Cell Structure and Microtubule Organisation During Gametogenesis of Ulva Mutabilis Føyn (Chlorophyta)

Botanica Marina 2017; 60(2): 123–135

Open Access

Christos Katsaros, Anne Weiss, Ira Llangos, Ioannis Theodorou and Thomas Wichard* Cell structure and microtubule organisation during gametogenesis of Ulva mutabilis Føyn (Chlorophyta)

DOI 10.1515/bot-2016-0073 upon additional removal of a swarming inhibitor accumu- Received 15 July, 2016; accepted 18 November, 2016; online first lated in the growth medium during gametogenesis. 11 January, 2017 Keywords: cell differentiation; gametogenesis; green macro Abstract: Cell structure and microtubule organisation algae; immunofluorescence; microtubule cytoskeleton. during gametogenesis of the green alga Ulva mutabilis was studied using light microscopy, transmission electron microscopy (TEM) and tubulin immunofluorescence. Introduction Microtubules in vegetative cells are organised in parallel bundles traversing the cortical cytoplasm. During Ulva mutabilis Føyn has attracted the interest of phycolo- gametogenesis, induced blade cells are transformed to gists since its first isolation by Føyn (1958), who estab- gametangia, depending on the maturity of the algae and lished this species as a laboratory organism in 1952. Many the removal of regulatory sporulation inhibitors. This dif- studies were published during the 1960s and 1970s mainly ferentiation is accompanied by formation of a conical cell on the ultrastructure of the thallus, cell division and mor- projection (papilla) towards the exterior of the thallus. phogenesis (Løvlie 1964, 1968, Løvlie and Bråten 1968, Microtubules form a clear, basket-like configuration con- 1970, Hoxmark and Nordby 1974, Fjeld and Løvlie 1976). verging towards the conical tip, but not reaching it. The A detailed description of the thallus of the develop conical microtubule structure stops below the tip, leav- mental mutant “slender”, which is often used for ing a circular “opening”. Parallel to the above, the cell experimental studies because of its shorter developmen- wall of the tip is differentiated, forming a “cap”. Nuclear tal cycle, was given first by Løvlie and colleagues (Løvlie divisions start at this stage, finally forming the nuclei of 1964, 1968, Fjeld and Løvlie 1976) and has been further future gametes. Cytokinesis takes place by membrane fur- analysed and confirmed by Wichard and Oertel (2010). An rowing and vesicle fusion, giving rise to 16 oval-shaped important analysis of the vegetative cell cycle by radioac- gametes. The conical microtubule organisation is gradu- tive labelling with 14C-uracil confirmed that the cell cycles ally depolymerised, and a cortical, intensely fluoresc- were synchronous and governed by a circadian rhythm ing microtubule bundle is formed in each gamete. At (Stratmann et al. 1996). this stage, the cap at the conical cell wall projection is Regarding cultivation, sexual reproduction repre- removed and the exit pore opens. The biflagellate gametes sents a fundamental phase linked to the production of remain initially motionless, connected by thin cytoplas- gametes. Blade cells of U. mutabilis excrete regulatory mic bridges. Finally, they are released to the environment factors into their cell walls and the environment. Upon removal of the sporulation inhibitors, the gametogenesis of the thalli can be artificially and synchronously initiated *Corresponding author: Thomas Wichard, Friedrich Schiller (Stratmann et al. 1996, Wichard and Oertel 2010). This was University Jena, Institute for Inorganic and Analytical Chemistry, Lessingstr. 8, 07743 Jena, Germany; and Jena School for Microbial also observed in Ulva linza L. (Vesty et al. 2015) and Ulva Communication, 07743 Jena, Germany, lactuca L. (Wichard and Oertel 2010). e-mail: [email protected] As fragmentation of the thalli washes out the regula- Christos Katsaros, Ira Llangos and Ioannis Theodorou: National and tory sporulation inhibitors of gametogenesis and zoospor- Kapodistrian University of Athens, Department of Biology, Section of ogenesis, it triggered the propagation of Ulva (Stratmann Botany, Athens 157 84, Hellas (Greece) Anne Weiss: Friedrich Schiller University Jena, Institute for Inorganic et al. 1996) and this was confirmed by observation of the and Analytical Chemistry, Lessingstr. 8, 07743 Jena, Germany; and proliferation of Ulva prolifera O.F. Mueller during green Jena School for Microbial Communication, 07743 Jena, Germany tides (Gao et al. 2010).

©2017, Christos Katsaros et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. 124 C. Katsaros et al.: Gametogenesis of Ulva

Significant cell-biological changes have already were completely discharged within a few minutes been observed during gametogenesis using scanning (Wichard and Oertel 2010, Vesty et al. 2015). electron microscopy (SEM) (Bråten 1971, Wichard and Although there are a large number of publications on Oertel 2010) and, more recently, metabolic changes have the development, genetics and physiology of U. mutabilis, been observed by matrix assisted laser desorption/ioni- the cell structure and the cytoskeleton organisation during sation mass spectrometric imaging (R.W. Kessler, A.C. gametogenesis have not yet been studied in detail. The Crecelius, U.S. Schubert, and T. Wichard, unpublished). only available information derives from the original ultra- Around noon of the second day after induction of game- structural studies of fertilisation and zygote formation togenesis, when the blade cells were irreversibly commit- (Bråten 1971), of meiosis and centriole behaviour (Bråten ted to the differentiation of gametangia, the chloroplasts and Nordby 1973), and from light microscopy of zoosporo- appeared reoriented, and papillae started growing at genesis and gametogenesis of Ulva (Nordby 1974). There- the outer surface of the blade cells (Figure 1). The papil- fore, gametogenesis of Ulva is a very intriguing process for lae converted, finally, into “capped” pores, while all understanding the function and dynamic changes of the progametes matured into 16 fully differentiated biflag- cytoskeleton and its role in the mechanism of gametangia ellate gametes overnight. In the morning, the “caps” formation and the gamete release. were successively dropped and the open exit pores The main aim of the present study was, therefore, to became visible. Although most pores seemed to be open elucidate the mechanism of gametogenesis by examining at this time and the gametangia were exposed to light, the fine structural changes occurring during this process the mature gametes remained completely motionless with particular attention to the cell wall differentiation because of the presence of a swarming inhibitor (SWI) in and the microtubule cytoskeleton organisation under the medium. After removal of the SWI, the gametangia standardised culture conditions.

Figure 1: Time course of induced gametogenesis and discharge of gametangia from Ulva mutabilis in relation to the swarming inhibitor (SWI) synthesis and action (summary of new and previous results). Gametogenesis was induced in Ulva mutabilis [mutant slender sl-G(mt +)]. The “determination phase” and “differentiation phase” are defined as described by Stratmann et al. (1996). The “swarming phase” is the time period when gamete release can be induced by light or a medium change, or may occur later spontaneously. (A) Regular vegetative G1 cell cycle phase in which gametogenesis can be induced by the removal of sporulation inhibitors SI-1 and SI-2 from the medium. (B) Regular vegetative S phase in which the genome is replicated normally or, after induction of gametogenesis, the period of SWI synthesis and excretion. (C) Next G1 phase after induction of gametogenesis. (D) Next S phase after induction of gametogenesis and accumulation of starch granules. (E) Time of irreversible commitment to gametangium differentiation. (F) Period of progamete formation, chloroplast reorientation and papilla initiation. (G) Period of progamete multiplication to 16 cells, and papilla maturation. (H) Period of gamete and pore cap maturation. (I) Period when the exit pores are open and when gamete release can be induced by light and/or depletion of SWI in the medium. (J) Period when the gametangia become insensitive to SWI and gamete release may occur spontaneously and asynchronously. Figure is from Wichard and Oertel (2010) with permission of © Wiley 2010. Images show the transformation of blade cells into gametangia. Microscopic images for stages a, f, h and i are shown (scale bars: 25 μm). C. Katsaros et al.: Gametogenesis of Ulva 125

Materials and methods pH 7.4) for 1 h. The samples were then washed thoroughly with MTB and the latter was gradually replaced by phos- phate-buffered saline (PBS; 137 mmol l − 1 NaCl, 0.7 mmol Algal material and cultivation − 1 − 1 − 1 l KCl, 5.1 mmol l Na2HPO4, 1.7 mmol l KH2PO4, 0.01% NaN , pH 7.4, v : v). After washing, the samples were Haploid gametophytes from the fast-growing develop- 2 transferred to the enzyme digestion solution, i.e. PBS mental mutant “slender” (sl) of Ulva mutabilis Føyn containing 2% cellulysin (219466, Lot No. B19227, Calbi- (mating type mt +) were cultured. The sl-G(mt +) mutant ochem-Novabiochem Corporation, San Diego, CA, USA), used was a derivative of the original sl strain, selected in 5% hemicellulase (H2125-150KU, Lot. No SLBH6404V, the laboratory, which had lost its capability for spontane- Sigma-Aldrich), 1% driselase (D-8037, Lot No. 19H0784, ous diploidization during parthenogenetic propagation Sigma-Aldrich), 2% pectinase (P-9179, Lot No. 53H0684, (Hoxmark 1975, Løvlie and Bryhni 1978). Ulva mutabilis Sigma-Aldrich), 2% macerozyme (M8002.0001, Lot No. was cultivated in Ulva culture medium (Stratmann et al. 010572.03, Duchefa Biochemie, Harrlem, The Netherlands) 1996) in a 17 : 7 h light/dark regime at 18°C with an illumi- and 3% PMSF (a protease inhibitor). After washing with nation of 80–120 μmol photons m − 2 s − 1 (50% GroLux, 50% PBS, the thallus pieces were gently squashed on cover- daylight fluorescent tubes; OSRAM, München, Germany) slips covered with poly-L-lysin. The samples were left to and no additional aeration. Further information about the dry and extraction of the pigments by Triton X-100 4% for materials used, the culture conditions and the prepara- 45 min at room temperature followed. After washing with tion of the seed stock can be found in Wichard and Oertel PBS, incubation with the first antibody YOL 1/34 (Serotec, (2010). All chemicals were purchased from Sigma-Aldrich Puchheim, Germany), diluted 1 : 100 in PBS with 1%, (v : v) (Taufkirchen, Germany) or Merck (Darmstadt, Germany). bovine serum albumin (BSA), continued overnight at room temperature. Washing again with PBS was followed by incubation with the secondary antibody anti-rat IgG FITC- Induction of gametogenesis conjugated (Sigma-Aldrich), diluted 1 : 100 in PBS-BSA for 2 h at 37°C in a dark and humid environment. The samples Intact mature thalli of the naturally occurring develop- were then washed with PBS and treated for DNA staining mental mutant “slender” of Ulva mutabilis were washed with Hoechst 33258 (Molecular Probes, Eugene, OR, USA) for 15 min with distilled water at noon and minced manu- in a final concentration of 5 μg ml − 1 in PBS for 10 min at ally into 3–5 mm fragments using an herb chopper (Zyliss, room temperature (Katsaros and Galatis 1992). Finally, the Zürich, Switzerland) prior to washing and distribution in cover-slips were mounted on slides with a drop of anti-fade fresh medium. The gametangia were mature in the morning substance (0.0024 g p-phenylen-diamine, 1 ml glycerol and of the third day after this medium change, and swarming 0.5 ml PBS). of the gametes was induced by an additional medium change and application of light. Thallus fragments were collected directly before and every 24 h after, induction of Transmission electron microscopy gametogenesis for tubulin immunofluorescence staining, TEM and light microscopy. The fragments were inspected In order to study the cell structure at different stages of under a microscope to determine the status of gametogen- gametogenesis, samples were taken at different times esis (Figure 1). The following protocols were used. after the induction of gametogenesis, fixed primarily with 2% glutaraldehyde and 2% formalin (v : v; Merck) in 0.1 m cacodylate buffer (Sigma-Aldrich) and then post- Tubulin immunofluorescence fixed with 2% osmium tetroxide (v : v; Sigma-Aldrich). The samples then underwent electron microscopy according The indirect immunofluorescence method was applied for to established protocols (Løvlie and Bråten 1970). the localization of tubulin, following a modified protocol described by Katsaros and Galatis (1992), i.e. small thallus pieces were fixed in 4% paraformaldehyde (PFA) and 0.125% glutaraldehyde in microtubule stabilising buffer Microscopic observation (MTB; v : v; 50 mmol l − 1 PIPES, 5 mmol l − 1 ethyleneglycol- bis(aminoethyl ether)-tetra-acetic acid: EGTA, 5 mmol l − 1 The immunofluorescence samples were examined under − 1 MgSO4·7H2O, 25 mmol l KCl, 4% NaCl, 2.5% polyvinyl pyr- epifluorescence with a Zeiss Axioplan™ microscope (Carl rolidone: PVP 25, v : v, 1 mmol l − 1 DL-dithiothreitol: DTT, Zeiss, Oberkochen, Germany). Pictures were recorded 126 C. Katsaros et al.: Gametogenesis of Ulva with AxioCam Mrc 5 (Carl Zeiss) using the AxioVision™ layers (Figures 3 and 4): the internal one is the wall of software. In order to ensure the accuracy of the results, each cell “itself”. This internal part consisted of a few at least 50 cells with the same microtubule cytoskeleton thin parallel bundles of fibrillar material (probably cel- were examined for each stage of gametogenesis. In par- lulose microfibrils) embedded in a large amount of amor- allel, gametogenesis was observed in living material by phous material. A second multilayered wall covered all the same Zeiss Axioplan™ microscope described above the cells externally. This external wall part consists of one or under a Leica DMIL LED microscope (Leica, Solms, electron-dense external thin layer and a number of other Germany) equipped with a digital camera DS-Fi2 (Nikon, internal layers, which bore thicker and electron-dense Düsseldorf, Germany). Thin sections were examined fibrillar bundles (Figure 4). under a Philips EM 300 transmission electron microscope. The microtubule cytoskeleton at this stage consisted of more or less parallel microtubule bundles arranged mainly in the cortical cytoplasm parallel to the plasma- Results lemma (Figure 9).

Interphase somatic cells Early stages of gametangial formation

The thallus appeared in cross-section to consist of two The first sign of the transformation of somatic cells to cell layers which separated easily forming a monolayer gametangia was observed 48 h after the induction of (Figure 2). The cells were orthogonal in shape with gametogenesis by the formation of a slight projection the dimensions 16–30 μm length and 8–18 μm width (papilla) towards the outside of the thallus. The cells (Figure 3). The undifferentiated cells of the develop- appeared polarised with most of the cytoplasm and the ing thallus were characterised by a more or less central chloroplast located at the tip and the vacuoles at the base, nucleus and a large cortical chloroplast with an obvious while the nucleus was located in a more central position pyrenoid. The pyrenoid was traversed by a single thy- (Figure 5). The external fraction of the cell wall appeared lakoid and surrounded by starch grains, while smaller to be differentiated. starch grains were observed among the thylakoid mem- The internal part of the cell wall pushed the layers branes. The cell wall was thick and consisted of several above which seemed to be breaking apart (Figures 5 and 6).

Figures 2–4: Vegetative cells of Ulva mutabilis before induction of gametogenesis. (2) Light micrograph of a semithin section showing a unilayered thallus. Note the difference in staining between external and internal cell wall layer. Scale bar: 20 μm. (3) Transmission electron micrograph of a thallus cell before induction of gametogenesis. The nucleus (N) has a relatively central position and the chloroplast (CHL) occupies a large part of the cytoplasm (CYT). Note the thick external wall. Scale bar: 3.6 μm. (4) Detail of external wall of a cell before induction of gametogenesis. Wall shows strict stratification, i.e. a thick internal amorphous layer, a median region with multiple fibrillar layers and an external dark-stained region (see brackets). Scale bar: 1 μm. C. Katsaros et al.: Gametogenesis of Ulva 127

Figures 5–8: Transmission electron micrographs of cells of Ulva mutabilis 48 h after the induction of gametogenesis. (5) The cell is polarised with most of the cytoplasm and organelles gathered at the tip area. The external cell wall has formed a projection towards the apical region. The cell wall appears differentiated at this area (N: nucleus). Scale bar: 2 μm. (6) Detail of the apical wall of the previous image, showing the loose structure of the wall and the formation of amorphous dark-stained material. Scale bar: 0.35 μm. (7) More advanced stage of a wall projection. The dark-stained material covers the projection, while its central part appears as a plug, consisting of amorphous material. The internal wall layer under the tip is considerably thickened and pushes the plug out (asterisk). Scale bar: 4 μm. (8) Cross-section of a cell with a plug-like cap. The dark-stained material is extended covering the cap. Scale bar: 0.33 μm.

The parallel layers of cellulose microfibrils were inter- The microtubule cytoskeleton was also drastically rupted and a round structure of amorphous material was changed during this stage. The cortical microtubule formed (Figure 7). Changes in the chemical composition of bundles disappeared and a new system of intensely fluo- the cell wall occurred at the same time, as an increase of rescent cortical microtubule bundles appeared running the thin external dark-stained wall layer could be observed parallel to the cell axis and converging towards the (Figures 6 and 7). The dark-stained material appeared to conical cell projection. Most of the microtubules were be extending gradually and covered all the round wall observed in the first stages of gametangial formation area, forming a “cap-like” structure (Figure 8). around the top of the conical projection (Figure 10). In 128 C. Katsaros et al.: Gametogenesis of Ulva

Advanced stages of gametangial formation – gamete formation

After the completion of gametangia formation, repeated mitotic divisions gave rise to 16 gametes in each gametangium. These divisions were accompanied by a reorganisation of the cytoplasm, with the loss of its previous polar organisation. No centrosomes or any similar polar structures were found organising the spindle, and the nuclear envelope remained intact during the first mitotic stages, breaking only at the poles (Figures 15 and 16). The cortical microtubule system of the previous stage disappeared and the spindle was organised by microtubules which entered the nucleoplasm through the polar gaps. During anaphase-telophase, the spindle was elongated and microtubule bundles appeared in the interzonal area. Cytokinesis started parallel to the mitotic divisions by furrowing membranes which extended between the daughter nuclei (Figures 17 and 18). Increased numbers of dictyosomes and endoplasmic reticulum (ER) membranes were observed close to the developing furrowing membranes (Figure 18). It was interesting that the cytokinetic membranes separating neighbouring and almost fully developed gametes in mature gametangia were not completed, leaving a narrow cytoplasmic bridge connecting them (Figures 19 and 21). The membrane surrounding these cytoplasmic bridges was lined by a dark-stained Figures 9–14: Immunolocalization of tubulin in developing gam- material (Figures 22 and 23). The microtubules also tra- etangia of Ulva mutabilis. versed the cortical cytoplasm of this area. Parallel to the (9) Interphase cell before induction of gametogenesis. The micro- mitotic divisions, a pair of centrosome-like structures was tubules are mainly peripherally arranged in more or less parallel formed de novo in each daughter cell. These structures bundles. Scale bar: 20 μm. (10) Early stage, 36–48 h after induction were rod-shaped and consisted of microtubule doublets of gametogenesis. Intensely fluorescent microtubule bundles traverse the cortical cytoplasm converging on a particular area (arrow in a circular configuration. The dark-stained material was localises the conical cell projection). Scale bar: 20 μm. (11) More connected with the base of these structures which were advanced stage, 48–60 h after induction of gametogenesis, showing usually not connected to the nuclei, but attached to the microtubules starting from a pointed site at basal part of gametan- external membrane of the gametangium (Figures 15 and μ Α gium. Scale bar: 20 m. (12) dvanced stage, 48–60 h after induc- 17). tion of gametogenesis, showing microtubules organised in a basket- like configuration, leaving a circular opening at the top (arrow). Scale The newly formed gametes had a round shape, which bar: 20 μm. (13) Gametangium, 60–72 h after induction of game- later became spindle-like, and were equipped with a pair togenesis. The microtubule-free area at the top appears broader than of flagella arising from a basal body (Figures 20 and 21). before (arrow). Scale bar: 20 μm. (14) Hoechst 33258 staining of the They were usually oriented with their long axes converg- nucleus of the cell in Figure 12. Scale bar: 20 μm. ing towards the conical aperture of the top. The microtubule cytoskeleton in the individual gametes consisted of more advanced stages, the microtubule cytoskeleton was cortical bundles radiating out of a particular point, which further changed, forming thin bundles which radiated out was the basal body of the flagellum. of a particular point at the base of the cell (Figure 11), tra- At this stage, the cell wall of the tip region was versed the cell periphery in a basket-like shape and con- further differentiated. The external layer of the cell wall verged to a circular area behind the top of the conical cell appeared broken and the internal layer underwent a par- projection (Figures 12 and 14). However, the microtubule ticular differentiation, with the deposition of additional bundles did not reach the top, leaving a circular opening layers of wall material forming a plug-shaped cap, closing between them (Figures 12 and 13). the opening of the external wall layers (Figure 24). The C. Katsaros et al.: Gametogenesis of Ulva 129

Figures 15–23: Transmission electron micrographs of developing gametangia and complete gametes of Ulva mutabilis. (15) Metaphase nucleus of developing gametangium. The nuclear envelope (NE) is still intact with gaps only at poles (arrows). Spindle microtubules (MT) traverse the nucleoplasm, but do not radiate out of the centrosome (C), which is attached to the external membrane. Scale bar: 0.5 μm. (16) Developing gametangium, in which first nuclear division is completed. Note that furrowing membranes (arrow) are growing between daughter nuclei (N). Scale bar: 2.6 μm. (17) Advanced stage of gamete formation, showing the furrowing membrane (arrow) development separating two daughter nuclei (N). The centrosome-like structures (C) are not connected to the nuclei, but to the external membrane. Scale bar: 0.7 μm. (18) Developing furrowing membranes and vesicles (probably of dictyosome origin, see white arrow). Note increased number of active dictyosomes (D) and membranes of ER close to developing furrow. Scale bar: 0.8 μm. (19) Fully developed gametangium before gamete release. Gametes are still connected to each other by thin cytoplasmic bridges (arrows). Scale bar: 3 μm. (20) Part of fully developed gamete at high magnification. Basal bodies (BB) of flagella are visible close to surrounding membrane. Scale bar: 0.3 μm. (21) Longitudinal section of gamete, showing nucleus, chloroplast with a pyrenoid (P) and starch grains (S) and one of the two flagella. Note that gamete is still connected to neighbouring gametes (arrows). Scale bar: 1 μm. (22 and 23) Detail of cytoplasmic bridges connecting neighbouring gametes. Note the dark-stained borders of cytoplasmic bridge and microtubules close to membrane. Scale bars: 0.5 and 0.1 μm. 130 C. Katsaros et al.: Gametogenesis of Ulva

Figures 24–26: Advanced stages of gametangia formation of Ulva mutabilis (before gamete release). (24) Cross-section of fully developed gametangium. Cell wall in conical projection appears broken and opening is plugged by cap of cell wall material. Scale bar: 2.8 μm. (25) Detail of cap. Cell wall appears differentiated with loose fibrillar layers embedded in amorphous matrix. Scale bar: 0.69 μm. (26) Exit pore of gametangium. Cap is not visible, but darkly stained amorphous material appears gathered in pore and below (arrow). Vesicles are observed among the gametes. Scale bar: 1.2 μm.

plug consisted of parallel layers of cellulose microfibrils showing a rather loose pattern (Figure 25). Interestingly, at this stage, a dark-stained amorphous material was visible under the opened exit pores (Figure 26).

Gamete release

After the completion of the flagella formation, the plug- shaped cap was removed and gamete discharge could take place through the opening of the release tube. Anti- tubulin immunofluorescence staining revealed that each gamete bore two flagella (Figures 27–30). The microtubule cytoskeleton of the gametes released consisted of thick, intensely fluorescent bundles surrounding the cell cortex (Figure 27). Apart from the cortical microtubules, thinner and shorter bundles and possibly single microtubules traversed the cytoplasm in other directions (Figures 29 and 30). All the microtubules of the gametes radiated out of Figures 27–30: Tubulin immunofluorescence in released gametes the flagellar basal body region. of Ulva mutabilis. (27) Released gamete bearing two equal flagella. Note that cortical microtubule bundles (tubulin fluorescence) extend from basal body towards the posterior. Scale bar: 20 μm. (28) Differential interfer- ence contrast microscopy-image of gamete in Figure 27. Scale Discussion bar: 20 μm. (29 and 30) Two gametes in which (apart from the two flagella) an intensely fluorescent microtubule bundle starting from The process of gametangia formation and gamete release flagellar basal body is directed to cytoplasm (arrows). Scale bar: in Ulva mutabilis is highly synchronised. A crucial role in 20 μm. C. Katsaros et al.: Gametogenesis of Ulva 131 this process is played by the presence of the sporulation polysaccharides. This architecture provides particular and swarming inhibitors (Stratmann et al. 1996, Wichard desiccation tolerance in Ulva. The capsular zoosporangial and Oertel 2010). By removal of the sporulation inhibitors wall and method of sporangial dehiscence (i.e. wall disso- gametogenesis can be artificially induced in the labora- ciation and the production of a mucilage plug) were char- tory. A particular divergence in the differentiation is fol- acters of the Ulvales sensu Stewart and Mattox (1975). lowed in order to transform somatic cells to gametangia. The differentiation of the external cell wall finally This process includes drastic changes in the cell struc- results in the formation of the exit pore for the release of ture. The polarisation of the cell, which is expressed by gametes. Similar observations have been reported during the rearrangement of the cell elements, is accompanied sporangia formation and meiotic divisions of U. mutabilis and probably controlled by the transformation of the sporophytes (Bråten and Nordby 1973). In this study, only microtubule cytoskeleton. It has been reported that inter- the final stage before the opening was presented showing phase cells of Ulva lactuca bear an impressive microtu- less densely packed and more randomly oriented fibrils bule system of intensely fluorescing microtubule bundles than in the rest of the cell wall. This was an indication which radiate from a cortical site close to the nucleus of the cell wall weakening before the opening of the exit and surround the chloroplast (Tsagkamilis and Katsaros pore. However, we found that the process of cell wall dif- 2007). However, it was not clear in this study whether ferentiation started very early, even 36 h after the induc- these cells were at a preliminary gametogenesis stage. tion of gametogenesis. The first sign was the change of the From both the old preliminary and the current findings, chemical composition of the cell wall. It seems that the it is clear that the microtubule organisation in interphase induction of gametogenesis triggered the activation of a cells of Ulva is different from land plants (Hashimoto mechanism of synthesis of new, rather amorphous wall 2015 and literature therein), therefore additional work material, which was guided to the top of the cell. This is needed to clarify the role of microtubules in cell wall material was concentrated gradually in a limited area, morphogenesis. The conical microtubule organisation interrupting the continuity of the cell wall layers. Bråten may facilitate the transport of cell elements to the top of and Nordby (1973) found membrane-bound vesicles at the the cell. A similar role of the microtubule cytoskeleton site of cell wall weakening. Such vesicles could probably has been reported in other polarised systems, such as transfer cell wall precursor materials which contributed in chlorenchyma cells of flowering plants (Chuong et al. to the bulging. Wichard and Oertel (2010) reported that 2006), bryophytes (Doonan et al. 1988), fungal hyphae wall papillae start growing at the outer surface of the cells (Roberson and Vargas 1994), green algae (Holzinger et al. when they were already irreversibly committed to gam- 2002) and brown algae (Karyophyllis et al. 1997, Katsaros etangium differentiation, the chloroplasts appeared reori- et al. 2006). However, actin has also been reported to par- ented and the first progametes had divided. The papillae ticipate in organelle movement (Grolig 1990, Higaki et al. converted into “capped” pores during the following night 2007, Blanchoin et al. 2010) . Therefore, it would be inter- (Figure 1). Using TEM, we found that the process of “cap” esting to examine the role of actin in the organisation of formation started quite early by the gradual differentia- the cytoplasm of the developing gametangia in future tion of the cell wall. studies. A third important finding of the present study was the The second interesting process occurring during reorganisation of the microtubule cytoskeleton during the gametogenesis is the differentiation of the cell wall. As gametangia formation and gamete release. The cortical, has already been mentioned, the cell wall of the gametan- basket-like microtubule configuration showed two focal gium consists of two electron-dense layers: an outer layer sites. The site at the bottom (internal side) of the cell is representing the vegetative cell wall and an inner layer or possibly functioning as a microtubule organising centre capsule. This structure has been described in sporangia of (MTOC). Since we did not find a centrosome in the inter- U. mutabilis (Løvlie and Bråten 1968) and in gametangia of phase cells of U. mutabilis that would organise micro U. intestinalis L. (McArthur and Moss 1979), as well as in tubules, we can assume that interphase microtubules the sporangial wall of Endophyton ramosum N.L. Gardner are organised by proteins which do not form a visible (Leonardi et al. 1997). A more detailed description of the structure, similar to the interphase of land plant cells, cell wall of Ulva compressa L. has been reported by Hol- and this supports the phylogenetic relationship between zinger et al. (2015). According to this study, the wall is green algae and higher plants (Meagher and Williamson multilayered, consisting of cellulose, callose and acidic 1994 , Wasteneys 2004). However, the fact that the nuclear 132 C. Katsaros et al.: Gametogenesis of Ulva envelope does not disassemble during mitosis, and the important role for the cytoplasmic bridges in the gamete spindle microtubules enter the nucleoplasm through the release mechanism of Ulva. This could also be combined polar gaps is characteristic of lower eukaryotes and algae with the period when the gametes are motionless due to rather than of land plants (Güttinger et al. 2009, Boettcher the presence of the SWI as well as with the fact that treat- and Barral 2013). As noted in the results, these microtu- ment with the non-competitive myosin-ATPase blocker bules converged to a circular area at the top of the cell. 2,3-butane-dione monoxime (BDM) caused complete This fits very well with the architecture of the gametan- inhibition of gamete release (Wichard and Oertel 2010). gium, meaning that microtubules reached the “neck” of The dark-stained material underlining the cytoplasmic the exit pore. From this particular organisation, it can be channel could be an actin ring. This ring may contribute to reasonably suggested that microtubules are implicated in the final closing of the gap and the separation of gametes, the gametangia maturation. thus, facilitating their synchronous release one after the In the following stages, repeated nuclear divisions other. occurred to form the 16 gametes. The absence of centro- After the complete formation of the gametes, their some-dependent MTOCs in the spindle poles of these release through the opened exit pore occurred. The mitoses suggests that the spindle was also organised microtubule cytoskeleton of each gamete consisted of without the involvement of the centrosomes. However, cortical bundles diverging from the basal body site. Two centrosome-like structures were formed parallel to the flagella rose from the basal body. An interesting observa- divisions. These structures were close to the nuclei, and tion during the gamete release was that the oval-shaped were not connected with them, but were attached to the gametes showed a kind of orientation with their long axis developing furrowing membrane. A similar process has converging towards the opening. This means that a spe- been described by Bråten and Nordby (1973) during the cific mechanism underlies this process. Experiments with sporogenesis of U. mutabilis. the myosin-ATPase blocker BDM have indicated that the An interesting phenomenon observed was the mechanism of gamete release depends potentially on a cytokinetic process during the gamete formation, i.e. the myosin-ATPase. Although the inhibiting concentration mechanism of separation of individual gametes. Since of 40 mmol l − 1 reported was high (Wichard and Oertel the ready-to-release gametes were surrounded only by 2010), it is similar to the inhibiting concentration found a membrane and not a cell wall, it was expected that a in diatoms, where gliding is based on an actin/myosin cytokinetic diaphragm would be developed between each system (Poulsen et al. 1999). pair of daughter nuclei. This process started quite early in The process of gamete release may involve a mecha- the present study, after telophase, with the furrowing of nism by which the gametes are mechanically extruded the plasma membrane (Figure 17). This mechanism differs out of the gametangium. This may occur by hydration of from that described in other multinucleate systems, such an extracellular substance (possibly callose-like) which as in endosperms of flowering plants, where cytoki- causes a considerable increase of its volume and, in turn, nesis took place after the complete formation of the pushes the gametes out of the gametangium. Leonardi daughter nuclei (Sørensen et al. 2002). The presence of an et al. (1997) studied the development and morphology of increased number of active dictyosomes and membranes sporangia and zoospores of the filamentous Ulvophyte of the endoplasmic reticulum (ER) close to the develop- Endophyton. They found that the sporangium was filled ing furrow suggests that vesicles from golgi and ER may with a mucilagenous fibrillar material in which mature have contributed to the development of this separating zoospores remained immersed until liberation. A thick diaphragm. layer of similar material covered the inner sporangial Interestingly, cytoplasmic connexions were observed cell wall and the upper apical part of the sporangium. in almost fully developed gametes constituting a kind of Our observations of dark-stained material below the exit network by which all the gametes were connected within pore of U. mutabilis gametangia (Figure 26) supported this the gametangium. These cytoplasmic bridges are similar hypothesis. In this context, the fragile inner perforated to the connexions between the fusing gametes of U. muta- seal reported by Wichard and Oertel (2010) could not be bilis observed by Bråten (1971). The cytoplasmic bridges in observed by TEM. Ulva may also be compared with those described in Volvox As all the above data have been extracted from the L. embryos by Green et al. (1981). They are formed as a naturally occurring developmental mutant “slender”, result of incomplete cytokinesis and it has been suggested future work should also include comparison with the that they play a role in generating the movements of the wild type alga. However, differences in regulatory effects, inversion process of Volvox. Therefore, we hypothesise an such as sporulation and swarming inhibitors, between C. Katsaros et al.: Gametogenesis of Ulva 133 morphotypes or even species have not been observed up References to now (Wichard and Oertel 2010, Vesty et al. 2015). Interestingly, under axenic conditions, Ulva forms Blanchoin, L., R. Boujemaa-Paterski, J.L. Henty, P. Khurana and callus-like cultures of blade cells which do not undergo C.J. Staiger. 2010. Actin dynamics in plant cells: a team effort from multiple proteins orchestrates this very fast-paced game. gametogenesis (Spoerner et al. 2012, Wichard 2015). Curr. Opin. Plant Biol. 13: 714–723. 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Impairment of cytoskeleton-dependent vesicle and Acknowledgments: This study was financed by the organelle translocation in green algae: combined use of a microfocused infrared laser as microbeam and optical National and Kapodistrian University of Athens (“Kap- tweezers. J. Microsc. 208: 77–83. odistrias” programme) to C.K and by the Deutsche Holzinger, A., K. Herburger, F. Kaplan and L.A. Lewis. 2015. Desicca- Forschungsgemeinschaft (DFG) through the Jena School tion tolerance in the chlorophyte green alga Ulva compressa: for Microbial Communication (German Excellence Ini- does cell wall architecture contribute to ecological success? tiative) and the Collaborative Research Center SFB 1127 Planta. 242: 477–492. “ChemBioSys” to A.W. and T.W. The authors would like to Hoxmark, R.C. 1975. Experimental analysis of life cycle of Ulva mutabilis. Bot. Mar. 18: 123–129. acknowledge networking support by the European Coop- Hoxmark, R.C. and O. Nordby. 1974. 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Stewart, K.D. and K.R. Mattox. 1975. Comparative cytology, evolution and classification of the green algae with some considera- Anne Weiss is a PhD candidate at the Institute for Inorganic and tion of the origin of other organisms with chlorophylls a and b. Analytical Chemistry of the Friedrich-Schiller-University Jena. She Bot. Rev. 41: 104–135. obtained her Diploma in Biology in the group of Molecular Botany Stratmann, J., G. Paputsoglu and W. Oertel. 1996. Differentia- at the University of Jena working on natural products in freshwater tion of Ulva mutabilis (Chlorophyta) gametangia and gamete microalgae. Her current research interest is the chemical commu- release are controlled by extracellular inhibitors. J. Phycol. 32: nication of bacteria-macroalgae interactions, bacterial dependent 1009–1021. development of Ulva sp. and aquaculture of macroalgae. C. Katsaros et al.: Gametogenesis of Ulva 135

Ira Llangos Thomas Wichard National and Kapodistrian University of Institute for Inorganic and Analytical Athens, Department of Biology, Section of Chemistry, Jena School for Microbial Botany, Athens 157 84, Hellas (Greece) Communication, Friedrich Schiller University Jena, 07743 Jena, Germany, [email protected]

Ira Llangos finished her undergraduate studies in the Department of Biology of the University of Athens. She conducted her Diploma Thomas Wichard is a research group leader at the Institute for thesis on the structure and microtubule organization during game- Inorganic and Analytical Chemistry of the Friedrich Schiller Univer- togenesis of Ulva mutabilis, supervised by Prof. Christos Katsaros. sity Jena. After he had been awarded a PhD in Biochemistry for his During that time she was an undergraduate Research Assistant in the studies at the Max Planck Institute for Chemical Ecology in Jena, he Section of Botany. She is currently enrolled in a postgraduate studies investigated the metal recruitment of nitrogen fixers at the Princeton program in Molecular Biology at the University of Tirana, Albania. Environmental Institute (USA). Now the main focus of his research group is to elucidate the mutualistic interactions between bacte- Ioannis Theodorou ria and the marine macroalga Ulva (“cross-kingdom-cross-talk”). National and Kapodistrian University of The group applies various methodologies in analytical chemistry, Athens, Department of Biology, Section of chemical ecology and molecular biology to understand the basis of Botany, Athens 157 84, Hellas (Greece) eco-physiological processes.

Ioannis Theodorou was an undergraduate research assistant in the group of Prof. Christos Katsaros in the Department of Biology of the University of Athens. He finished his undergraduate studies in the Department of Biology and is interested in the evolution and development of photosynthetic organisms. At the moment, he is continu- ing his postgraduate studies in the Master’s programme Molecular Life Sciences at Friedrich Schiller University of Jena.