Nejvíce citovaný článek - PubMed ID 20922544
Chlamydomonas reinhardtii: duration of its cell cycle and phases at growth rates affected by light intensity
The extremophilic unicellular red microalga Galdieria sulphuraria (Cyanidiophyceae) is able to grow autotrophically, or mixo- and heterotrophically with 1% glycerol as a carbon source. The alga divides by multiple fission into more than two cells within one cell cycle. The optimal conditions of light, temperature and pH (500 µmol photons m-2 s-1, 40 °C, and pH 3; respectively) for the strain Galdieria sulphuraria (Galdieri) Merola 002 were determined as a basis for synchronization experiments. For synchronization, the specific light/dark cycle, 16/8 h was identified as the precondition for investigating the cell cycle. The alga was successfully synchronized and the cell cycle was evaluated. G. sulphuraria attained two commitment points with midpoints at 10 and 13 h of the cell cycle, leading to two nuclear divisions, followed subsequently by division into four daughter cells. The daughter cells stayed in the mother cell wall until the beginning of the next light phase, when they were released. Accumulation of glycogen throughout the cell cycle was also described. The findings presented here bring a new contribution to our general understanding of the cell cycle in cyanidialean red algae, and specifically of the biotechnologically important species G. sulphuraria.
- Klíčová slova
- Galdieria, cell cycle, cell division, growth, light intensity, red algae, synchronization, temperature, trophic regimes,
- MeSH
- buněčný cyklus fyziologie MeSH
- heterotrofní procesy fyziologie MeSH
- kultivované buňky MeSH
- mikrořasy cytologie růst a vývoj MeSH
- Rhodophyta cytologie růst a vývoj MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Multiple fission is a cell cycle variation leading to the production of more than two daughter cells. Here, we used synchronized cultures of the chlorococcal green alga Parachlorella kessleri to study its growth and pattern of cell division under varying light intensities. The time courses of DNA replication, nuclear and cellular division, cell size, total RNA, protein content, dry matter and accumulation of starch were observed at incident light intensities of 110, 250 and 500 µmol photons m-2s-1. Furthermore, we studied the effect of deuterated water on Parachlorella kessleri growth and division, to mimic the effect of stress. We describe a novel multiple fission cell cycle pattern characterized by multiple rounds of DNA replication leading to cell polyploidization. Once completed, multiple nuclear divisions were performed with each of them, immediately followed by protoplast fission, terminated by the formation of daughter cells. The multiple fission cell cycle was represented by several consecutive doublings of growth parameters, each leading to the start of a reproductive sequence. The number of growth doublings increased with increasing light intensity and led to division into more daughter cells. This study establishes the baseline for cell cycle research at the molecular level as well as for potential biotechnological applications, particularly directed synthesis of (deuterated) starch and/or neutral lipids as carbon and energy reserves.
- Klíčová slova
- Parachlorella kessleri, cell cycle pattern, deuterated lipid, deuterated starch, deuterium, energy reserves, growth processes, light intensity, reproduction events,
- MeSH
- buněčné kultury * MeSH
- buněčný cyklus * MeSH
- Chlorophyta růst a vývoj MeSH
- světlo * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including Chlamydomonas reinhardtii, a well-established model organism in cell cycle studies, are no exception. Chlamydomonas reinhardtii, a green unicellular alga of the Chlorophyceae class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in C. reinhardtii. Synchronous cultures of C. reinhardtii were cultivated in growth medium containing 70 or 90% D2O. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of C. reinhardtii, contradicting the role of the "sizer" in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.
- Klíčová slova
- Chlamydomonas reinhardtii, cell cycle, cell division, commitment point, deuterium, heavy water, multiple fission, stress,
- MeSH
- buněčné dělení účinky léků MeSH
- buněčný cyklus účinky léků MeSH
- Chlamydomonas reinhardtii růst a vývoj metabolismus MeSH
- deuterium škodlivé účinky chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- deuterium MeSH
An increase in temperature can have a profound effect on the cell cycle and cell division in green algae, whereas growth and the synthesis of energy storage compounds are less influenced. In Chlamydomonas reinhardtii, laboratory experiments have shown that exposure to a supraoptimal temperature (39 °C) causes a complete block of nuclear and cellular division accompanied by an increased accumulation of starch. In this work we explore the potential of supraoptimal temperature as a method to promote starch production in C. reinhardtii in a pilot-scale photobioreactor. The method was successfully applied and resulted in an almost 3-fold increase in the starch content of C. reinhardtii dry matter. Moreover, a maximum starch content at the supraoptimal temperature was reached within 1-2 days, compared with 5 days for the control culture at the optimal temperature (30 °C). Therefore, supraoptimal temperature treatment promotes rapid starch accumulation and suggests a viable alternative to other starch-inducing methods, such as nutrient depletion. Nevertheless, technical challenges, such as bioreactor design and light availability within the culture, still need to be dealt with.
- Klíčová slova
- Chlamydomonas reinhardtii, cell cycle, microalgae, pilot-scale production, starch, supraoptimal temperature,
- MeSH
- biomasa * MeSH
- bioreaktory MeSH
- buněčný cyklus MeSH
- Chlamydomonas reinhardtii metabolismus MeSH
- fotobioreaktory * MeSH
- kultivační média MeSH
- mikrořasy MeSH
- průmyslová mikrobiologie metody MeSH
- škrob metabolismus MeSH
- světlo MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kultivační média MeSH
- škrob MeSH
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.
- Klíčová slova
- Desmodesmus quadricauda, cell cycle, confocal Raman microscopy, guanine, lipids, microalgae, polyphosphate, starch,
- 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
- Názvy látek
- guanin MeSH
- lipidy MeSH
- polyfosfáty MeSH
- škrob MeSH
Temperature is one of the key factors affecting growth and division of algal cells. High temperature inhibits the cell cycle in Chlamydomonas reinhardtii. At 39 °C, nuclear and cellular divisions in synchronized cultures were blocked completely, while DNA replication was partly affected. In contrast, growth (cell volume, dry matter, total protein, and RNA) remained unaffected, and starch accumulated at very high levels. The cell cycle arrest could be removed by transfer to 30 °C, but a full recovery occurred only in cultures cultivated up to 14 h at 39 °C. Thereafter, individual cell cycle processes began to be affected in sequence; daughter cell release, cell division, and DNA replication. Cell cycle arrest was accompanied by high mitotic cyclindependent kinase activity that decreased after completion of nuclear and cellular division following transfer to 30 °C. Cell cycle arrest was, therefore, not caused by a lack of cyclin-dependent kinase activity but rather a blockage in downstream processes.
- Klíčová slova
- Chlamydomonas reinhardtii, DNA replication, cell cycle arrest, cell size, cyclin-dependent kinase, starch accumulation, supraoptimal temperature, synchronized cultures,
- MeSH
- bílkoviny řas metabolismus MeSH
- buněčné kultury metody MeSH
- Chlamydomonas reinhardtii cytologie fyziologie MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- down regulace MeSH
- fyziologický stres MeSH
- kontrolní body buněčného cyklu * MeSH
- regulace genové exprese u rostlin MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bílkoviny řas MeSH
- cyklin-dependentní kinasy 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.
BACKGROUND: Nitrogen starvation is known to cause drastic alterations in physiology and metabolism leading to the accumulation of lipid bodies in many microalgae, and it thus presents an important alternative for biofuel production. However, despite the importance of this process, the molecular mechanisms that mediate the metabolic remodeling induced by N starvation and especially by stress recovery are still poorly understood, and new candidates for bioengineering are needed to make this process useful for biofuel production. RESULTS: We have studied the molecular changes involved in the adaptive mechanisms to N starvation and full recovery of the vegetative cells in the microalga Chlamydomonas reinhardtii during a four-day time course. High throughput mass spectrometry was employed to integrate the proteome and the metabolome with physiological changes. N starvation led to an accumulation of oil bodies and reduced Fv/Fm.. Distinct enzymes potentially participating in the carbon-concentrating mechanism (CAH7, CAH8, PEPC1) are strongly accumulated. The membrane composition is changed, as indicated by quantitative lipid profiles. A reprogramming of protein biosynthesis was observed by increased levels of cytosolic ribosomes, while chloroplastidic were dramatically reduced. Readdition of N led to, the identification of early responsive proteins mediating stress recovery, indicating their key role in regaining and sustaining normal vegetative growth. Analysis of the data with multivariate correlation analysis, Granger causality, and sparse partial least square (sPLS) provided a functional network perspective of the molecular processes. Cell growth and N metabolism were clearly linked by the branched chain amino acids, suggesting an important role in this stress. Lipid accumulation was also tightly correlated to the COP II protein, involved in vesicle and lysosome coating, and a major lipid droplet protein. This protein, together with other key proteins mediating signal transduction and adaption (BRI1, snRKs), constitute a series of new metabolic and regulatory targets. CONCLUSIONS: This work not only provides new insights and corrects previous models by analyzing a complex dataset, but also increases our biochemical understanding of the adaptive mechanisms to N starvation in Chlamydomonas, pointing to new bioengineering targets for increased lipid accumulation, a key step for a sustainable and profitable microalgae-based biofuel production.
- Publikační typ
- časopisecké články MeSH
Synchronized cultures of the green alga Chlamydomonas reinhardtii were grown photoautotrophically under a wide range of environmental conditions including temperature (15-37 °C), different mean light intensities (132, 150, 264 μmol m⁻² s⁻¹), different illumination regimes (continuous illumination or alternation of light/dark periods of different durations), and culture methods (batch or continuous culture regimes). These variable experimental approaches were chosen in order to assess the role of temperature in the timing of cell division, the length of the cell cycle and its pre- and post-commitment phases. Analysis of the effect of temperature, from 15 to 37 °C, on synchronized cultures showed that the length of the cell cycle varied markedly from times as short as 14 h to as long as 36 h. We have shown that the length of the cell cycle was proportional to growth rate under any given combination of growth conditions. These findings were supported by the determination of the temperature coefficient (Q₁₀), whose values were above the level expected for temperature-compensated processes. The data presented here show that cell cycle duration in C. reinhardtii is a function of growth rate and is not controlled by a temperature independent endogenous timer or oscillator, including a circadian one.
