J. Ocean Univ. China (Oceanic and Coastal Sea Research) DOI 10.1007/s11802-012-1946-2 ISSN 1672-5182, 2012 11 (3): 383-388 http://www.ouc.edu.cn/xbywb/ E-mail:[email protected]
Nuclear Transition Between the Conjunction Cells of Phaeodactylum tricornutum Bohlin (Bacillariophyta)
LI Si1), PAN Kehou2), *, ZHU Baohua2), and ZHANG Lin2)
1) Key Laboratory of Marine Genetics and Breeding of Chinese Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China
2) Key Laboratory of Mariculture of Chinese Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China
Abstract Phaeodactylum tricornutum is one of the important marine diatoms for oceanic primary production. Its reproduction has profound significance in the life cycle; however, the nuclear behavior during its sexual reproduction was not clear. In this study, we observed the nuclear transition and determined its correlation with cell conjunction. It was found that two cells jointed at their apices first and swung and aligned each other immediately, and nucleus from one cell was able to transfer into another one during cell con-jugation. The cell pairs conjugated for nuclear transition were different from those formed in mitosis in hypovalve thickness and cellular arrangement. Our findings proved the existence of sexual reproduction in P. tricornutum.
Key words Phaeodactylum tricornutum; sexual reproduction; nuclear transition
1 Introduction The marine diatom Phaeodactylurn tricornutum Boh-
lin was isolated and identified by Allen and Nelson in 1910, and so far about 10 axenic strains have been iso-lated and genetically characterized (de Martino et al., 2007). Usually, P. tricornutum exhibits three morpho-logical types, fusiform, triradiate and oval, each has a unique abundance in a community (Wilson and Lucas, 1942). P. tricornutum appears in fusiform shape in sea-water; however it becomes oval when cultured on solid medium and gradually converts into fusiform and some-times triradiate when retransferred into liquid medium (Barker, 1935). It was believed that morphological con-version of P. tricornutum was caused by the marine envi-ronment condition (Wilson, 1946; Borowitzka and Vol-cani, 1978; de Martino et al., 2007), and many morpho-logical changes have been observed previously (Johansen, 1991; Gutenbrunner et al., 1994; de Martino et al., 2011). In contrast, few studies of the nuclear behavior during the reproduction have been conducted.
Sexual reproduction associated with auxosporulation has been documented in diatoms (Mann 1993), and the mating process has been observed in other diatoms (Chepurnov et al., 2002; Mann and Chepurnov, 2005; Amato et al., 2005). Unfortunately, direct evidence of nu-clear transition in P. tricornutum was not available. Here
the nuclear behavior of P. tricornutum will be studied to find direct evidence of nuclear transition in its life cycle.
2 Material and Methods P. tricornutum (MACC B228) was provided by Key
Laboratory of Mariculture of Chinese Ministry of Educa-tion, Ocean University of China. It was characterized as Chinese strain Pt10 by de Martino et al. (2007). P. tri-cornutum was cultured in 500 mL glass erlenmeyer flasks with initial inoculation of 8 × 105
cell mL-1 at 22℃±1℃ and 27.5–37.5 μmol photons m-2 s-1 with a rhythm of 12 h light and 12 h dark. The culture was shaken 3 times a day with flask position shifted simultaneously. The alga was fixed with 2.5% glutaraldehyde and stained with 0.5 mg
mL-1 propidium iodide in phosphate-buffered saline (pH 7.4) supplemented with 0.09% sodium azide, and ob-served once a day since inoculation. The nuclei of stained P. tricornutum cells were yellow sparkling spots in the green fluorescent field under a Nikon ECLIPSE 50 i mi-croscope (Nikon Instruments Co., Ltd, Shanghai, China) implemented with Pixera Pro150ES Digital Camera sys-tem (Pixera Corporation, USA). The alga at a density of above 1.2 × 107
cell mL-1 was fixed with 4% glutaraldehyde for 2 h, washed 3 times with 1.8% sucrose buffer (pH 7.4), fixed in acetone containing 1% osmium tetroxide (pH 7.4) for 3 d, washed 3 times with 1.8% sucrose buffer (pH 7.4) again, dehydrated in a graded ethanol series (10%, 20%, 30%, 50%, 70% and 100% (twice)) and 100% acetone (twice), embedded in Epon812 epoxy resin and sliced for
LI et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (3): 383-388
384
transmission electron microscopic observation. All opera-tions were carried out at 4℃. The slices were stained with uranylacetate and lead citrate and observed under a Hi-tachi H-7000 transmission electron microscope (TEM).
3 Results 3.1 Conjugation of Cells
Cell conjugation occurred during the whole culturing period. Before cytoplasmic exchange, two cells attached
each other, contacted and jointed at their apices. A dent formed in bigger apex, into which smaller one inlaid (Figs.1a and f). The jointed cells swung (Fig.1b) and aligned together (Fig.1g). The attachment at apices usu-ally disappeared after alignment (Figs.1h and i), while it was still reserved by some conjugated cells (Fig.1j). The attachment endured a great tension, which bent the fusi-form arms (Fig.1j). Occasionally, the third cell was in-volved in the conjugation either at the middle of the two apices (Figs.1c and d) or at one apex alone (Fig.1e).
Fig.1 Conjugation of P. tricornutum cells. a and b, two cells hooked at apices; c and d, three cells (a cell pair and a free cell) jointed together at different positions; e, an extra conjugation of three cells; f, a cell pair similar to that of e; g, a typical cell pair attaching each other at two apices; h and i, a typical cell conjugation attaching at the center of cells at different positions; j, an extra conjugation of three cells; scale bars = 10 µm.
3.2 Nuclear Transition and Possible Cytoplasmic Exchange
Before nuclear transition, each conjugated cell con-tained only one nucleus which appeared near the center of girdle where cells conjugated each other (Figs.2a, b). Then the nucleus in one cell (positive) moved into the other (passive) (Figs.2c, d), staying there independently (Figs.2g, h). Coexistence of two nuclei in one cell after transition was distinguishable from that of two nuclei generated in mitosis. The latter paralleled with apical axis (Fig.3c) while the former did not (Fig.2g). The fusion of two nuclei occurred soon after nuclear transition, and sometimes the empty positive cell could be observed (Figs.2g and h). After transition, only one oval cell was yielded (Figs.2i, j, k, l and m), which was immediately expanded and released (Figs.2i, j and k). The spherical cells were also found near the oval cells (Figs.2n, o and p). Nuclear transition is associated with cytoplasmic ex-change, which was not clearly observed in this study.
3.3 Structural Comparison of Cell Pairs Conjugated
for Nuclear Transition and Those Formed in Mitosis
Mitosis of P. tricornutum produces cell pairs morpho-logically similar to the conjugated cells. These two types of cell pairs were not distinguishable in bright field (Fig.3e). They were apparently different from each other in green fluorescent field and under TEM (Figs.3e and c) although their cellular diameters were similar to each other (Figs.3a and b).
The thickness of hypovalve of conjugated cells was dif-ferent from that of normal cells formed in mitosis. After mitosis, a new valve was generated with two ends of newly developed valve (sectional plane of the narrow girdle view) contacted with the epivalve of the mother cell and spread beyond the hypovalve of the mother cell (Fig.4a). Two daughter cells shared the same hypovalve (Fig.4a). Cytoplasm of daughter cells was separated. The newly developed hypovalve divided into two (Fig.4b),
LI et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (3): 383-388
385
and one of them contacted with the hypovalve of the mother cell (Fig.4b). Once new hypovalves developed completely, a spacer was left between two daughter cells (Fig.4c). Daughter cells were ruptured between mother epivalve and hypovalve (Fig.4d). The gap between mother epivalve and hypovalve became bigger (Fig.4e), yielding a cell pair finally (Fig.4f). The newly developed
hypovalve was thinner than the epivalve and hypovalve of matured cells (Fig.3a). Therefore, once conjugated cells matured, their hypovalves were the same in thick-ness as epivalves. The thickness difference between newly developed hypovalves and mature epivalve was critical in distinguishing the cell pairs conjugated for nu-clear transition from the cell pairs formed after mitosis.
Fig.2 Nuclear transition of P. tricornutum cells and formation of oval cells. a, a pair of cells with sparkling nuclei; b, the same pair of cells in bright field; c and d, nuclear transition observed in the same pair of cells; e and f, a pair of conju-gated cells after nuclear transition; i, j and k, oval cell in a ruptured passive cell; n, o and p, the spherical cell; k and l, a passive cell containing two nuclei; l and m, an oval cell was released. Scale bars = 10 µm (a, b, c, d, e, f, g, h, i, j and k) or 5 µm (l, m, n, o and p). a, c, d, e, g, i, m and n were taken from green fluorescent field; while b, f, j, l and p were taken from bright field, and h and k were taken from contrast field.
LI et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (3): 383-388
386
Fig.3 Comparison of normal cells with those formed in mitosis. a, a normal P. tricornutum cell; b, c, d and e, two daughter cells of mitosis contained in old valves; d and e, the same pair of cells. Scale bars = 1 µm (a, b) or 10 µm (c, d and e). a, b and c, transmission electron micrographs; d, in green fluorescent field; e, in bright field. a and b, sectional plane of narrow girdle view; c, sectional plane of broad girdle view; ep, epivalve; g, girdle band; hyp, hypovalve; lg, lipid granule; m, mi-tochondria; n, nucleus; pl, plastid; pyr, intraplastidial pyrenoid.
Fig.4 Valve arrangement and movement of daughter cells after mitosis. TEM photos were sectional planes of narrow gir-dle view. a through f, the procedure of two daughter cells separating from each other. Scale bars = 1 µm. ep, epivalve; g, girdle band; hyp, hypovalve; lg, lipid granule; n, nucleus; nval, new valve; nhyp, new hypovalve; pl, plastid; pyr. intra-plastidial pyrenoid.
Cells conjugated for nuclear transition were also dis-tinguishable from those formed immediately after mitosis in cellular arrangement. Cell pairs formed during mitosis overlapped the epivalves at the girdle band of mother cell (Fig.4f, Figs.5b, c and d). The cellular hypovalves of these cell pairs were separated by a spacer (Fig.3b, Figs.4c, e, f, Figs.5b, c and d: white arrow). This cellular arrangement was termed as cis position (Fig.6a). In con-
trast, cells conjugated for nuclear transition arranged their cells in trans position (Fig.6b). This arrangement short-ened the distance between cells; the epivalve of one cell seamlessly contacted with the hypovalve of the other (Fig.5a: black arrow) (Fig.5a: white arrow). The contact-ing site (white arrow) showed the fusion of cell wall and the trace of vanished part of hypovalve of the cell on the right (Fig.5a).
LI et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (3): 383-388
387
Fig.5 Structural comparison of cells conjugated for nuclear transition and those formed in mitosis. TEM photos were sec-tional planes of narrow girdle view. a, exchange of cytoplasm between conjugated cells; b, c, d, paired cells after mitosis. Scale bars = 1 µm. v, vacuole.
Fig.6 Schematic representation of the cellular arrangement of conjugated cells for nuclear transition and those after mito-sis. a, cell pair formed after mitosis, cis position; b, cell pair conjugated for nuclear transition.
4 Discussions It is clear that nuclear transition appeared during the
cellular conjunction of P. tricornutum, indirectly indicat-ing the exchange of genetic substances and the existence of sexual reproduction in this alga. Pattern of cellular conjunctions and valve arrangement after mitosis elabo-rated in this study were new complementary acknowl-edgement of the cellular characteristics of P. tricornutum.
The difference in the thickness of hypovalve and relative positions between cells can be applied to distinguishing cell pairs that conjugated for nuclear transition from those formed in mitosis.
Diatom forms cell pairs and even cellular chains in different lengths (Smetacek, 1999). This may result from the fusion of adjacent cells (Borowitzka et al., 1977) in girdle regions poor in silica but rich in organic material (Francius et al., 2008). We have observed the fusion of
LI et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2012 11 (3): 383-388
388
conjunctive cells (Fig.5a), which have been documented in previous studies. We found that cell fusion in the girdle region was more frequent than that in the remaining part. Two cells attached to each other at the central region when hooked at the apices of arms, soon leaving the api-ces free and transferring nuclear materials (Figs.2c and d). We found that one oval cell, instead of two, was released (Figs.2i, j, k, l and m), which was different from the re-ported (Lewin et al., 1958; Gutenbrunner et al., 1994; de Martino et al. 2007, 2011). Our observations supported the pattern IIA proposed by Hustedt (Mann, 1993). Fur-ther study was needed to decipher the mechanism of nu-clear transition between/among adjacent cells as were previously studied in other diatoms (e.g., Chepurnov et al., 2002; Neidium et al., 2005; Amato et al., 2005).
Acknowledgements This study was supported by the State Basic Research
and Development Program of China (973 Program) (2011- CB200901), the Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province (BS2010SW037) and the Opening Research Project of Experimental Marine Biology Laboratory, Institute of Oceanology, Chinese Academy of Sciences.
References Allen, E. J., and Nelson, E. W., 1910. On the artificial culture of
marine plankton organisms. Journal of the Marine Biological Association of the United Kingdom (New Series), 8 (5): 421- 474.
Amato, A., Orsini, L., D’Alelio, D., and Montresor, M., 2005. Life cycle, size reduction patterns, and ultrastructure of the pennate planktonic diatom Pseudo-nitzschia delicatissima (Bacillariophyceae). Journal of Phycology, 41 (5): 542-556, DOI: 10.1111/j.1529-8817.2005.00080.x.
Barker, H. A., 1935. Photosynthesis in diatoms. Archives of Microbiology, 6 (1): 141-156.
Borowitzka, M. A., Chiappino, M. L., and Volcani, B. E., 1977. Ultrastructure of a chain-forming diatom Phaeodactylum tri-cornutum. Journal of Phycology, 13 (2): 162-170.
Borowitzka, M. A., and Volcani, B. E., 1978. The polymorphic diatom Phaeodactylum tricornutum: ultrastructure of its mor-
photypes. Journal of Phycology, 14 (1): 10-21. Chepurnov, V. A., Mann, D. G., Vyverman, W., Sabbe, K., and
Danielidis, D. B., 2002. Sexual reproduction, mating system, and protoplast dynamics of Seminavis (Bacillariophyceae). Journal of Phycology, 38 (5): 1004-1019, DOI: 10.1046/j. 1529-8817.2002.t01-1-01233.x
De Martino, A., Bartual, A., Willis, A., Meichenin, A., Villazán, B., Maheswari, U., and Bowler, C., 2011. Physiological and molecular evidence that environmental changes elicit mor- phological interconversion in the model diatom Phaeodac- tylum tricornutum. Protist, 162 (3): 462-481.
De Martino, A., Meichenin, A., Shi, J., Pan, K., and Bowler, C., 2007. Genetic and phenotypic characterization of Phaeo- dactylum tricornutum (Bacillariophyceae) accessions. Journal of Phycology, 43 (5): 992-1009, DOI: 10.1111/j.1529-8817. 2007.00384.x.
Francius, G., Tesson, B., Dague, E., Martin-Jézéquel, V., and Dufrêne, Y. F., 2008. Nanostructure and nanomechanics of live Phaeodactylum tricornutum morphotypes. Environmen- tal Microbiology, 10 (5): 1344-1356.
Gutenbrunner, S. A., Thalhamer, J., and Schmid, A. M. M., 1994. Proteinaceous and immunochemical distinctions between the oval and fusiform morphotypes of Phaeodactylum tricornutum (Bacillariophyceae). Journal of Phycology, 30 (1): 129-136.
Johansen, J. R., 1991. Morphological variability and cell wall composition of Phaeodactylum tricornutum (Bacillariophy-ceae). Great Basin Naturalist, 51 (4): 310-315.
Lewin, J. C., Lewin, R. A., and Philpott, D. E., 1958. Observa-tions on Phaeodactylum tricornutum. Journal of General Mi-crobiology, 18 (2): 418-426.
Mann, D. G., 1993. Patterns of sexual reproduction in diatoms. Hydrobiologia, 269-270 (1): 11-20, DOI: 10.1007/BF00027- 999.
Mann, D. G., and Chepurnov, V. A., 2005. Auxosporulation, mating system, and reproductive isolation in Neidium (Bacil-lariophyta). Phycologia, 44 (3): 335-350.
Smetacek, V., 1999. Diatoms and the ocean carbon cycle. Pro-tist, 150 (1): 25-32.
Wilson, D. P., 1946. The triradiate and other forms of Nitzschia Closterium (Ehrenberg) Wm. Smith, Forma Minutissima of Allen and Nelson. Journal of the Marine Biological Associa-tion of the United Kingdom, 26: 235-270, DOI: 10.1017/ S002531540001211x.
Wilson, D. P., and Lucas, C. E., 1942. Nitzschia cultures at hull and at Plymouth. Nature, 149: 331-331.
