Autospore Release Research Articles

CsubMADS1, a lag phase transcription factor, controls development of polar eukaryotic microalga Coccomyxa subellipsoidea C-169.

MADS-box transcription factors (TFs) have not been functionally delineated in microalgae. In this study, the role of CsubMADS1 from microalga Coccomyxa subellipsoidea C-169 has been explored. Unlike Type II MADS-box proteins of seed plants with MADS, Intervening, K-box, and C domains, CsubMADS1 only has MADS and Intervening domains. It forms a group with MADS TFs from algae in the phylogenetic tree within the Type II MIKCC clade. CsubMADS1 is expressed strongly in the lag phase of growth. The CsubMADS1 monomer does not have a specific localization in the nucleus, and it forms homodimers to localize exclusively in the nucleus. The monomer has two nuclear localization signals (NLSs): an N-terminal NLS and an internal NLS. The internal NLS is functional, and the homodimer requires two NLSs for specific nuclear localization. Overexpression (OX) of CsubMADS1 slows down the growth of the culture and leads to the creation of giant polyploid multinucleate cells, resembling autospore mother cells. This implies that the release of autospores from autospore mother cells may be delayed. Thus, in wild-type (WT) cells, CsubMADS1 may play a crucial role in slowing down growth during the lag phase. Due to starvation in 2-month-old colonies on solid media, the WT colonies produce mucilage, whereas OX colonies produce significantly less mucilage. Thus, CsubMADS1 also negatively regulates stress-induced mucilage production and probably plays a role in stress tolerance during the lag phase. Taken together, our results reveal that CsubMADS1 is a key TF involved in the development and stress tolerance of this polar microalga.

Modification of cell volume and proliferative capacity of Pseudokirchneriella subcapitata cells exposed to metal stress

The impact of metals (Cd, Cr, Cu and Zn) on growth, cell volume and cell division of the freshwater alga Pseudokirchneriella subcapitata exposed over a period of 72h was investigated. The algal cells were exposed to three nominal concentrations of each metal: low (closed to 72h-EC10 values), intermediate (closed to 72h-EC50 values) and high (upper than 72h-EC90 values). The exposure to low metal concentrations resulted in a decrease of cell volume. On the contrary, for the highest metal concentrations an increase of cell volume was observed; this effect was particularly notorious for Cd and less pronounced for Zn. Two behaviours were found when algal cells were exposed to intermediate concentrations of metals: Cu(II) and Cr(VI) induced a reduction of cell volume, while Cd(II) and Zn(II) provoked an opposite effect. The simultaneous nucleus staining and cell image analysis, allowed distinguishing three phases in P. subcapitata cell cycle: growth of mother cell; cell division, which includes two divisions of the nucleus; and, release of four autospores. The exposure of P. subcapitata cells to the highest metal concentrations resulted in the arrest of cell growth before the first nucleus division [for Cr(VI) and Cu(II)] or after the second nucleus division but before the cytokinesis (release of autospores) when exposed to Cd(II). The different impact of metals on algal cell volume and cell-cycle progression, suggests that different toxicity mechanisms underlie the action of different metals studied. The simultaneous nucleus staining and cell image analysis, used in the present work, can be a useful tool in the analysis of the toxicity of the pollutants, in P. subcapitata, and help in the elucidation of their different modes of action.

Induction time of Fe-SOD synthesis and activity determine different tolerance of two Desmodesmus (green algae) strains to chloridazon: A study with synchronized cultures

Cells of two Desmodesmus armatus strains (276-4a and 276-4d) grown asynchronously in batch cultures after 24-h treatment revealed different tolerance to chloridazon (photosynthetic herbicide) applied at a concentration of 3.45mgL−1. To find time- and cell cycle-dependent biochemical reasons leading to such a difference, a population of young autospores of both strains synchronized by a light/dark (14/10) regime were exposed to chloridazon at the initiation of the light period. Chloridazon reduced the growth and number of divisions of cell strain 276-4d. In consequence, at the end of the dark phase the number of released autospores was reduced by 50% compared with the control. In contrast, the growth and reproductive processes of cell strains 276-4a was unaffected. Moreover, chloridazon treatment speeded up cell development, as a result of which the release of autospores took this process observed in the control cells over. There is a relationship between photosynthetic activity response to chloridazon and time-dependent changes in Fe-SOD content and activity. The energy trapped in the reaction centre (RC) was similar in both strains, but the amount of energy absorbed by RCs was twice as high in strain 276-4d as in 276-4a. In consequence, non-photochemical energy dissipation occurring in the cells of 276-4d strain far exceed the value obtained for 276-4a strain. The control cells of both strains differed significantly in the content of FSD 1 and FSD 2 proteins, whereas the differences in Fe-SOD isoforms activities were slight. The 8-fold increase in SOD content in CHD treated cells of strain 276-4a was associated with the transience of photosynthetic efficiency impairment. In CHD treated cells of strain 276-4d, neither activity of Fe-SOD nor FSDs protein content was instantly affected. Different response of developing cells of two Desmodesmus strains to CHD is influenced by the inherent features of cells; the short time required to induce stress adaptive mechanism involving chloroplastic Fe-SOD activity and biosynthesis in the cells of CHD tolerant strain 276-4a seems to play the key role, being an overriding on the high, but not induced in response to stress, FSD protein level and activity in cells of strain 276-4d.

Relationships between growth, development and photosynthetic activity during the cell cycle of Desmodesmus armatus (Chlorophyta) in synchronous cultures

The ultrastructural changes observed under an electron microscope, growth and biomass production expressed as the dry matter:cell volume ratio (DM:CV), as well as the photosynthetic activity characterized by chlorophyll fluorescence, of Desmodesmus armatus have been monitored in a synchronized light:dark (14 h:10 h) regime cell cycle. The timing of the commitment points triggering the reproductive processes and their termination were characterized by the commitment and autospore release curves, respectively. During the light period, each cell attained three commitment points in the 3rd, 6th and 11th hour, which resulted in the formation of eight autospores released from the parent cell during the dark period of the cell cycle. The successive commitment points occurred at times when DM:CV reached extreme values. The maximum DM:CV in the middle of the light period (5–6 h) coincided with the maximum effective quantum efficiency of PS II (ΦPS II). At this time, bands of several thylakoids could be seen in some areas of chloroplast, which were closer to each other and more contrasted than in other phases of the cell cycle. The highest efficiency of PS II antennae () in the 3rd hour occurred in cells with the lowest DM:CV ratio. The photochemical quenching of fluorescence (qP) increased slightly, whereas non-photochemical quenching (NPQ) rapidly decreased during the first 3 h of the cell cycle. The main contributor to NPQ was its energy-dependent component (qE). In the 11th hour, cells started to accumulate starch grains and lipid droplets, which were partly (starch) or completely (lipids) utilized during the first few hours of the next cell cycle. In conjunction with the ultrastructural changes, two functionally different phases of the cell cycle could be distinguished: the vegetative (about 10 h) and the reproductive (about 4 h) phases, and the relationships between growth, reproduction and photosynthesis in the Desmodesmus cell cycle are discussed.

Revision of the classification system of Radiococcaceae FOTT ex KOMÁREK (except the subfamily Dictyochlorelloideae) ( Chlorophyta)

Radiococcaceae (Chlorophyta) is a large and widespread family in terrestrial and freshwater habitats. The taxonomy of this family is difficult because no coherent system of generic criteria exists, fact which led to many species transfers from one genus to another with as a consequence many synonyms. Based on traditional criteria and a study of Radiococcaceae isolated from soils we propose to rearrange the generic system according to only several, easily observable features which are: 1) capacity to form spherical shaped vegetative cells, 2) presence or absence of pyrenoid, 3) number of autospores (only/mainly two or only/mainly four-eight), 4) behaviour of the sporangial cell wall during autospore release (rupture or gelatinisation). As additional secondary criteria are used 5) the capacity to form tubular branched colonies (for separation of the genus Palmodictyon), 6) number of chloroplasts (for definition of the genera Planktosphaeria and Phacomyxa), 7) capacity to form plate-like sarcinoid colonies (for the separation of the genera Dispora and Crucigloea). According to these principles 25 genera (including 10 new genera) comprising about 80 described species are recognised. The system is artifical, but allows to more easily recognise genera and species and also an easier allocation of new taxa.

Viability of dried cells, and survivability and reproduction under water stress, low light, heat, and UV exposure in Chlorella vulgaris

Dried Chlorella vulgaris Beyerinck cells were 100% viable when stored in desiccators over fused CaCl 2 for 2 days either in light or dark, but the viability decreased to1% after 30 days at 20 C in light. Neither dried nor wet cells remained viable after10 days at 0 C in dark. Tolerance of C. vulgaris cells to dry storage might be dueeither to their unicellular nature or to the chemical composition of cell wall containing sporopollenin. Osmotic regulation did not seem to occur, as the alga was salt-sensitive. Autospores or young vegetative cells were more sensitive than autospore mother cells to both physical and physiological water stress, imposed respectively by growing them on media with a high agar content or in salinized liquid media. The rupture of autospore mother cells and release of autospores did not occur even in100 mM NaCl. The vegetative cells remained viable in darkness at 22 1 C for 45days, but the formation of autospore mother cells required light intensities of more than 10 mmol m –2 s –1 . The vegetative cells were sensitive to heat or UV.

Photoacclimation of Chlorella vulgaris to Red Light from Light‐Emitting Diodes Leads to Autospore Release Following Each Cellular Division

AbstractThe detailed light requirement for photosynthesis and photoautotrophic cell growth can be assessed using solid state technology. Advanced light‐emitting diodes (LEDs), constructed with double‐power double‐heterostructure (DDH) gallium aluminum arsenide (GaAlAs) chips, were examined for their ability to support mass culture of the eucaryotic alga Chlorella vulgaris. LEDs with peak emittance of 680 nm (with half‐power band width of 20 nm) were used as a sole light source for the cultivation of C. vulgaris. Fluorescent light (FL) served as a control. The final cell mass and specific cellular growth rate under LEDs were comparable to those obtained under full‐spectrum light (FL). The narrow‐spectrum monochromatic red light was found to reduce the average cell volume from 60 μm3 to 30 μm3, and to make the size distribution and the per cell DNA distribution narrower, but did not affect the total biomass production. By switching light sources, the two distinct cell population states (obtained under red LEDs and FL, respectively) were found to be interchangeable. Two parametric flow cytometric analyses showed that the cells grown under red LED light had a more uniform DNA content at all cell sizes, as compared to cells grown under FL. These results show that the critical cell size for releasing autospores under red LED is smaller than that under FL. The number of autospores in one mother cell when grown under LED light appeared to be two, so that the mother cells break up after only one round of DNA replication. Although the solid state LED light source altered the cell cycle behavior of C. vulgaris, it can be used as an effective light source for autotrophic growth. Use of LEDs therefore promises to advance the current state of algal photobioreactors due to their efficiency, smallness, reliability, long lifetime, and desirable light characteristics.

Metabolic response of Chlorella emersonii to the herbicide sulfometuron methyl

Although it is clear that acetohydroxy acid synthase (AHAS; EC 4.1.3.18) is the target for sulfonylurea herbicides such as sulfometuron methyl (SMM), there is considerable uncertainty as to the mechanism(s) by which inhibition of AHAS inhibits or kills cells. We have further studied the mode of action of SMM, and its effects on metabolism and physiology in the unicellular green alga Chlorella emersonii var. emersonii. Addition of SMM to cells synchronized to a cycle of 16 h light-8 h dark showed that they were very sensitive to SMM toxicity in the first 16 h of the cell cycle, during which cell mass, protein and DNA increased. The increase in protein, DNA and chlorophyll was halted rapidly after SMM addition. Sulfometuron methyl prevented cell division even if added late in the light stages, when most of the protein and DNA were already synthesized, but did not affect cell division and autospore release if added after protein and DNA synthesis were complete. This suggests that SMM interferes with processes involved in preparation for division, beyond what would be expected if the cells were starved of the branched-chain amino acids needed as precursors for synthesis of proteins in general. The accumulation of α-ketobutyrate (αKB) in the cells in response to addition of SMM, and its possible role in the growth inhibition, was also investigated (in continually illuminated cultures). Intracellular αKB accumulated rapidly within 30 min of SMM addition, but declined nearly to basal levels in several hours. This paralleled the decrease and subsequent recovery of extractable AHAS activity. Despite this, growth of the algal culture did not recover. We suggest that metabolites formed by misincorporation of αKB in place of α-ketoisovalerate (e.g., in the ketopantoate hydroxymethyl transferase reaction) might be responsible for the persistence of growth inhibition. We note that an important difference between the effect of SMM and that observed with externally added αKB is that the ratio between intracellular αKB and α-ketoisovalerate is expected to be high in the first case, but not necessarily in the second.

TRIGLYCERIDE ACCUMULATION AND FATTY ACID PROFILE CHANGES IN CHLORELLA (CHLOROPHYTA) DURING HIGH pH‐INDUCED CELL CYCLE INHIBITION1

ABSTRACTAlkaline pH stress resulted in triglyceride (TG) accumulation in Chlorella CHLOR1 and was independent of medium nitrogen or carbon levels. Based on morphological observations, alkaline pH inhibited autospore release, thus increasing the time for cell cycle completion. Autospore release has been postulated to coincide with TG utilization within the microalgal cell division cycle. The alkaline pH stress affected lipid accumulation by inhibiting the cell division cycle prior to autospore release and, therefore, prior to TG utilization. Cells inhibited in this manner showed an increase in TG accumulation but a decrease in both membrane lipid classes (glycolipid and polar lipid). Unlike TG fatty acid profiles, membrane lipid fatty acid profiles were not stable during TG accumulation. The membrane profiles became similar to the TG, i.e. less unsaturated than in the membrane lipids of unstressed control cells.

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and β-1,4-mannanase activity coeluted in gel filtration and were separated from β-D-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization ≧6. The soluble degradation products of the cell wall consisted of 92-96% mannose and 4-8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.

Protein N-glycosylation and its possible role in the cell cycle of Chlorella

Crude microsomal membranes prepared from Chlorella vulgaris catalyze the synthesis of the dolichol-bound monosaccharides Dol-P-Man, Dol-P-Glc, and Dol-PP-GlcNAc. The mannosyl transferase catalyzing the transfer of mannose from GDP-Man to Dol-P increases continuously in the light-phase of the cell cycle in parallel to total protein. The rate of in vivo incorporation of [ 14C]glucosamine into glycoproteins increases during the total Chlorella cell cycle; during early S-phase the relative increase based on total protein is lowest. Tunicamycin inhibits glucosamine incorporation into trichloroacetic acid (TCA) insoluble material by 60%, whereas protein-synthesis is not affected. The release of autospores is almost completely inhibited by tunicamycin (20 μg/ml), when the antibiotic is added to a synchronous culture before the S-phase. Glycoproteins seem to be required for the initiation of DNA-synthesis in Chlorella, as has been postulated previously for other cells.

Polyamine Biosynthetic Enzymes in the Cell Cycle of Chlorella

During the life cycle of Chlorella vulgaris Beijerinck var vulgaris fa. vulgaris growing synchronously, the specific activity of ornithine decarboxylase peaked at the 2nd hour of the cycle, whereas that of arginine decarboxylase changed only slightly, increasing towards the end of the cycle. The endogenous level of putrescine and spermidine on a per cell basis increased gradually up to the 8th hour of the cycle, and declined thereafter. Thus, the peak of ornithine decarboxylase activity and the polyamine increase preceded both DNA replication (which took place between the 6th and 8th hours of the cycle) and autospore release (which started at the 8th hour). A 2-fold increase in the light intensity caused doubling of the DNA content, resulting in doubling of the number of autospores per mother cell. It also brought about a 2-fold increase in the specific activity of ornithine decarboxylase and polyamine content, the peaks being at the same hour of the cycle under high and low light intensities. The increase in cell number and polyamine content in a Chlorella culture grown under high light intensity was inhibited by alpha-difluoromethyl ornithine, a specific inhibitor of ornithine decarboxylase, this inhibition being partially reversed by putrescine.It is suggested that in C. vulgaris the sequence of events which relates polyamine biosynthesis to cell division is as follows: increased ornithine decarboxylase activity, accumulation of polyamines, DNA replication, and autospore release.

Studies on the control of cell division in a green alga,Ankistrodemus gracilids (Chlorococcales)

The proliferation patterns of the green alga,Ankistrodesmus gracilis were investigated by electron microscopy. The multichotomically dividing phase, namely the type I phase was characterized by multichotomical nuclear division, whereas the dichotomically dividing phase, i.e., the type II phase was related to dichotomical nuclear division. Type I phase was controlled by a lower cell density and a diurnal light-dark rhythm of LD 14:10. Type II phase was induced by a cell density of more than 1.0×107 cells/ml. Independent of the 2 types of proliferation patterns, the diurnal light-dark rhythms related to the autospore release from a mother cell. The diurnal light-dark rhythm of LD 14∶10 induced the scattered release of autospores. On the other hand, a rhythm of LD 18∶6 induced the release of a couple of tightly adhered autospores from a mother cell which had been observed apparently as a type II cell by light microscopy.