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ISEP XIV |
1 Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70503-2451, USA
2 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803-0001, USA
3 Department of Oceanography and Coastal Sciences, Louisiana State University, Baton Rouge, LA 70803-0001, USA
Correspondence
Russell L. Chapman
chapman{at}lsu.edu
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Published online ahead of print on 25 October 2002 as DOI 10.1099/ijs.0.02561-0.
The GenBank/EMBL/DDBJ accession numbers for the phragmoplastin sequences of Bazzania trilobata, Cephaleuros virescens (South Africa), Cephaleuros virescens (USA) and Trentepoholia iolithus are respectively AY146407–AY146410.
Present address: Department of Biological Sciences, The University of Alabama, PO Box 870345, 425 Scientific Collections Bldg, Tuscaloosa, AL 35487-0345, USA. 
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), Charales (Pickett-Heaps, 1967) and Zygnematales (Fowke & Pickett-Heaps, 1969a, b; Grolig, 1992); it has not been documented in Chlorokybales or Klebsormidiales. Generally, phragmoplast-mediated cell division is not found in taxa belonging to the other lineage of the green plant dichotomy, the chlorophytes.
Some green algae in the order Trentepohliales belie this neat dichotomy, however. Although the taxonomic affiliation of this subaerial green algal group was once problematic, ultrastructural (Sluiman, 1989) and molecular phylogenetic studies of the 18S rRNA (Zechman et al., 1990; López-Bautista et al., 2002) have positioned the order firmly inside the ulvophycean clade, a mainly marine algal group related to the chlorophycean (rather than charophycean) lineage of green algae. Chapman & Henk (1986), however, documented phragmoplast-type cell division in Cephaleuros parasiticus Karsten, and immunofluorescence cytochemical and ultrastructural studies (Chapman et al., 2001) confirmed the presence of a phragmoplast-type cytokinesis in Trentepohlia odorata (Wiggers) Whitrock.
The presence of a phragmoplast-type cytokinesis in just a few algae of the chlorophycean lineage remains an unexplained evolutionary event, leading Chapman et al. (2001) to ask: is the phragmoplast-mediated cell division in the Trentepohliales the same as (i.e. homologous to) that in the streptophytes or is it a functionally similar but different (i.e. non-homologous) cellular process? Although major developmental patterns in the phragmoplast-mediated cytokinesis of the Trentepohliales and the land plants are similar, important differences have been reported (Chapman et al., 2001). Based on the particular temporal separation of karyokinesis and cytokinesis, the double cone microtubular structure and the centripetal coalescence of phragmoplast vesicles found in the Trentepohliales and not shared by the streptophytes, one could suggest parallel evolution of phragmoplast-mediated cytokinesis in the Trentepohliales.
Gu & Verma (1996) reported that a plant dynamin-like (PDL) protein isolated from soybean was associated with cell-plate formation during cytokinesis. They termed this PDL protein ‘phragmoplastin’ and concluded that it is associated with exocytic vesicles that deposit cell-plate materials during cytokinesis. Later studies (Gu & Verma, 1997) suggested that phragmoplastin was concentrated in the area where membrane fusion is active and that phragmoplastin participates in an early membrane fusion event during cell-plate formation. Zhang et al. (2000) demonstrated that phragmoplastin polymerizes into spiral coiled structures, suggesting that it may assemble into helical arrays wrapping around and squeezing vesicles into vesicle–tubule–vesicle structures during cell-plate formation. In a recent review, Verma (2001) named phragmoplastin as a marker for the phragmoplast and initiation of the cell plate.
Therefore, the key to understanding the evolutionary history of the phragmoplast cytokinetic system may lie in the study of phragmoplastin. Previous research on phragmoplastin has only involved higher land plants, so the important first step is to determine whether the phragmoplastin gene is also found in more basal taxa. In this research note, we report the results of preliminary research designing primers and amplifying the phragmoplastin gene from three algal taxa and one liverwort.
Phragmoplastin sequences were obtained from GenBank for two isolates of Glycine max (U25547 and U36430), Arabidopsis thaliana (L36939) and Nicotiana tabacum (AJ244024). DNA samples were obtained from Bazzania trilobata (L.) S. Gray (liverwort) (provided by Yin-Long Qui; collected by Karen Renzaglia, s.n.) and Chlamydomonas sp. D66. Plant samples were Trentepohlia iolithus (L.) Wallroth from Ireland and two isolates of Cephaleuros virescens Kunze in Fries; one from the LSU campus, Baton Rouge, USA, and isolate SAG 119.80 from Natal, South Africa.
Total cellular DNA was extracted from Trentepohlia iolithus and Cephaleuros virescens samples as described in the handbook for DNA isolation from plant tissue from the DNeasy Plant Mini kit (Qiagen). The nucleic acid pellet was dissolved in sterile distilled water. DNA samples were stored at -20 °C. The integrity of DNA preparations was checked by electrophoresis in agarose gels stained with ethidium bromide.
DNA amplification by PCR of the phragmoplastin gene was accomplished by using primers designed from the conserved regions of the phragmoplastin gene of Glycine max (Gu & Verma, 1996). Template DNA was amplified in 0·2 ml thin-wall PCR tubes in a total reaction volume of 12 µl. Each amplification reaction consisted of 2 µl template DNA, 0·625 µl of two flanking primers, 1·25 µl each of MgCl2 (25 mM), Thermophilic DNA polymerase 10x buffer and 1 mM dNTPs, 0·1 µl Taq DNA polymerase (New England Biolabs) and sterile distilled water. Negative controls without DNA templates were included.
Amplification cycles were controlled in a GeneAmp PCR System 2400 (Perkin-Elmer) thermocycler. An initial denaturation step at 95 °C for 3 min was followed by five cycles, each consisting of 1 min at 95 °C, primer annealing at 40 °C for 1 min and an extension step at 68 °C for 2 min. Thirty-five cycles followed consisting of 1 min at 95 °C, primer annealing at 42 °C for 1 min and an extension step at 70 °C for 2 min.
Amplified products and a standard 1 kb DNA ladder (Life Technologies) were subjected to electrophoresis on 0·8 % agarose gels stained with ethidium bromide and analysed for correct length, yield and purity. PCR products were purified through a low-melting-point agarose gel (SeaPlaque agarose BMA). Embedded PCR products were viewed under UV illumination, separated with sterile razor blades and transferred to 0·5 ml centrifuge tubes. Agarose digestion was performed in a thermocycler for 5 min at 70 °C followed by 42 °C with 0·5 µl Gelase for 2 h. PCR products were checked for concentration in an agarose gel against a DNA ladder (see above).
Purified PCR amplification products were sequenced with the same PCR primers using Big-Dye terminator cycle-sequencing ready reactions (Applied Biosystems) in an ABI 3100 Prism automated sequencer. DNA sequences were captured as both text and colour-coded electrophoregrams. Sequence data were visualized using Sequencher and as NBRF files. An alignment was performed using Sequencher and visually inspected using MacClade 4 (Maddison & Maddison, 2002); the alignment is available upon request from the authors. Phylogenetic analyses were performed using the computer program PAUP* 4.0b10 (Swofford, 2002) for phylogenetic reconstruction. Given the number of taxa under analysis (Swofford et al., 1996), an exhaustive search from the parsimony criterion was selected (Nei & Kumar, 2000). As a measure of internal support, a bootstrap resampling technique was used (Graur & Li, 2000) based on 10 000 resampling replications with a 50 % majority-rule consensus tree selected. Tree length and consistency (CI) and retention (RI) indexes were also calculated.
The successfully designed primers were based on conserved regions of the phragmoplastin sequence of Glycine max (GenBank accession no. U25547). The forward primer (FRAFOR; 5'-CCTAATCTCTTTGGTCAACAAAAT-3', 24 bases) anneals to Glycine max positions 183–206. The reverse primer (FRAREV; 5'-GGTTGACCCTCTACAGCTACT-3', 21 bases) anneals to Glycine max positions 636–616.
PCR products were obtained for Trentepohlia iolithus, two isolates of Cephaleuros virescens and Bazzania trilobata. PCR products, when compared to a standard DNA ladder, showed a size of 
500 bp, the expected size for the section of the phragmoplastin gene under amplification. All sequences obtained were submitted to BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), and the results confirmed the similarity to phragmoplastin sequences previously deposited in GenBank.
Efforts to amplify the phragmoplastin gene from Chlamydomonas sp. failed, despite attempts using many different PCR conditions. Although there are other possible reasons for the negative results, they may be related to the mode of cell division in Chlamydomonas, which is characterized by phycoplast-mediated cytokinesis. Chlamydomonas is assigned to the Volvocales, an order that molecular phylogenetic studies (Buchheim et al., 1990) have positioned in the class Chlorophyceae (chlorophyte lineage rather than streptophyte lineage). This result in Chlamydomonas is interesting; if phragmoplastin is functioning mainly in the coalescence of vesicles during phragmoplast-mediated cytokinesis, its presence would not be necessary during cytokinesis in Chlamydomonas, which does not involve vesicle coalescence. It would be appropriate to look for this gene in phycoplast-containing algae that do form cell plates (such as Stigeoclonium and Oedogonium) and particularly in Spirogyra, a genus in the streptophyte lineage that undergoes cleavage, cell-plate formation and vesicle fusion (McIntosh et al., 1995), as well as all those related members of the Zygnematales (desmids and filamentous forms).
Phragmoplastin sequences from the green algae Trentepohlia iolithus and Cephaleuros virescens and the liverwort Bazzania trilobata as well as the higher land plant sequences from GenBank (Glycine, Arabidopsis and Nicotiana) were aligned and visually inspected. Land plants, represented by Glycine, Arabidopsis, Nicotiana and Bazzania, formed a group that was aligned relatively easily. The green algal sequences showed more variation, and they aligned better within the group than with the land plant sequences. Two Cephaleuros virescens isolates, one from the USA and another from South Africa, showed substantial differences in their phragmoplastin sequences despite belonging to the same species. This interesting result is not surprising. Analysis of the 18S rDNA sequences of the two samples, as well as another set of isolates from Taiwan and South Africa, indicates that this taxon may consist of different species sharing a convergent morphology (López-Bautista et al., 2002).
The parsimony analysis performed on this preliminary database is shown in Fig. 1. Although distance methods (neighbour-joining) with bootstrap resampling were also applied (data not shown), they did not resolve the tree; in contrast, parsimony methods did resolve the phylogenetic tree fully. As expected, the isolates of Glycine max form a group closely related to Arabidopsis thaliana and Nicotiana tabacum. Cephaleuros virescens from Baton Rouge, LA, USA, and from South Africa form another group closely related to Trentepohlia iolithus. The liverwort Bazzania trilobata forms an intermediate clade between the higher land plants and the green algal group.
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stated that clues to the evolution of the mitotic and cytokinetic apparatus in higher plants are found in simple land plants. We believe, however, that the solution to this puzzle resides further down the taxonomic scale, in the charophycean green algae that are basal to the streptophyte lineage and in the green algae of the chlorophyte lineage. The charophycean taxa Spirogyra and Mougeotia, for example, have shown a perhaps ‘primitive’ phragmoplast-mediated cytokinesis different from that of land plants; Coleochaete has even been reported to exhibit two types of cytokinesis, the typical phragmoplast and a variant, in the same thallus (Marchant & Pickett-Heaps, 1973; Brown et al., 1994; but see also comments on polarized cytokinesis in Doty et al., 2002). The chlorophycean Trentepohliales, with their phragmoplast-mediated cytokinesis, exhibit the ‘wrong’ type of cell division for their phylogenetic lineage! Exploration of the phragmoplastin gene may unveil the evolutionary history of the development of the phragmoplast and shed some light on the early events of colonization of the land by the green algae.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown, R. C., Lemmon, B. E. & Graham, L. E. (1994). Morphogenetic plastid migration and microtubule arrays in mitosis and cytokinesis in the green alga Coleochaete orbicularis. Am J Bot 81, 127–133.[CrossRef]
Buchheim, M. A., Turmel, M., Zimmer, E. A. & Chapman, R. L. (1990). Phylogeny of Chlamydomonas (Chlorophyta) based on cladistic analysis of nuclear 18S rRNA sequence data. J Phycol 26, 689–699.[CrossRef]
Chapman, R. L. & Henk, C. (1986). Phragmoplast in cytokinesis of Cephaleuros parasiticus (Chlorophyta) and a comparison with C. virescens vegetative cells. J Phycol 22, 83–88.
Chapman, R. L., Borkhsenious, O., Brown, R. C., Henk, M. C. & Waters, D. A. (2001). Phragmoplast-mediated cytokinesis in Trentepohlia: results of TEM and immunofluorescence cytochemistry. Int J Syst Evol Microbiol 51, 759–765.[Abstract]
Doty, K. F., Baldwin, J. L. & Cook, M. E. (2002). Cytokinesis in the charophycean alga Coleochaete (Charophyceae). In Abstracts of the Botany 2002 Annual Meeting, p. 17. Madison, WI: University of Wisconsin.
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Grolig, F. (1992). The cytoskeleton of the Zygnemataceae. In The Cytoskeleton of the Algae, pp. 165–194. Edited by D. Menzel. Boca Raton, FL: CRC Press.
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López-Bautista, J. M., Waters, D. A. & Chapman, R. L. (2002). The Trentepohliales revisited. Constancea 83.1 (http://ucjeps.berkeley.edu/constancea/83/lopez_etal/trentepohliales.html)
Maddison, D. R. & Maddison, W. P. (2002). MacClade 4: Analysis of Phylogeny and Character Evolution, version 4.0. Sunderland, MA: Sinauer Associates.
Marchant, H. J. & Pickett-Heaps, J. D. (1973). Mitosis and cytokinesis in Coleochaete scutata. J Phycol 9, 461–471.[CrossRef]
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Pickett-Heaps, J. D. (1967). Ultrastructure and differentiation in Chara (fibrosa). II. Mitosis. Aust J Biol Sci 20, 883–894.
Sluiman, H. (1989). The green algal class Ulvophyceae: an ultrastructural survey and classification. Cryptogam Bot 1, 83–84.
Swofford, D. L. (2002). PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), version 4. Sunderland, MA: Sinauer Associates.
Swofford, D. L., Olsen, G. J., Waddell, P. J. & Hillis, D. M. (1996). Phylogenetic inference. In Molecular Systematics, 2nd edn, pp. 407–514. Edited by D. M. Hillis, C. Moritz & B. K. Mable. Sunderland, MA: Sinauer Associates.
Verma, D. P. S. (2001). Cytokinesis and building of the cell plate in plants. Annu Rev Plant Physiol Plant Mol Biol 52, 751–784.[CrossRef][Medline]
Zechman, F. W., Theriot, E. C., Zimmer, E. A. & Chapman, R. L. (1990). Phylogeny of the Ulvophyceae (Chlorophyta): cladistic analysis of nuclear encoded rRNA sequence data. J Phycol 26, 700–710.[CrossRef]
Zhang, Z., Hong, Z. & Verma, D. P. S. (2000). Phragmoplastin polymerizes into spiral coiled structures via intermolecular interaction of two self-assembly domains. J Biol Chem 275, 8779–8784.
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