2022 Volume 87 Issue 1 Pages 17-22
Multicellular marine algae have a simple body structure composed of just a small number of basic cell types. However, the mechanism of their cellular differentiation and multicellular morphogenesis remains mostly unknown. The multicellular leafy seaweed Gayralia oxysperma is composed of blade cells and rhizoid cells. In axenic culture, this species loses its typical multicellular morphology because, upon shaking, the two cell types separate to form a loose mass composed of the two cell types. This fragile cell mass was used to isolate each cell type for culture experiments and examination of their growth properties. Artificially synthesized thallusin, a morphogenesis-promoting factor, was also tested for its effect on the cells. The isolated blade cells are divided to generate daughter cells displaying various differentiation patterns, whether in the presence or absence of thallusin. None of the tested rhizoid cells divided but they elongated greatly to over 1 mm in length during 10 d of culture. These observations indicate that blade cells maintain their totipotency, while the rhizoid cells lose their abilities to divide and differentiate into other cell types. This result is in contrast to findings with the previously well-studied Ulva, which shares similarities with Gayralia in leafy morphology and early development but its rhizoid and stem cells retain their totipotency, while its blade cells do not. Therefore, the morphogenesis system in Gayralia seems to be fundamentally different from that of Ulva and requires further investigation.
Marine green macroalgae Gayralia (Ulotrichales) and Ulva (Ulvales) are ulvophytes in the Class Ulvophyceae. They have a simple leafy structure composed of only a few cell types such as blade cells and rhizoid cells. In early development, Gayralia germinates to form a uniseriate filament, becomes saccate, and then opens into a blade-like thallus with a single cell layer (Wilkinson 2007). Ulva, also, undergoes saccate development but does not open, becoming a thallus two cells thick or tubes one cell thick (De Clerck et al. 2018).
In axenic culture conditions, both genera lose their typical leafy morphology and develop into an amorphous aggregate of cells (Tatewaki et al. 1983, Spoerner et al. 2012). However, when they are exposed to certain bacterial strains or grown in a conditioned medium to which is added a filtrate of bacterial culture, their complete morphogenesis is observed. Thallusin is the only identified algal morphogenesis inducer (morphogen). It was isolated from an epiphytic marine bacterium strain, YM2-23 (Matsuo et al. 2003, Alsufyani et al. 2020). Thallusin induces development of the thallus in G. oxysperma and Ulva spp. at extremely low concentrations, between 1 fg mL−1 and 1 ag mL−1 (Matsuo et al. 2005). Natural thallusin was originally extracted in limited amounts from the culture medium of the YM2-23 strain, but thallusin and various analogs have now been artificially synthesized and tested for their algal morphogen activity (Yamamoto et al. 2018). However, the essential mechanism by which thallusin induces algal morphogenesis remains unknown.
Currently, U. compressa (syn. U. mutabilis) is the generally accepted model organism for studies of algal growth, development, and morphogenesis (Wichard 2015, Wichard et al. 2015) and its genome has been sequenced (De Clerck et al. 2018). According to Spoerner et al. (2012), the wild U. compressa strain is composed of only three differentiated cell types which are blade, stem, and rhizoid cells, and its complete growth and morphogenesis can be induced by co-culture in combination with two particular species of bacteria. The stem and rhizoid cells are totipotent, like germ cells, but the blade cells are not. As long as stem cells are flanked by a differentiated blade or rhizoid cells, their division is suppressed. Therefore, in order to examine the potential growth and differential abilities of the different cell types, isolation of each cell type is required. However, this is difficult to achieve in Ulva because even in axenic conditions the tissue develops into a callus-like morphotype composed of cells firmly attached to each other. In contrast, axenic G. oxysperma becomes a colony-like mass consisting of loosely aggregated blade and rhizoid cells with a fragile structure that falls apart upon shaking (Tatewaki et al. 1983). The developmental characteristics of Gayralia make it easy to isolate each cell type as single cells.
Here, the axenic Gayralia cell mass is used as a source to separate each cell type and examine their potential growth characteristics by following the development of the isolated single cells in culture. In addition, synthesized thallusin was tested for its effects on these cells.
A leafy green thallus was collected at site S4 named by Hiraoka and Higa (2016) in the Shimanto River estuary, Japan (32°56′17″N, 132°59′30″E) on February 1, 2018. The thallus sample was identified as G. oxysperma because it was asexually reproduced by negatively phototactic biflagellate neutral zoids termed ‘zoosporoids’ by Bliding (1968), developed saccate, and then opened into a blade-like thallus in culture. These characters are taxonomically diagnostic for G. oxysperma (Wilkinson 2007).
Synchronous sporulation was induced by a culture of thallus fragments of 1–2 cm in length, which were placed in a glass beaker with 500 mL of natural seawater-based ES medium (Andersen et al. 2005) in an incubator maintained at 20°C on a 12 : 12 h LD cycle with illumination with LED light at 100–200 µmol photons m−2 s−1 (ISLM-500X300NWWWW-TK; CCS, Kyoto, Japan). Light intensity was measured using a photon sensor (Li-Cor Biosciences; Lincoln, USA). Under these conditions, numerous zoosporoids were released from the reproductively mature fragments within 4 days. For axenic culture, the zoids were isolated from accompanying bacteria and any contaminants by using their phototactic behavior, modified from the protocol for Ulva gamete purification by Califano and Wichard (2018). Using a glass pipette, a dense zoid suspension was carefully placed inside a semicylindrical glass trough {20 cm length, 6 cm in diameter, Fig. 1A containing 15 mL sterilized artificial seawater [Marine Art SF-1; Tomita Pharmaceutical, Naruto, Japan, see Supplementary Table 2 in Hiraoka et al. (2020)]} , irradiated at one end with LED light at 100 µmol photons m−2 s−1. The zoids swam from the light-irradiated side to the opposite side because of their negative phototaxis. Immediately, densely gathered zoids were transferred to another similar container. By swimming two or more times through the sterilized seawater, the zoids were isolated from bacteria and other cell types. All containers and seawater were previously autoclaved at 121°C for 15 min, and the purification was performed at a clean bench.
To isolate the blade and rhizoid cells, the purified zoids were cultured under aseptic conditions and placed (20 to 30 in number) at 20°C for 48 h in the dark in a sterile plastic dish (9 cm diameter) with 40 mL of supplemented artificial seawater (SAS; sterilized artificial seawater supplemented with enrichment stock solution of the ES medium on prescription). Settled zoids became spherical cells which were regarded as germ cells for the present study.
The dish of germ cells was transferred to a rotary shaker at 50 rpm (Neo Shaker; As One, Osaka, Japan) under a weak white LED light (＜50 µmol photons m−2 s−1, 12 : 12 h LD cycle, LED5000KRa90; Iida Lighting, Kyoto, Japan) for more than 10 d. In this condition, the germ cells produced a colony-like mass consisting of loosely aggregated blade cells with many rhizoids, as previously reported by Tatewaki et al. (1983). The cell mass was transferred to a cell culture flask (VTC-F25V ‘Vent Cup,’ 25 cm2; As One, Osaka, Japan) containing 15 mL SAS and cultured under the same conditions for 4 d.
Typical round cells without any protuberances and elongated cells with a protuberance were selected from the well-developed cell mass as blade and rhizoid cells, respectively (Fig. 1B). Single germ cells, blade cells or rhizoid cells were introduced into each well of 24-well microtiter plates (Iwaki, Shizuoka, Japan) with each well containing 1 mL SAS alone or SAS containing 1,000 fmol mL−1 thallusin introduced through a 0.22-µm filter (Millipore; Billerica, USA). The thallusin was synthesized according to Yamamoto et al. (2014). The well plates were shaken on a rotary shaker at 50 rpm under a 12 : 12 h LD cycle with white LED light at 100 µmol photons m−2 s−1 at 20°C for the germ, blade, and rhizoid cells and the fourth preparation with rhizoid cells at 25°C. Light intensity was measured at the upper surface of the well plates.
Morphological changes of each cell type were observed with an inverted microscope (ECLIPSETs2; Nikon, Tokyo, Japan) at 48-h intervals for 10 d. The length of the rhizoid cells was measured at 1 d and after 10 d of culture using image analysis software (WinROOF; Mitani, Tokyo, Japan). Axenicity for the used samples was tested by plating aliquots of their cell and medium suspension on marine broth agar (Marine Agar 2216; Difco, Detroit, USA) and checking for the absence of bacterial colony formation after 7 d of incubation at 20°C.
Fluorescence microscopyFor fluorescence microscopic observations, SYBR Green I nucleic acid stain (TaKaRa Bio, Tokyo, Japan), diluted 1 : 100,000 with dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Industries, Ltd., Osaka, Japan), was added to blade and rhizoid cells. After staining for 15 min, the cells were observed under blue excitation (470–490 nm) with a fluorescence microscope (BX51; Olympus, Tokyo, Japan).
Statistical analysisObserved developmental patterns in the three cell types were classified (see Results) and differences between the presence or absence of thallusin in a frequency distribution of the developmental patterns were evaluated by the Chi-square test. Differences in the length increase of rhizoid cells between 20°C and 25°C and between the presence or absence of thallusin were evaluated by Welch’s t-test.
Developmental processes of the three cell types in the morphogenesis test were classified into five types as follows (Table 1, Fig. 2):
| Cell type | Temperature (°C) | Thallusin | Cell division | No cell division | ||||
|---|---|---|---|---|---|---|---|---|
| I. Blade cell and rhizoid cell | II. Two blade cells | III. Two rhizoid cells | IV. Elongation | V. Stop | ||||
| Germ cell | 20 | + | 100 | 0 | 0 | 0 | 0 | n=100 |
| 20 | − | 100 | 0 | 0 | 0 | 0 | n=100 | |
| Blade cell | 20 | + | 51 | 8 | 24 | 10 | 7 | n=100 |
| 20 | − | 46 | 11 | 20 | 13 | 10 | n=100 | |
| Rhizoid cell | 20 | + | 0 | 0 | 0 | 50 | 0 | n=50 |
| − | 0 | 0 | 0 | 50 | 0 | n=50 | ||
| 25 | + | 0 | 0 | 0 | 50 | 0 | n=50 | |
| − | 0 | 0 | 0 | 50 | 0 | n=50 | ||
All the germ cells were divided asymmetrically until 4 d, regardless of the addition of thallusin, to form a blade cell and a rhizoid cell (Fig. 3A). The generated blade cells continuously divided, growing steadily in the longitudinal direction to form an erect filament. In the presence of thallusin, the filament developed into a foliate structure maintaining the rhizoid-thallus growth axis (Fig. 3B). In the absence of thallusin, the serial cells lost the division orientation and became loose aggregates of blade cells and rhizoid cells (Fig. 3C).
Development of the isolated blade cells could be classified into all five types based on observations of morphological changes during 10 d in culture (Fig. 4). Through cell divisions, the blade cells generated more blade cells, both blade cells and rhizoid cells, or rhizoid cells. In some cases, the blade cells elongated without cell division and became rhizoid cells. The addition of thallusin did not cause the blade cells to undergo multicellular morphogenesis as observed in the germ cells. There was no significant difference in the occurrence of the five morphogenesis types in the blade cells between the additive group with or without thallusin (p＞0.05).
None of the isolated rhizoid cells divided but continuously lengthened their projecting part (Fig. 5). The rhizoid cells of approximately 360 µm in initial length reached a maximum total length of 1,600 µm at 25°C, growing 4.4 times their initial length after 10 d of culture. As there was a significant difference in growth between 20°C and 25°C (p＜0.05), the higher temperature accelerated the elongation. There was no significant difference in the growth of the rhizoid cells in the presence or absence of thallusin (p＞0.05). Stained with SYBR Green I, the rhizoid and blade cells each have one nucleus and several organelle nucleoids (Fig. 6). The nucleus of rhizoid cells was observed at the tip of the projection.
The present study confirmed the differences in growth and differentiation between the isolated blade and rhizoid cells of G. oxysperma. The rhizoid cell was characterized by extensive elongation activity without either mitosis or cell division. In contrast, the blade cells showed various kinds of division and differentiation, generating either blade cells, rhizoid cells, or both types via cell division. These observations indicate that blade cells have totipotency. Although morphologically identical round-shaped cells were selected as the blade cells for use in experiments, they appear to have been in various stages of the differentiation process. For further developmental research, a molecular approach will be necessary to detect the internal status of the blade cells. Interestingly, in Ulva the blade cells do not have totipotency but rhizoid cells and stem cells do (Spoerner et al. 2012). The cell types with totipotency are therefore fundamentally different for Gayralia and Ulva, and the morphogenesis of Gayralia is distinct from that of Ulva.
In previous research on Gayralia morphogenesis, it has been unclear which cell types are affected by thallusin or morphogenesis-inducing bacteria (Matsuo et al. 2003, 2005). It is shown here that thallusin is effective on germ cells newly raised from zoosporoids but has little or no effect on the blade and rhizoid cells isolated from a well-developed cell mass. Newborn germ cells have an initial state for cell division and differentiation. However, as the cells isolated from the cell mass underwent several more cell divisions, apparently their differentiation status progressed. Thallusin may enable effective induction of multicellular morphogenesis only in undifferentiated cells such as young germ cells.
In Ulva, thallusin has been reported to induce the development of basal rhizoids and healthy cell wall formation (Alsufyani et al. 2020). However, these two functions of thallusin were not observed in Gayralia in the present study. Although Ulva does not obviously develop rhizoid cells in axenic conditions (Alsufyani et al. 2020), our axenic Gayralia produced rhizoid cells with high elongation activity even in the absence of thallusin. Also, Ulva forms unusual cell walls with spherical transparent protrusions under axenic conditions without thallusin (Spoerner et al. 2012, Alsufyani et al. 2020). However, such unusual protrusions in cell walls have not been found in axenic Gayralia (Tatewaki et al. 1983). That was also confirmed in the absence of thallusin in the experiments reported here. There is a structural difference in the sulfated polysaccharides which are a key component of the cell walls of Ulva (Ulvales) and Gayralia (Ulotrichales) (Ciancia et al. 2020). Rhamnan sulfate is the major sulfated polysaccharide of the order Ulotrichales, and it has a significantly higher viscosity than ulvan, which is the major sulfated polysaccharide of the order Ulvales (Tsubaki et al. 2016). Their differences in rhizoid development and cell wall formation are perhaps associated with such rheological differences in major components of their cell walls.
It has been considered that thallusin-dependent morphogenesis could be a common feature of the orders Ulotrichales and Ulvales (Matsuo et al. 2005). However, Alsufyani et al. (2020) have recently suggested that, as with the hormones of Plantae, thallusin possesses distinct functions in algal development according to the underlying developmental mechanisms of the recipient. The present study supports this suggestion.
This work was supported by the JST-OPERA Program (Grant Number JPMJOP1832) and the Kochi University Biomass Refinery of Marine Algae research project. The authors would like to thank Dr. Masanobu Kawachi (National institute for environmental studies, Japan) for technical supports of fluorescence microscopy.
