Botanica Marina 2015; 58(3): 229–238 Chikako Nagasato*, Makoto Terauchi, Atsuko Tanaka and Taizo Motomura Development and function of plasmodesmata in zygotes of Fucus distichus Abstract: Brown algae have plasmodesmata, tiny tubu- Introduction lar cytoplasmic channels connecting adjacent cells. The lumen of plasmodesmata is 10–20 nm wide, and it takes a Multicellular organisms such as animals, fungi, land simple form, without a desmotubule (the inner membrane plants, and brown algae have specific cellular connections. structure consisting of endoplasmic reticulum in the plas- These structures connect the cytoplasm of adjacent cells modesmata of green plants). In this study, we analyzed and provide a route for cell-cell communication. Green the ultrastructure and distribution of plasmodesmata plants, including land plants and certain species of green during development of Fucus distichus zygotes. The first algae (reviewed in Robards and Lucas 1990, Raven 1997), cytokinesis of zygotes in brown algae is not accompanied and brown algae (Bisalputra 1966) possess plasmodes- by plasmodesmata formation. As the germlings develop, mata. These are cytoplasmic canals that pass through the plasmodesmata are found in all septal cell walls, includ- septal cell wall and connect adjacent cells. Plasmodes- ing the first cell division plane. Plasmodesmata are formed mata in green plants and brown algae share similar char- de novo on the existing cell wall. Pit fields, which are clus- acteristics; however, plasmodesmata in brown algae lack ters of plasmodesmata, were observed in germlings with a desmotubule, a tubular strand of connecting endoplas- differentiated cell layers. Apart from the normal plas- mic reticulum (ER) that penetrates the plasmodesmata modesmata, these pit fields had branched plasmodes- of land plants (Terauchi et al. 2012). The diameter of the mata that appeared to arise from the lateral preexisting plasmodesmata in land plants is 20–50 nm, but in brown ones. Fluorescent tracers with different molecular sizes algae, it is 10–20 nm. were microinjected to examine the size exclusion limit Intercellular connections in land plants are divided of molecules for transit through the plasmodesmata. into primary and secondary plasmodesmata based on the Fluorescein isothiocyanate (FITC)-dextran of 3 kDa size time of their formation (reviewed in Ehlers and Kollmann was spread over the germlings, and 10 kDa FITC-dextran 2001, Burch-Smith et al. 2011). Primary plasmodesmata was tracked only in the rhizoid. The size exclusion limit are formed during cytokinesis as simple straight forms, was < 10 kDa for the thallus but < 40 kDa for the rhizoid. whereas secondary plasmodesmata arise in an existing cell wall, irrespective of cytokinesis. Formation of the primary Keywords: brown algae; cell differentiation; microinjec- plasmodesmata in land plants has been well documented tion; pit field; plasmodesmata. (Hepler 1982). The ER is trapped in the growing cell plate, eventually forming the plasmodesmata. Secondary plas- DOI 10.1515/bot-2014-0082 modesmata have simple, branched, or twined forms Received 6 November, 2014; accepted 29 April, 2015 (Ehlers and Kollmann 2001, Burch-Smith et al. 2011). The occurrence of these structures is divided into “de novo” and “modified” types. De novo secondary plasmodesmata are simple structures indistinguishable from the primary plasmodesmata, and thus, their formation is difficult to monitor. Modified secondary plasmodesmata have *Corresponding author: Chikako Nagasato, Muroran Marine branching forms of Y-, V-, X-, and H-shapes or a twined Station, Field Science Center for Northern Biosphere, Hokkaido form (Ehlers and Kollmann 2001, Burch-Smith et al. 2011). University, Muroran 051-0013, Japan, Branched plasmodesmata are formed by lateral branching e-mail: [email protected] from the preexisting simple plasmodesmata (Burch-Smith Makoto Terauchi: Research Center for Inland Seas, Kobe University, et al. 2011), whereas twined plasmodesmata are formed by Kobe 657-8501, Japan Atsuko Tanaka and Taizo Motomura: Muroran Marine Station, insertion of new plasmodesmata into existing ones during Field Science Center for Northern Biosphere, Hokkaido University, cell wall expansion (Faulkner et al. 2008). Furthermore, Muroran 051-0013, Japan the H-shaped plasmodesmata are considered to be formed 230 C. Nagasato et al.: Plasmodesmata in Fucus by the development of a central cavity connecting twined are regulated by a divalent ion such as Ca2+ (Erwee plasmodesmata. In addition, branched plasmodesmata and Goodwin 1983) and by degeneration/synthesis of may form twined simple plasmodesmata intermediates callose deposited at the constricted neck region of the (Burch-Smith et al. 2011). The presence of secondary plas- plasmodesmata (Radford and White 2001, Zavaliev modesmata is correlated with growth, development, and et al. 2011, 2013). In brown algae, symplastic transport response to extracellular signals. has been elucidated using radioisotopes (Schmitz and In brown algae, a new septal cell wall is created by Lobban 1976, Amat and Srivastava 1985). However, the growth of a new cell partition membrane and deposition maximum size of molecules that can pass through plas- of cell wall components (Nagasato and Motomura 2002, modesmata and the regulation system for transport, Nagasato et al. 2010, 2014). Although plasmodesmata for- such as that described in land plants, remain unclear. To mation in brown algae has been observed in the cytoki- our knowledge, only one study has been conducted on nesis of vegetative cells, previous studies have shown the movement of the fluorescent tracer through the plas- that the first cytokinesis in zygotes of brown algae is not modesmata by microinjection in the fucoid zygotes of accompanied by the formation of plasmodesmata. During Fucus spiralis (Bouget et al. 1998), showing that 10 kDa cytokinesis in the apical meristem of Dictyota dichotoma, molecules could pass through the plasmodesmata in plasmodesmata formation is initiated by invaginations of Fucus germlings. the membranous sacs, transitional membranous struc- In this study, we observed the ultrastructure of Fucus tures in the formation of a new cell partition membrane distichus at each developmental stage, in order to resolve (Terauchi et al. 2012), which penetrate the membranous open questions about brown algal plasmodesmata, the sacs. The region of newly formed plasmodesmata during presence of secondary plasmodesmata, and the occur- cytokinesis later becomes a pit field (Terauchi et al. 2012). rence of pit fields. Moreover, to understand the function Recently, we reported that the complexity of body plan of plasmodesmata, we investigated the SEL of plasmodes- in brown algae influences the number and arrangement mata by microinjecting fluorescent tracers with different of plasmodesmata (Terauchi et al. 2015). The distribu- molecular sizes. tion of plasmodesmata was examined in brown algal species showing uniseriate and multiseriate filamen- tous thalli and complex multicellular thalli and was compared between sporophytes (complex multicellular Materials and methods thalli) and gametophytes (uniseriate filament) of Sac- charina japonica. The plasmodesmata in uniseriate and Culture multiseriate filamentous brown algae were found to be sparsely distributed, whereas species with complex mul- Mature thalli of the monoecious species Fucus distichus ticellular thallus had pit fields (Terauchi et al. 2015). This Linnaeus were collected at Oinaoshi, Muroran, Hok- pattern was also found in the alternation of generations kaido, Japan (42°30′N, 140°97′E), from April to June 2014. of S. japonica. It is expected that plasmodesmata prob- The receptacles were washed with filtered seawater and ably occur secondarily in the first cell division plane, and wiped with paper towels. They were placed in Petri dishes pit fields in species with complex multicellular thallus and kept overnight at 18°C under continuous light from are formed alongside cellular differentiation. However, fluorescent lamps at 30–40 μmol·m-2·s-1. Before the release in brown algae, the existence of secondary plasmodes- of eggs and sperm, the receptacles were transferred to a mata and the occurrence of pit fields during development cold, dark place for 2 h. The release of eggs and sperm in the complex multicellular species have not been con- and fertilization were performed as described in previ- firmed until now. ous studies (Abe 1970, Wakana and Abe 1992, Motomura In green plants, inorganic ions, metabolites, RNAs, 1994). Cooled seawater was poured into dishes containing and proteins are transported through plasmodesmata the receptacles to induce the release of gametes and ferti- (Lucas et al. 1995, McLean et al. 1997, Kim et al. 2005). The lization. The zygotes were set on a gold carrier (flat speci- size exclusion limit (SEL), which has been measured by men carrier, Leica Microsystems, Wetzlar, Germany) for microinjection of fluorescent tracers with different sizes high-pressure freezing or on a glass slide with a TaKaRa is 1–3 kDa, depends on the species and cell types (Erwee slide seal (Takara, Shiga, Japan) and incubated at 18°C and Goodwin 1983, Goodwin 1983, Terry and Robards with Provasoli’s enriched seawater (PES) medium (Prov- 1987, Kempers et al. 1993, Radford and White 2001, asoli 1968) containing 40 mg·l-1 chloramphenicol prior to 2011). Molecular movement and SEL in plasmodesmata microinjection. C. Nagasato et al.: Plasmodesmata in Fucus 231 High-pressure freezing