Identification of early biomarkers in proteomic profiles of the phaeophyte Saccharina japonica proximal to and beneath the front of bryozoan colonies

Introduction

The edible brown seaweed Saccharina japonica has been cultured widely in East Asia since the 1970s. The seaweed thalli are a popular health seafood and used as abalone feed. In 2015, 442,771 t (wet weight) of S. japonica were produced by farming; an additional 10 t (wet weight) were harvested from natural populations in Korea (Korea Fisheries Association 2016). Epiphytism is a major problem in seaweed aquaculture, as it reduces the product quality and yield. Bryozoans, hydroids, amphipods, copepods, and gastropods are some of the most abundant epiphytes on the blades of seaweeds (Peteiro and Freire 2013). Bryozoan and hydroid colonies on S. japonica thalli reduced the seaweed tissue viability by 69% and 77%, respectively, compared with healthy tissues (Getachew et al. 2015a,b). Unsightly, coarse epiphytic colonizers also markedly reduce the commercial value of the colonized thalli. One of the most common epiphytic colonizers on S. japonica thalli is the encrusting bryozoan Membranipora membranacea. It is a colonial animal that filter-feeds on bacteria, flagellates, diatoms, small planktonic organisms, and decayed organic material (de Burgh and Fankboner 1978). The planktotrophic bryozoan larvae settle on kelp thalli and give rise to large colonies covering the surface of the seaweed. Besides being highly calcified, bryozoan colonies contain extraordinary amounts of crude ash and harmful arsenic exceeding the limit of the provisional tolerable weekly intake by humans (Getachew et al. 2015a). The colonies of M. membranacea also reduce the rate of photosynthesis 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 environmental changes/stresses on the biological activities of seaweeds at biochemical and molecular levels. Although the biochemical and physical mechanisms are not clear, after removing the bryozoans, seaweed tissues beneath the colonies exhibited elevated potassium, iodine, and docosahexaenoic acid levels and reduced copper, chromium, and cadmium levels compared with the distal healthy tissues (Getachew et al. 2015a). When epiphytic 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-regulated proteins in the proximal and colony-front tissues can be used as biomarkers in future research for early diagnosis or improving strain resistance to epiphytic infestation.

In this paper, early bryozoan colonization marker proteins from proximal and colony-front tissue sections of colonized S. japonica thalli were identified and their primary roles in cellular activities are discussed.

Materials and methods

Seaweed, bryozoans, and reagents

Fresh thalli of the late harvest Saccharina japonica (J.E. Areschoug) C.E. Lane, C. Mayes, Druehl et G.W. Saunders were collected from Gijang aquaculture farm, Busan, Korea in June 2015 and 2016. A voucher specimen was deposited in the author’s laboratory (Y.K. Hong). The seaweed tissues were washed and cleaned with autoclaved seawater. They were acclimatized in an aquarium tank with in situ light and temperature conditions for 3 days. Different tissue sections of the thalli covered by the bryozoan Membranipora membranacea were selected (Figure 1), and colonies were gently scraped off with a stiff plastic sheet. Thallus tissue proximal to the bryozoan colony (i.e. the 1-cm zone beyond the boundary of the colony) and tissue from the colony-front (i.e. the narrow zone under the newly formed front of the colony after removing the bryozoans) were immediately freeze dried (SFD-SM, Samwon Freezing Engineering Co., Busan, Korea), ground to a fine powder, and kept at –70°C before analysis. Distal healthy tissues located at least 30 cm from the colony were treated in the same way and used as a control. Most of reagents used in this study were of analytical grade from Sigma-Aldrich Co., St. Louis, MO, USA.

Figure 1:

The Saccharina japonica thallus sections defined as colony-front and thallus tissue proximal to Membranipora membranacea colonies.

Colony-front tissues were collected from the narrow zone under the newly formed front of the colony after removing the bryozoans. Thallus tissue proximal to the bryozoan colony was obtained from the 1-cm zone outside the boundary of the colony.

Results

The crustose bryozoan Membranipora membranacea frequently colonizes the late-harvested thalli of aquacultured Saccharina japonica. To identify proteins that are markers of early colonization or initial cue proteins upon colonization, we separated the thallus tissue proximal to bryozoan colonies, tissue at the colony front, and distal healthy thallus tissues to isolate the proteins that responded to M. membranacea colonization. The protein isolation from each tissue was replicated and optimized to confirm the differentially displayed protein profiles. For the thallus tissue proximal to bryozoan colonies, tissue at the colony front, and distal healthy thallus tissues, 151, 151, and 110 protein spots, respectively, were detected on two-dimensional gel plates (Figure 2). Of the 151 spots in the thallus tissue proximal to bryozoan colonies, 149 spots showed different expression levels compared with distal healthy tissues; 99, 50, and two spots were up-, down-, and similarly regulated, respectively. Of the 99 up-regulated spots, 41 had spot intensities more than twice those from the distal tissues. Of the 50 down-regulated spots, the spot intensities of seven were less than half those in the distal tissues. From the 48 spots that differed markedly in intensity between tissues (41 up-regulated+7 down-regulated), we selected 35 (33 up-regulated+2 down-regulated) spots that appeared constantly in replicated experiments for protein analysis. In a database search of proteins from algae, land plants, and bacteria, we identified 17 (14 up-regulated+3 down-regulated) spots, of 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. Sixteen of them were up-regulated and three were down-regulated. On searching the NCBI and EMBL databases, 18 and 17 proteins, respectively, were confidently identified. Sixteen proteins were present in both databases.

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.

The 19 identified proteins were F-type H-ATPase beta subunit (F-ATPase β), chloroplast ATP synthase CF1 alpha chain (ATPase α), chloroplast ATP synthase beta subunit (ATPase β), signal-induced proliferation-associated 1-like protein 1 isoform X9 (SIPA1L1), sporulation-specific protein 15-like isoform X4 (SSP15), glutamyl-tRNA reductase (GluTR), mitochondrial serine-pyruvate aminotransferase (SP-NH₂ transferase), keratin type I cytoskeletal 9 (KRT9), cytochrome c oxidase 2, zinc finger protein 3 (ZFP3), meiotic W68, actin, cytochrome P450, protein kinase domain-containing protein (protein kinase), thioredoxin domain-containing protein 3 (TXNDC3), expansin 6 (EXPA6), hypothetical NCU01829, RNA editing complex protein (RNA editing protein), and LRR/NB-ARC domains-containing disease resistance protein (NB-ARC protein). Among the identified proteins, three up-regulated proteins (F-ATPase β, ATPase α, and ATPase β) were primarily expressed only in the thallus tissue proximal to bryozoan colony and expressed at low levels in the distal healthy tissues (Figure 3). These ATP synthesis-related proteins showed markedly increased spot intensities; approximately 3198-fold higher in proximal tissues than in healthy tissues. Proteins that exhibited higher spot intensities in proximal tissues than in distal tissues included sporulation-specific SSP15 (1395-fold higher), proliferation-associated SIPA1L1 (1314-fold higher), cytoskeleton-associated KRT9 (1059-fold higher), amino acid metabolism-associated SP-NH₂ transferase (525-fold higher), and photosynthesis-related GluTR (286-fold higher). Eight proteins (cytochrome c oxidase 2, ZFP3, meiotic W68, actin, cytochrome P450, CIPK20 protein kinase, TXNDC3, and EXPA6) involved in ATP synthesis, protein stabilization, cell division, cytoskeleton, oxidoreduction, signal transduction, oxidoreduction, and cell wall loosening, respectively, were significantly up-regulated by 4–10-fold in thallus tissue proximal to bryozoan colonies (Figure 4). Three down-regulated proteins (NCU01829, RNA editing protein, and NB-ARC protein), which were present mostly in healthy tissues but were rare in thallus tissue proximal to bryozoan colonies, were significantly down-regulated by 0.0–0.2-fold in the thallus tissue proximal to bryozoan colonies (Figure 5). Of the 19 proteins identified from a homology-based cross-species database, we identified four proteins related to ATP synthesis, two proteins related to oxidoreduction, two proteins related to the cytoskeleton, and one protein each related to proliferation, sporulation, photosynthesis, amino acid metabolism, protein stabilization, cell division, signal transduction, cell wall loosening, hypothetical protein, RNA editing, and disease resistance.

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.

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.

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.

Of the 151 spots in the colony-front tissues, 69, 75, and seven spots were up-, down-, and similarly regulated, respectively (Figure 2). Of the 69 up-regulated spots, the spot intensities of 40 were more than twice those in the distal healthy tissues. Of the 75 down-regulated spots, the spot intensities of 17 spots were less than half those in the distal tissues. From among 57 of these spots (40 up-regulated+17 down-regulated), we subjected 29 (16 up-regulated+13 down-regulated) that appeared constantly in replicated experiments to protein analysis. A database search of proteins from algae, land plants, and bacteria identified 14 (10 up-regulated+4 down-regulated) spots, of which spot no. 14 was a mixture of three proteins. Table 2 lists the identities of these 16 proteins with their MW, pI, and functions. Twelve proteins were significantly up-regulated and four were down-regulated. Searches of the NCBI and EMBL databases identified 15 and 14 proteins, respectively; 13 proteins were present in both databases. The 16 identified proteins were F-ATPase β, ATPase α, ATPase β, SIPA1L1, GluTR, SP-NH₂ transferase, 50S ribosomal protein L1P (Rpl1P), KRT9, serine/arginine-rich splicing factor (SRSF), NB-ARC protein, rho GTPase-activating protein 26 isoform X4 (ARHGAP26 protein), two-component response regulator (PilR), hypothetical NCU01829, hypothetical CBY14049.1, serine/threonine-protein kinase Nek2 isoform X1 (NEK2 protein kinase), transmembrane protein (TP). Of the identified proteins, nine up-regulated proteins (F-ATPase β, ATPase α, ATPase β, SIPA1L1, GluTR, SP-NH₂ transferase, Rpl1P, KRT9, and SRSF) were mostly expressed only in the colony-front tissues and were rare in the distal healthy tissues (Figure 3). Of these 9 up-regulated proteins, the ribosomal Rpl1P protein showed markedly increased spot intensity (approximately 5724-fold higher) in colony-front tissues than in healthy tissues. The spot intensity of the ATPase mixture (F-ATPase β, ATPase α, and ATPase β) was higher by 2475-fold. The SP-NH₂ transferase and KRT9 spot intensities were 1213- and 1059-fold higher, respectively. The spot intensities of splicing factor SRSF, SIPA1L1, and GluTR were higher by 273-, 192-, and 157-fold, respectively. Three proteins (NB-ARC, ARHGAP26, and PilR) involved in disease resistance and signal transduction were significantly up-regulated by 3–5-fold in the colony-front tissues (Figures 4 and 5). Four down-regulated proteins (hypothetical NCU01829, hypothetical CBY14049.1, NEK2, and TP) were significantly down-regulated by 0.3-fold in the colony-front tissues (Figure 5). Of the 16 proteins identified from a homology-based cross-species database, we found four proteins related to signal transduction, three related to ATP synthesis, two hypothetical proteins, and one protein related to each of proliferation, photosynthesis, amino acid metabolism, protein synthesis, cytoskeleton, stress control, and disease resistance.

The SSP15 protein was specifically up-regulated by 1395-fold only in the thallus tissue proximal to bryozoan colonies, while little was expressed at the colony front or in distal healthy tissues (Figure 3). Therefore, SSP15 can be used as a specific marker for the early warning response of S. japonica to bryozoan colonization. The up-regulated proteins Rpl1P and SRSF were specifically up-regulated only in the colony-front tissues by 5724- and 273-fold, respectively, and were rare in the thallus tissue proximal to bryozoan colonies and distal healthy tissues (Figure 3). Seven up-regulated proteins [ATPase mixture (F-ATPase β, ATPase α, and ATPase β), SIPA1L1, GluTR, SP-NH₂ transferase, and KRT9] were up-regulated in the thallus tissue proximal to bryozoan colonies and bryozoan-front tissues by 157–2475-fold, but were rare in the distal healthy tissues. In particular, the ATPase mixture was strongly up-regulated both in the thallus tissue proximal to bryozoan colonies and colony-front tissues by 3198- and 2475-fold, but was rare in the distal healthy tissues (Figure 3). The ATPase proteins may also serves as bryozoan colonization cue proteins induced in thallus tissue proximal to bryozoan colonies and colony-front sections of the colonized S. japonica thalli.

Discussion

Proteomics has been used to reveal complex plant-insect interactions that help identify candidate genes involved in the defense response of plants to herbivory (Wu and Baldwin 2010). Protein profiling using proteomics can also be used to identify biomarkers that are up- or down-regulated in response to parasite colonization. Proteomic profiles of the edible brown seaweed Saccharina japonica have been determined for temperature changes (Yotsukura et al. 2012) and different incubation conditions (Kim et al. 2011). Previously, we examined the proteomic profiles of S. japonica upon bryozoan (Getachew et al. 2014) and hydrozoan (Getachew et al. 2016) infestation. As of yet, however, no study has evaluated the effects of epiphytic bryozoan infection on the proteomic profiles of the S. japonica thallus tissue proximal to bryozoan colonies and at the colony front. Identifying these initial colonization-induced proteins would provide markers for the early detection of epiphyte infestation or help to improve the resistance of S. japonica to infestation.

In higher plants, sagebrush becomes more resistant to herbivores after exposure to volatile cues from neighbors damaged by clipping or natural herbivores (Karban and Shiojiri 2009). Cue compounds were required for systemic-induced resistance among branches on an individual sagebrush, while vascular connections were insufficient. Once a bryozoan, a colonial filter-feeding epiphyte, settles on a seaweed blade, it undergoes bipolar growth in circular colonies that continuously increase in diameter (Iyengar and Harvell 2002). Although no cues from colonized or damaged seaweed tissues are known, we postulated that signals might be provided by the bryozoans or colonized tissues. To examine the signal transduction of colonization, we prepared tissue sections from thallus tissue proximal to the bryozoan colonies and at the front of the colonized tissues, and then compared them with distal healthy tissues as a control.

The up-regulated protein SSP15 (pI 5.2, 51 kDa) was expressed in large quantities only in the thallus tissue proximal to bryozoan colonies, while little was expressed at the colony front or in distal healthy tissues. This protein is a potential marker specifically expressed in the thallus tissue proximal to bryozoan colonies. SSP15 is predicted to be a sporulation-specific protein 15-like isoform X4 with a Mascot protein score of 75 (Protein scores greater than 83 are significant; p<0.05; Phongpa-Ngan et al. 2011). There is no clear information on how SSP15 is related to seaweed sporulation or epiphytic infestation. It may be associated with spindle pole bodies throughout the life cycle and play an indispensable role in the initiation of spore membrane formation (Ikemoto et al. 2000). Spore walls are constructed by accumulating wall materials in the lumen of forespore membranes (Tanaka and Hirata 1982). Thick walls confer resistance to the seaweed spores towards various environmental stresses. Epibiotic crust colonization reduced spore release from fertile blades (Saier and Chapman 2004). High expression of this protein in the early phase of colonization (i.e. in the thallus tissue proximal to bryozoan colonies) might indicate the seaweed’s response to maintain normal sporulation and growth. 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 determine the underlying molecular mechanisms.

The ATPase mixture (F-ATPase β, ATPase α, and ATPase β) was strongly expressed in both the thallus tissue proximal to bryozoan colonies and at the colony front, but not in the distal healthy tissues. F-ATPase β or the F-type H⁺-transporting ATPase beta subunit is an ATP synthase that catalyzes ATP synthesis and hydrolysis at the expense of the transmembrane electrochemical proton potential difference in chloroplasts, mitochondria, and bacteria (Pedersen and Carafoli 1987). The beta DELSEED region, which is the part of subunit β that has the amino acid sequence of Asp-Glu-Leu-Ser-Glu-Glu-Asp, is highly conserved in all ATP synthases (Hara et al. 2001). The DNA sequence of this fragment may be used to detect or quantify F-ATPase β with the DNA/RNA polymerase chain reaction. In plant chloroplasts, the proton motive force is generated by primary photosynthetic proteins and not the respiratory electron transport chain (Rühle and Leister 2015). There is a substrate-binding site on both ATPase α and ATPase β; those on the beta subunits are catalytic, while those on the alpha subunits are regulatory. The overall structure and catalytic mechanism of the chloroplast ATP synthases are similar to those of the mitochondrial enzymes.

The up-regulated protein Rpl1P (pI 4.3, 24 kDa) was specifically expressed only in the colony-front tissues (approx. 5724-fold), while it was rare in the thallus tissue proximal to the bryozoan colonies and distal healthy tissues. Rpl1P is predicted to be plastid 50S ribosomal protein L1P. It was up-regulated in the inner parts of bryozoan colonies (Getachew et al. 2014). The stresses caused by high light, salt, and reactive oxygen radicals induce SRSF in plants (Yang et al. 2014). The up-regulation of SRSF in the colony-front tissues may help to increase the response to colonization. It was also up-regulated in the inner parts of hydrozoan colonies (Getachew et al. 2016). Therefore, the Rpl1P and SRSF proteins are potential markers of colonization by bryozoans and other epiphytes. Identifying the early cue proteins or inducers related to defense mechanism or stress control could help to improve the resistance of S. japonica strains against pathogens, parasites, and environmental stresses.