Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.
- MeSH
- buněčná stěna chemie MeSH
- buněčný cyklus * MeSH
- časové faktory MeSH
- Chlorophyta cytologie růst a vývoj MeSH
- guanin analýza MeSH
- lipidová tělíska metabolismus MeSH
- lipidy analýza MeSH
- mikroskopie * MeSH
- polyfosfáty analýza MeSH
- Ramanova spektroskopie * MeSH
- škrob analýza MeSH
- velikost buňky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
A terrestrial green microalga was isolated at Ås, in Akershus County, Norway. The strain corresponded to a coccoid chlorophyte. Morphological characteristics by light and electron microscopy, in conjunction with DNA amplification and sequencing of the 18 s rDNA gene and ITS sequences, were used to identify the microalgae. The characteristics agree with those of the genus Coelastrella defined by Chodat, and formed a sister group with the recently described C. thermophila var. globulina. Coelastrella is a relatively small numbered genus that has not been observed in continental Norway before; there are no previous cultures available in collections of Norwegian strains. Gas chromatography analyses of the FAME-derivatives showed a high percentage of polyunsaturated fatty acids (44-45%) especially linolenic acid (C18:3n3; 30-34%). After the stationary phase, the cultures were able to accumulate several carotenoids as neoxanthin, pheophytin a, astaxanthin, canthaxanthin, lutein, and violaxanthin. Due to the scarcity of visual characters suitable for diagnostic purposes and the lack of DNA sequence information, there is a high possibility that species of this genus have been neglected in local environmental studies, even though it showed interesting properties for algal biotechnology.
- MeSH
- biologické pigmenty analýza MeSH
- biotechnologie MeSH
- Chlorophyta klasifikace cytologie genetika MeSH
- druhová specificita MeSH
- feofytiny analýza MeSH
- fylogeneze * MeSH
- karotenoidy analýza MeSH
- kyselina alfa-linolenová analýza MeSH
- mastné kyseliny analýza MeSH
- mikrořasy klasifikace cytologie genetika izolace a purifikace MeSH
- ribozomální DNA MeSH
- RNA ribozomální 18S genetika MeSH
- xanthofyly MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Norsko MeSH
Most cells divide into two daughter cells; however, some green algae can have different division patterns in which a single mother cell can sometimes give rise to up to thousands of daughter cells. Although such cell cycle patterns can be very complex, they are governed by the same general concepts as the most common binary fission. Moreover, cell cycle progression appears to be connected with size, since cells need to ensure that their size after division will not drop below the limit required for survival. Although the exact mechanism that lets cells measure cell size remains largely unknown, there have been several prominent hypotheses that try to explain it.
The progression of the cell cycle in green algae dividing by multiple fission is, under otherwise unlimited conditions, affected by the growth rate, set by a combination of light intensity and temperature. In this study, we compared the cell cycle characteristics of Desmodesmus quadricauda at 20 °C or 30 °C and upon shifts between these two temperatures. The duration of the cell cycle in cells grown under continuous illumination at 20 °C was more than double that at 30 °C, suggesting that it was set directly by the growth rate. Similarly, the amounts of DNA, RNA, and bulk protein content per cell at 20 °C were approximately double those of cells grown at the higher temperature. For the shift experiments, cells grown at either 20 °C or 30 °C were transferred to darkness to prevent further growth, and then cultivated at the same or the other temperature. Upon transfer to the lower temperature, fewer nuclei and daughter cells were produced, and not all cells were able to finish the cell cycle by division, remaining multinuclear. Correspondingly, cells placed in the dark at the higher temperature divided faster into more daughter cells than the control cells. These differences correlated with shifts in the preceding cyclin-dependent kinase activity, suggesting that cell cycle progression was not related to growth rate or cell biomass but correlated with cyclin-dependent kinase activity.
A simultaneous application of negative phase contrast and polarization microscopy was used to study the internal structure of microbial cells. Negative phase contrast allowed us to display the fine cell structures with a refractive index of light approaching that of the environment, e.g., the cytoplasm, and converted an invisible phase image to a visible amplitude one. In the polarizing microscope, cross-polarizing filters, together with first-order quartz compensator and a turntable, showed maximum birefringence of individual structures. Material containing algae was collected in ponds in the villages Sýkořice and Zbečno (Protected Landscape Area Křivoklátsko). Objects were studied in a laboratory microscope (Carl Zeiss Jena, type NfpK), equipped with a basic body In Ph 160 with an exchangeable module Ph, LOMO St. Petersburg turntable mounted on a centering holder of our own construction and a Nikon D 70 digital SLR camera. Anisotropic granules were found only in the members of two orders of algae (Euglenales, Euglenophyceae and Chlorococcales, Chlorophyceae). They always showed strong birefringence and differed in both number and size. An important finding concerned thin pellicles in genus Euglena (Euglenales, Euglenophyceae) which exhibited weak birefringence. In genus Pediastrum (Chlorococcales, Chlorophyceae), these granules were found only in living coenobium cells. In contrast, dead coenobium cells contained many granules without birefringence-an important finding. Another important finding included birefringent lamellar structure of the transverse cell wall and weak birefringence of pyrenoids in filamentous algae of genus Spirogyra (Zygnematales, Conjugatophyceae). It was clearly displayed by the negative phase contrast and has not been documented by other methods. This method can also record the very weak birefringence of the frustule of a diatom of genus Pinnularia (Naviculales, Bacillariophyceae), which was further reinforced by the use of quartz compensator-an important finding. Simultaneous use of negative phase contrast and polarization microscopy allowed us to study not only birefringent granules of storage substances in microorganisms, but also the individual lamellae of the cell walls of filamentous algae and very thin frustule walls in diatoms. These can be visualized only by this contrast method, which provides a higher resolution (subjective opinion only) than other methods such as positive phase contrast or relief contrast.
- MeSH
- anizotropie MeSH
- biologie buňky přístrojové vybavení MeSH
- buněčná stěna chemie MeSH
- Chlorophyta chemie cytologie MeSH
- cytologické techniky metody MeSH
- cytoplazma chemie MeSH
- dvojitý lom MeSH
- Euglenida chemie cytologie MeSH
- mikroskopie fázově kontrastní * MeSH
- polarizační mikroskopie * MeSH
- rozsivky chemie cytologie MeSH
- Zygnematales chemie cytologie MeSH
- Publikační typ
- časopisecké články MeSH
The family Oocystaceae (Chlorophyta) is a group of morphologically and ultrastructurally distinct green algae that constitute a well-supported clade in the class Trebouxiophyceae. Despite the family's clear delimitation, which is based on specific cell wall features, only a few members of the Oocystaceae have been examined using data other than morphological. In previous studies of Trebouxiophyceae, after the establishment of molecular phylogeny, the taxonomic status of the family was called into question. The genus Oocystis proved to be paraphyletic and some species were excluded from Oocystaceae, while a few other species were newly redefined as members of this family. We investigated 54 strains assigned to the Oocystaceae using morphological, ultrastructural and molecular data (SSU rRNA and rbcL genes) to clarify the monophyly of and diversity within Oocystaceae. Oonephris obesa and Nephrocytium agardhianum clustered within the Chlorophyceae and thus are no longer members of the Oocystaceae. On the other hand, we transferred the coenobial Willea vilhelmii to the Oocystaceae. Our findings combined with those of previous studies resulted in the most robust definition of the family to date. The division of the family into three subfamilies and five morphological clades was suggested. Taxonomical adjustments in the genera Neglectella, Oocystidium, Oocystis, and Ooplanctella were established based on congruent molecular and morphological data. We expect further taxonomical changes in the genera Crucigeniella, Eremosphaera, Franceia, Lagerheimia, Oocystis, and Willea in the future.
Coccoid green algae traditionally classified in Dictyochloropsis have a complex, reticulate chloroplast, when mature, without a pyrenoid. They occupy remarkably diverse ecological niches as free-living organisms or in association with lichen-forming fungi and were recently shown to form two distinct lineages within Trebouxiophyceae. We used a polyphasic approach to revise the taxonomy of the genus. Based on phylogenetic analysis of the 18S rRNA gene, and detailed morphological investigation using comparative conventional light and confocal microscopy, we have assigned these lineages to two genera, Dictyochloropsis and Symbiochloris gen. nov. We have reconsidered the diagnostic generic features as follows: Dictyochloropsis comprises only free-living algae with a reticulate chloroplast, forming lobes in a parallel arrangement at some ontogenetic stages, and which reproduce only by means of autospores. This agrees with Geitler's original diagnosis of Dictyochloropsis, but not with the later emendation by Tschermak-Woess. Consequently, the species of Dictyochloropsis sensu Tschermak-Woess are assigned to Symbiochloris, with new combinations proposed. Symbiochloris encompasses free-living and/or lichenized algae with lobed chloroplasts and that reproduce by forming zoospores characterized by two subapical isokont flagella that emerge symmetrically near the flattened apex. In addition, using coalescent-based approaches, morphological characters and secondary structure of ITS transcripts, we inferred species boundaries and taxonomic relationships within the newly proposed genera. Two species of Dictyochloropsis and nine species of Symbiochloris are delimited, including the newly described species D. asterochloroides, S. handae, S. tropica, and S. tschermakiae. Our results further support the non-monophyly of autosporine taxa within Trebouxiophyceae.
Phosphorus is an essential element for life on earth and is also important for modern agriculture, which is dependent on inorganic fertilizers from phosphate rock. Polyphosphate is a biological polymer of phosphate residues, which is accumulated in organisms during the biological wastewater treatment process to enhance biological phosphorus removal. Here, we investigated the relationship between polyphosphate accumulation and electron-dense bodies in the green alga Parachlorella kessleri. Under sulfur-depleted conditions, in which some symporter genes were upregulated, while others were downregulated, total phosphate accumulation increased in the early stage of culture compared to that under sulfur-replete conditions. The P signal was detected only in dense bodies by energy dispersive X-ray analysis. Transmission electron microscopy revealed marked ultrastructural variations in dense bodies with and without polyphosphate. Our findings suggest that the dense body is a site of polyphosphate accumulation, and P. kessleri has potential as a phosphate-accumulating organism.
- MeSH
- barvení a značení MeSH
- biologické modely MeSH
- Chlorophyta cytologie růst a vývoj metabolismus ultrastruktura MeSH
- elektrony * MeSH
- fosfáty metabolismus MeSH
- lipidy chemie MeSH
- polyfosfáty metabolismus MeSH
- sekvenční analýza RNA MeSH
- síra metabolismus MeSH
- transkriptom genetika MeSH
- zobrazování trojrozměrné MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A synchronous population of cells is one of the prerequisites for studying cell cycle processes such as DNA replication, nuclear and cellular division. Green algae dividing by multiple fission represent a unique single cell system enabling the preparation of highly synchronous cultures by application of a light-dark regime similar to what they experience in nature. This chapter provides detailed protocols for synchronization of different algal species by alternating light-dark cycles; all critical points are discussed extensively. Moreover, detailed information on basic analysis of cell cycle progression in such cultures is presented, including analyses of nuclear, cellular, and chloroplast divisions. Modifications of basic protocols that enable changes in cell cycle progression are also suggested so that nuclear or chloroplast divisions can be followed separately.
- MeSH
- barvení a značení metody MeSH
- buněčné dělení MeSH
- buněčné kultury metody MeSH
- buněčný cyklus MeSH
- Chlamydomonas reinhardtii cytologie genetika růst a vývoj MeSH
- Chlorophyta cytologie genetika růst a vývoj MeSH
- chloroplasty genetika MeSH
- DNA rostlinná genetika MeSH
- fotoperioda * MeSH
- frakcionace buněk metody MeSH
- replikace DNA MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Green algae dividing by multiple fission comprise unrelated genera but are connected by one common feature: under optimal growth conditions, they can divide into more than two daughter cells. The number of daughter cells, also known as the division number, is relatively stable for most species and usually ranges from 4 to 16. The number of daughter cells is dictated by growth rate and is modulated by light and temperature. Green algae dividing by multiple fission can thus be used to study coordination of growth and progression of the cell cycle. Algal cultures can be synchronized naturally by alternating light/dark periods so that growth occurs in the light and DNA replication(s) and nuclear and cellular division(s) occur in the dark; synchrony in such cultures is almost 100% and can be maintained indefinitely. Moreover, the pattern of cell-cycle progression can be easily altered by differing growth conditions, allowing for detailed studies of coordination between individual cell-cycle events. Since the 1950s, green algae dividing by multiple fission have been studied as a unique model for cell-cycle regulation. Future sequencing of algal genomes will provide additional, high precision tools for physiological, taxonomic, structural, and molecular studies in these organisms.
