Evidence for a G-Protein Regulated Adenylate Cyclase and a Ca2/Calmodulin Controlled Phosphodiesterase in the Phytoflagellate Chlorogonium

In the green flagellate Chlorogonium elongatum the cAMP concentration of the cells depends on the culture conditions of the alga. Autotrophically cultured cells possess three times more cAMP than cells cultured heterotrophically (in the dark with acetate). The activity of adenylate cyclase in a membrane fraction of Chlorogonium is distinctly stimulated by GTPγS. Such a stimulation is generally regarded as an indication of the participation of heterotrimeric G-proteins in this regulatory process. The activity of phosphodiesterase is stimulated in vitro by calmodulin and inhibited by trifluoperazin and EGTA.     

Short-term variation in extracellular phosphatase activity: possible limitations for diagnosis of nutrient status in particular algal populations

Species-specific extracellular phosphatase activity (PA) was studied in natural phytoplankton with an emphasis on short-term variation in PA and its consequences for the diagnosis of inorganic phosphorus (P) deficiency. Extracellular PA was measured over 3 days at 4-h intervals to compare variations in algal PA in two distinct aquatic environments: in the Římov reservoir and acidified Plešné Lake. PA was directly detected in an epifluorescence microscope using fluorescently labelled enzyme activity; the fluorescence signal was quantified using image analysis. High short-term variation was found both in the total PA of the samples and specific PA of Ankyra ancora (G. M. Smith) Fott, Monoraphidium dybowski (Wolosz.) Hind. and Kom.-Legn., and Chlorogonium fusiforme Matwienko. The specific PA of these species varied between zero and 157 fmol cell⁻¹ h⁻¹. The PA of Chlorogonium fusiforme showed a significant relationship with the time of day. During the study period, populations of the certain species were present that differed in physiological status and activity due to diurnal or random changes caused by mixing in the reservoir or lake, or both. Therefore, species-specific extracellular PA should be interpreted carefully, as an indicator of P deficiency in freshwater phytoplankton if based on limited number of samplings.     

TAXONOMIC STUDY OF TWO NEW GENERA OF FUSIFORM GREEN FLAGELLATES,TABRIS GEN. NOV. AND HAMAKKO GEN. NOV. (VOLVOCALES,CHLOROPHYCEAE)1

On the basis of LM, we isolated strains of two species of fusiform green flagellates that could be assigned to former Chlorogonium (Cg.) Ehrenb. One species, “Cg.”heimii Bourr., lacked a pyrenoid in its vegetative cells and required organic compounds for growth. The other was similar to Cg. elongatum (P. A. Dang.) Francé and “Cg.”acus Nayal, but with slightly smaller vegetative cells. Their molecular phylogeny was also studied based on combined 18S rRNA, RUBISCO LSU (rbcL), and P700 chl a‐apoprotein A2 (psaB) gene sequences. Both species were separated from Chlorogonium emend., Gungnir Nakada and Rusalka Nakada, which were formerly assigned to Chlorogonium. They were accordingly assigned to new genera, Tabris Nakada gen. nov. and Hamakko (Hk.) Nakada gen. nov. as T. heimii (Bourr.) Nakada comb. nov. and Hk. caudatus Nakada sp. nov., respectively. Tabris is differentiated from other genera of fusiform green flagellates by its vegetative cells, which only have two apical contractile vacuoles and lack a pyrenoid in the chloroplast. Hamakko, on the other hand, is distinguishable by the fact that its pyrenoids in vegetative cells are penetrated by flattened thylakoid lamellae.     

The Mitochondrial Genome of Chlorogonium elongatum Inferred from the Complete Sequence

The 22,704-bp circular mitochondrial DNA (mtDNA) of the chlamydomonad alga Chlorogonium elongatum was completely cloned and sequenced. The genome encodes seven proteins of the respiratory electron transport chain, subunit 1 of the cytochrome oxidase complex (cox1), apocytochrome b (cob), five subunits of the NADH dehydrogenase complex (nad1, nad2, nad4, nad5, and nad6), a set of three tRNAs (Q, W, M), and the large (LSU)- and small (SSU)-subunit ribosomal RNAs. Six group-I introns were found, two each in the cox1, cob, and nad5 genes. In each intron an open reading frame (ORF) related to maturases or endonucleases was identified. Both the LSU and the SSU rRNA genes are split into fragments intermingled with each other and with other genes. Although the average A + T content is 62.2%, GC-rich clusters were detected in intergenic regions, in variable domains of the rRNA genes, and in introns and intron-encoded ORFs. A comparison of the genome maps reveals that C. elongatum and Chlamydomonas eugametos mtDNAs are more closely related to one another than either is to Chlamydomonas reinhardtii mtDNA. Received: 3 November 1997 / Accepted: 12 January 1998     

PHYLOGENY OF CHLAMYDOMONAS (CHLOROPHYTA) BASED ON CLADISTIC ANALYSIS OF NUCLEAR 18S rRNA SEQUENCE DATA1

The genus Chlamydomonas Ehrenberg may contain as many as 450 described species. Morphological, physiological and molecular data show that variation among some Chlamydomonas species can he great, leading to speculation that multiple, generic-level lineages exist within this genus. The most recent systematic studies of Chlamydomonas have led to proposals of nine distinct morphological and 15 distinct sporangial autolysin groups. Partial sequences from the nuclear small subunit rRNAs from 14 Chlamydomonas species representing 12 autolysin and four morphological groups, and from three flagellates thought to he related to Chlamydomonas were determined in a phylogenetic study of relationships among these algae. Sequence comparisons among some Chlamydomonas species revealed differences comparable to the sequence divergence between soybeans and cycads. Cladistic analysis of the sequence data suggests that multiple lineages exist among species of Chlamydomonas. Some of these lineages represent alliances of both Chlamydomonas and non-Chlamydomonas taxa; thus, the current taxonomy does not reflect natural, or monophyletic, groups. Collectively, these lineages may represent distinct families or even orders.     

Regulation of the Synthesis of Isocitrate Lyase and the Corresponding mRNA in the Green Alga Chlorogonium

In the unicellular green alga Chlorogonium elongatum the level of isocitrate lyase (ICL), the rate of its synthesis and the level of ICL-mRNA measured by in vitro translation are considerably increased after addition of acetate to the culture medium of autotrophically precultured cells. Almost identical increases are obtained independently of whether the cells are cultured after the addition of acetate in the dark (heterotrophically) or in the light (mixotrophically). Transfer of heterotrophic cells to autotrophic conditions results in a fast decrease of ICL-mRNA and ICL protein, while a transfer to mixotrophic conditions causes no alterations in both molecular species. Therefore the concentration of ICL and its translatable mRNA is controlled only by acetate and is unaffected by light.     

TOWARD A NATURAL SYSTEM OF SPECIES IN CHLOROGONIUM (VOLVOCALES, CHLOROPHYTA): A COMBINED ANALYSIS OF MORPHOLOGICAL AND rbcL GENE SEQUENCE DATA

The taxonomy of species of Chlorogonium (Volvocales, Chlorophyta) was studied based on comparative light and electron microscopy and DNA sequence data of 23 strains from five major algal culture collections. All of the 23 strains showed pyrenoids under photoautotrophic conditions, but 17 of the 23 exhibited marked reduction in size of pyrenoids, or pyrenoids were absent under photoheterotrophic conditions. The strains could clearly be delineated into six species, C. euchlorum, C. elongatum, C. fusiforme, C. capillatum, C. neglectum, and C. kasakii on the basis of differences in cell shape, number of contractile vacuoles, number and stability of pyrenoids, and ultrastructure of pyrenoids and stigmata. This distinction of species based on morphology was also supported by analyses of rbc L gene sequences. The later strongly showed that each species, C. euchlorum (seven strains), C. elongatum (three strains), and C. capillatum (10 strains), forms a robust clade. Although some morphological differences were noted within different strains of C. euchlorum and C. capillatum, these features were regarded as strain-specific because they were not reflected in the rbc L gene phylogenies. In addition, the rbc L gene trees strongly suggested that C. neglectum and C. kasakii are closely related to each other, consistent with the similarity of the ultrastructure of pyrenoids and stigmata between the two species. However, C. kasakii can be distinguished clearly from C. neglectum by its multiple pyrenoids in the chloroplast and acute anterior and posterior ends in the vegetative cell.     

MOLECULAR PHYLOGENY,ULTRASTRUCTURE, AND TAXONOMIC REVISION OF CHLOROGONIUM (CHLOROPHYTA): EMENDATION OF CHLOROGONIUM AND DESCRIPTION OF GUNGNIR GEN. NOV. AND RUSALKA GEN. NOV.1

We examined the molecular phylogeny and ultrastructure of Chlorogonium and related species to establish the natural taxonomy at the generic level. Phylogenetic analyses of 18S rRNA and RUBISCO LSU (rbcL) gene sequences revealed two separate clades of Chlorogonium from which Chlorogonium (Cg.) fusiforme Matv. was robustly separated. One clade comprised Cg. neglectum Pascher and Cg. kasakii Nozaki, whereas the other clade included the type species Cg. euchlorum (Ehrenb.) Ehrenb., Cg. elongatum (P. A. Dang.) Francé, and Cg. capillatum Nozaki, M. Watanabe et Aizawa. On the basis of unique ultrastructural characteristics, we described Gungnir Nakada gen. nov. comprising three species: G. neglectum (Pascher) Nakada comb. nov., G. mantoniae (H. Ettl) Nakada comb. nov., and G. kasakii (Nozaki) Nakada comb. nov. We also emended Chlorogonium as a monophyletic genus composed of Cg. euchlorum, Cg. elongatum, and Cg. capillatum. Because Cg. fusiforme was distinguished from the redefined Chlorogonium and Gungnir by the structure of its starch plate, which is associated with pyrenoids, we reclassified this species as Rusalka fusiformis (Matv.) Nakada gen. et comb. nov.     

MORPHOLOGY AND PAEDOGAMOUS SEXUAL REPRODUCTION IN CHLOROGONIUM CAPILLATUM SP. NOV. (VOLVOCALES, CHLOROPHYTA)1

Morphology and sexual reproduction in Chlorogonium capillatum Nozaki, Watanabe & Aizawa sp. nov. (Volvocales, Chlorophyta) originating from Miyatoko Mire, Japan, were studied under controlled laboratory conditions. Vegetative cells of this new species were fusiform with blunt anterior and posterior ends, and they had a massive parietal chloroplast and numerous contractile vacuoles distributed throughout the protoplast. Several to many pyrenoids were randomly distributed in the chloroplast, but they disappeared under the light microscope when grown photoheterotrophically. During asexual reproduction, the first division took place transversely without a preceding rotation of the parental protoplast. In sexual reproduction, the parental protoplast divided successively to form 32 or 64 small, biflagellate isogametes. After gametogenesis, the gametes did not escape from the parental cell (gametangial) wall, within which pairs of the adjoining gametes fused to form quadriflagellate zygotes. Such zygotes were then released from the parental cell wall and developed into hypnozygotes, which at maturity developed numerous thin spines or hairs on the zygote wall. On zygote germination, four biflagellate germ cells were released from the zygote wall separately. This type of gametic union, "paedogamy," has not previously been described in the green algae except for Chlorococcum echinozygotum Starr . Chlorogonium capillatum can be clearly distinguished from other described species of Chlorogonium by its numerous contractile vacuoles and blunt anterior and posterior ends in vegetative cells as well as by its unique sexual reproduction, in which paedogamous conjugation occurs, and numerous thin spines or hairs that develop on the hypnozygote walls .