Cell death upon H(2)O(2) induction in the unicellular green alga Micrasterias

In the present study, we investigated whether the unicellular green alga Micrasterias denticulata is capable of executing programmed cell death (PCD) upon experimental induction, and which morphological, molecular and physiological hallmarks characterise this. This is particularly interesting as unicellular freshwater green algae growing in shallow bog ponds are exposed to extreme environmental conditions, and the capacity to perform PCD may be an important strategy to guarantee survival of the population. The theoretically 'immortal' alga Micrasterias is an ideal object for such investigations as it has served as a cell biological model system for many years and details on its growth properties, physiology and ultrastructure throughout the cell cycle are well known. Treatments with low concentrations of H(2)O(2) are known to induce PCD in other organisms, resulting in severe ultrastructural changes to organelles, as observed in TEM. These include deformation and part disintegration of mitochondria, abnormal dilatation of cisternal rims of dictyosomes, occurrence of multivesicular bodies, an increase in the number of ER compartments, and slight condensation of chromatin. Additionally, a statistically significant increase in caspase-3-like activity was detected, which was abrogated by a caspase-3 inhibitor. Photosynthetic activity measured by fast chlorophyll fluorescence decreased as a consequence of H(2)O(2) exposure, whereas pigment composition, except for a reduction in carotenoids, was the same as in untreated controls. TUNEL positive staining and ladder-like degradation of DNA, both frequently regarded as a hallmark of PCD in higher plants, could only be detected in dead Micrasterias cells.     

PCD and autophagy in the unicellular green alga Micrasterias denticulata

Programmed cell death (PCD) plays a central role in normal plant development and is also induced by various biotic and abiotic stress factors. In the unicellular freshwater green alga Micrasterias denticulata morphological and biochemical hallmarks such as the appearance of autophagosomes, increased production of ROS and degradation of genomic DNA into small fragments (“DNA laddering”) indicate PCD. Our data not only demonstrate that Micrasterias is capable of performing PCD under salt stress, but also that it is triggered by the ionic and not osmotic component of salinity. Additionally, results from the present and previous studies suggest that different inducers may lead to different cell death pathways in one and the same organism.     

Physiological and structural changes in the chloroplast of the green alga Micrasterias denticulata induced by UV-B simulation

Exposure of postmitotic growing and non-growing cells of the unicellular green alga Micrasterias denticulata to different UV-B cut-off wavelengths together with simulated sunlight in a sun simulator has revealed a marked resistence of the algae against strong irradiation. While down to a cut-off wavelength of 284 nm irradiated during the most sensitive stage of cell development chloroplast ultrastructure remains unaffected, severe changes in arrangement and structure of stroma and grana thylakoids occur only at the lowest cut-off wavelengths of 280 and 275 nm. The structural alterations end up in a more or less complete desintegration of grana and stroma thylakoids with the remaining membraneous structures appearing in negative staining thus indicating drastic changes in membrane composition. Photosynthetic activity determined by chlorophyll fluorescence (ratio of variable to maximal fluorescence) and oxygen evolution responded more sensitively to UV-B irradiation. With decreasing UV cut-off wavelengths and prolonged incubation a decrease of photochemistry of PS II occured reaching its lowest values after 60 min at 275 and 280 nm. Oxygen production was even maintained under strong UV irradiation with a cut-off wavelenght of 275 nm up to 15 min. With prolonged UV-B treatment any activity was lost. HPLC separations of pigments exhibited the appearance of break-down products (mainly derivatives of chl b and chl a) with decreasing cut-off wavelength and increasing exposure time. The xanthophyll cycle pigments seemed to be unaffected at least for an irradiation period of 60 to 90 min at low UV cut-offs. Possible mechanisms of UV stress avoidance or protection are discussed with regard to the varying altitudes of the natural habitats of the algae.     

Nitric oxide suppresses growth and development in the unicellular green alga Micrasterias denticulata

Nitric oxide (NO), a key molecule in inter- and intracellular signalling, is implicated in developmental processes, host defense, and apoptosis in higher plants. We investigated the effect of NO on development in the unicellular green alga, Micrasterias denticulata, using two different NO donors, S-nitroso-N-acetyl-dl-penicillamine (SNAP) and sodium nitroprusside (SNP). Investigations at the light microsopic level revealed that both NO donors suppressed cell growth. Ultrastructural analyses were performed with SNAP- as well as SNP-treated cells and, additionally, with the control compound N-acetyl-d-penicillamine (NAP). Cells incubated with NO donors lacked a secondary wall and dictyosomal function was impaired, whereas NAP-treated cells showed no difference in development and organelle structure compared to control cells. Moreover, cisternae of the Golgi stacks were slightly involute and no vesicles were pinched off after SNAP and SNP incubation. The NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, potassium salt) abrogated the effect of SNP, thus confirming that inhibition of cell growth is due to nitric oxide. Addition of iodoacetic acid, an inhibitor of cysteine-containing enzymes, like glyceraldehyde-3-phosphate dehydrogenase (GAPDH), evoked similar effects on cell growth and secondary wall formation as obtained by treatment with NO donors. Therefore, we hypothesize that NO inhibits activity of enzymes involved in the secretory pathway, such as GAPDH, via S-nitrosylation of the cysteine residue and, consequently, modulates cell growth in M. denticulata.     

Kinesin-like proteins are involved in postmitotic nuclear migration of the unicellular green alga Micrasterias denticulata

The unicellular green alga Micrasterias denticulata performs a two-directional postmitotic nuclear migration during development, a passive migration into the growing semicell, and a microtubule mediated backward migration towards the cell centre. The present study provides first evidence for force generation by motor proteins of the kinesin family in this process. The new kinesin specific inhibitor adociasulfate-2 causes abnormal nuclear displacement at 18 microM. AMP-PNP, a non hydrolyseable ATP analogue or the general ATPase inhibitors calyculin A and sodium orthovanadate also disturb nuclear migration. In addition kinesin-like proteins are detected by means of immunoblotting using antibodies against brain kinesin, plant derived antibodies to kinesin-like proteins and a calmodulin binding kinesin-like protein. Immunoelectron microscopy suggests a correlation of conventional kinesin-like proteins, but not of the calmodulin binding kinesin-like protein to the microtubule apparatus associated with the migrating nucleus.     

The effects of continuous illumination on the function and structure of Micrasterias torreyi Bail. (Conjugatophyceae)

Abstract. The division rate of Micrasterias torreyi cells grown under continuous illumination first accelerated but soon slowed down, and the cells lost their ability to divide after about 1 month. During the treatment the cells became pale green, the pyrenoids became fewer in number and defects appeared in the chloroplasts. After 1 month, the cells also soon died, even when subjected to intermittent illumination. The most striking structural alterations were found in the chloroplasts: the starch granules lost their typical structure, the lamellae were damaged and numerous electron dense precipitates appeared in the chloroplasts. The precipitates were similar to those formed in cells treated with supraoptimal external calcium concentrations and X-ray microanalysis showed that the precipitates were rich in calcium in both cases. The results suggest that light controls and activates the Ca²⁺ uptake in the plasma membrane as well as in the chloroplast envelope, that the large sized chloroplasts of Micrasterias are effective in regulation of cytoplasmic Ca²⁺ concentration, and that the injuries caused by continuous illumination may be largely due to the accumulation of Ca²⁺ in the chloroplasts.     

A freshwater green alga under cadmium stress: Ameliorating calcium effects on ultrastructure and photosynthesis in the unicellular model Micrasterias

Cadmium is a highly toxic heavy metal pollutant arising mainly from increasing industrial disposal of electronic components. Due to its high solubility it easily enters soil and aquatic environments. Via its similarity to calcium it may interfere with different kinds of Ca dependent metabolic or developmental processes in biological systems. In the present study we investigate primary cell physiological, morphological and ultrastructural responses of Cd on the unicellular freshwater green alga Micrasterias which has served as a cell biological model system since many years and has proved to be highly sensitive to any kind of abiotic stress. Our results provide evidence that the severe Cd effects in Micrasterias such as unidirectional disintegration of dictyosomes, occurrence of autophagy, decline in photosystem II activity and oxygen production as well as marked structural damage of the chloroplast are based on a disturbance of Ca homeostasis probably by displacement of Ca by Cd. This is indicated by the fact that physiological and structural cadmium effects could be prevented in Micrasterias by pre-treatment with Ca. Additionally, thapsigargin an inhibitor of animal and plant Ca(2+)-ATPase mimicked the adverse Cd induced morphological and functional effects on dictyosomes. Recovery experiments indicated rapid repair mechanisms after Cd stress.     

Glutamate dehydrogenase of the unicellular green alga Scenedesmus acutus

The coenzyme-non-specific glutamate dehydrogenase (EC 1.4.1.3) from Scenedesmus acutus in inhibited by p-hydroxymercuribenzoate only in the deamination reaction. From this result and from its stability in the presence of urea it is concluded that this enzyme exhibits and equilibrium between three conformations: aminating and deaminating conformations induced by NADH-2-oxoglutarate and NAD⁺-glutamate, respectively, and the “native” conformation in the absence of substrates.     

Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida

Summary Cortical microtubules and cortical microfilaments were visualized in cells ofMicrasterias pinnatifida treated by freeze-substitution, and the pattern of their distribution was reconstructed from serial sections. Most cortical microtubules accompanied the long microfilaments that ran parallel to the microtubules. Cortical microfilaments not accompanied by the microtubules were also found. They were short and slightly curved. Both types of cortical microfilament were not grouped into bundles, and were 6–7 nm in diameter, a value that corresponds to the diameter of filaments of F-actin.     

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.     

Scale-invariant pattern in the alga Micrasterias

Spatial structures arise in a variety of different physical, chemical and biological systems. A striking example is found during morphogenesis in the single-celled alga Micrasterias, where cell extensions called lobes branch repeatedly to produce a highly regular, apparently self-similar pattern. Lobe outgrowth in Micrasterias is thought to be controlled by the local accumulation of growth determinants at the lobe tips. These tip-growth sites undergo successive spatial bifurcations, leading to the recursively branched, final cell form. I have tested for scale invariance of this form, by measuring the distribution of tips as a function of position along the cell perimeter in mature Micrasterias cells of four different species. This tip distribution should reflect the steady-state distribution of growth determinants at the end of the spatial bifurcation process. For each cell measured, the distribution of tips resembled a Cantor set with three levels of constant, nested scaling. Significantly, roughly the same scale factor (3.0) was found at each scaling level in individual cells, and among different cells in each of the four species measured. These data suggest that scaling by this factor is intrinsic to the pattern formation process in Micrasterias.     

Abscisic acid-dependent algal morphogenesis in the unicellular green alga Haematococcus pluvialis

To study the physiological role of abscisic acid (ABA) in the unicellular green alga Haematococcus pluvialis, we investigated the effect of ABA on both algal morphogenesis and carotenogenesis in liquid and plate cultures. When ABA was added to vegetative cells of H. pluvialis, red mature cyst cells with enhanced carotenogenesis rapidly appeared on agar plates in Petri dishes. We considered these conditions as drought stress. In plate culture, the morphological change from vegetative to cyst cells was prevented by the inhibitor of chloroplastic protein synthesis, chloramphenicol (CP), resulting in algal death. Exogenous ABA caused recovery of algal encystment even in the presence of CP. The relationship between ABA concentration and morphogenesis in H. pluvialis showed that a decrease in ABA coincided with cyst formation. In contrast, immature cyst cells underwent maturation accompanied by enhanced carotenogenesis in either the presence of CP or the absence of ABA. Therefore, ABA might regulate algal morphogenesis from vegetative to cyst cells, but not carotenogenesis in cyst cells of H. pluvialis. Furthermore, endogenous active oxygen species generated under drought stress were involved in all algal events, including ABA biosynthesis, encystment, and enhanced carotenogenesis. These results indicate that ABA, induced by oxidative stress, could function as a stress hormone in algal morphogenesis in H. pluvialis under drought stress.     

Photobleaching in the unicellular green alga Dunaliella parva 19/9

The change in the pigment composition of the unicellular alga Dunaliella parva 19/9 during exposure to high light (4000 mol m^-2 s^-1) has been investigated. During photobleaching the carotenoids were lost at a greater rate than the chlorophylls. In these photoinhibitory conditions, -carotene and especially the minor carotenes, - and -carotene, were more susceptible to oxidative destriction than the xanthophylls. Lutein, the major carotenoid present, was the most stable of the carotenoids in these conditions. In addition to the direct photobleaching of carotenoids and chlorophylls, high light treatment induced the de-epoxidation of violaxanthin to antheraxantin and zeaxanthin. Small amounts of zeaxanthin were present in cells prior to illumination but the amount increased 2.4 fold following high light treatment. The effects of extremes of temperature during exposure to high light intensities were also investigated. The destruction of chlorophylls was found to be more temperature sensitive than that of the carotenoids. The general pattern of loss for the individual carotenoids was similar to that found at 25°C, i.e., the carotenes were more readily degraded than the xanthophylls.     

The effects of continuous illumination on southern and northern Micrasterias strains

Different strains of Micrasterias (Chlorophyta, Conjugatophyceae); M. rotata (Grev.) Ralfs ex. Ralfs and M. denticulata Breb. ex. Ralfs var. angulosa (Hantzsch) W. & G. S. West from northern and southern Finland were treated with continuous illumination in order to study the cellular effects of the treatment and whether the tolerance to continuous light of the northern Finnish strains is related to the different daylenght conditions in northern and southern areas. During the growing season the Finnish strains normally live in long-day conditions or even in continuous light (between 60 and 70°N), and they also tolerated continuous illumination in the laboratory. Ultrastructural changes were found especially in the chloroplasts, where formation of calcium precipitates of different forms and sizes and also formation of plastoglobuli containing lipids appeared. However, even in 4-week treatments the ultrastructure of cells of these northern strains was not totally disrupted, contrary to what was found in southern M. torreyi , studied earlier. Southern and northern strains tolerated continuous illumination in different ways. They seem to differ from each other physiologically, and the differences are possibly located in their ionic metabolism and regulation. The injuries sustained during continuous illumination of Micrasterias may largely be caused by the accumulation of Ca²⁺ in cytoplasm and organelles, especially in the chloroplasts.     

Architecture and evolution of subtelomeres in the unicellular green alga Chlamydomonas reinhardtii

In most eukaryotes, subtelomeres are dynamic genomic regions populated by multi-copy sequences of different origins, which can promote segmental duplications and chromosomal rearrangements. However, their repetitive nature has complicated the efforts to sequence them, analyse their structure and infer how they evolved. Here, we use recent genome assemblies of Chlamydomonas reinhardtii based on long-read sequencing to comprehensively describe the subtelomere architecture of the 17 chromosomes of this model unicellular green alga. We identify three main repeated elements present at subtelomeres, which we call Sultan, Subtile and Suber, alongside three chromosome extremities with ribosomal DNA as the only identified component of their subtelomeres. The most common architecture, present in 27 out of 34 subtelomeres, is a heterochromatic array of Sultan elements adjacent to the telomere, followed by a transcribed Spacer sequence, a G-rich microsatellite and transposable elements. Sequence similarity analyses suggest that Sultan elements underwent segmental duplications within each subtelomere and rearranged between subtelomeres at a much lower frequency. Analysis of other green algae reveals species-specific repeated elements that are shared across subtelomeres, with an overall organization similar to C. reinhardtii. This work uncovers the complexity and evolution of subtelomere architecture in green algae.