were resuspended in 500 µL of TAP medium and incubated for 24 h with agitation on a gyratory shaker (140 rpm, 50 μmol/m2/s). The cell suspension was subjected to centrifugation (1,500 × g for 10 min) to collect the supernatants. The supernatants were transferred to another tube, and the pelleted cells were washed twice with 500 μL of TAP medium and then lysed in 500 μL of 1× CCLR (Promega). Each 5-μL aliquot of the supernatant and cell lysate was mixed with 5 μL of the Nano-Glo Luciferase Assay Reagent (Promega), and luminescent intensity was measured, as described above. 2.9. In-gel luminescence analysis C. reinhardtii transformants were cultured in 4 mL of TAP medium for 10 days and subjected to centrifugation (2,500 × g, 15 min) to collect the supernatant. The supernatant (culture medium) was freeze-dried and the lyophilizate was dissolved in 100 μL of 20 mM Tris-HCl (pH 7.2) as the supernatant fraction. Pelleted cells were washed twice with 200 μL of 20 mM Tris-HCl (pH 7.2), resuspended in 100 μL of 20 mM Tris-HCl (pH 7.2), and sonicated on ice 4 times for 30 s each. The insoluble materials were removed from the lysate by centrifugation (12,000 × g, 30 min), and the soluble extract was collected as the intracellular fraction. Five microliters of the supernatant and intracellular fractions were subjected to SDS-PAGE [5%–20% Tris-glycine polyacrylamide native gel (e-PAGEL; ATTO)]. The gel was rinsed with Milli-Q water and sprayed with 1/2× Nano-Glo Luciferase Assay Reagent

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(Promega). The luminescent image was visualized using ImageQuant LAS4000 (GE Healthcare Life Sciences).

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2.10. Plasmid construction for the transformation of E. coli The sequence of CL581-23 was amplified by PCR using CL581-23-F and CL581-23-R primers and assembled with the inverse-PCR products that had been amplified using the pCold-inv-F and pCold-inv-R primer set and the pColdI::Halo vector (unpublished plasmid) as a template; this process resulted in the creation of the pColdI::Halo-CL581-23 plasmid. The MBP sequence was amplified by PCR using MBP-F and MBP-R primers and pMAL-C2E (NEB) as a template and assembled with the inverse-PCR products of the pColdI::Halo-CL581-23 plasmid, which had been amplified using the pCold-Halo-inv primer set; this process resulted in the creation of the pColdI::MBP-CL581-23 plasmid. The sequences for all of the constructed plasmids were verified by DNA sequencing. The oligonucleotides used for these procedures are listed in Table S1.

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2.11. Expression and purification of recombinant CL581-23 proteins The pColdI::MBP-CL581-23 plasmid was transformed into the E. coli BL21(DE3) strain (HIT21; RBC Bioscience). The transformants were cultured at 37°C in 5 mL of LB medium containing 50 μg/mL ampicillin until the OD600 reached 0.4–0.5. After incubation on ice for 5 min and at 15°C for 30 min, expression of the recombinant protein was induced by the addition of 0.5 mM IPTG for 24 h at 15°C with agitation. The cultured cells were harvested by centrifugation (15,000 × g, 2 min), washed twice with 20 mM Tris-HCl (pH 7.2), and resuspended in 200 μL of 20 mM Tris-HCl (pH 7.2). The cell suspension was sonicated on ice 4 times for 30 s each using a Handy Sonic UR-21P (Tomy Seiko), and the cell debris was removed by centrifugation (12,000 × g, 30 min, 4°C). An aliquot of the supernatant (2.5 μL) was separated by SDS-PAGE (15% polyacrylamide gel, e-PAGEL; ATTO), and the expressed proteins were visualized using Coomassie Brilliant Blue staining. Purification of the recombinant proteins was performed using amylose magnetic beads (NEB), in accordance with the manufacturer’s instructions. In brief, 200 μL of the supernatant was mixed with the washed magnetic beads and incubated at 4°C for 1 h with agitation. A magnet was used to separate the supernatant from the mixture. After washing the bead pellet 3 times with MBP column buffer [200 mM NaCl, 1 mM EDTA, 1 mM DTT, 20 mM Tris-HCl (pH 7.4)], 50 μL of MBP column buffer containing 10 mM maltose was applied to the bead pellet and incubated for 10 min at 4°C with agitation. A magnet was used to separate the supernatant from the rest of the mixture. This elution step was repeated twice.

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3. Results 3.1. Characterization of the CL58 gene sequence The EST information, which was derived from C. W80 cells cultured under normal (0.5 M NaCl) and salt-stressed (1.5 M NaCl) conditions (Miyasaka et al., 2016; 2000), was utilized to explore a novel gene showing high mRNA expression. Among 960 unigenes, a gene of unknown function, which was designated CL58, was identified (eighth out of 960 unigenes) (Table S2). Among all genes with a large number of EST clones, the CL58 gene, whose function was unknown, was unique because most of the others were housekeeping genes. (Table S2). Based on this EST information, a full-length sequence of the CL58 cDNA clone was obtained from C. W80 total RNA using 5′-RACE, 3′-RACE, and RT-PCRbased full-length cloning, demonstrating that the cDNA sequence of the CL58 gene contained a 489-bp open-reading frame, which is predicted to encode a protein of 163 amino acid residues, with a mass of 18 kDa and a pI of 6.3 (Fig. 1A). The genomic DNA of the coding region of the CL58 gene comprised five exons (Fig. S1). The CL58 protein sequence was predicted to be a secreted protein with a 23-amino-acid secretory signaling sequence at the N-terminus, as determined using protein localization prediction programs, WoLF PSORT (Horton et al., 2007), TargetP (Emanuelsson et al., 2000), or PredAlgo (Tardif et al., 2012) (Fig. 1A). A BLASTP search of the CL58 protein sequence revealed its similarity to a large number of microbial CHRD domain-containing proteins with unknown functions (Fig. 1B). CHRD is a domain that was identified in both an inhibitor of bone morphogenic protein (BMP), chordin, and bacterial proteins, but its function remains unknown (Hyvönen, 2003). Actually, the Pfam program (Finn et al., 2016) was used to identify the presence of a CHRD domain between amino acids 33 and 154 of the deduced CL58 protein sequence (Fig. 1A). A similarity search of the Protein Data Bank using protein blast indicated that amino acids 72 to 94 of the CL58 protein were 48% identical to the partial structure of cytochrome C-551 from Pseudomonas stutzeri; however, the CL58 protein is unlikely to be a hemebinding protein because it has no heme-binding motif.

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3.2. Expression of the CL58 gene in response to stress To assess the function of the CL58 gene, changes in the mRNA levels after stress (high salinity, MV, hydrogen peroxide, Cu toxicity, and low temperature) were analyzed by quantitative real-time PCR. Cu toxicity (750 μM CuSO4 for 8 h) and low temperature (4°C for 8 h) conditions induced approximately 10fold and 5-fold increases in transcript levels, respectively (Fig. 2). This induction of gene expression under low temperature and toxic Cu conditions suggests that transcript levels of the CL58 gene are under the control of ROS; oxidative stress induced by 150 μM MV, but not by 1 mM hydrogen peroxide, caused a slight increase in CL58 transcript levels (Fig. 2). The expression analysis suggests that the CL58 gene is related to the observed stress responses in the C. W80 strain.

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3.3. Localization of CL58 using heterologous expression in C. reinhardtii The deduced amino acid sequence of the CL58 protein contains a predicted secretory signaling sequence (Fig. 1A). To assess whether the CL58 protein is secreted, the CL58 gene was heterologously expressed in a species closely related to C. W80, C. reinhardtii, and the localization of the CL58 protein was analyzed using transgenic cell lines. For this purpose, the CL58 gene was expressed as a fusion gene with NanoLuc (Hall et al., 2012), and the codon-optimized NanoLuc gene for C. reinhardtii was used in this study. Because the expression levels of the transgene are known to be generally low in C. reinhardtii, we expected this construct to be useful for the rapid screening of transgenic lines expressing recombinant proteins due to its high sensitivity. A series of transgenes (Fig. 3A) under the control of the PsaD promoter were inserted into the nuclear genome of C. reinhardtii strain C-9 using a high-efficiency electroporation method (Yamano et al., 2013), and positive transgenic lines expressing the recombinant proteins were screened by measuring the NanoLuc activity of each clone. More than 90% of the total NanoLuc activity was detected in the culture supernatant while less than 10 % was in the intracellular fraction in transgenic lines that express the CL58-NLuc fusion protein in a manner similar to those that express the NanoLuc fused with a secretory signaling sequence of the C. reinhardtii ars1 gene (ars1sp-NLuc) (Fig. 3B). This result was in contrast with that of transgenic lines that express NanoLuc (Fig. 3B). The N-terminal 25 amino acids of CL58 (CL581-25-NLuc) were sufficient for the detection of NanoLuc activity in the culture supernatant (Fig. 3A and B). Transformants expressing a CL58-NLuc mutant that lacks the N-terminal 25 amino acids, which contain the predicted signaling peptide, had negligible NanoLuc activity detected in culture medium (data not shown). An in-gel luminescence analysis demonstrated the expression of the recombinant proteins at the expected sizes in the intracellular fraction and the culture media (Fig. 3C).

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3.4. Expression and purification of the recombinant CL58 protein in E. coli We could not find any apparent phenotypic differences between the C. reinhardtii transgenic lines expressing CL58-NLuc and NanoLuc (data not shown). Therefore, we next aimed to purify the recombinant CL58 protein for its prospective functional characterization, such as the identification of interacting proteins using pull-down assays. When the CL58 protein lacking the signaling sequence was expressed as a fusion with a His-tag in either the pET28a (Novagen) or the pQE2 (Qiagen) vector or as a fusion with a His-tag and a Halo-tag (Promega) in the pColdI vector (Takara) in E. coli BL21(DE3) cells, the recombinant proteins were found in insoluble aggregates in all cases (data not shown). In contrast, when CL58 was expressed as a fusion with MBP in the pColdI vector (Takara), the recombinant protein was found in the soluble fraction, and thus could easily be purified using amylose magnetic beads as a single band of the expected size (Fig. 4). We speculate that the fusion with MBP, due to the well-known potential of MBP to enhance the solubility of a fused protein, and an induction of expression under low incubation temperatures might drastically improve the solubility of the recombinant proteins in E. coli. Although this study could not clearly disclose the function of CL58 protein, this recombinant protein might be one of the cues for further functional analysis by its use for the identification of interacting proteins using affinity purification followed by mass spectrometry, which is expected to provide a substantial amount of information about the function of the CL58 protein.

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4. Discussion The genes contributing to abiotic stress resistance in extremophilic microalgae may help improve the abiotic stress tolerance of plants. Indeed, some genes from extremophilic microalgae have been shown to confer stress resistance to plants (Hirooka et al., 2009; Waditee et al., 2005; Yoshimura et al., 2004). In this study, we focused on a novel gene of unknown function named CL58, which was explored based on the EST information in the halotolerant marine alga C. W80 generated by Miyasaka et al. (2016; 2000). The deduced amino acid sequence of CL58 contains a CHRD domain (Fig. 1A), which was found in the protein chordin and in a number of microbial proteins (Hyvönen, 2003). Some algae, including a freshwater strain of C. reinhardtii, have genes encoding proteins containing a CHRD domain, but their homology with the CL58 protein is not high. Instead, the CL58 protein sequence shows high homology with proteins in marine bacteria like Ferrimonas marina and Alteromonas macleodii, implying that the CL58 protein is related to marine bacteria. Although the role of the CL58 gene in the stress resistance of C. W80 remains unexplained, our data suggest that it encodes a secreted protein whose expression is induced in response to stress, including high Cu and low temperature conditions. CL58 proteins have a signaling peptide that can function in C. reinhardtii (Fig. 1A and Fig. 3), similar to some microbial CHRD domain proteins, which have putative secretory signaling sequences (Hyvönen, 2003). Chordin, which has CHRD domains, is also known to be a secreted protein and binds to bone morphogenetic proteins (BMPs), which are a group of signaling molecules belonging to the transforming growth factor- (TGF-) superfamily (Piccolo et al., 1996; Reversade and De Robertis, 2005); in Bacillus cereus and B. anthracis, a protein containing a CHRD domain is suggested to regulate the formation of the exosporium and affect both spore size and shape (Fazzini et al., 2010). Despite these insights, however, the function of the CHRD domain has largely remained unresolved. We show that the expression of the CL58 gene is regulated in response to high Cu and low temperature conditions (Fig. 2), which are known to induce oxidative damage by generating ROS in plants and algae (Sharma and Dietz, 2009; Stoiber et al., 2013; Suzuki and Mittler, 2006). Therefore, the expression of this gene might be under the control of ROS and/or it might play a role in an antioxidant defense mechanism. The function of the CL58 protein is currently unknown. Little is known about secretory proteins in green algae, but several secretory proteins whose levels are changed in response to environmental conditions including stresses are becoming clear. For example, high-CO2-inducible 43 kDa protein/Feassimilation 1 (H43/FEA1) is a well-known extracellular protein in C. reinhardtii, whose expression is induced at the transcriptional level by high CO2 concentrations (Hanawa et al., 2007), iron deficiency (Allen et al., 2007), and excess cadmium level (Rubinelli et al., 2002). In addition, a proteomic analysis found that 22 of 129 extracellular proteins, including gametogenesis-related proteins and hydroxyprolinerich glycoproteins, show an increase in their levels under high CO2 concentrations in C. reinhardtii (Baba et al., 2011). Besides being found in C. reinhardtii, changes in the profiles of extracellular proteins in response to UV-B and temperature stresses were also observed in the cyanobacterium Synechocystis salina and the green alga Chlorella vulgari (Rakleova et al., 2014). Interestingly, differences of secreted proteases under UV-B and temperature stresses were observed between the Antarctic and more stresssensitive mesophilic strains (Rakleova et al., 2014). Overall, recent studies are clarifying the fact that some secretory proteins show changes in their levels in response to environmental cues, but their physiological roles remain largely unknown. Nevertheless, these insights and our data suggest the existence of an important relationship between stress responses and secreted proteins. In conclusion, we isolated a gene of unknown function from a halotolerant green alga (C. W80) and suggest that this gene encodes a stress-responsive secreted protein with a CHRD domain. Functional studies of the CL58 protein might provide insights in the fields of stress response and secreted proteins and eventually contribute to the generation of transgenic plants with increased tolerance to various stresses.

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Acknowledgements We thank Dr. Yoshiki Nishimura (Kyoto University) for providing the plasmid. This work was supported by JSPS KAKENHI Grant Number 25550063.

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Authors’ contributions R.I., S.N., and K.T. performed experiments and analyzed the data. H.M. designed experiments, analyzed the data, and co-wrote the manuscript. N.K. performed the in silico analysis of the CL58 sequence and edited the manuscript. S.T., K.F., and H.M. designed and performed the experiments, supervised the project, and edited the manuscript. K.H. supervised the project and edited the manuscript. All of the authors approved the final manuscript.

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Conflicts of interest: none

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Fig. 1. Sequence analysis of the C. W80 CL58 gene. (A) Nucleotide and deduced amino acid sequences of the CL58 gene. The putative secretory signaling peptide is underlined in the amino acid sequence, and the CHRD domain is shown in bold. (B) Sequence alignment of the deduced CL58 protein sequence and other representative CHRD domain-containing proteins with high homology to that protein (E-value < 10−12). The indicated proteins are as follows: 1) CL58 (the N-terminal 31 amino acids are omitted), 2) WP_067664535 of Ferrimonas marina, 3) WP_039225533 of Alteromonas macleodii, 4) WP_046231847 of Paenibacillus algorifonticola, and 5) WP_003336126 of Brevibacillus laterosporus. The darker colors show the highest degree of conservation. Fig. 2. Changes in the transcript levels of the CL58 gene in response to stresses. The data shown are the values relative to those of GAPDH. Data are the means  SD (n = 3).

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Fig. 3. Determination of NanoLuc localization. (A) Heterologous expression of recombinant proteins in C. reinhardtii. NLuc: NanoLuc; Ars1sp-NLuc: the secretory signaling peptide of C. reinhardtii ars1 fused with NLuc; CL58-NLuc: full-length CL58 fused with NLuc; CL581-25-NLuc: CL58 containing the putative signaling peptide (amino acid residues 1–25) fused with NLuc. (B) Comparison of NanoLuc activities in the intracellular fraction and culture media (secreted protein fraction) for the transgenic lines expressing the indicated proteins. NanoLuc activities of the intracellular fraction (white) and culture media (gray) were measured using a luminometer, and the results are indicated as the percent of total activity for each recombinant protein. The data represent the average percentage of three independent transgenic lines. (C) SDS-PAGE in-gel luminescence analysis. In-gel NanoLuc activity was detected for proteins prepared from culture media (M) and the intracellular fraction (I) of the transgenic lines expressing the indicated recombinant proteins.

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Fig. 4. Expression and purification of the recombinant CL58 protein lacking the signaling peptide. E. coli cells harboring the expression vector for MBP-CL5824-163 were cultured without (lane 1) or with (lane 2) IPTG, and equal volumes of those cell lysates were subjected to SDS-PAGE. MBP-CL5824-163 was purified using amylose magnetic beads and applied to lane 3 (6.5 μg). The Coomassie Brilliant Bluestained gel is shown. The arrow shows the position of the MBP-CL5824-163 band (~59 kDa).

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CCLR : Cell Culture Lysis Reagent CL58 : cluster58 EST : expressed sequence tag GAPDH : glyceraldehyde 3-phosphate dehydrogenase MBP : maltose-binding protein MOM : modified Okamoto medium NanoLuc : NLuc ROS : reactive oxygen species TAP : Tris-acetate-phosphate

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Abbreviations list

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Highlights  The CL58 gene was isolated in a marine green alga  CL58 mRNA levels increased under high copper concentration and cold condition  Transgenic Chlamydomonas reinhardtii expressing the CL58 gene were created  CL58 protein contained a secreted peptide that functions in C. reinhardtii  Recombinant CL58 protein was expressed as a soluble protein in E. coli and purified