**2. Results**

#### *2.1. Effect of Photooxidative Stresses on the Tested Strains*

Scenedesmaceae strains were obtained from culture collections, as described previously [11]. *S. obtusus* and *C. striolata* were selected because they are the type species of the genera *Scenedesmus* and *Coelastrella*, respectively. Other strains were chosen as the relatively well-used research strains in the genus *Coelastrella*. According to the locality information in the literature, these strains were isolated from non-stressed environmental conditions, such as lakeside and peat bog [13]. The test strains showed good growth in the medium used for the Ki-4 and Oki-4N strains [10]; therefore, these strains were subjected to photooxidative stress after growing under non-stressed conditions.

Strains Ki-4 and Oki-4N showed the optimum expression level of AstaP when they were grown under 0.7 M and 0.5 M NaCl stress with high light exposure conditions (800 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1), respectively, where the color of cells changed from green to orange [9,10]. The tested Scenedesmaceae strains' cell color turned from green to white under the same stress conditions as Ki-4; therefore, we examined the upper limits of salt concentrations for each strain under the same high light intensity. Figure 1 shows the change in cell color of the strains after one to two weeks from the start of stress treatments using the upper limit of salt concentrations for each strain under high light exposure conditions. *S. obtusus* and *S. obliquus* turned green to white under 0.3 M NaCl and 0.25 M NaCl with high light exposure conditions; therefore, these strains were stressed by 0.25 M and 0.2 M NaCl, respectively, under the high light exposure conditions. Other strains were stressed with 0.5 M NaCl (for *C. vacuolata*) or 0.4 M NaCl (for *S. costatus* and *C. striolata*) with high light exposure.

A 1.0 g aliquot of wet cells stressed by salt with a high light exposure was suspended in 9.0 mL of 50 mM Tris-HCl buffer, pH 7.5. Cells were broken by a bead beater instrument, and the aqueous supernatants were obtained after ultracentrifugation. Strain Ki-4 and Oki-4N produced orange supernatants with broad absorption peak at 484 nm (Table 1) [9,10]. Except for *S. obliquus*, the test strains produced pale orangish or yellowish supernatants. The color of aqueous supernatants from the Scenedesmaceae strains were difficult to detect; therefore, the supernatants were concentrated by using a protein concentrator, and the colors and the absorption spectrum for each strain were shown in Figure 2. The amount of pigment produced per aliquot of cells from each test strain was estimated to be less than 5–10 times lower than that of Ki-4 and Oki-4N (Table 1).

These orange supernatants were passed through gel-filtration column chromatography, and two major peaks were detected (Figure 3). The first peaks from each test strain were detected around the HPLC retention time of 18 min, which were estimated to have a molecular weight greater than 670 kDa, and the absorption peaks were observed at approximately 454, 484, and 674 nm. These data indicated that the first peaks contained chlorophyll, which resembled that of the first peak of the strain Oki-4N described in our previous study [10]. The second major peak in each strain was detected at the HPLC retention time of 28 min (*S. obtusus*) or 30–35 min, which showed a broad absorption peak at 484 nm. In our previous study, AstaP orthologs were classified into two groups: glycosylated large-sized AstaPs and non-glycosylated small-sized AstaPs. The difference in the second peak retention time indicated variations in molecular weight among the test strains.

**Figure 1.** Survival of test strains under salt stress conditions with high light exposure. Salt concentrations for each test strains were decided based on the upper limit of salt concentrations with high light (w/HL, 800 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1). Each test strain was cultivated under non-stressed conditions and stressed by adding salt w/HL for one to two weeks as described in Materials and Methods.

**Table 1.** Measurements of the amount of aqueous pigments after ultracentrifugation of cell extracts.


**Figure 2.** Absorption spectrum and the color of aqueous cell extracts. Aqueous cell extracts from photooxidative stressed cells were obtained after ultracentrifugation and concentrated by using a protein concentrator. ×1, ×10, ×20 means the rate of concentration by using a protein concentrator.

**Figure 3.** Separation of carotenoid pigments by gel-filtration column chromatography. Aqueous cell extracts obtained after ultracentrifugation were loaded into the gel-filtration column chromatography.

The protein fractions from the second peak in each test strain were collected, and the binding pigments were extracted as described previously [9,10]. The elution profiles were compatible with those from the strains Ki-4 and Oki-4N (Figure 4), and the pigments from each strain were identified to be astaxanthin and adonixanthin based on C18-HPLC retention time and absorption spectrum. These results indicated that the test strains also produced AstaP-like water-soluble proteins under the photooxidative stress conditions.

#### *2.2. Effect of Photooxidative Stress Response on Model Microalgae*

In our previous study, AstaP orthologs are found in the genome annotation data of *Chlamydomonas reinhardtii* and *Chlorella variabilis* [8]. In this study, we analyzed the production of aqueous pigments from two strains under photooxidative stress conditions. *Cmy. reinhardtii* and *Crl. variabilis* turned green to white under 0.25 M and 0.5 M NaCl conditions with high light intensity, respectively. Therefore, photooxidative stresses were archived under 0.2 M and 0.4 M NaCl, respectively, with high light for six days. The cell-free extracts from photooxidative-stressed cells were obtained by a bead beater, ultracentrifuged, and their color was pale yellow. A yellow fraction was detected using gel filtration HPLC of the aqueous supernatant at 18 min in *Crl. variabilis* but not in *Cmy. reinhardtii*. The yellow fraction of *Crl. variabilis* showed absorption peaks at 454, 484, and 674 nm, indicating the presence of chlorophyll-binding protein similar to that of other Scenedesmaceae strains. These data suggested that the production of aqueous carotenoid-binding protein was not detectable in these model microalgae.

**Figure 4.** C18-HPLC elution profiles of the binding carotenoids from the collected Peak2 fractions after gel filtration column chromatography. Ax, astaxanthin; Ad, adonixanthin; Lt, lutein; and Ca, canthaxanthin.

#### *2.3. Purification and Characterization of Water-Soluble Astaxanthin-Binding Protein from S. costatus SAG 46.88*

In order to characterize the existence of AstaP orthologs in the test strains, we chose *S. costatus* for further purification of astaxanthin binding protein because of its relatively high expression level (Table 1). The aqueous orange fraction obtained by gel-filtration column chromatography from the stressed *S. costatus* CFE was further purified by isoelectric focusing. An orange band that migrated to the position corresponding to pI 9 was detected (Figure 5A), and the purity of the excised orange protein band was determined by SDS-PAGE (Figure 5B). A single band appeared as an apparent molecular mass of 27 kDa, and the N-terminal amino acid sequence was determined to be AVPEAKTT, which showed partial homology to the N-terminal sequence of Ki-4-AstaP (ATPKANAT). The binding pigments of the purified protein by isoelectric focusing were analyzed by C18-HPLC and were astaxanthin and adonixanthin, which were identical to those of Ki-4 AstaP [9]; therefore, we named this protein ScosAstaP.

**Figure 5.** Purification and characterization of AstaP from *S. costatus*. ( **A**) isoelectric focusing of aqueous pigments. The circle indicates the migrated orange bands after the electrophoresis; (**B**) the migrated orange band in A excised and electrophoresed by SDS-PAGE. The purified protein is shown by an arrow. Molecular mass standards are shown (kDa); ( **C**) two-dimensional electrophoresis of the total proteins of *S. costatus*. *S. costatus* was cultivated under high light conditions and subjected to 0.4 M NaCl for six days. An arrow indicates the excised spot for N-terminal sequencing.

> Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was performed to detect the photooxidative stress-inducible spots in *S. costatus*, and a large spot was detected in the area of alkaline pI after exposure to salt stress with high light (Figure 5). The Nterminal amino acid sequence (AVPEAKTT) from the large spot coincided with that of the purified protein. These results indicated that the orange protein was an AstaP ortholog inducible under the photooxidative stress conditions.

#### *2.4. AstaP Ortholog in S. costatus Is a Fasciclin-Like Glycosylated Protein*

To obtain the gene encoding ScosAstaP, a cDNA library was constructed from the stressed *S. costatus* mRNA. The gene encoding the N-terminal amino acid sequence was found in the de novo sequencing data of the cDNA library, and the full-length cDNA was obtained by PCR using the *S. costatus* cDNA library as a template. The deduced amino acid sequence from a gene encoding ScosAstaP showed 62% identity with Ki-4 AstaP (Figure 6A). Bioinformatics analyses revealed that ScosAstaP conserved 20 amino acid residues of the N-terminal hydrophobic signal sequence and two fasciclin-like H1 and H2 domains. These protein primary structures are similar to Ki-4-AstaP; however, only one putative N-glycosylation Asn-x-Thr site was detected in ScosAstaP compared to the five sites in Ki-4 AstaP (Figure 6A). The calculated molecular mass of the mature protein deduced from ScosAstaP cDNA, excluding 20 amino acid residues of the N-terminus signal peptide, was 21.4 kDa. The reason for the difference of calculated molecular weight and the measured molecular weight (apparent molecular weight from SDS-PAGE was 27 kDa) is supposedly due to the glycosylation of ScosAstaP like as Ki-4 AstaP (Ki-4 AstaP was 21.2 kDa of the calculated molecular mass and the apparent molecular weight from SDS-PAGE was 33 kDa). Northern blot analysis indicated that the gene encoding ScosAstaP was inducible by photooxidative stress (Figure 7).


**Figure 6.** Structural comparison of Ki-4 AstaP and *S. costatus* AstaP orthologs. (**A**) comparison of the deduced amino acid sequences of Ki-4 AstaP and ScosAstaP. N-terminal signal peptides are shown in red font. The experimentally detected N-terminal sequence is highlighted in green. Potential sites for N-linked glycosylation are boxed (black: Ki-4 AstaP, red: ScosAstaP); (**B**) C18-HPLC elution profile of the binding carotenoids from purified Ki-4 AstaP [9] and the purified ScosAstaP. Ax, astaxanthin; Ad, adonixanthin; Lt, lutein; and Ca, canthaxanthin.

**Figure 7.** Northern blots of total RNA probed with the gene encoding AstaP orthologs from *S. costatus, C. vacuolata, Cmy. reinhardtii, and Crl. variabilis*. Strains were cultivated under photooxidative stress conditions. 0, just before the start of stress, 1–2, after one and two days of stress. Gene accession numbers are shown in parentheses. Ethidium-bromide staining of the ribosomal RNA (rRNA) to confirm the equal loading is shown below the autoradiogram.

#### *2.5. Identification of AstaP Orthologs in C. vacuolata and Two Model Microalgae*

*C. vacuolata* showed two peaks of astaxanthin-binding protein by gel-filtration chromatography (Figure 3); therefore, the presence of two types of AstaP orthologs was predicted. Two cDNA sequences that encode AstaP orthologs were found in the cDNA sequence data from the photooxidative-stressed *C. vacuolata* cDNA library. Both fulllength cDNA clones were amplified by PCR, and nucleotide sequences were confirmed. The deduced amino acid sequence indicated that *C. vacuolata* possesses two types of AstaP orthologs. These two proteins showed different pI (calculated pI were 6.7 and 9.7), and we named them CvacAstaP1 and CvacAstaP2, respectively. Both orthologs possess N-glycosylation sites and N-terminal hydrophobic signal peptides for cell surface secretion, and they showed 44% identity with each other (Supplementary Figures S1 and S2). ScosAstaP1 had ten putative N-linked glycosylation sites, whereas ScosAstaP2 had only two putative N-glycosylation sites. The number of N-glycosylation sites was expected to affect the difference in molecular weight detected by gel-filtration column chromatography, as shown in Figure 3.

AstaP orthologs were found in the model algae *Cmy. reinhardtii* and *Crl. variabilis,* and phylogenetic analysis was performed in our previous study. Both cDNAs were amplified by PCR and confirmed the coincidence of the deduced amino acid sequence in the database (Supplementary Figure S1). To determine the gene expression profiles of AstaP orthologs in model algae and *C. vacuolata*, the genes encoding *Cmy. reinhardtii*, *Crl. variabilis*, and two AstaP orthologs from *C. vacuolata* were amplified and analyzed these expression profiles by Northern blot analysis. Both AstaP orthologs were induced by photooxidative stress treatments.

The *Scenedesmus actus* AstaP ortholog was identified as a translation product of a gene induced by Cr stress [14], but its binding of astaxanthin has not been determined.

Phylogenetic analysis revealed that the AstaP orthologs were classified into three groups: glycosylated basic pI, glycosylated acidic pI, and non-glycosylated acidic pI (Figure 8).

**Figure 8.** Neighbor-joining phylogenetic tree of the deduced sequence of the AstaP orthologs. A livewort homologue (Marchantia\_polym: *Marchantia polymorpha*) was used as an outgroup. Chlamydomonas rein: *Chlamydomonas reinhardtii*, Chlolella\_vari: *Chlorella variabilis*, Monoraphidium\_negl: *Monoraphidium neglectum*, Scenedesmus\_acut: *Scenedesmus acutus*, Botryococcus\_bra: *Botryococcus braunii*, Coccomyxa\_sub: *Coccomyxa subellipsoidea*, Fragilariopsis\_cyl: *Fragilariopsis cylindrus*, Chrysochromulina sp: *Chrysochromulina tobinii*, Phaeodactylum\_trico: *Phaeodactylum tricornutum*, Volvox\_carteri: *Volvox carteri,* and Galdieria\_sulph: *Galdieria sulphuraria*. Accession numbers for each protein are shown in parentheses. The bootstrap values >50 are indicated at the branch points.
