Proteomic Profiles and Ultrastructure of Regenerating Protoplast of Bryopsis Plumosa (Chlorophyta)

Research Article

Algae 2016, 31(4): 379-390 https://doi.org/10.4490/algae.2016.31.11.31 Open Access

Proteomic profiles and ultrastructure of regenerating protoplast of Bryopsis plumosa (Chlorophyta)

Tatyana A. Klochkova1,2, Min Seok Kwak1 and Gwang Hoon Kim1,* 1Department of Biology, Kongju National University, Kongju 32588, Korea 2Kamchatka State Technical University, Petropavlovsk-Kamchatsky 683003, Russia

When a multinucleate cell of Bryopsis plumosa was collapsed by a physical wounding, the extruded protoplasm ag- gregated into numerous protoplasmic masses in sea water. A polysaccharide envelope which initially covered the protoplasmic mass was peeled off when a cell membrane developed on the surface of protoplast in 12 h after the wounding. Transmission electron microscopy showed that the protoplasmic mass began to form a continuous cell membrane at 6 h after the wounding. The newly generated cell membrane repeated collapse and rebuilding process several times until cell wall developed on the surface. Golgi bodies with numerous vesicles accumulated at the peripheral region of the rebuilding cell at 24 h after the wounding when the cell wall began to develop. Several layers of cell wall with distinctive electron density developed within 48-72 h after the wounding. Proteome profile changed dramatically at each stage of cell rebuilding process. Most proteins, which were up-regulated during the early stage of cell rebuilding disappeared or reduced significantly by 24-48 h. About 70-80% of protein spots detected at 48 h after the wounding were newly appeared ones. The expression pattern of 29 representative proteins was analyzed and the internal amino acid sequences were obtained using mass spectrometry. Our results showed that a massive shift of gene expression occurs during the cell- rebuilding process of B. plumosa.

Key Words: Bryopsis plumosa; cell rebuilding; cell wall; proteome; protoplast; ultrastructure

INTRODUCTION

Some coenocytic green algae are able to rebuild a new based plasma membrane, and formation of a cell wall cell from the remnants of destroyed cell in sea water (Kim et al. 2001, 2002). (Tatewaki and Nagata 1970, Kobayashi and Kanaizuka The generation of cell membrane on the surface of ag- 1985, Pak et al. 1991, Kim and Klochkova 2004, Bottalico glutinated protoplasmic mass is the most crucial event et al. 2008). Several very distinctive sequential steps of of the cell rebuilding process in Bryopsis plumosa (Hud- this cell rebuilding process have been revealed from de- son) Agardh. Previous study using Nile Red as fluorescent tailed cellular and molecular studies: agglutination of probe for intracellular lipids showed that formation of cell organelles into protoplasmic masses in sea water, cell membrane occurred during the first 6 h from the mo- formation of a primary polysaccharide envelope on their ment of protoplasm extrusion into seawater (Kim et al. surface, replacement of this primary envelope by a lipid- 2001). During this whole time period, the primary poly-

This is an Open Access article distributed under the Received October 6, 2016, Accepted November 31, 2016 terms of the Creative Commons Attribution Non-Com- Corresponding Author mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which * permits unrestricted non-commercial use, distribution, and reproduction E-mail: [email protected] in any medium, provided the original work is properly cited. Tel: +82-41-8508504, Fax: +82-41-8508479

saccharide envelope that enclosed protoplasts acted like MATERIALS AND METHODS a cell membrane, showing semi-permeability and selec- tive transport of materials. Calcofluor White staining Plant material and laboratory culture showed that cell wall development occurred in 24-48 h after the wounding (Kim et al. 2001, 2002, Klochkova et Vegetative plants of B. plumosa were collected from Ka- al. 2003). Ultrastructure of the regenerating protoplast of chon, southern coast of Korea and maintained as unialgal B. plumosa has been reported (Pak et al. 1991), but de- cultures in IMR medium (Kim et al. 2006). Plants were tailed regeneration processes of cell membrane and cell grown at 23°C with 12 : 12 h L : D cycle and 15 µmol m-2 wall are still unknown. As each step of protoplast forma- s-1 light intensity. Protoplasts were prepared as described tion is very distinct in terms of time intervals and requires in Kim et al. (2001) and grown at the same culture condi- a cascade of specific cellular events, one can expect that tions as above. different groups of genes are regulated at each step. Proteomics is one of the most powerful tools in modern Sample preparation for protein extraction biology to study the differential display of gene expression since proteins are often directly related to biological To understand the changes of proteome profiles during processes and cellular function (Pandey and Mann 2000, cell rebuilding process five 2-DE maps were prepared us- Tyers and Mann 2003). Two-dimensional polyacrylamide ing following samples: 1) control vegetative plants before gel electrophoresis (2-DE), which can separate complex wounding (0 h); 2) 3-h-old protoplasts: the time when protein mixtures, was used to identify protein changes membrane “patches” begin to incorporate into a primary that occur normally during development and tissue dif- polysaccharide envelope; 3) 12-h-old protoplasts: plasma ferentiation, as a result of disease, drug treatment, envi- membrane development is complete; 4) 24-h-old proto- ronmental change, etc. (Celis et al. 1999a, 1999b, Span- plasts: cell wall formation is in progress; and 5) 48-h-old didos and Rabbitts 2002). In phycology, this branch of protoplasts: cell wall is complete and cell begins to show “omics” has been widely applied, as evident from a large polarity. Culture medium was removed from the algal number of publications accumulating recently (e.g., thalli (2 g) with a tissue paper and they were frozen in liq- Kim et al. 2008, Choi et al. 2015). Proteomic approach uid nitrogen and used for protein extraction. has been applied in Bryopsis studies by Yamagishi et al. The wet weight of algal thalli used for protoplast prepa- (2004) who performed 2-DE analysis of the hybrid plants rations was 36 g. Protoplasts were prepared as described that developed from hybrid protoplasts of B. plumosa ob- in Kim et al. (2001) and grown in 100 × 10-mm glass Petri tained by mixing protoplasm of its gametophyte and spo- dishes containing IMR medium with 50 mg L-1 ampicillin rophyte. Most proteins synthesized by the hybrid plants to avoid bacterial contamination. Protoplasts were har- were identical to those of normal sporophytes of B. plu- vested at 3, 12, 24, and 48 h after formation using a fine mosa (Yamagishi et al. 2004). As they used mature plants glass pipette, transferred to 1-mL plastic tubes and cen- for the proteomic study, the changes of proteome profiles trifuged at 1,500 ×g for 10-15 sec. IMR medium was re- at early stage of cell rebuilding was not observed. moved from the tubes and precipitated protoplasts were Ultrastructure of rebuilding cell was observed over a frozen in liquid nitrogen and stored at -75°C. We avoided time course to determine exact timing of cell membrane mechanical disruption of the protoplasts during harvest- regeneration and cell wall development in B. plumosa. ing and centrifugation procedures as much as possible. To study proteome profiles during the cell rebuilding To prepare samples for 2-DE, materials were washed process, we constructed 2-DE protein maps before and twice in ice-cold phosphate buffer saline (PBS) and lysed after collapse of its giant cell. Proteins which showed sig- in sample buffer, composed of 7 M urea (Sigma, St. Louis, nificant expression changes at each stage were chosen MO, USA), 2 M thiourea (Sigma), 4% (w/v) 3-(3-cholami- and characterized using matrix assisted laser desorption dopropy) dimethymmonio-1-propanesulfonate (CHAPS, ionization-time of flight mass spectrometry (MALDI-TOF Sigma), 1% (w/v) dithiothreitol (DTT, Sigma), 2% (v/v) MS). pharmalyte (Amersham Biosciences, Uppsala, Sweden), and 1 mM benzamidine (Sigma). Proteins were extracted for 1 h at room temperature with vortexing. After centrifugation at 15,000 ×g for 1 h at 15°C, insoluble material was discarded and soluble fraction was used for 2-DE. Protein loading was normalized by Bradford assay (Bradford et al. 1976). https://doi.org/10.4490/algae.2016.31.11.31 380 Klochkova et al. Cell-Rebuilding Process of Bryopsis plumosa

Two dimensional electrophoresis gel pieces with 50% aqueous acetonitrile. After concen-

tration, the peptide mixture was desalted using C18Zip- Immobilized pH gradient dry strips were equilibrated Tips (Millipore Co., Bedford, MA, USA), and peptides were for 12-16 h with 7 M urea, 2 M thiourea, containing 2% eluted in 1-5 µL of acetonitrile. An aliquot of this solution CHAPS, 1% DTT, and 1% pharmalyte, and were then load- was mixed with an equal volume of a saturated solution ed with 200 µg of sample. Isoelectric focusing was per- of α-cyano-4-hydroxycinnamic acid (Sigma) in 50% aque- formed at 20°C using a Multiphor II electrophoresis unit ous acetonitrile, and 1 µL of mixture was spotted onto a and EPS 3500 XL power supply (Amersham Biosciences) target plate. following manufacturer’s instruction. The voltage was lin- early increased from 150 to 3,500 V during 3 h for sample Mass spectrometry determination and similarity entry followed by constant 3,500 V, with focusing com- searches plete after 96 kVh. Prior to the second dimension, strips were incubated for 10 min in equilibration buffer (50 mM Protein analysis was performed using an Ettan MAL- Tris⋅Cl buffer of pH 6.8, 6 M urea, 2% sodium dodecyl sul- DI-TOF MS (Amersham Biosciences). Peptides were fate [SDS], and 30% glycerol; Sigma), first time with 1% evaporated with a N2 laser at 337 nm using a delayed ex- DTT and second time with 2.5% iodoacetamide (Sigma). traction approach. They were accelerated with 20 kV in- Equilibrated strips were inserted onto sodium dodecyl jection pulse for time-of-flight analysis. Each spectrum sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was the cumulative average of 300 laser shots. Spectra gels (20-24 cm, 10-16%). SDS-PAGE was performed using were calibrated with trypsin auto-digestion ion peak m/z Hoefer DALT 2-DE system (Amersham Biosciences) fol- (842.510, 2,211.1046) as internal standards. The search lowing manufacturer’s instruction. 2-DE gels were run at program ProFound, developed by the Rockefeller Univer- 20°C for 1.7 kVh. Thereafter, the gels were silver-stained sity (http://prowl.rockefeller.edu/prowl-cgi/profound. as described by Oakley et al. (1980), except that fixing exe) was used for protein identification by peptide mass and sensitization step with glutaraldehyde were omit- fingerprinting (Zhang and Chait 2000). These procedures ted. Minimum three copies using total proteins extracted were performed in Korea Basic Science Institute (KBSI, from different material were analyzed for the proteome Daejeon, Korea). Additional search was performed using profiles of each stage. publically available BLASTp (protein-protein BLAST) op- tion in the National Center for Biotechnology Informa- Image analysis tion (NCBI) database (NCBI 2016).

Quantitative analysis of digital images was carried out Microscopy using the PDQuest software (version 7.0; BioRad, Hercu- les, CA, USA) according to the manufacturer’s protocols. For time-lapse video microscopy, protoplasts were Quantity of each spot was normalized by total valid spot placed on a glass slide and a coverslip was lowered and intensity. Protein spots were selected for the significant sealed with VALAP (1 : 1 : 1; vaseline / lanolin / paraffin) expression variation deviated over two fold in its expres- melted on a hot plate at 70°C. The slide preparations were sion level, compared with control or normal sample. examined and recorded using a digital imaging time- lapse recorder (AllBT, Daejeon, Korea) mounted on the Enzymatic digestion of protein in-gel microscope. For transmission electron microscopy (TEM), proto- Protein spots were enzymatically digested in-gel in a plasts were prepared on 2% agar. The protoplasts partially manner similar to that described by Shevchenko et al. embedded on agar gels were fixed in specially developed (1996), using modified porcine trypsin (Promega, Madi- fixative solution (1% para-formaldehyde, 2.5% glutaral- son, WI, USA). Gel pieces were washed with 50% aceto- dehyde, 2.5% dimethyl sulfoxide, 10% sucrose, and 0.05% nitrile (Sigma) to remove SDS, salt and stain, dried to re- CaCl2 in 0.5 M cacodylate buffer) for 2 h. Samples were move the solvent and then rehydrated with trypsin (8-10 then rinsed with the same buffer and post-fixed with %1 ng µL-1) and incubated for 8-10 h at 37°C. The proteolytic osmium tetroxide at 4°C for 2 h. Thereafter, they were reaction was terminated by addition of 5 µL of 0.5% triflu- rinsed out with PBS buffer and were dehydrated in a grad- oroacetic acid (Sigma). Tryptic peptides were recovered ed ethanol series with 10% increments (each step was 10 by combining the aqueous phase from several extracted min), embedded in Spurr’s epoxy resin and polymerized

381 http://e-algae.org Algae 2016, 31(4): 379-390

A B C D

E F G H

I J K L

Fig. 1. Time-lapse video microscopic observations on Bryopsis plumosa protoplast during the first 3 h after formation from the extruded protoplasm in seawater. (A-L) Primary polysaccharide envelope can break and re-form 2-3 times during the first 2 h (arrows). Scale bar represents: 10 μm. overnight at 72°C oven (Polysciences Inc., Warrington, PA, tions with time-lapse video microscopy showed that not USA). Resin-embedded samples were sectioned with a only the primary envelope but also the newly generated diamond knife, and thin sections stained with uranyl ac- plasma membrane collapsed at least 2-3 (6) times during etate for 30 min and 2% (w/v) lead citrate for 10 min were the first 12 h after protoplast formation Fig( . 1E-L). Each viewed and photographed on a JEM-1011 transmission time it reformed the membrane again; however, activity electron microscope (JEOL, Tokyo, Japan). to reform slowed down each time, and some protoplasts could not reform anymore and degenerated. TEM showed that some discontinuous “patches” of a RESULTS membrane began to appear on the surface of protoplasmic mass at 3 h after its formation (Fig. 2A), and complete When the protoplasm was extruded from the cut-open plasma membrane was formed by 6-12 h after the wound- B. plumosa thalli into seawater, the cell organelles agglu- ing (Fig. 2B & C). Previously, Calcofluor White staining tinated into small protoplasmic masses at first. During showed that accumulation of cell wall material started the next 10-20 min each protoplasmic mass contracted, at 6-8 h after protoplast formation (Kim et al. 2001), but became spherical and enclosed by a primary polysac- distinctive ultrastructure of cell wall was first observed at charide envelope, forming a compact protoplast-like 24 h after the wounding. An accumulation of Golgi bodies structure (Fig. 1A-D). This primary protoplast repeatedly and numerous vesicles was observed along the periph- ruptured and regenerated the structure during the first eral region of the protoplast at 20-24 h after the wound- 3 h with active rotation of the cell organelles. Observa- ing. The deposited cell-wall material became more distin-

https://doi.org/10.4490/algae.2016.31.11.31 382 Klochkova et al. Cell-Rebuilding Process of Bryopsis plumosa

A B

C D

E F

Fig. 2. Transmission electron micrographs of Bryopsis plumosa protoplasts showing changes of their outer surface structure per different times after formation. (A) During the first 1 h after formation, protoplast is not covered with a lipid-based cell membrane and it is enclosed with an amorphous polysaccharide envelope. By 3 h after protoplast formation, some discontinuous membranous “patches” began to appear on its surface, which are interrupted with areas without membrane (arrows). (B & C) The plasma membrane is fully formed in the period between the 3rd and 12 h of the protoplast's life (arrows). (D) A thin cell wall is visible on the surface of protoplast at 24 h after its formation (arrows). (E) One- layered cell wall at 48 h after protoplast formation in seawater (arrow). (F) Thick three-layered cell wall at 72 h after protoplast formation. Three distinct layers are shown with arrows and numbers. c, chloroplast; cw, cell wall; ER, endoplasmic reticulum; n, nucleus. Scale bars represent: A & D, 2 μm; B, C, E & F, 1 μm.

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Fig. 3. Proteomic maps of the vegetative plants of Bryopsis plumosa (0 h) and protoplasts at 3, 12, 24, and 48 h after formation. Arrows and numerals point to some differentially expressed proteins (see Table 1 for details). IEF, isoelectric focusing.

https://doi.org/10.4490/algae.2016.31.11.31 384 Klochkova et al. Cell-Rebuilding Process of Bryopsis plumosa

Fig. 4. Enlarged images, showing comparison of intensities in the expression of some representative differentially expressed proteins detected on the proteomic maps of the vegetative plants of Bryopsis plumosa (0 h) and protoplasts at 3, 12, 24, and 48 h after formation. (A) Protein spot

2211 identified as chaperone protein pmfD precursor. (B) Protein spot 6617 (unidentified). Mr and pI value of that protein spot were 67.6 and pH 5.5, respectively. (C) Protein spots 8110 and 8209 identified as alanine racemase and glycoprotein UL9, respectively.

guishable on ultrathin sections, and a layer of materials protein spots in control (0 h), but only 259 proteins were with numerous vesicles was clearly visible on the surface detected throughout the cell rebuilding process (3-48 of the developing protoplast (Fig. 2D). After 48 h, multi- h). Quantitative analysis showed that 59.8% of the pro- layered thick cell wall (2-2.5 µm) developed. Each layer of teins that appeared in control were significantly (<3 fold the cell wall showed different electron density suggesting change) down-regulated and 10.8% of the proteins were that the developing cell deposited different types of cell- up-regulated in 24-h-old protoplasts. About 70-80% of wall materials during the rebuilding process (Fig. 2E & F). proteins detected at 48 h were not detected at all in the initial stage (0-12 h). As a result, the proteome profile at Protein profile in vegetative plants, protoplasts, 48 h after the wounding was almost completely different and newly formed cells from those of control and early stage of protoplast formation (Fig. 3). The number of detected proteins increased Proteome profiles of B. plumosa changed dramati- over time course; total number of detected proteins at 24 cally in short time intervals during the cell rebuilding and 48 h was 1,860 and 1,915 spots, respectively; 25-36% process (Fig. 3). Most proteins that appeared in control increase compared to those in control (1,420 spots) and disappeared during the process and many new proteins in 3 h (1,234 spots). These results (dramatic change in specifically appeared at each stage of protoplast forma- proteome profile and increase of detected protein spots tion (Fig. 4). The silver-stained gels showed 1,350-1,420 in late stage of protoplast formation) were repeated three

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times using different materials. It is noteworthy that the difficulties in fixation of this fragile structure (Pak et al. protein spots strongly expressed in early stage disap- 1991). We could stably fix the structure of regenerating peared over time course and the proteome profile of protoplast using a fixative containing 10% sucrose and 48-h-old protoplasts was filled with numerous weak pro- agar-embedding method, which enabled us to determine tein spots (Fig. 3). the exact time of cell membrane and cell wall develop- Internal amino acid sequence was obtained from 49 ment. The protoplasmic mass was surrounded with a chosen proteins which were specifically up-regulated at continuous cell membrane at 6 h after the wounding, but each cell-rebuilding stage and 29 proteins were annotat- the collapse and rebuilding of developing protoplast was ed (Table 1). Only 21 proteins were annotated with >80% observed until 12 h after the wounding, suggesting that similarity, and 6 proteins were annotated the same with there are mechanisms holding the cell organelles togeth- two approaches, ProFound program and BLASTp. Most er until whole cell-rebuilding process is complete with identified proteins belonged to catalytic enzymes in- the development of cell wall. volved in ordinary cell metabolism (Table 1). In general, A lectin involved in the aggregation of the extruded cell the number of analyzed proteins was too small to under- organelles in seawater and initial formation of the proto- stand any proteome profiles in detail. plasmic mass was isolated from B. plumosa and named as bryohealin (Kim et al. 2006, Yoon et al. 2008). This lectin is universal not only for other populations of B. plumosa DISCUSSION (NCBI accession No. BAI43481), but also for other species of Bryopsis, as it was found in B. hypnoides Lamouroux Cell rebuilding process of Bryopsis plumosa is com- (Xu et al. 2012) and B. maxima Okamura ex Segawa (NCBI posed of several distinctive events, and development of accession No. BAI94586). Bryohealin contains an anti- plasma membrane and cell wall are the most crucial steps biotic domain and its function may be protection of the to complete the process (Kim et al. 2001). The exact time newly generated protoplasts from bacterial contamina- of each developmental step was determined for the first tion (Klochkova et al. 2005, Yoon et al. 2008). Several addi- time using ultrastructural analysis. Observations with tional lectins involved in the aggregation of expelled cell time-lapse video microscopy showed that the rebuilding organelles were isolated from B. plumosa and confirmed cell may undergo the collapse of cell membrane at least that a lectin-carbohydrate complementary system medi- 2-3 (6) times during the first 12 h. A comparative pro- ates agglutination of extruded cell organelles (Han et al. teomic experiment was designed based on these times, 2010, 2011, 2012, Jung et al. 2010). Niu et al. (2009) sug- and the results revealed a striking shift of proteome pro- gested that the aggregation of cell organelles might be file before and after the development of cell membrane. proton-gradient dependent, because it was inhibited by About 60% of proteins detected in control vegetative cell nigericin. Xu et al. (2012) reported that a 43 kDa lectin disappeared or significantly reduced at late stage of cell appeared at 3 h after protoplasm extrusion into seawater rebuilding, and about 70-80% (over 1,000 spots) of pro- and then disappeared at 6 h from western blotting analy- teins detected at 48 h were newly appeared. Such a mas- sis. These data are confusing, because the amino acid se- sive proteomic shift is very rare in any organisms. The quence of their “novel” lectin from B. hypnoides is 100% male and female gametophytes of red algae, Bostrychia identical to bryohealin from B. plumosa, clearly implying radicans and B. moritziana, show only 5-48 differential these are the same proteins. Bryohealin has the molecu- proteins out of 699-921 total protein spots (Kim et al. lar weight of 54 kDa and it usually shows little migration 2008). The proteome shift observed during cell-rebuild- change on 2-DE gel (Kim et al. 2006, Yoon et al. 2008). It is ing of B. plumosa is similar to that reported in germinat- more likely that the regenerating protoplast is using lec- ing seeds of higher plants which is driven by an epigen- tins which were produced before the cell rupture to hold etic modification Gallardo( et al. 2001). cell organelles together until cell wall development. Time-lapse video microscopy showed that there was The ultrastructure of cell wall on rebuilt protoplast of an active relocation of cell organelles inside protoplas- B. plumosa was observed for the first time. Golgi bodies mic mass surrounded with primary envelop. This active with numerous vesicles were observed at the peripheral movement of protoplasmic mass often caused the col- region of the rebuilding cell at 24 h after the wounding. lapse of developing protoplasts at early stage of cell re- Cell wall showed distinctive layers with different electron building process. There are very few ultrastructural stud- density at 48-72 h after the wounding. Previous studies ies on cell rebuilding process of B. plumosa because of showed that the newly built cell showed polarity at 48 h

https://doi.org/10.4490/algae.2016.31.11.31 386 Klochkova et al. Cell-Rebuilding Process of Bryopsis plumosa - - - - program ProFound program dase ted-1 like 2 (KNAP2) lytic subunit precursor / thiamine monophos phate kinase domain subunit iron-sulfur according to search with to search according Similar protein candidate protein Similar Putative ascorbate peroxi Putative knot protein Homeobox cata H+-ATPase Vacuolar RNA helicase DEAD box EF-Tu Chloroplast orf1AF466203_3-putative pmfD protein Chaperone Thymidine phosphorylase Unknown Unknown FOG: PAS/PAC COG2202: Unknown B6-F complex Cytochrome Unknown UL9 Glycoprotein ) % identity ( NCBI / protein NCBI / protein Accession No. in No. Accession ADF56044 / 92 XP009337133 / 100 WP021757877 / 91 KMP09324 / 75 - XP_008624170 / 75 / 100 ALT68774 WP_029096030 / 100 WP_018729200 / 100 WP_035088285 / 80 WP_008328067 / 83 XP_007749571 / 100 XP015818526 / 83 WP_062042390 / 82 KLJ10955 / 83 WP_035792075 / 100 WP_044239244 / 88 WP_015801651 / 88 KZZ96035 / 83 - - - - b search in NCBI search according to BLASTp according knotted-1-like 1 CIRG_09494 YYE_02288 autotrans membrane domain- porter barrel ATP- containing protein, dependent DNA helicase PcrA hypothetical protein A1O5_10807 with throm loproteinase bospondin motifs 14 isoform X1 ine O-methyltransferase permeasetransporter Similar protein candidate protein Similar Ascorbate peroxidase protein Homeobox Aminopeptidase protein Hypothetical Unknown protein Hypothetical outer protein, Adhesin-like / protein Hypothetical protein Hypothetical A disintegrin and metal protein Hypothetical Protein-S-isoprenylcyste protein Hypothetical ABC Alpha-galactosidase, Oxidoreductase 0 0 0 a 35.4 88.4 44.2 6.7 6.4 35.4 61.7 2.1 58.7 24.1 48 h 48.65 10 0 33.4 69.1 17.6 67.8 33.1 82.4 60.9 28.7 53.5 37.2 5.6 53.7 24 h 10 58 39.9 25.1 28.2 77.9 35.7 95.3 51.7 81.9 95.7 73.9 47.7 92.9 54.3 12 h 87 96 0 3 h 95.9 5.1 30 100 100 100 100 100 100 100 100 100 100 100 Relative intensity of protein spot intensity of protein Relative 100 100 100 100 22 33 0 h 50.6 67.8 46.7 61.1 32.5 45.6 30.3 46.9 2.9 sequence Bryopsis plumosa Internal amino acid Internal LPDATKGLDHLR SSGTSRETSK GAGYPNTGNTNA VPETTNNNPK NSGHGWGWTNNCNPN NMSEENNPYNR NGIAEFN / FYAITTGAGWR ANWVAVYDGR EPEPVYW AATQNPSEPGNR GGCPEFFWVGSPY VPYSPEGTETNGS SIYWNER NAPDNFTR NPGAPDASNENA weight weight (kDa) / (kDa) 48.6 / 5.4 29.9 / 5.8 80.5 / 5.4 54.0 / 5.0 54.2 / 5.0 29.3 / 5.3 38.2 / 4.7 17.2 / 5.1 25.4 / 5.4 38.9 / 5.3 31.8 / 5.3 15.5 / 6.1 17.3 / 7.0 33.9 / 6.0 33.5 / 6.4 Molecular Molecular isoelectric point (pH) Expression level and molecular characteristics of some differentially displayed proteins extracted from 2-DE gels of the vegetative plants (0 h and control), protoplasts (3, 12, and 24 h), protoplasts control), (0 h and plants vegetative extracted 2-DE gels of the from proteins displayed and molecular characteristics of some differentially level Expression 1. No. spot Protein Protein 6414 7114 6712 4401 4411 6109 2211 5004 6111 6201 6207 8001 8011 8203 8209 Table (48 h) of cells and newly formed

387 http://e-algae.org Algae 2016, 31(4): 379-390 - - - - - program ProFound program doreductase uridylyltransferase foldase) (lipase activator protein) subunit plex iron-sulfur lase-1,5-bisphosphate / oxygenase carboxylase tein M related thase B according to search with to search according Similar protein candidate protein Similar Unknown synthetase Prolyl-tRNA Unknown Unknown oxi Pyruvate-flavodoxin Galactose-1-phosphate (lipase Lipase chaperone Alanine racemase B6-F com Cytochrome Elongation factor Tu subunit of ribuLarge Ornithine decarboxylase II sporulation pro Stage tRNA pseudouridine syn ) % identity ( NCBI / protein NCBI / protein Accession No. in No. Accession AAW45864 / 89 AAW45864 WP_026246990 / 100 WP_056521873 / 100 XP_012266911 / 100 SAK75260 / 82 XP_005649261 / 100 KID84064 / 90 / 82 ABV48732 KIR35501 / 100 XP_003063104 / 91 XP_009350076 / 100 P26958 / 100 OBQ92157 / 100 WP_010867670 / 100 NP_587861 / 83 - - - b search in NCBI search according to BLASTp according porter protein uncharacterized isoform LOC105692344 X1 containing protein subunit, plex iron-sulfur precursor chloroplast chloroplastic-like / sphate carboxylase subunit large oxygenase (chloroplast) Similar protein candidate protein Similar Monosaccharide trans Monosaccharide Amidinotransferase / protein Hypothetical Chitin binding domain- NAD(P)-binding protein synthase Beta-ketoacyl factor C6 transcription protein Cytoplasmic b6/f com Cytochrome Elongation factor G-2, Ribulose-1,5-bispho Ornithine decarboxylase protein Sporulation Tyrosyl-DNA Tdp1 phosphodiesterase a 52.6 53.2 40.5 55.6 23.9 26.3 44.1 60.1 78.1 89.7 48 h 0 0 85 100 46.7 74.6 73.6 15.1 37.6 45.3 28.5 24 h 67 58 50 0 100 100 100 100 100 100 100 100 100 100 100 100 100 12 h 6.4 11.1 38.2 88.1 3 h 0.5 96.3 88.5 68.6 91.8 92.7 91.8 68.7 63.8 71.7 2.1 36.1 0 0 Relative intensity of protein spot intensity of protein Relative 0 h 3.3 4.7 6.6 90.8 36.6 27.4 32.7 35.8 88.9 61.4 54.1 44.9 0 25 sequence Internal amino acid Internal YCPETAGTPHR EGHSIAGAHP / NPCIENI LGFVAAEAR VSPILSGNYQF IPTFGGNGFVGSR THPTGAFTGTGGNPP SGWFGPNAVWNR GGCPEFFWTFPTM VLFSPWTETDF GITISTSHVEYDTPTR EVTLGFVDLMR GMVGNAGVIKSEVVLISK LFMLIFLNNTR LTTSSVGEYGNPR weight weight (kDa) / (kDa) 39.5 / 4.5 31.2 / 4.6 41.3 / 5.1 26.8 / 5.3 30.3 / 5.4 35.0 / 5.3 31.9 / 5.4 29.9 / 6.5 15.7 / 5.4 52.7 / 4.8 12.6 / 4.5 50.9 / 4.7 26.3 / 5.6 Molecular Molecular isoelectric point (pH) Continued 101.9 / 4.5

1. No. spot Protein Protein 2203 2209 4314 6102 6112 6208 6216 8110 6009 3406 2005 2416 2803 7105 Proteins that appeared similar in both search options are shown with highlighting. shown options are similar in both search appeared that Proteins The time interval when particular proteins are the most up-regulated is shown with highlighting. with highlighting. time interval is shown The the most up-regulated when particular are proteins Table and BLASTp. program ProFound approaches, the same with two annotated which were proteins, indicate Bold letters two-dimensional2-DE, polyacrylamide gel electrophoresis. a b

https://doi.org/10.4490/algae.2016.31.11.31 388 Klochkova et al. Cell-Rebuilding Process of Bryopsis plumosa

after the wounding (Kim et al. 2001, Kim and Klochkova ACKNOWLEDGEMENTS 2004). It may be a coincidence, but further investigation using this cell-rebuilding model system may reveal the re- This work was supported by a National Research Foun- lationship between the development of cell wall and the dation of Korea Grant (NRF-2015M1A5A1041804) funded occurrence of polarity in developing cell. to GHK. This research was also supported by Golden Our results showed the “pros and cons” of proteomic Seed Project, Ministry of Agriculture, Food and Rural Af- methods. The ‘pros’ of proteomic methods are simple and fairs, and a grant from Marine Biotechnology Program fast profiling of gene expression on a 2-DE gel. The general (PJT200620, genome analysis of marine organisms and change of proteome profile over time course was clearly development of functional applications) funded by Min- demonstrated on each 2-DE gel and the pattern was easy istry of Oceans and Fisheries, Korea. to understand at a glance. However, detailed analysis on differentially displayed proteins was disappointing. The ‘cons’ of proteomic methods are the difficulty in getting REFERENCES amino acid sequence from each protein spots (Kim et al. 2008). Although proteomics is a powerful tool to study the Aitken, A. 1996. 14-3-3 and its possible role in co-ordinating differentially expressed proteins involved in various bio- multiple signalling pathways. Trends Cell Biol. 6:341- logical processes, it offers very limited access to the ac- 347. tual characteristics of weakly expressed proteins (Pandey Bottalico, A., Felicini, G. P., Delle Foglie, C. I. & Perrone, C. and Mann 2000, Tyers and Mann 2003). Less than 5% of 2008. 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F., Hein, B., represent the specific gene regulation at each stage of cell Wolf, H. & Celis, A. 1999a. Proteomics and immunohis- rebuilding process. It seems not worthy to discuss possi- tochemistry define some of the steps involved in the ble role of the identified proteins in protoplast formation squamous differentiation of the bladder transitional except for two chaperones that were up-regulated at the epithelium: a novel strategy for identifying metaplastic early stage of protoplast formation. The common feature lesions. Cancer Res. 59:3003-3009. of lipase chaperone, also called as lipase foldase or lipase Celis, J. E., Ostergaard, M., Rasmussen, H. H., Gromov, P., activator protein, is to assist in the protein folding. They Gromova, I., Varmark, H., Palsdottir, H., Magnusson, N., are also involved in the translocation of proteins across Andersen, I., Basse, B., Lauridsen, J. B., Ratz, G., Wolf, H., the membranes (Smith et al. 1998, Feldman and Frydman Orntoft, T. 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