Influences of hydrozoan colonization on proteomic profiles of the brown alga Saccharina japonica

Introduction

The brown alga Saccharina japonica (J.E. Areschoug) C.E. Lane, C. Mayes, Druehl et G.W. Saunders has been extensively cultivated in Korea, China, and Japan since the 1970s. Thalli of the seaweed are popular as health food. The amount of S. japonica produced by aquaculture in 2013 was 373,264 t (wet weight), and an additional 4 t (wet weight) was collected from natural populations in Korea (Korea Fisheries Association 2014). Fouling by epiphytic animal colonizers on the seaweed thalli has become a major challenge for the seaweed industry, and reducing this fouling during the cultivation period has been difficult. Several fouling species occur in temperate waters, and the stoloniferous hydrozoan Obelia geniculata (Linnaeus, 1758) is one of commonest epiphytic species. Its planktotrophic larvae settle on the kelp blades of S. japonica, where they develop into extensive colonies. Over the last two decades, there were severe outbreaks especially on late-harvested S. japonica (Park and Hwang 2012). Such colonization is considered an important issue since it results in unsightly, coarse and indelicate epiphytic contaminants, and means that the harvested S. japonica has no commercial value. Hydrozoans contain high amounts of zinc (1.7 g kg^-1 dry weight) at levels higher than the recommended range in food (Getachew et al. 2015a). As evaluated for food or fodder, hydrozoans and/or hydrozoan-colonized tissues seriously reduce the quality of the seaweed, so that the epiphytic hydrozoans must be removed from the seaweed blade prior to its use. Although hydroid infestation on seaweed has been known for a long time, most studies so far have focused on the biology of the hydrozoans. The hydrozoan O. geniculata is one of the sessile animal epiphytes distributed throughout the world, except in the high Arctic and Antarctic seas (Cornelius 1990). The life cycle includes free-living medusa, planula, and sessile colonial polyp stages (Slobodov and Marfenin 2004). The hydrozoans are one of the most voracious groups among the passive benthic suspension feeders. They are able to ingest a large quantity of heterogeneous diet including invertebrate eggs and fecal pellets (Orejas et al. 2000). The photoprotein obelin is responsible for the bioluminescence of the hydroid O. geniculata (Markova et al. 2002).

Upon epiphytic colonization, the host seaweed can respond physiologically, which may lead to changes in biochemical composition at the protein level in the thalli. Protein profiling using proteomics can be used to identify marker proteins that are up- or down-regulated in response to environmental stresses or parasite colonization. Recently, proteomic profiles from S. japonica were identified with seasonal changes (Yotsukura et al. 2010) and different incubation conditions (Kim et al. 2011). The proteomic profiles (Getachew et al. 2014) and biochemical composition (Getachew et al. 2015b) of S. japonica upon bryozoan colonization were also analyzed. As yet, however, no report has evaluated the effects of epiphytic O. geniculata infection on the proteomic profile of the edible brown seaweed S. japonica. We have, therefore, investigated differentially expressed proteins in S. japonica tissues in response to O. geniculata colonization and their primary roles in cellular activities.

Materials and methods

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 (pI) 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.

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.

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.

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 hydrozoan-colonized 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.

Discussion

From the hydrozoan-colonized tissues of Saccharina japonica, 107 protein spots were separated by 2-DE. Among them, 77 and 28 spots were distictively up- and down-regulated, respectively. With bryozoan colonization, 145 protein spots were detected from 2-DE (Getachew et al. 2014). Among them, 69 and 32 spots were up- and down-regulated, respectively. When blades of S. japonica were collected in different seasons, summer blades showed 67 up- and 28 down-regulated spots compared to winter samples (Yotsukura et al. 2010). Three and six distinctive protein spots were up- and down-regulated, respectively, when S. japonica was incubated at pH 9.5 and at pH 8.5 (Kim et al. 2011). Upon hydrozoan infection, we found similar numbers of protein spots with significantly different expressions compared to bryozoan infection or seasonal changes. Some protein spots showed lower observed pI values than their theoretical values. The shifting of observed pI values often correlated with post-translational modifications including phosphorylation (Zhu et al. 2005). Multiple phosphorylations in one protein may result in a significant pI decrease.

Most identified proteins from hydrozoan-colonized tissues were related to functions of stress control, signal transduction and photosynthesis. For example, Cdc46/Mcm5, found mostly in the colonized tissue, belongs to a family of proteins which are essential for initiation of DNA replication (Dalton and Whitbread 1995). The helicase activity of Cdc46/Mcm5 is required to unwind the energetically stable duplex DNA in an ATP-dependent manner (Tuteja 1997). Genes encoding for the helicases are up-regulated under the influence of various environmental stresses (Tuteja et al. 2011). Thus, the up-regulation of Cdc46/Mcm5 upon hydrozoan infection may help S. japonica to survive under the epiphytic stress. GluTR, the protein up-regulated by 41-fold in the bryozoan-colonized tissues compared with healthy tissues (Getachew et al. 2014), was also found mostly (277-fold) in the hydrozoan-colonized tissues. Tetrapyrrole biosynthesis for chlorophyll occurs through a ubiquitous and highly conserved pathway that provides many molecules involved in light harvesting, energy transfer, and signal transduction (Molina et al. 1999). The presence of more GluTR in the colonized tissue may help the host to maintain an adequate metabolite flux for its precursor 5-aminolevulinic acid formation, which may compensate for the loss of tetrapyrroles upon colonization. Photosynthesis-related MCP and CsoS were up-regulated three-fold in the colonized tissues. Epiphytic colonies interfere with mineral and nutrient uptake by imposing a mechanical barrier (Hurd et al. 2000). Thus, S. japonica needs an enhanced carbon dioxide concentrating mechanism to compensate for the lower availability of CO₂. The bioC was up-regulated in the colonized tissues. Biotin is a cofactor of carboxylases, which catalyze carboxylation reactions in crucial metabolic processes, such as the synthesis and catabolism of amino acids, fatty acids and isoprenoids (Nikolau et al. 2003). Mutation of genes is observed resulting in the accumulation of hydrogen peroxide and the down-regulation of genes involved in stress responses (Li et al. 2012). Thus, the up-regulation of bioC in hydrozoan-colonized tissues may help S. japonica to detoxify the hydrogen peroxide produced by the colony stress. Environmental stresses also induce SRSF in plants (Yang et al. 2014). The SRSF is known as an alternative splicing factor or pre-mRNA-splicing factor. Spliced mRNAs were up-regulated in response to high light, salt and oxidative stress (Tanabe et al. 2007). The up-regulation of SRSF in the colonized tissue may help to increase the response mechanism against the colony stress. The PilR was up-regulated (11-fold) in the hydrozoan-colonized tissues, and also up-regulated (38-fold) in the bryozoan-colonized tissues (Getachew et al. 2014). The PilR system is composed of two elements, a histidine kinase and a response regulator involving a phosphorelay system. In Arabidopsis thaliana, histidine kinase 1 mRNA was more abundant in roots than in other tissues under conditions of high salinity and low temperature, suggesting that the enzyme is necessary for the efficient sensing of environmental signals (Urao et al. 2000). The up-regulation upon epiphytic infection may allow the host to become sensitive to environmental signals, which possibly leads to increased resistance against epiphytes.

In contrast, some proteins involved in signal transduction, stress control, protein metabolism, photosynthesis, defense responses, cell growth and the cytoskeleton were diminished in the hydrozoan-colonized S. japonica tissues. For example, PGK (EC 2.7.2.3) was found mostly in healthy tissues but was rare in hydrozoan-colonized tissues. It is a key enzyme in the Calvin cycle and glycolysis/gluconeogenesis, which catalyzes the transfer of a phosphoryl group to 3-phosphoglycerate and the reverse. In a drought-susceptible genotype of sunflower, a decrease of PGK was observed (Castillejo et al. 2008). Down-regulation of this protein in the colonized tissues may suggest that the energetic metabolism of the tissue under hydrozoan colonization is repressed. EXPA6 is a class of cell wall proteins that mediates cell wall loosening by disrupting hydrogen bonds between cellulose and matrix glycans (Han et al. 2012). In most cases, silencing expansin gene expression inhibits plant growth, whereas excessive expression leads to faster growth. Over-expression of the expansin gene has increased seedling growth in rice (Li et al. 2013). The down-regulation of EXPA6 in hydrozoan-colonized tissues may indicate repression of S. japonica cell growth under hydrozoan colony. IF3 was almost absent upon hydrozoan infection. Translational modulation emerges as a key step in plants to adapt to the challenges imposed by biotic and abiotic threats (Echevarría-Zomeño et al. 2013). The down-regulation of IF3 in colonized tissues made the host’s resistance to hydrozoan infection weaker. CaMK2N2 was also found mostly in healthy tissue but not in hydrozoan-colonized tissues. Calcium is a universal secondary messenger that regulates a range of cellular and physiological processes in plants (Zhang and Lu 2003). It has vital roles in response to various signals, including light, mechanical disturbances, abiotic stress, and pathogen elicitors. Changes in cytosolic calcium are sensed by a group of Ca²⁺-binding proteins, including CaMK in signal transduction. Plants over-expressing these genes can increase their stress tolerances. Thus, the down-regulation of the CaMK2N2 (a CaMK inhibitor) in hydrozoan-colonized tissues may help S. japonica to increase signal transduction and stress resistance. In higher eukaryotes, loss of cytoplasmic ribosomal proteins results in a reduced growth rate as well as developmental defects (Szakony and Byrne 2011). The down-regulation of rpl1P in hydrozoan-colonized tissues might indicate a reduction of S. japonica cell growth under colonization. Additionally, signal regulatory TP, Hsp90, GAPα, defense-responding DUOX2 and F3H proteins, TARS and Prf1 required for protein metabolism, UCP3 and vBPO7 involved in stress control, PPOX in photosynthesis, and an actin-binding protein interaptin were diminished by less than half in the hydrozoan-colonized S. japonica tissues. Decreased expression of these proteins may make the host S. japonica more susceptible to the hydrozoan infection. Specific proteins present in S. japonica that are induced in hydrozoan- but not in bryozoan-colonized tissues were Cdc46/Mcm5, bioC and SRSF as up-regulated proteins; PGK, EXPA6, CaMK2N2, rpl1P, TP, DUOX2, UCP3, vBPO7 and a mixture of Prf1 and interaptin as down-regulated proteins (Getachew et al. 2014). Common response factors of the host against hydrozoan and bryozoan infections were GluTR, PilR and a mixture of MCP and CsoS as up-regulated proteins; IF3, PPOX, Hsp90, GAPα, TARS and F3H as down-regulated proteins. Finally, clarifying the initial cue protein or inducer related to signal transduction, stress control, and/or defense mechanism would support strain improvement of S. japonica with regard to resistance against parasites, pathogens and environmental stress. Isolation of the initial cue proteins in early colonization is now in progress.