Protoplast Production from Sphacelaria Fusca (Sphacelariales, Phaeophyceae) Using Commercial Enzymes
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https://doi.org/10.15433/ksmb.2020.12.1.050 ISSN 2383-5400 (Online) Protoplast Production from Sphacelaria fusca (Sphacelariales, Phaeophyceae) Using Commercial Enzymes Jose Avila-Peltroche 1, Boo Yeon Won 2* 1Graduate student, Department of Life Science, Chosun University, Gwangju 61452, Republic of Korea 2Researcher, Department of Life Science, Chosun University, Gwangju 61452, Republic of Korea (Received 14 May 2020, Revised 3 June 2020, Accepted 3 June 2020) Abstract Sphacelaria is a filamentous brown algal genus that can be epibiotic on macroalgae, marine plants, and sea turtles. Its important role in benthic ecosystems, exposure to different stressors (e.g., grazing), and use as a model organism make Sphacelaria ideal for assessing physiological responses of organisms to environmental inputs. Single-cell RNA sequencing is a powerful new probe for understanding environmental responses of organisms at the molecular (transcriptome) level, capable of delineating gene regulation in different cell types. In the case of plants, this technique requires protoplasts (“naked” plant cells). The existing protoplast isolation protocols for Sphacelaria use non-commercial enzymes and are low-yielding. This study is the first to report the production of protoplasts from Sphacelaria fusca (Hudson) S.F. Gray, using a combination of commercial enzymes, chelation, and osmolarity treatment. A simple combination of commercial enzymes (cellulase Onozuka RS, alginate lyase, and driselase) with chelation pretreatment and an increased osmolarity (2512 mOsm/L H 2O) gave a protoplast yield of 15.08 ± 5.31 × 10 4 protoplasts/g fresh weight, with all the Sphacelaria cell types represented . Driselase had no crucial effect on the protoplast isolation. However, the increased osmolarity had a highly significant and positive effect on the protoplast isolation, and chelation pretreatment was essential for optimal protoplast yield. The protocol represents a significant step forward for studies on Sphacelaria by efficiently generating protoplasts suitable for cellular studies, including single-cell RNA sequencing and expression profiling. Keywords : Phaeophyceae , Commercial enzymes, Sphacelaria fusca , Protoplast isolation as the isopod Dynamene magnitorata [7], spider crab Leucippa pentagona [2], and the “key herbivore” The filamentous brown alga genus, Sphacelaria, is Diadema antillarum [8]. It is known that grazers can a common epiphyte on macroalgae and marine plants control the proliferation of epiphytic algae and thus they [1,2], as well as epizoic on sea turtles [3]. It provides help to avoid a decrease in the performance of the host food and habitat for animals, playing an important role [9]. Sphacelaria can be also be infected in the wild by in coastal benthic communities [4-6]. Sphacelaria repre- fungal parasites such as the chytrid Chytridium poly- sents an important component of the diet of grazers such siphonae [10]. Abiotic factors (e.g. intertidal elevation) * Corresponding author This is an open-access journal distributed under the terms of the Creative Phone: +82-62-230-7161 Fax: +82-62-230-7467 Commons Attribution Non-Commercial License E-mail: [email protected] (http://creativecommons.org/licenses/by-nc/4.0/ ) - 50 - can affect the distribution of Sphacelaria in its host, as it has been reported for other epiphytic filamentous Isolation and culture of the strain brown algae [11]. Besides Sphacelaria has been used Sphacelaria fusca (MBRB0073TC18209C1) was as a model organism for plant morphogenesis due to collected by hand from Sargassum muticum in Jindo its apical growth and ease of cultivation [12-14]. Thus, Island, Jeollanam-do, Korea, on July 27, 2017. Apical Sphacelaria is a suitable organism for exploring differ- filaments of S. fusca were cultured in 12 well plates ent stressors and environmental inputs, both in con- containing PES medium [29] under 14:10-h light/dark trolled and non-controlled conditions. photoperiod at 20°C with light intensity 40 μmol Transcriptome analysis can help to understand the mo- photons/m 2/s of blue LED (DyneBioCo.Korea). The lecular basis of physiological responses to environmental medium was renewed weekly. After 3 months , plants stressors [15]. Recently, single-cell RNA sequencing has were transferred to 100×40 mm Petri dishes and were emerged as a novel approach to measure transcriptome cultured under the same culture conditions. Two months with high resolution using different cell types of animal later, plants accumulated much biomass and were cells [16]. However, the presence of cell walls has hin- transferred into 1-L flat-bottomed round flasks filled with dered the application of this approach to plant cells [17]. 1-L PES medium under aeration. Light intensity was One way to overcome this problem is using protoplasts, 40-60 μmol photons/m 2/s of white fluorescent light. The which are plant cells whose cell wall has been removed air was sterilized using 0.22-μ m surfactant-free cellulose by enzymatic methods [18]. The high amounts of proto- acetate (SFCA) syringe filters (Corning,Germany). The plasts are more important than their regeneration ability medium was renewed every 2 weeks. in single-cell RNA sequencing [17,19]. Moreover, they represent an important biotechnological tool for ge- Identification of the culture strain nome-editing and gene-silencing technologies [20], as Taxonomic identification was performed using well as, for the production of useful secondary metabo- morphological characters [30] from cultures maintained lites, such as dictyopterenes from the seaweed in 60×15 mm Petri dishes without agitation. Dictyopteris prolifera [21]. Photomicrographs were taken using a Leica inverted In brown algae, protoplast isolation has been reported microscope (DMi8; Leica, Germany) equipped with a in 29 species [18,22-25]. To date, protoplasts from Leica DFC450C camera. Genomic DNA extraction, Sphacelaria have only been isolated in low amounts us- PCR amplification, DNA purification, and sequencing ing commercial and non-commercial enzymes for mi- were performed as previously described [31,32] using crotubule analysis during regeneration [26,27]. The pro- cultured samples. The plastid-encoded RuBisCo spacer toplast isolation becomes more expensive and time-con- region was amplified using the primer combinations G1 suming because these non-commercial enzymes need to and G2 [33]. The amplified region sequences were be extracted from marine herbivores or microorganisms compared to the GenBank nucleotide database using the [28]. Thus, protoplast isolation protocols using commer- BLAST program [34]. cial enzymes are important for the application of sin- gle-cell RNA sequencing in Sphacelaria . Protoplast isolation In this study, we report for the first time the protoplast The commercially available cell wall lytic enzymes isolation from Sphacelaria fusca using a simple mix of used for this study included cellulase Onozuka RS commercial enzymes. Also, we tested the effect of os- (Yakult Co. Ltd., Japan), alginate lyase, and driselase molarity and driselase inclusion on protoplast pro- from Basidiomycetes sp. (Sigma-Aldrich, USA). duction to determine the best conditions for this process. Different enzyme combinations and conditions are - 51 - shown in Table 1. measurements for each repetition. In protoplast preparations, cell types were identified by protoplast Table 1. Combinations and concentrations of enzyme mixtures for protoplast isolation from Sphacelaria fusca. size and the presence or absence of physodes [26]. Composition of enzyme Commercial mixtures Viability and cell wall removal enzymes A B C D The viability of protoplasts and cell wall removal was assessed by the red chlorophyll autofluorescence and Cellulase Onozuka RS 1 1 1 1 (%) staining with calcofluor white M2R (Sigma-Aldrich, Alginate lyase (U/mL) 4 4 4 4 Driselase (%) 1 - 1 - USA), respectively, as previously described [25]. Osmolarity 1X 1X 1.6X 1.6X 1X : 1570 mOsm/L H 2O; 1.6X: 2512 mOsm/LH 2O Portoplast regeneration Protoplasts were washed three times with enzymatic Protoplast isolation was carried out as previously solution by centrifugation for 10 min at 100× g. Cells described [35,36] with some modifications [25]. were dispensed into 2 mL of regeneration media (PES Briefly, approximately 100– 300 mg plants from 1-L with 285 mM NaCl and 5 mM CaCl 2) and cultured round flasks (3-day-old cultures) were incubated in a at 20°C in the dark and at initial protoplast density of 0.22-μ m filter-sterilized enzymatic solution (400 mM 9 x 10 3 protoplasts/mL plus antibiotic mix (50 mg/L NaCl, 130 mM MgCl 2·6H 2O, 22 mM MgSO 4, 160 mM penicillin G, 25 mg/L streptomycin and 5 mg/L KCl, 2 mM CaCl 2, and 10 mM MES; pH6; 1570 chloramphenicol). After 1 day in the dark, osmotic mOsm/L H 2O) containing cellulase Onozuka RS and pressure was reduced slowly by adding 400 µL of PES. alginate lyase, either with or without driselase, at 20°C Cultures were exposed to 1-2 μmol photons m 2/s with shaking at 70 rpm for 6h in the dark. The 14:10-h light/dark photoperiod. Osmotic pressure was osmolarity of the enzymatic solution was tested in two further reduced during the next 2 days by adding 800 levels: normal osmolarity (1X=1570 mOsm/L H 2O) and µL of PES each day. Cultures were finally exposed to 2 increased osmolarity (1.6X=2512 mOsm/L H 2O). 10 μmol photons m /s under the same photoperiod. The Osmolarity was increased by increasing the component medium was renewed every week thereafter. concentrations in the enzymatic solution keeping their same proportions. Chelation pre-treatment was Statistical analysis conducted with a calcium-chelating solution [665 mM To evaluate the effect of driselase addition and NaCl, 30 mM MgCl 2·6H 2O, 30 mM MgSO 4, 20 mM osmolarity on protoplast yield a generalized linear KCl, and 20 mM ethyleneglycol– bis(β– amino – model with a binomial negative error distribution ethylether)–N,N,N ′,N ′–tetraacetic acid tetrasodium salt (GLM.nb) was used as a traditional method for (EGTA Na– 4) as the calcium chelator; pH5.5] for 20 handling overdispersed data.