Saccharina japonica (Park and Hwang, 2012)

Getachew, Paulos, Nam, Bo-Hye, Young Cho, Ji & Hong, Yong-Ki, 2016, Influences of hydrozoan colonization on proteomic profiles of the brown alga Saccharina japonica, Botanica Marina (Warsaw, Poland) 59 (2), pp. 2-3 : 86-87

Saccharina japonica , hydrozoan, and reagents

Fresh blades of late-harvested Saccharina japonica were collected from the Gijang aquaculture farm, Busan, Korea in June 2013 and 2014. A voucher specimen was deposited in the author’s laboratory (Y. K. Hong). The seaweed tissues were washed and cleaned with autoclaved seawater. Colonies of O. geniculata were gently scraped off with a stiff plastic sheet. Healthy tissues located at least 30 cm from the colony were used as a control. Both colonized tissues (blade tissues remaining beneath the colony after the removal of hydrozoans) and healthy tissues collected from many thalli were immediately freeze-dried ( SFD-SM, Samwon Freezing Engineering Co., Busan, Korea), ground to a fine powder, and kept at -70°C before analysis. Most reagents used in this study were of analytical grade from Sigma-Aldrich Co. , St. Louis, MO, USA.

Protein electrophoresis

Protein preparation followed the previous methods of Getachew et al. (2014). Briefly, the seaweed powder (0.5 g) was homogenized in ten volumes of a lysis solution. Proteins were extracted for 1 h, and used for two-dimensional gel electrophoresis (2-DE). For 2-DE, immobilized pH gradient dry strips (4–10 NL IPG, 24 cm; Genomine, Pohang, Korea) were equilibrated for 14 h, and loaded with 200 µg samples. Isoelectric focusing was performed at 20°C using a Multiphor II electrophoresis unit (GE Healthcare, Little Chalfont, UK). The voltage was increased linearly from 150 to 3500 V over 3 h for sample entry, and the focusing was considered to be complete after 96 kVh. Prior to the second dimension, strips were incubated twice for 10 min each in an equilibration buffer. Equilibrated strips were then inserted onto sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels (20 × 24 cm, 10–16%). The SDS-PAGE was run at 20°C for 1700 Vh and silver-stained without fixing, followed by sensitization with glutaraldehyde ( Oakley et al. 1980).

Quantitative analysis

To evaluate the change in intensity of each protein spot on the 2-D gels, quantitative analysis of digitized images was carried out using PDQuest software (version 7.0; Bio-Rad, Hercules, CA, USA). The quantity of each spot was normalized by total valid spot intensity. Protein spots were selected for significant differences in expression of over two-fold or less than half of spot intensity ratio compared with the control or healthy tissues.

Protein digestion and identification

Protein spots were enzymatically digested in gel by the method of Shevchenko et al. (1996) using porcine trypsin (Promega, Madison, WI, USA). Gel pieces were washed with 50% acetonitrile, vacuum-dried, and incubated with trypsin (9 ng µl-1) in 50 mM ammonium bicarbonate, pH 8.7 for 9 h at 37°C. For the identification of proteins, samples were analyzed using a 4700 Proteomics Analyzer with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)/TOF™ ion optics (Applied Biosystems, Foster City, CA, USA). Sequence tag searches were performed via a National Center for Biotechnology Information (NCBI) search using the program Mascot (Matrix Science Ltd., London, UK) and a European Molecular Biology Laboratory (EMBL) search using MS BLAST ( Shevchenko et al. 2001).

Results

The stoloniferous hydrozoan Obelia geniculata was widespread on blades of late-harvested Saccharina japonica . Blade parts with hydrozoan-colonies and healthy tissues were used to isolate proteins induced by O. geniculata infection. The protein isolation from tissues was replicated and optimized to confirm the differently expressed protein profiles. In the hydrozoan-colonized tissues, 107 protein spots were detected on a 2-DE gel plate, while 75 spots were detected in the healthy tissues ( Figure 1 ). Out of the 107 spots in colonized tissues, 105 had different expression levels between the healthy and colonized tissues; 77 and 28 spots were up- and down-regulated, respectively, upon O. geniculata colonization. Out of the 77 up-regulated spots, 30 had more than twice the spot intensity compared with the healthy tissues. Out of the 28 down-regulated spots, 22 had less than half the spot intensity compared with the healthy tissues. Among these 52 (30+22) spots, 38 clear and abundant spots, which appeared constantly in replicated experiments, were selected and subjected to protein analysis. Through a database search of proteins from algae, land plants, and bacteria, 21 spots were identified, of which two were mixtures of two proteins. The identities of these 23 proteins and their molecular weight (MW), isoelectric point (p I) values, and functions are summarized in Table 1. Among them, 7 and 16 identified proteins were significantly up-and down-regulated, respectively. By searching the NCBI and EMBL databases, 17 and 18 proteins, respectively, were confidently identified. Twelve proteins overlapped.

The identified 23 proteins were cell-division cycle 46/ minichromosome maintenance protein 5 (Cdc46/Mcm5), glutamyl-tRNA reductase (GluTR), microcompartments protein (MCP), carboxysome shell peptide (CsoS), biotin synthesis protein (bioC), serine/arginine-rich splicing factor (SRSF), two-component response regulator (PilR), chloroplast phosphoglycerate kinase (PGK), expansin 6 (EXPA6), translation initiation factor 3 (IF3), calcium/ calmodulin-dependent protein kinase II inhibitor 2 (CaMK2N2), 50S ribosomal protein L1P (rpl1P), transmembrane protein (TP), protoporphyrinogen oxidase (PPOX), dual oxidase 2 like (DUOX2), PIH1 domain-containing protein 2 (Hsp90), GTPase-activating protein alpha (GAPα), threonyl-tRNA synthetase (TARS), flavanone 3-hydroxylase (F3H), uncoupling protein 3 (UCP3), vanadium-dependent bromoperoxidase 7 (vBPO7), peptide chain release factor 1 (Prf1), and interaptin in a database search of both NCBI and EMBL. Among the identified proteins, two up-regulated proteins (Cdc46/Mcm5 and GluTR) were mostly expressed only in the hydrozoan-colonized tissues but were rare in the healthy tissues ( Figure 2 ). The Cdc46/Mcm5, related to stress control, showed sharply increased spot intensity or protein amount; approximately 1308-fold more in colonized tissues than in healthy tissues. The spot intensity of photosynthesis-related GluTR also increased sharply by approximately 277-fold. Five proteins (MCP, CsoS, bioC, SRSF and PilR) in photosynthesis, stress control, and signal transduction were significantly up-regulated by approximately 3–11-fold in hydrozoan-colonized tissues ( Figure 3 ). Meanwhile, five down-regulated proteins (PGK, EXPA6, IF3, CAMK2N2 and rpl1P), which were found mostly in healthy tissues but were rare in hydrozoan-colonized tissues, were related to photosynthesis, cell growth, stress control and signal transduction in a database search ( Figure 4 ). Eleven proteins (TP, PPOX, DUOX2, Hsp90, GAPα, TARS, F3H, UCP3,) vBPO7, Prf1, and interaptin), related to signal transduction, defense response, protein metabolism, stress control, photosynthesis and the cytoskeleton were significantly down-regulated by 0.1–0.5-fold in the hydrozoan-colonized tissues ( Figure 5 ). From the 23 proteins identified through a homology-based cross-species database, we found that six proteins were related to stress control, five proteins to signal transduction, five proteins to photosynthesis, two proteins to protein metabolism, two proteins to defense response, two proteins to cell growth, and one protein to the cytoskeleton.

Figures

Figure 1: Two-dimensional gel electrophoresis profiles of late-harvested Saccharina japonica. (A) Healthy tissues. (B) Hydrozoan-colonized tissues. The separated proteins were visualized by silver staining.

Figure 2: A close-up view of 2-dimensional electrophoresis gels showing the identified up-regulated proteins (indicated by arrows) found mostly in hydrozoan-colonized tissues but rare in healthy tissues. (A) Healthy tissues. (B) Hydrozoan-colonized tissues.

Figure 3: A close-up view of 2-dimensional electrophoresis gels showing the identified up-regulated proteins (indicated by arrows) altered by hydrozoan colonization. (A) Healthy tissues. (B) Hydrozoan-colonized tissues.

Figure 4: A close-up view of 2-dimensional electrophoresis gels showing the identified down-regulated proteins (indicated by arrows) found mostly in healthy tissues but rare in hydrozoancolonized tissues. (A) Healthy tissues. (B) Hydrozoan-colonized tissues.

Figure 5: A close-up view of 2-dimensional electrophoresis gels showing the identified down-regulated proteins (indicated by arrows) altered by hydrozoan colonization. (A) Healthy tissues. (B) Hydrozoan-colonized tissues.