- Research article
- Open Access
Variations in morphology and PSII photosynthetic capabilities during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta)
© Xie et al; licensee BioMed Central Ltd. 2010
- Received: 14 September 2009
- Accepted: 28 April 2010
- Published: 28 April 2010
Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae.
In the tetraspores, we observed two types of division, cruciate and zonate, and both could develop into multicellular bodies (disks). During the first 84 hours, tetraspores divided several times, but the diameter of the disks changed very little; thereafter, the diameter increased significantly. Scanning electron microscopy observations and analysis of histological sections revealed that the natural shape of the disk remains tapered over time, and the erect frond grows from the central protrusion of the disk. Cultivation of tissue from excised disks demonstrated that the central protrusion of the disk is essential for initiation of the erect frond. Photosynthetic (i.e., PSII) activities were measured using chlorophyll fluorescence analysis. The results indicated that freshly released tetraspores retained limited PSII photosynthetic capabilities; when the tetraspores attached to a substrate, those capabilities increased significantly. In the disk, the PSII activity of both marginal and central cells was similar, although some degree of morphological polarity was present; the PSII photosynthetic capabilities in young germling exhibited an apico-basal gradient.
Attachment of tetraspores to a substrate significantly enhanced their PSII photosynthetic capabilities, and triggered further development. The central protrusion of the disk is the growth point, may have transfer of nutritive material with the marginal cells. Within the young germling, the hetero-distribution of PSII photosynthetic capabilities might be due to the differences in cell functions.
- Apical Cell
- Morphological Polarity
- Marginal Cell
- Cleavage Furrow
- Normal Disk
Red algae are primitive photosynthetic eukaryotes that are widely distributed around the world, and they are ideal subjects for studies of photosynthesis and development. Red algae strongly resemble to cyanobacteria: Both are characterised by the presence of phycobilisomes, which consist of phycobiliproteins, which function as a photosynthetic antenna system with chlorophyll a . Red algae exhibit alternation of generations. The spores, which are produced from sporophytes, are large spherical cells that can develop rapidly into mature individuals under laboratory conditions. Furthermore, the release of spores can be controlled easily by photoperiod [2, 3].
Guiry  previously described the germination patterns of red algae spores as consisting of five representative types: Nemalion, Gelidium, Naccaria, Ceramium, and Dumontia. Nemalion-type and Gelidium-type germination are very similar to each other and, are less common among the red algae than the other types . The Naccaria-type is common in Bonnemaisoniales and in many of the primitive members of the Gigartinales . The Ceramium-type, which exhibits a bipolar germination pattern, is found in Bangiophycidae and Ceramiales [7–9]. The Dumontia-type generally occurs in advanced members of Gigartinales, Rhodymeniales and Gracilariales [6, 10–13]. Of the five representative types, the first four share some common features. After attachment to a substrate, the spore first forms one or more protuberance(s), from which the apical cell and a rhizoid are produced. In the Dumontia type of germination, the spore develops into a mass of small cells within the original spore wall, and that multicellular body then grows into a disk that stems from the division of marginal cells of the mass. Using scanning electron microscopy (SEM), Chen and Taylor (1976) investigated Dumontia-type development in Condrus crispus and concluded that the extracellular sheath is very important for its development . Thus, this type of germination differs from the other four types.
Species of Gracilaria, whose spores exhibit Dumontia-type germination, are economically important algae, and thus they have received a lot of attention from researchers [14–17]. The polysiphonia-type life history of Gracilaria consists of two isomorphic generations (diploid tetrasporophyte and haploid gametophyte) [18–20], as well as a parasitic heteromorphic carposporophyte generation. Tetraspores released from the tetrasporophyte play a crucial role in the life cycle of Gracilaria and have been investigated extensively [19, 21, 22]. Although spore germination has been studied intensively [11, 23, 24], few studies have focused on the detailed morphological and photosynthetic changes at the single-cell level that occur during the early development of Gracilaria tetraspores. In this study, we investigated the early developmental properties of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss, including morphological changes and changes in the photosynthetic capabilities of PSII.
Two types of division in G. vermiculophyllatetraspores
Changes in diameters of tetraspores and disks
The development pattern of the tetraspores of G. vermiculophylla, along with other species of Gracilaria , falls within the framework of the Dumontia-type germination described above. This scenario differs significantly from the development of conchospores in Porphyra (Rhodophyta) described by Fan et al. (2008), in which the conchospores elongate when the first cell division occurs. With further divisions of conchospores, the longitudinal length of the conchospores increased .
Changes in PSII photosynthetic capabilities of tetraspores and disks during development
In freshly released tetraspores that were in suspension for about 8 hours, both Fv/Fm and Y (II) remained very low, suggesting that they were dormant and maintained only suspended metabolism. After attachment, their photosynthetic capabilities increased dramatically (Figure 4) and were accompanied by rapid division. Thus, the attachment of tetraspores enhanced their photosynthetic capabilities of PSII. In addition, only attached tetraspores began to divide, whereas none of the suspended tetraspores did. Therefore, attachment must play a great role in triggering further development. The promoting effect of attachment on further development was reported for the conchospores of Porphyra (Rhodophyta), as well, because only attached conchospores could develop normally. Similar results have been reported for algae, such as some species of Rhodophyta [26, 27], Phaeophyta , and Chlorophyta . Thus, it seems that attachment is essential for spores to develop.
Initiation of the erect frond
These results demonstrate that the central protrusion of the disk plays a crucial role in the disk stage of development of tetraspores; it is the growth point of the disk and it controls the initiation of the erect frond. In contrast, the marginal cells of the disk grow centrifugally and always attach to the substrate. It is noteworthy that a degree of morphological polarity is present in the marginal cells and the central cell. However, this polarity is not reflected in the photosynthetic capabilities of PSII according to our results (Figure 9). Thus, we believe that the marginal cells not only function as a holdfast to keep the thalli stable in the onrushing tide, which was described previously by Li , but that they also support the growth of the central protrusion by transition photosynthate, since the pit connection, from which nutrients are transferred from hosts cells to their parasites [31, 32], is generally occurred among the cells of disk in some species in the genus Gracilaria .
Repair of the excised disk
These results together with the results of both SEM and analysis of serial paraffin slices validate the premise that the central protrusion of the disk is responsible for the initiation of the erect frond. These data also illustrate the potential regeneration capabilities of the disk. Li (1999)  described this phenomenon was described in Gracilariopsis lemaneiformis, they found that suspended small pieces cut from the disk could grow into a spherical mass of cells and then initiate many fresh erect fronds around it. In our study, the excised disk remained attached and bore some degree of polarity; thus the initiation of the erect frond always occurred from the central protrusion of the new disk (Figure 11). However, the disk has enormous potential for regeneration, so it might be feasible to take the advantage of this capability for propagation in farming.
Heterogeneous distribution of photosynthetic capabilities along the length of the young germling
This phenomenon of heterogeneous distribution of photosynthetic ability could be attributed to differences in cell functions within the young germling. Gracilaria has uniaxial construction, and the manner of growth is by the division of the apical cell. The daughter cells divided from the apical cell and then elongate and enlarge , forming an apico-basal gradient in cell age and cell volume. This gradient corresponds to the distribution of the photosynthetic activities of PSII. The rapid consecutive division of apical cells may result in the incomplete assembly of PSII, which could possibly explain the low photosynthetic activities of apical cells (Figure 14). However, after division, the daughter cells would in turn expand through the disposition of large amounts of wall material, which could be synthesized by high efficient photosynthesis (i.e. high value of Fv/Fm and Y (II)). Meanwhile, the occurrence of the hetero-distribution of PSII photosynthetic capabilities and the pit connections between the thallus cells of Gracilaria , imply that nutritive materials are transferred from basal cells to apical cells to promote their rapid division.
The PSII activities of tetraspores increased significantly after the tetraspores attached to the substrate. Attachment is likely triggered their rapid division, and consequently further development was initiated. Through one of two division types, (cruciate or zonate type), attached tetraspores could developed into tapered disk, whose central protrusion was the growth point that controlled the initiation of the erect frond. Although some degree of morphological polarity was present in the disk, the chlorophyll fluorescence analysis results showed that PSII photosynthetic capabilities within the disk were similar for all parts of the disk. When the tetraspores developed into young germlings, the PSII activities varied among the different parts of the germling.
Mature reproductive tetrasporophytes of G. vermiculophylla were collected from the intertidal zone of Zhan Shan, Qingdao (36°05'N, 120°18'E), China. Fronds were rinsed three times in sterilized seawater to remove epiphytes from the thallus surface.
Release and culture of tetraspores
For spore release, the rinsed tetrasporophytes were cultivated in sterilized Petri dishes containing PES medium  at 20°C under a photo regime of 12 hours light: 12 hours dark with light intensity of 30 μmol m-2 s-1. The fronds were removed as soon as the tetraspores were released in abundance. The culture conditions of the tetraspores were the as same as those of the fronds. The culture medium was renewed every 2 days.
Cultivation of excised disks
Disks, which developed from single-cell tetraspores 2 weeks after release, were excised in situ. The remaining part adhering to the slide was cultivated continuously in the same constant culture conditions. Morphological and photosynthetic changes in the remainders were photographed and described at 24 hours intervals.
Scanning Electronic Microscopy (SEM)
Developing disks were observed by SEM, and the samples for SEM were treated as follows: Disks were detached from the slides and fixed with 0.1 M PBS (pH 7.2) containing 5% glutaraldehyde for 120 min. The samples then were rinsed four times with 0.1 M PBS, resuspended in an ethanol gradient for dehydration, and dried with a carbon dioxide critical point drier (HPC-2) (Hitach Co., Japan). An ion coater (IB-3) (Eiko Engineering, Ibaraki, Japan) was used to sputter metals onto the samples' surface. The samples then were observed and photographed using a KYKY2800B SEM (Kyky, Beijing, China).
Preparation of serial paraffin slices
Detached disks of various sizes were fixed in 4% formaldehyde solution for 12 hours. They then were dehydrated in 70%, 83%, and 95% ethanol for 1 hour each, followed by 100% ethanol for 30 min. Before embedding in paraffin, the samples were treated as follows: ethanol/xylene (1:1) for 1 hour, 100% xylene for 30 min, xylene/paraffin for 20 min, and 100% paraffin for 20 min. The paraffin-embedded disks were sliced at a thickness of 10 μm with a Leica 2016 microtome (Leica Instruments Ltd., Shanghai, China).
Measurement of photosynthetic parameters
Photosynthetic capability was determined by analysis of chlorophyll fluorescence, which was measured using a microscopy-PAM apparatus (Walz GmbH, Effeltrich, Germany). Imagingwin 2.21d software was used to set the parameters and analyze the fluorescence data. Before measurement, the samples, which included single-cell tetraspores, disks, and young germlings detached from the substrate, were kept in the dark for 15 min. The dark fluorescence yield, F0, was determined under low measuring light, and then a saturation pulse was applied to assess the maximal fluorescence (Fm). The Fm yield in illuminated samples was denoted as Fm'. The maximal PSII quantum yield (Fv/Fm) was calculated using the equation: Fv/Fm = (Fm - F0)/Fm. The effective PSII quantum yield was calculated as follows: Y (II) = (Fm' - F)/Fm', where the F is real time fluorescence yield. All measurements were performed at room temperature.
The data were analyzed with SPSS 13.0 (SPSS Inc., Chicago, IL). Statistical significance was tested by one-way ANOVA. All values were expressed as means ± s.d. The graphs were plotted with OriginPro 8.0 (OriginLab Corporation, Northampton, USA).
The work was supported by grants from the National Natural Science Foundation of China (30830015), the 863 Project of China (No. 2006AA10A413) and Project for Agriculture Transformation (2007GB24910418).
- Mimuro M: Photo capture, exciton migration and trapping and fluorescence emission in cyanobacteria and red algae. Chlorophyll a fluorescence: a signature of photosynthesis. Edited by: Papageorgiou GC, Govindjee. 2004, The Netherlands: Springer, 173-195.View ArticleGoogle Scholar
- Breeman AM, Hoopen At: The mechanism of daylength perception in the red alga Acrosymphyton purpuriferum. Journal of Applied Phycology. 1986, 23: 36-42.View ArticleGoogle Scholar
- Dickson LG, Waaland JR: Porphyra nereocystis: a dual-daylength seaweed. Planta. 1985, 165: 548-553. 10.1007/BF00398102.View ArticlePubMedGoogle Scholar
- Guiry MD: Sporangia and spores. Biology of the Red Algae. Edited by: Cole KM, Sheath RG. 1990, New York: Cambridge university press, 347-376.Google Scholar
- Gabrielson PW, Garbary D: Systematics of red algae (Rhodophyta). Critical Reviews in Plant Sciences (USA). 1986, 3: 325-366. 10.1080/07352688609382215.View ArticleGoogle Scholar
- Coomans RJ, Hommersand MH: Vegetative growth and organization. Biology of the Red Algae. Edited by: Cole KM, Sheath RG. 1990, New York: Cambridge University Press, 275-304.Google Scholar
- Cole K, Conway E: Studies in the Bangiaceae: reproductive modes. Bot Mar. 1980, 23: 545-553.Google Scholar
- Fritsch FE: The Structure and Reproduction of the Algae. 1945, Combridge: Combridge University PressGoogle Scholar
- Dixon PS: Biology of the Rhodophyta. 1973, New York: Hafner PressGoogle Scholar
- Mshigeni KE, Lorri WSM: Spore germination and early stages of development in Hypnea musciformis (Rhodophyta, Gigartinales). Marine Biology. 1977, 42: 161-164. 10.1007/BF00391567.View ArticleGoogle Scholar
- Ye N, Wang H, Wang G: Formation and early development of tetraspores of Gracilaria lemaneiformis (Gracilaria, Gracilariaceae) under laboratory conditions. Aquaculture. 2006, 254: 219-226. 10.1016/j.aquaculture.2005.08.026.View ArticleGoogle Scholar
- Chen LCM, Taylor ARA: Scanning electron microscopy of early sporeling ontogeny of Chondrus crispus. Canadian Journal of Botany. 1976, 54: 672-678. 10.1139/b76-071.View ArticleGoogle Scholar
- Rietema H, Klein AWO: Environmental control of the life cycle of Dumontia contorta (Rhodophyta) kept in culture. Mar Ecol Prog Ser. 1981, 4: 23-29. 10.3354/meps004023.View ArticleGoogle Scholar
- Marinho-Soriano E, Bourret E: Effects of season on the yield and quality of agar from Gracilaria species (Gracilariaceae, Rhodophyta). Bioresource Technology. 2003, 90: 329-333. 10.1016/S0960-8524(03)00112-3.View ArticlePubMedGoogle Scholar
- Li H, Yu X, Jin Y, Zhang W, Liu Y: Development of an eco-friendly agar extraction technique from the red seaweed Gracilaria lemaneiformis. Bioresource Technology. 2008, 99: 3301-3305. 10.1016/j.biortech.2007.07.002.View ArticlePubMedGoogle Scholar
- Blunden G, Guiry MD, Blunden G: Seaweed resources in Europe: uses and potential. 1991, Chichester, J. Wiley and sonsGoogle Scholar
- Gurgel CFD, Fredericq S: Systematics of the Gracilariaceae (Gracilariales, Rhodophyta): a critical assessment based on rbcL sequence analyses. Journal of Phycology. 2004, 40: 138-159. 10.1111/j.1529-8817.2004.03070.x.View ArticleGoogle Scholar
- Engel C, Gaggiotti OE, Destombe C, Valero M: Population dynamics and stage structure in a haploid-diploid red seaweed, Gracilaria gracilis. Journal of Ecology. 2001, 89: 436-450. 10.1046/j.1365-2745.2001.00567.x.View ArticleGoogle Scholar
- Polifrone M, De Masi F, Gargiulo GM: Alternative pathways in the life history of Gracilaria gracilis (Gracilariales, Rhodophyta) from north-eastern Sicily (Italy). Aquaculture. 2006, 261: 1003-1013. 10.1016/j.aquaculture.2006.08.026.View ArticleGoogle Scholar
- Ogata E, Matsui T, Nakamura H: The life cycle of Gracilaria verrucosa (Rhodophyceae, Gigartinales) in vitro. Phycologia. 1972, 11: 75-80. 10.2216/i0031-8884-11-1-75.1.View ArticleGoogle Scholar
- Plastino EM, Oliveira FEC: Deviations in the life-history of Gracilaria sp. (Rhodophyta, Gigartinales), from Coquimbo, Chile, under different culture conditions. Hydrobiologia. 1988, 164: 67-74. 10.1007/BF00014350.View ArticleGoogle Scholar
- Destombe C, Valero M, Vernet P, Couvet D: What controls haploid-diploid ratio in the red alga, Gracilaria verrucosa?. Journal of Evolutionary Biology. 1989, 2: 317-338. 10.1046/j.1420-9101.1989.2050317.x.View ArticleGoogle Scholar
- Jones WE: Effect of Spore Coalescence on the Early Development of Gracilaria verrucosa (Hudson) Papenfuss. Nature. 1956, 426-427. 10.1038/178426b0.Google Scholar
- Terada R, Yamamoto H: A taxonomic study on two Japanese species of Gracilaria: Gracilaria shimodensis sp. nov. and Gracilria blodgettii (Gracilariales, Rhodophyta). Phycological Research. 2000, 48: 189-198. 10.1111/j.1440-1835.2000.tb00215.x.View ArticleGoogle Scholar
- Fan X, Wang G, Li D, Xu P, Shen S: Study on early-stage development of conchospore in Porphyra yezoensis Ueda. Aquaculture. 2008, 278: 143-149. 10.1016/j.aquaculture.2008.02.037.View ArticleGoogle Scholar
- Bouzon ZL, Ouriques LC, Oliveira EC: Spore adhesion and cell wall formation in Gelidium floridanum (Rhodophyta, Gelidiales). Journal of Applied Phycology. 2006, 18: 287-294. 10.1007/s10811-006-9028-8.View ArticleGoogle Scholar
- Pueschel CM: Ultrastructure of tetrasporogenesis in Palmaria palmata (Rhodophyta). Journal of Phycology. 1979, 15: 409-424.View ArticleGoogle Scholar
- Baker JRJ, Evans LV: The ship fouling alga ectocarpus I ultrastructure and cytochemistry. Protoplasma. 1973, 77: 1-13. 10.1007/BF01287289.View ArticleGoogle Scholar
- Callow ME, Evans LV: Studies on the ship-fouling alga Enteromorpha III. cytochemistry and autoradiography of adhesive production. Protoplasma. 1974, 80: 15-27. 10.1007/BF01666348.View ArticleGoogle Scholar
- Li R: Study on characteristics of creeping tissue of Gracilariopsis lemaneiformis. Oceanologia et Limnologia Sinica. 1999, 30: 41-44.Google Scholar
- Wetherbee R, Quirk HM: The fine structure and cytology of the association between the parasitic red alga Holmsella australis and its red algal host Gracilaria furcellata. Protoplasma. 1982, 110: 153-165. 10.1007/BF01283318.View ArticleGoogle Scholar
- Wetherbee R, Quirk HM: The fine structure of secondary pit connection formation between the red algal alloparasite Holmsella australis and its red algal host Gracilaria furcellata. Protoplasma. 1982, 110: 166-176. 10.1007/BF01283319.View ArticleGoogle Scholar
- Wang S, Pei L, Duan D: Rhodophyta. Research on the ultrastructure of economic algae in China. 2004, Zhejiang Science & Technology Press, 151-159.Google Scholar
- Dixon PS: Cell enlargement in relation to the development of thallus form in Florideophyceae. European Journal of Phycology. 1971, 6: 195-205. 10.1080/00071617100650221.Google Scholar
- McLachlan J: Growth media-marine. Handbook of phycological methods. Culture methods and growth measurements. Edited by: Stein JR. 1979, Cambridge: Cambridge University Press, 25-51.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.