Botanica Marina 2018; 61(2): 149–159

Paulos Getachew, Bo-Hye Nam and Yong-Ki Hong* Identification of early biomarkers in proteomic profiles of the phaeophyte japonica proximal to and beneath the front of bryozoan colonies https://doi.org/10.1515/bot-2017-0065 Received 8 September, 2017; accepted 27 February, 2018; online first Introduction 15 March, 2018 The edible brown Saccharina japonica has Abstract: The sessile bryozoan membran- been cultured widely in East Asia since the 1970s. The acea frequently colonizes the phaeophyte Saccharina seaweed thalli are a popular health seafood and used japonica. Identifying early colonization markers using as abalone feed. In 2015, 442,771 t (wet weight) of proteomics could assist in the early detection of epiphytic S. ­japonica were produced by farming; an additional 10 t contamination. Different sections of thallus tissue proxi- (wet weight) were harvested from natural populations in mal to the bryozoan (i.e. the 1-cm zone beyond the bound- (Korea Fisheries­ Association 2016). Epiphytism is ary of the ) and tissue from the colony-front (i.e. the a major problem in seaweed aquaculture, as it reduces narrow zone under the newly formed front of the colony the product quality and yield. Bryozoans, hydroids, after removing the bryozoans) were separated. From the amphipods, copepods, and gastropods are some of the proteomic profiles of S. japonica, we detected 151 protein most abundant epiphytes on the blades of spots (99 up-, 50 down-, and 2 similarly regulated) from (Peteiro and Freire 2013). Bryozoan and hydroid colonies proximal tissues and 151 spots (69 up-, 75 down-, and on S. japonica thalli reduced the seaweed tissue viability 7 same-regulated) from colony-front tissues. Hundred and by 69% and 77%, respectively, compared with healthy ten spots were detected from distal healthy thallus tissue, tissues (Getachew et al. 2015a,b). Unsightly, coarse epi- used as a control. The protein SSP15 was specifically up- phytic colonizers also markedly reduce the commercial regulated in the proximal tissues by ca. 1395-fold, while it value of the colonized thalli. One of the most common exhibited little expression at the colony-front and in dis- epiphytic colonizers on S. japonica thalli is the encrusting tal healthy tissues. ATPases were markedly up-regulated bryozoan Membranipora membranacea. It is a colonial in both the proximal and colony-front tissues by 3198- that filter-feeds on , flagellates, , and 2475-fold, respectively. Rpl1P and SRSF proteins were small planktonic organisms, and decayed organic mate- specifically up-regulated only in colony-front tissues by rial (de Burgh and Fankboner 1978). The planktotrophic 5724- and 273-fold, respectively. Therefore, these proteins bryozoan larvae settle on thalli and give rise to large may be used as specific biomarkers for the early detec- colonies covering the surface of the seaweed. Besides tion of bryozoan colonization on each tissue type of the being highly calcified, bryozoan colonies contain seaweed. extraordinary amounts of crude ash and harmful arsenic exceeding the limit of the provisional tolerable weekly Keywords: biomarker; bryozoans; MALDI-TOF/MS; prot- intake by humans (Getachew et al. 2015a). The colonies eomics; Saccharina japonica. of M. membranacea also reduce the rate of photosynthe- sis by reducing pigment concentrations (Hepburn et al. 2006), decrease the ammonium uptake rate (Hurd et al. 2000), and reduce spore release from fertile blades (Saier and Chapman 2004). It is important to understand the impacts of environ- *Corresponding author: Yong-Ki Hong, Department of mental changes/stresses on the biological activities of Biotechnology, Pukyong National University, Namgu, Busan 48513, seaweeds at biochemical and molecular levels. Although Korea, e-mail: [email protected] the biochemical and physical mechanisms are not clear, Paulos Getachew: Department of Biotechnology, Pukyong National after removing the bryozoans, seaweed tissues beneath University, Namgu, Busan 48513, Korea; and Center for Food Science the colonies exhibited elevated potassium, iodine, and and Nutrition, Addis Ababa University, P. O. Box 1176, Addis Ababa, Ethiopia docosahexaenoic acid levels and reduced copper, chro- Bo-Hye Nam: Biotechnology Research Division, National Institute of mium, and cadmium levels compared with the distal Fisheries Science, Gijang-gun, Busan 46083, Korea healthy tissues (Getachew et al. 2015a). When epiphytic 150 P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies bryozoans colonize kelp thalli, the bryozoans also affect protein expression by inducing signal transduction or a response in the host seaweed. We previously found that 14 proteins were up-regulated and seven were down- regulated in bryozoan-colonized tissues (i.e. the entire colonized area after removing the bryozoans; Getachew et al. 2014). Therefore, we postulated that the proximal and colony-front tissues may be directly or indirectly influenced by secretions from the epiphytic bryozoans, similar to mechanisms of self-recognition that affect plant communication and defense (Karban and Shiojiri 2009). The molecular impact and up- and down-regu- lated proteins in the proximal and colony-front tissues can be used as biomarkers in future research for early Figure 1: The Saccharina japonica thallus sections defined as diagnosis or improving strain resistance to epiphytic colony-front and thallus tissue proximal to Membranipora mem- infestation. branacea colonies. In this paper, early bryozoan colonization marker Colony-front tissues were collected from the narrow zone under the newly formed front of the colony after removing the bryozoans. proteins from proximal and colony-front tissue sections Thallus tissue proximal to the bryozoan colony was obtained from of colonized S. japonica thalli were identified and their the 1-cm zone outside the boundary of the colony. primary roles in cellular activities are discussed. Protein electrophoresis

Protein was prepared by the methods of Getachew et al. Materials and methods (2014). Briefly, the seaweed powder (0.5 g) in 5 ml lysis solution was extracted for 1 h, and used for two-dimen- Seaweed, bryozoans, and reagents sional gel electrophoresis (2-DE). The sodium dodecyl sul- fate-polyacrylamide gel electrophoresis gels (20 × 24 cm, Fresh thalli of the late harvest Saccharina japonica 10%–16%) were silver-stained and followed by sensitiza- (J.E. Areschoug) C.E. Lane, C. Mayes, Druehl et G.W. tion with glutaraldehyde. Saunders were collected from Gijang aquaculture farm, Busan, Korea in June 2015 and 2016. A voucher specimen Quantitative analysis was deposited in the author’s laboratory (Y.K. Hong). The seaweed tissues were washed and cleaned with auto- To evaluate the change in intensity of each protein spot claved seawater. They were acclimatized in an aquar- on the 2-DE gels, quantitative analysis of digitized images ium tank with in situ light and temperature conditions was performed using PDQuest software (version 7.0; Bio- for 3 days. Different tissue sections of the thalli covered Rad, Hercules, CA, USA). The quantity of each spot was by the bryozoan Membranipora membranacea were normalized by total valid spot intensity. Protein spots on selected (Figure 1), and colonies were gently scraped off each 2-DE gel plate were selected for significant differ- with a stiff plastic sheet. Thallus tissue proximal to the ences in expression of over two-fold or less than half of bryozoan colony (i.e. the 1-cm zone beyond the bound- spot intensity ratio compared with the control or distal ary of the colony) and tissue from the colony-front (i.e. healthy tissues. the narrow zone under the newly formed front of the colony after removing the bryozoans) were immediately freeze dried (SFD-SM, Samwon Freezing Engineering Protein digestion and identification Co., Busan, Korea), ground to a fine powder, and kept at –70°C before analysis. Distal healthy tissues located Protein spots were enzymatically digested in gel by the at least 30 cm from the colony were treated in the same method of Shevchenko et al. (1996) using porcine trypsin way and used as a control. Most of reagents used in this (9 ng μl−1; Promega, Madison, WI, USA) for 9 h at 37°C. study were of analytical grade from Sigma-Aldrich Co., For the identification of proteins, samples were analyzed St. Louis, MO, USA. using a 4700 Proteomics Analyzer with matrix-assisted P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies 151 laser desorption ionization-time of flight (MALDI-TOF)/ in intensity between tissues (41 up-regulated + 7 down- TOF™ mass spectrometer (Applied Biosystems, Foster regulated), we selected 35 (33 up-regulated + 2 down- City, CA, USA). Sequence tag was identified via a National regulated) spots that appeared constantly in replicated Center for Biotechnology Information (NCBI) search using experiments for protein analysis. In a database search of Mascot (Matrix Science Ltd, London, UK) and a European proteins from , land plants, and bacteria, we iden- Molecular Biology Laboratory (EMBL) search using MS tified 17 (14 up-regulated + 3 down-regulated) spots, of BLAST (Shevchenko et al. 2001). which spot no. 14 was a mixture of three proteins. Table 1 lists the identities of these 19 proteins with their molecular weight (MW), isoelectric point (pI) values, and functions. Results Sixteen of them were up-regulated and three were down- regulated. On searching the NCBI and EMBL databases, 18 The crustose bryozoan Membranipora membranacea and 17 proteins, respectively, were confidently identified. frequently colonizes the late-harvested thalli of aqua- Sixteen proteins were present in both databases. cultured Saccharina japonica. To identify proteins that The 19 identified proteins were F-type H-ATPase beta are markers of early colonization or initial cue proteins subunit (F-ATPase β), chloroplast ATP synthase CF1 alpha upon colonization, we separated the thallus tissue proxi- chain (ATPase α), chloroplast ATP synthase beta subunit mal to bryozoan colonies, tissue at the colony front, and (ATPase β), signal-induced proliferation-associated 1-like distal healthy thallus tissues to isolate the proteins that protein 1 isoform X9 (SIPA1L1), sporulation-specific responded to M. membranacea colonization. The protein protein 15-like isoform X4 (SSP15), glutamyl-tRNA reduc- isolation from each tissue was replicated and optimized tase (GluTR), mitochondrial serine-pyruvate aminotrans- to confirm the differentially displayed protein profiles. For ferase (SP-NH2 transferase), keratin type I cytoskeletal the thallus tissue proximal to bryozoan colonies, tissue at 9 (KRT9), cytochrome c oxidase 2, zinc finger protein 3 the colony front, and distal healthy thallus tissues, 151, (ZFP3), meiotic W68, actin, cytochrome P450, protein 151, and 110 protein spots, respectively, were detected on kinase domain-containing protein (protein kinase), two-dimensional gel plates (Figure 2). Of the 151 spots in thioredoxin domain-containing protein 3 (TXNDC3), the thallus tissue proximal to bryozoan colonies, 149 spots expansin 6 (EXPA6), hypothetical NCU01829, RNA editing showed different expression levels compared with distal complex protein (RNA editing protein), and LRR/NB-ARC healthy tissues; 99, 50, and two spots were up-, down-, domains-containing disease resistance protein (NB-ARC and similarly regulated, respectively. Of the 99 up-regu- protein). Among the identified proteins, three up-regu- lated spots, 41 had spot intensities more than twice those lated proteins (F-ATPase β, ATPase α, and ATPase β) were from the distal tissues. Of the 50 down-regulated spots, primarily expressed only in the thallus tissue proximal to the spot intensities of seven were less than half those in bryozoan colony and expressed at low levels in the distal the distal tissues. From the 48 spots that differed markedly healthy tissues (Figure 3). These ATP synthesis-related

Figure 2: Two-dimensional gel electrophoresis profiles of the late-harvested Saccharina japonica. (A) Distal healthy S. japonica tissue. (B) S. japonica thallus tissue proximal to the bryozoan colony. (C) S. japonica tissue at the bryozoan colony front. The separated proteins were visualized by silver staining. Numbers attached to the arrows refer to the spot number listed in Tables 1 and 2. 152 P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies Photosynthesis Sporulation Proliferation ATP synthesis ATP Cell wall loosening wall Cell Oxidoreduction ATP synthesis ATP Signal transduction Signal Oxidoreduction ATP synthesis ATP Disease resistance Disease Cytoskeleton Cell divisionCell Function RNA editing Protein stabilization Protein ATP synthesis ATP Hypothetical Hypothetical protein Cytoskeleton Amino acid Amino acid metabolism

3.8 7.6 4.7 0.0 8.6 4.2 0.1 4.2 0.2 10.0 10.4 286.1 524.8 1394.5 1313.5 3197.7 1059.4 intensity Ratio of spot spot of Ratio

– – 717 301 963 2629 3328 2360 3684 1567 3850 3649 1846 2265 1052 3923 2951 1944 2449 score MS BLAST BLAST MS

Q63QF1 – – YP006639069 P23116 XP_005023889 YP006639015 ABA94760 BAL05124 CBJ32298 EOY10154 EKC38741 XP002034594 CCC89336 ELW64862 Accession ID Accession (EMBL) XP004195374 XP956613 XP005584242 XP780986

– 93 75 79 76 85 84 56 82 62 77 84 81 109 125 150 161 105 104 score Mascot

gi | 209516552 gi | 565381387 gi | 512949794 gi | 164428214 gi | 546226132 – gi | 483507784 gi | 403066526 gi | 218186108 gi | 298710991 gi | 354961713 gi | 567893315 gi | 77745053 gi | 209945748 gi | 343473184 gi | 444724252 gi | 448083510 Accession ID Accession (NCBI) gi | 55956899 gi | 313225411

48 51 86 33 63 37 50 63 86 63 86 24 56 86 24 56 56 63 56 Mass (kDa)

p I 6.8 5.2 4.6 4.4 7.1 4.3 5.2 4.9 4.3 4.8 4.3 4.9 4.3 4.8 4.7 4.8 4.6 4.3 4.3

/Burkholderia H160 /Burkholderia sp. Burkholderia K96243 pseudomallei Solanum tuberosum Solanum Heterocephalus glaber Heterocephalus OR74A crassa Neurospora Sinorhizobiu meliloti Sinorhizobiu japonica pinnatifida/Saccharina Undaria Anas platyrhynchos Anas Oryza sativa sativa Indica Group/Oryza Group Japonica Saccharina japonica Saccharina Phanerochaete chrysosporium Phanerochaete Ectocarpus siliculosus Ectocarpus Saccharina japonica/Crassostrea gigas japonica/Crassostrea Saccharina Citrus clementina/Theobroma cacao clementina/Theobroma Citrus Drosophila simulans Drosophila Tupaia chinensis Tupaia IL3000 congolense Trypanosoma CBS 7064 CBS farinose Millerozyma Homo sapiens/Macaca fascicularis Homo sapiens/Macaca Oikopleura dioica/Strongylocentrotus dioica/Strongylocentrotus Oikopleura purpuratus

transferase 2 SP-NH GluTR SSP15 SIPA1L1 NCU01829 EXPA6 ATPase β ATPase TXNDC3 CIPK20 protein kinase protein CIPK20 ATPase α ATPase Cytochrome P450 Cytochrome F-ATPase β F-ATPase Actin Mixture NB-ARC protein NB-ARC Meiotic W68 Meiotic ZFP3 RNA editing protein Cytochrome c oxidase 2 oxidase c Cytochrome KRT9 Protein name Protein

24 17 16 15 Down-regulated proteins in the thallus tissue proximal to bryozoan colonies bryozoan to proximal tissue in the thallus proteins Down-regulated 12 11 9 7 The spot intensities are expressed as the ratio of the intensity in the proximal tissue to that in the healthy tissue. in the healthy that to tissue in the proximal the intensity of the ratio as expressed are intensities The spot 6 5 Up-regulated proteins found mostly in the thallus tissue proximal to bryozoan colonies, but rare in healthy tissue in healthy rare but colonies, bryozoan to proximal tissue in the thallus mostly found proteins Up-regulated 14 18 3 2 13 Up-regulated proteins in the thallus tissue proximal to bryozoan colonies bryozoan to proximal tissue in the thallus proteins Up-regulated 1 26 Spot Spot no Saccharina japonica. Saccharina late-harvested of tissue healthy in distal and colonies bryozoan to proximal tissue in the thallus identified 1: Proteins Table P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies 153

Figure 3: A close-up view of two-dimensional gels showing the proteins (arrows) up-regulated mostly in the thallus tissue proximal to the bryozoan colony and at the colony front, but present at only very low levels in distal healthy tissues. (A) Distal healthy Saccharina japonica tissue. (B) S. japonica thallus tissue proximal to the bryozoan colony. (C) S. japonica tissue at the bryozoan colony front. proteins showed markedly increased spot intensities; thallus tissue proximal to bryozoan colonies (Figure 5). Of approximately 3198-fold higher in proximal tissues than the 19 proteins identified from a homology-based cross- in healthy tissues. Proteins that exhibited higher spot database, we identified four proteins related to intensities in proximal tissues than in distal tissues ATP synthesis, two proteins related to oxidoreduction, included sporulation-specific SSP15 (1395-fold higher), two proteins related to the cytoskeleton, and one protein proliferation-associated SIPA1L1 (1314-fold higher), each related to proliferation, sporulation, photosynthesis, cytoskeleton-associated KRT9 (1059-fold higher), amino amino acid metabolism, protein stabilization, cell divi- acid metabolism-associated SP-NH2 transferase (525- sion, signal transduction, cell wall loosening, hypotheti- fold higher), and photosynthesis-related GluTR (286-fold cal protein, RNA editing, and disease resistance. higher). Eight proteins (cytochrome c oxidase 2, ZFP3, Of the 151 spots in the colony-front tissues, 69, 75, and meiotic W68, actin, cytochrome P450, CIPK20 protein seven spots were up-, down-, and similarly regulated, kinase, TXNDC3, and EXPA6) involved in ATP synthesis, respectively (Figure 2). Of the 69 up-regulated spots, the protein stabilization, cell division, cytoskeleton, oxi- spot intensities of 40 were more than twice those in the doreduction, signal transduction, oxidoreduction, and distal healthy tissues. Of the 75 down-regulated spots, the cell wall loosening, respectively, were significantly up- spot intensities of 17 spots were less than half those in regulated by 4–10-fold in thallus tissue proximal to bryo- the distal tissues. From among 57 of these spots (40 up- zoan colonies (Figure 4). Three down-regulated proteins regulated + 17 down-regulated), we subjected 29 (16 up- (NCU01829, RNA editing protein, and NB-ARC protein), regulated + 13 down-regulated) that appeared constantly which were present mostly in healthy tissues but were in replicated experiments to protein analysis. A database rare in thallus tissue proximal to bryozoan colonies, search of proteins from algae, land plants, and bacteria were significantly down-regulated by 0.0–0.2-fold in the identified 14 (10 up-regulated + 4 down-regulated) spots, 154 P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies

Figure 4: A close-up view of two-dimensional gels showing the proteins (arrows) that were up-regulated by bryozoan colonization. (A) Distal healthy Saccharina japonica tissue. (B) S. japonica thallus tissue proximal to the bryozoan colony. (C) S. japonica tissue at the bryozoan colony front. of which spot no. 14 was a mixture of three proteins. and SRSF) were mostly expressed only in the colony- Table 2 lists the identities of these 16 proteins with their front tissues and were rare in the distal healthy tissues MW, pI, and functions. Twelve proteins were significantly (Figure 3). Of these 9 up-regulated proteins, the ribosomal up-regulated and four were down-regulated. Searches Rpl1P protein showed markedly increased spot intensity of the NCBI and EMBL databases identified 15 and 14 (approximately 5724-fold higher) in colony-front tissues proteins, respectively; 13 proteins were present in both than in healthy tissues. The spot intensity of the ATPase databases. The 16 identified proteins were F-ATPase β, mixture (F-ATPase β, ATPase α, and ATPase β) was higher

ATPase α, ATPase β, SIPA1L1, GluTR, SP-NH2 transferase, by 2475-fold. The SP-NH2 transferase and KRT9 spot inten- 50S ribosomal protein L1P (Rpl1P), KRT9, serine/arginine- sities were 1213- and 1059-fold higher, respectively. The rich splicing factor (SRSF), NB-ARC protein, rho GTPase- spot intensities of splicing factor SRSF, SIPA1L1, and activating protein 26 isoform X4 (ARHGAP26 protein), GluTR were higher by 273-, 192-, and 157-fold, respectively. two-component response regulator (PilR), hypothetical Three proteins (NB-ARC, ARHGAP26, and PilR) involved NCU01829, hypothetical CBY14049.1, serine/threonine- in disease resistance and signal transduction were signifi- protein kinase Nek2 isoform X1 (NEK2 protein kinase), cantly up-regulated by 3–5-fold in the colony-front tissues transmembrane protein (TP). Of the identified proteins, (Figures 4 and 5). Four down-regulated proteins (hypo- nine up-regulated proteins (F-ATPase β, ATPase α, ATPase thetical NCU01829, hypothetical CBY14049.1, NEK2, and

β, SIPA1L1, GluTR, SP-NH2 transferase, Rpl1P, KRT9, TP) were significantly down-regulated by 0.3-fold in the P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies 155

Figure 5: A close-up view of two-dimensional gels showing the proteins (arrows) that were down-regulated by bryozoan colonization. (A) Distal healthy Saccharina japonica tissue. (B) S. japonica thallus tissue proximal to the bryozoan colony. (C) S. japonica tissue at the bryozoan colony front.

colony-front tissues (Figure 5). Of the 16 proteins identi- bryozoan colonies and colony-front sections of the colo- fied from a homology-based cross-species database, we nized S. japonica thalli. found four proteins related to signal transduction, three related to ATP synthesis, two hypothetical proteins, and one protein related to each of proliferation, photosynthe- Discussion sis, amino acid metabolism, protein synthesis, cytoskel- eton, stress control, and disease resistance. Proteomics has been used to reveal complex plant-insect The SSP15 protein was specifically up-regulated by interactions that help identify candidate genes involved 1395-fold only in the thallus tissue proximal to bryozoan in the defense response of plants to herbivory (Wu and colonies, while little was expressed at the colony front or Baldwin 2010). Protein profiling using proteomics can in distal healthy tissues (Figure 3). Therefore, SSP15 can be also be used to identify biomarkers that are up- or down- used as a specific marker for the early warning response regulated in response to parasite colonization. Proteomic of S. japonica to bryozoan colonization. The up-regulated profiles of the edible brown seaweed Saccharina japonica proteins Rpl1P and SRSF were specifically up-regulated have been determined for temperature changes (Yot- only in the colony-front tissues by 5724- and 273-fold, sukura et al. 2012) and different incubation conditions respectively, and were rare in the thallus tissue proximal (Kim et al. 2011). Previously, we examined the proteomic to bryozoan colonies and distal healthy tissues (Figure 3). profiles of S. japonica upon bryozoan (Getachew et al. Seven up-regulated proteins [ATPase mixture (F-ATPase 2014) and hydrozoan (Getachew et al. 2016) infestation.

β, ATPase α, and ATPase β), SIPA1L1, GluTR, SP-NH2 As of yet, however, no study has evaluated the effects of transferase, and KRT9] were up-regulated in the thallus epiphytic bryozoan infection on the proteomic profiles of tissue proximal to bryozoan colonies and bryozoan-front the S. japonica thallus tissue proximal to bryozoan colo- tissues by 157–2475-fold, but were rare in the distal healthy nies and at the colony front. Identifying these initial colo- tissues. In particular, the ATPase mixture was strongly up- nization-induced proteins would provide markers for the regulated both in the thallus tissue proximal to bryozoan early detection of epiphyte infestation or help to improve colonies and colony-front tissues by 3198- and 2475-fold, the resistance of S. japonica to infestation. but was rare in the distal healthy tissues (Figure 3). The In higher plants, sagebrush becomes more resistant ATPase proteins may also serves as bryozoan coloniza- to herbivores after exposure to volatile cues from neigh- tion cue proteins induced in thallus tissue proximal to bors damaged by clipping or natural herbivores (Karban 156 P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies Signal transduction Signal Signal transduction Signal Hypothetical protein Hypothetical Hypothetical protein Hypothetical Signal transduction Signal Signal transduction Signal Disease resistance Disease Cytoskeleton Stress control Protein synthesis Protein Amino acid metabolism Amino acid Function ATP synthesis ATP ATP synthesis ATP ATP synthesis ATP Proliferation Photosynthesis

0.3 0.3 0.3 0.3 2.5 3.9 4.7 273.1 524.8 286.1 1059.4 5723.9 3197.7 1313.5 intensity Ratio of spot spot of Ratio

– – 71 88 201 301 963 2762 1944 1568 2449 1038 3649 3684 3328 2629 score MS BLAST BLAST MS

Q845W1 XP006123907 – XP956613 L22436 XP005996168 EOY10154 XP001737907 XP005584242 Q67JS9 XP780986 Accession ID Accession (EMBL) CBJ32298 YP006639015 YP006639069 – Q63QF1

– 79 86 84 87 79 56 79 88 81 79 93 104 150 125 109 score Mascot

– gi | 558165793 gi | 313239068 gi | 164428214 gi | 335420350 gi | 553134484 gi | 567893315 gi | 471206245 gi | 55956899 gi | 317123107 gi | 313225411 Accession ID Accession (NCBI) gi | 298710991 gi | 403066526 gi | 546226132 gi | 512949794 gi | 209516552

32 28 29 33 51 30 24 15 63 24 56 63 63 63 86 48 Mass (kDa)

p I 5.7 4.7 4.6 4.4 5.6 5.7 4.3 4.8 4.3 4.3 4.3 4.3 4.3 4.3 4.6 6.8

Burkholderia multivorans Burkholderia Pelodiscus sinensis Pelodiscus Oikopleura dioica Oikopleura OR74A crassa Neurospora E1L3A / shabanensis Salinisphaera aeruginosa Pseudomonas /Latimeria chalumnae OR74A /Latimeria crassa Neurospora SAW760 dispar IP1 /Entamoeba invadens Entamoeba cacao clementina/Theobroma Citrus Homo sapiens/Macaca fascicularis Homo sapiens/Macaca DSM marianensis Thermaerobacter thermophilum 12885 /Symbiobacterium Oikopleura dioica/Strongylocentrotus purpuratus dioica/Strongylocentrotus Oikopleura Taxonomy Ectocarpus siliculosus Ectocarpus Saccharina japonica Saccharina Undaria pinnatifida/Saccharina japonica pinnatifida/Saccharina Undaria Heterocephalus glaber Heterocephalus pseudomallei H160 /Burkholderia sp. Burkholderia K96243

transferase 2 TP NEK2 protein kinase NEK2 protein CBY14049.1 NCU01829 PilR ARHGAP26 protein SRSF protein NB-ARC KRT9 Rpl1P Protein name Protein Mixture F-ATPase β F-ATPase ATPase α ATPase ATPase β ATPase SIPA1L1 GluTR SP-NH

The spot intensities are expressed as the ratio of the intensity in the front tissue to that in the healthy tissue. in the healthy that to tissue in the front the intensity of the ratio as expressed are intensities The spot 23 22 21 Down-regulated proteins in colony-front tissue in colony-front proteins Down-regulated 12 20 19 27 tissue in colony-front proteins Up-regulated 18 26 25 . japonica Saccharina late-harvested of tissue healthy distal and tissue in the colony-front identified 2: Proteins Table no Spot Up-regulated proteins found mostly in colony-front tissue, but rare in healthy tissue in healthy rare but tissue, in colony-front mostly found proteins Up-regulated 14 15 17 24 P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies 157 and Shiojiri 2009). Cue compounds were required for DELSEED region, which is the part of subunit β that has systemic-induced resistance among branches on an the amino acid sequence of Asp-Glu-Leu-Ser-Glu-Glu-Asp, individual sagebrush, while vascular connections were is highly conserved in all ATP synthases (Hara et al. 2001). insufficient. Once a bryozoan, a colonial filter-feeding The DNA sequence of this fragment may be used to detect epiphyte, settles on a seaweed blade, it undergoes bipolar or quantify F-ATPase β with the DNA/RNA polymerase growth in circular colonies that continuously increase in chain reaction. In plant chloroplasts, the proton motive diameter (Iyengar and Harvell 2002). Although no cues force is generated by primary photosynthetic proteins and from colonized or damaged seaweed tissues are known, not the respiratory electron transport chain (Rühle and we postulated that signals might be provided by the bryo- Leister 2015). There is a substrate-binding site on both zoans or colonized tissues. To examine the signal trans- ATPase α and ATPase β; those on the beta subunits are duction of colonization, we prepared tissue sections from catalytic, while those on the alpha subunits are regula- thallus tissue proximal to the bryozoan colonies and at the tory. The overall structure and catalytic mechanism of the front of the colonized tissues, and then compared them chloroplast ATP synthases are similar to those of the mito- with distal healthy tissues as a control. chondrial enzymes. The up-regulated protein SSP15 (pI 5.2, 51 kDa) was The up-regulated protein Rpl1P (pI 4.3, 24 kDa) was expressed in large quantities only in the thallus tissue specifically expressed only in the colony-front tissues proximal to bryozoan colonies, while little was expressed (approx. 5724-fold), while it was rare in the thallus at the colony front or in distal healthy tissues. This protein tissue proximal to the bryozoan colonies and distal is a potential marker specifically expressed in the thallus healthy tissues. Rpl1P is predicted to be plastid 50S tissue proximal to bryozoan colonies. SSP15 is predicted to ribosomal protein L1P. It was up-regulated in the inner be a sporulation-specific protein 15-like isoform X4 with a parts of bryozoan colonies (Getachew et al. 2014). The Mascot protein score of 75 (Protein scores greater than 83 stresses caused by high light, salt, and reactive oxygen are significant; p < 0.05; Phongpa-Ngan et al. 2011). There radicals induce SRSF in plants (Yang et al. 2014). The is no clear information on how SSP15 is related to seaweed up-regulation of SRSF in the colony-front tissues may sporulation or epiphytic infestation. It may be associated help to increase the response to colonization. It was with spindle pole bodies throughout the life cycle and also up-regulated in the inner parts of hydrozoan col- play an indispensable role in the initiation of spore mem- onies (Getachew et al. 2016). Therefore, the Rpl1P and brane formation (Ikemoto et al. 2000). Spore walls are SRSF proteins are potential markers of colonization by constructed by accumulating wall materials in the lumen bryozoans and other epiphytes. Identifying the early of forespore membranes (Tanaka and Hirata 1982). Thick cue proteins or inducers related to defense mechanism walls confer resistance to the seaweed spores towards or stress control could help to improve the resistance various environmental stresses. Epibiotic crust coloniza- of S. japonica strains against pathogens, parasites, and tion reduced spore release from fertile blades (Saier and environmental stresses. Chapman 2004). High expression of this protein in the early phase of colonization (i.e. in the thallus tissue proxi- Acknowledgements: This work was supported by a mal to bryozoan colonies) might indicate the seaweed’s grant from the National Institute of Fisheries Science response to maintain normal sporulation and growth. (R2018021), Republic of Korea. Therefore, this predicted sporulation-specific protein is a potential marker protein specific for the thallus tissue proximal to early bryozoan colonization. Further studies should characterize the SSP15 of S. japonica in detail and References determine the underlying molecular mechanisms. de Burgh, M.E. and P.V. Fankboner. 1978. A nutritional association The ATPase mixture (F-ATPase β, ATPase α, and between the bull kelp Nereocystis luetkeana and its epizootic ATPase β) was strongly expressed in both the thallus bryozoans Membranipora membranacea. 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Mar. 47: aquatic biosciences by the Tokyo University of Fisheries, Japan for 265–271. work on molecular immunology of the flat fish Paralichthys oliva- Shevchenko, A., M. Wilm, O. Vorm and M. Mann. 1996. Mass spec- ceus. Recently, she has been taking part in the Genome Project of trometric sequencing of proteins from silver-stained poly- Marine Organisms funded by the Ministry of and Fisheries, acrylamide gels. Anal. Chem. 68: 850–858. Korea. P. Getachew et al.: Proteomics of Saccharina japonica induced by bryozoan colonies 159

Yong-Ki Hong Department of Biotechnology, Pukyong National University, Namgu, Busan 48513, Korea [email protected]

Yong-Ki Hong is a professor of seaweed biotechnology and bio- chemistry at Pukyong National University, Korea. He was awarded a PhD in seaweed biotechnology by the University of California, Santa Barbara for his work on differential display technique, and a PhD in microbiology by the Kyungpook National University, Korea for his work on plasmid transformation. Since 1993, he has focused his research on the isolation of biologically active substances (memory enhancers, anti-inflammatory agents, antifoulants, algicidal sub- stances, etc.) from seaweed. He was a college dean, editor-in-chief, and President of the Asian-Pacific Society for Applied Phycology.