THE EFFECTS OF SELECTED INHIBITORS ON CELLULOSE MICROFIBRIL ASSEMBLY IN BOERGESENIA FORBESII (CHLOROPHYTA) PROTOPLASTS1

Protoplasts of Boergesenia forbesii (Harvey) were treated with inhibitors of protein synthesis in order to investigate their effects on cellulose synthesis. Cellulose synthesis was reversibly inhibited by 10 μM cycloheximide as assayed by fluorescence microscopy of Tinopal binding to cellulose. Freeze fracture and image analysis of cycloheximide- treated cells indicated a reduction in the number of intramembrane particles; however, the terminal synthesizing complexes remained at all times. Treatment with 10 μM actinomycin D, when applied during the first hour of protoplast formation, irreversibly inhibits cellulose synthesis and terminal complex formation. De novo protein synthesis is required for cell wall regeneration by protoplasts. The data suggest that the structural subunits visualized in the terminal complex do not undergo significant turnover, but that there may exist an essential proteinaceous component of cellulose synthesis which must be continually renewed.     

COORDINATNE NUCLEAR AND CHLOROPLAST DIVISION IN UNILOCULAR SPORANGIA OF LAMINARIA ANGUSTATA (LAMINARIALES,PHAEOPHYCEAE)1

Changes in the number of nuclei and chloroplasts were examined during the process of unispore formation in unilocular sporangia of Laminaria angustata. Just before meiosis, eight chloroplasts were always present in unilocular sporangial mother cells. The number of chloroplasts remained constant through meiosis. After the resulting four nuclei divided again (third nuclear division), a close association between a nucleus and a chloroplast developed among each of the eight nuclei and eight chloroplasts. The eight chloroplasts divided ahost synchronously before the synchronous division of the eight nuclei. Following the 16 nucleate stage with 16 chloroplasts and the final 32 nucleate stage with 32 chloroplasts, 32 unispores, each with a nucleus and a chloroplast, were fomd in unilocular sporangza of L. angustata. Immunofluorescence microscopy using an anti-centrin antibody showed that two anticentrin-stained structures (as future mitotic poles) occurred adjacent to each of the premitotic four nuclei, and each spot was located near a chloroplast. Therefore, after the third division, each of the eight nuclei established close contact with a chloroplast presumably mediated by the centrosomes.     

LOW-TEMPERATURE-INDUCED SYNTHESIS OF α-CAROTENE IN THE MICROALGA DUNALIELLA SALINA (CHLOROPHYTA)

The microalga Dunaliella salina (Teo.) is well known as an accumulator of β-carotene (β,β-carotene) when subjected to growth-limiting conditions (e.g. exposure to high irradiances). In addition, the carotenoid α-carotene (β,ε-carotene) may also be synthesized and subsequently accumulated by this alga under specific growth conditions. The main factor in stimulating the synthesis of this carotene was determined to be exposure to lower than optimum temperatures for algal growth. A 7.5-fold increase in the levels of α-carotene was observed when the temperature was decreased from 34 to 17° C, whilst levels of β-carotene were unaltered. The accumulation of α-carotene was unaffected by irradiance, although its isomeric composition was greatly altered by light levels. The proportion of 9- cis α-carotene increased from 15% to 45% of total α-carotene when the irradiance was decreased from 260 to 50 μmol·m⁻²·s⁻¹. Exposure to higher irradiances had little influence on the isomeric composition of this carotenoid. A reduction in growth temperature did not influence the isomeric composition of α-carotene. Nutrient status (nitrogen and phosphate) had no effect on either the content or isomeric composition of α-carotene accumulated by D. salina.     

STRUCTURAL FEATURES OF NUCLEAR GENES IN THE CENTRIC DIATOM THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)

Thalassiosira weissflogii (Grun.) Fryxell et Hasle is one of the more commonly studied centric diatoms, and yet molecular studies of this organism are still in their infancy. The ability to identify open reading frames and thus distinguish between introns and exons, coding and noncoding sequence is essential to move from nuclear DNA sequences to predicted amino acid sequences. To facilitate the identification of open reading frames in T. weissflogii , two newly identified nuclear genes encoding β-tubulin and t  -complex polypeptide (TCP)-γ, along with six previously published nuclear DNA sequences, were examined for general structural features. The coding region of the nuclear open reading frames had a G + C content of about 49% and could readily be distinguished from noncoding sequence due to a significant difference in G + C content. The introns were uniformly small, about 100 base pairs in size. Furthermore, the 5' and 3' splice sites of introns displayed the canonical GT/AG sequence, further facilitating recognition of noncoding regions. Six of the nuclear open reading frames displayed relatively little bias in the use of synonymous codons, as exemplified by the cDNAs encoding β-tubulin and TCP-γ. Two open reading frames displayed strong bias in the use of particular codons (although the codons used were different), as exemplified by the cDNA encoding fucoxanthin chlorophyll a/c binding protein. Knowledge of codon bias should facilitate, for example, design of degenerate PCR primers and potential heterologous reporter gene constructs.     

HIGH LOCALIZATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXULASE/OXYGENASE IN THE PYRENOIDS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA), AS REVEALED BY CRYOFIXATION AND IMMUNOGOLD ELECTRON MICROSCOPY

The distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the chloroplasts of the unicellular green alga Chlamydomonas reinhardtii Dangeard was examined using cryotechnique and conventional fixation for immunogold electron microscopy. Both methods provided essentially identical results, although somewhat higher densities of gold particles indicating Rubisco molecules were recognized in the pyrenoids of cryofixed cells. The gold particles were highly concentrated in the pyrenoid matrix within the chloroplasts. Even when considering the vast difference in volume between the pyrenoid and the rest of the Chloroplast, more than 99% of the total Rubisco labeling in the chloroplast was calculated to be present in the pyrenoid matrix. High localization of Rubisco in the pyrenoid matrix was also recognized regardless of cell age, based on immunofluorescence microscopy of the same en bloc samples. These results are inconsistent with a recent immunocytochemical study employing cryotechnique in which more than 90% of the total Rubisco was recognized in the thylakoid region (thylakoid membranes and stroma) of C. reinhardtii cells. Rubisco highly localized in the pyrenoid matrix may take part in active photosynthetic CO₂ fixation and/or the CO₂ concentrating mechanism .     

A Rhizobium meliloti mutant, lacking a functional γ-aminobutyrate (GABA) bypass, is defective in glutamate catabolism and symbiotic nitrogen fixation

Abstract A Rhizobium meliloti mutant, CMF1 2:38, was isolated which was specifically defective in the degradation of glutamate as sole carbon and nitrogen source. Biochemical analysis of CMF1 2:38 revealed a reduction in succinic semialdehyde dehydrogenase (SSDH) activity, the third enzyme of the γ-aminobutyrate (GABA) bypass. Evidence is presented which suggests that the Tn 5-induced mutation in CMF1 2:38 exists in a regulatory gene governing the expression of both NAD and NADP-linked SSDH activity. CMF1 2:38 nodulated alfalfa plants, but was reduced in its nitrogen fixation activity and biomass accumulating ability relative to the wild-type strain. The results presented in this study indicate that the GABA bypass is a major mechanism of glutamate degradation in R. meliloti CMF1 and that glutamate catabolism via this pathway may play an important role in the symbiotic nitrogen fixation process.     

THE EFFECT OF 4-AMINO HEX-5-YNOIC ACID (γ-ACETYLENIC GABA, γ-ETHYNYL GABA) A CATALYTIC INHIBITOR OF GABA TRANSAMINASE, ON BRAIN GABA METABOLISM IN VIVO

Abstract— 4-Amino hex-5-ynoic acid (γ-acetylenic GABA, γ-ethynyl GABA, RM171.645), a catalytic inhibitor of GABA transaminase in vitro , induces a rapid, long-lasting dose-dependent decrease of GABA transaminase activity and, to a lesser extent, of glutamate decarboxylase activity in the brains of rats and mice when given by a peripheral route. The GABA concentration in whole brain increases up to 6-fold over control values. The action of γ-acetylenic GABA is relatively specific, as no in vivo inhibition of brain aspartate and alanine transaminase activities could be detected. Furthermore, the amount of radioactive drug bound to the protein fraction of brain homogenate is of the same order of magnitude as that of the GABA transaminase present, as calculated from total GABA transaminase activity, molecular weight and specific activity of the pure enzyme. γ-Acetylenic GABA illustrates the usefulness of a catalytic irreversible enzyme inhibitor in altering neurotransmitter metabolism in vivo .     

TRANSIENT AMMONIUM UPTAKE IN THE MACROALGA ULVA LACTUCA (CHLOROPHYTA): NATURE,REGULATION, AND THE CONSEQUENCES FOR CHOICE OF MEASURING TECHNIQUE1

The nature of transient ammonium uptake by the macroalga Ulva lactuca L. was studied from the depletion of ammonium after single additions of ammonium to batch cultures. The experiments were carried out by the application of two different experimental setups: the “multiple flask” and the “perturbation” techniques. Uptake rate was nonlinear with time, and three distinct, succeeding phases of uptake were identified: 1) “surge” uptake, i.e. transiently enhanced uptake that lasted for a few hours only, followed by 2) “internally” controlled uptake, a relatively constant phase occurring at high substrate concentrations, and finally 3) the “externally” controlled uptake phase, which was substrate-dependent and occurred at low substrate concentrations. Surge uptake occurred over a broad range of substrate concentrations but was concentration-dependent and, so, equalled externally controlled uptake rates at substrate concentrations below 3–10 μM. The transient nature of ammonium uptake rate seemed related to rapid changes in small intracellular pools of inorganic nitrogen or amino acids rather than to changes in total N content of the algae. The transient nature of ammonium uptake has important implications for the measurement of uptake rates when either of the two standard methods, the multiple flask and the perturbation technique, are used, and I recommend that a combination of the two methods be used for future uptake experiments.     

A STUDY OF CELL WALL AND FLAGELLA FORMATION DURING CELL DIVISION IN THE SCALY GREEN ALGA,SCHERFFELIA DUBIA (CHLOROPHYTA)1

The thecate green flagellate Scherffelia dubia (Perty) Pascher divides within the parental cell wall into two progeny cells. It sheds all four flagella before cell division, and the maturing progeny cells regenerate new walls and flagella. By synchronizing cell division, we observed mitosis, cytokinesis, cell maturation, flagella extension, and cell wall formation via differential interference contrast microscopy of live cells and serial thin‐section EM. Synthesis of thecal and flagellar scales is spatially and temporally strictly separated. Flagellar scales are collected in a pool during late interphase. Before prophase, Golgi stacks divide, flagella are shed, the parental theca separates from the plasma membrane, and flagellar scales are deposited on the plasma membrane near the flagellar bases. At prophase, Golgi bodies start to synthesize thecal scales, continuing into interphase after cytokinesis. During cytokinesis, vesicles containing thecal scales coalesce near the cell posterior, forming a cleavage furrow that is initially oriented slightly diagonal to the longitudinal cell axis but later becomes transverse. After the progeny nuclei have moved into opposite directions, resulting in a “head to tail” orientation of the progeny cells, theca biogenesis is completed and flagellar scale synthesis resumes. Progeny cells emerge through a hole near the posterior end of the parental theca with four flagella of about 8 μm long. The precise timing of flagellar and thecal scale synthesis appears to be an evolutionary adaptation in a scaly green flagellate for the thecal condition, necessary for the evolution of the phycoplast and thus multicellularity in the Chlorophyta.     

PURIFICATION AND CHARACTERIZATION OF A LECTIN,BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) 1

When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin–carbohydrate complementary system. Two sugars, N‐acetyl‐d ‐glucosamine and N‐acetyl‐d ‐galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate‐labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N‐acetyl‐d ‐galactosamine and N‐acetyl‐d ‐glucosamine in the crude extract. One‐step column purification using N‐acetyl‐d ‐glucosamine‐agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross‐linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The N‐terminal 15 amino acid sequence of the lectin was Ser–Asp–Leu–Pro–Thr–X–Asp–Phe–Phe–His–Ile–Pro–Glu–Arg–Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa“bryohealin.”     

EVOLUTIONARY RELATIONSHIPS AMONG MULTIPLE MODES OF CELL DIVISION IN THE GENUS NANNOCHLORIS (CHLOROPHYTA) REVEALED BY GENOME SIZE, ACTIN GENE MULTIPLICITY, AND PHYLOGENY

A single cell divides to multiply, but not all cells follow the same pattern of division. We studied cell division in seven strains from six species belonging to the genus Nannochloris Naumann and classified their modes of cell division into three types: binary fission ( N. bacillaris Naumann), budding ( N. coccoides Naumann), and autosporulation resulting in the formation of two to four daughter cells ( N. maculata Butcher, N . sp. SAG 251-2, N. atomus Butcher CCAP 251/7 and SAG 14.87, and N. eucaryotum [Wilhelm et al.] Menzel and Wild). To determine the evolutionary relationships among these multiple modes of cell division, we investigated the strains' genome sizes, copy number of actin genes, and phylogeny. The genome sizes were determined by counter-clamped homogeneous electric fields electrophoresis and fluorimetry. The genomes are very small and range from 12.6 Mbp ( N. maculata ) to 47.4 Mbp ( N. atomus SAG 14.87). The genomes of Nannochloris species seem to be among the smallest for free-living eukaryotes. Nannochloris bacillaris (binary fission), N. coccoides (budding), Nannochloris sp. (two-cell type of autosporulation), and N. eucaryotum (multicell type of autosporulation) contain a single actin gene, whereas N. maculata (two-cell type of autosporulation) and two strains of N. atomus (two-cell type of autosporulation) contain two actin genes. This suggests that the actin gene was duplicated in this eukaryote, which has a very small genome. Phylogenetic analyses of partial actin gene sequences suggest that autosporulation is the ancestral mode of cell division.     

IMMUNOLOCALIZATION OF AN α-1,4-GLUCAN LYASE IN YOUNG AND MATURE PARTS OF THE RED ALGA GRACILARIOPSIS SP. (RHODOPHYTA)

The enzyme α-1,4-glucan lyase (EC 4.2.2.13) was studied in cells of young and mature parts of the red alga Gracilariopsis sp. by using immunogold labeling in ultrastructural studies. In young tissues, the α-1,4-glucan lyase was observed at two different sites: around the starch granules in the cytosol and in the stroma of the chloroplast. In mature tissues, the α-1,4-glucan lyase was present only in the chloroplasts. The possible role of this starch-degrading enzyme in red algae is discussed.     

EFFECTS OF NH4+ ASSIMILATION ON DARK CARBON FIXATION AND β-1,3-GLUCAN METABOLISM IN THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

The effects of NH₄⁺ assimilation on dark carbon fixation and β-1,3-glucan metabolism in the N-limited marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) were investigated by chemical analysis of cell components and incorporation of ¹⁴C-bicarbonate. The diatom was grown in pH-regulated batch cultures with a 14:10 h LD cycle until N depletion. The cells were then incubated in the dark with ¹⁴C-bicarbonate, but without a source of N for 2 h, then in the dark with 63 μmol·L⁻¹ NH₄⁺ for 3 h. Without N, the cellular concentration of free amino acids was almost constant (∼4.5 fmol·cell⁻¹). Added NH₄⁺ was assimilated at a rate of 12 fmol·cell⁻¹·h⁻¹, and the cellular amino acid pool increased rapidly (doubled in <1 h, tripled in <3 h). The glutamine level increased steeply (45× within 3 h), and the Gln/ Glu ratio increased from 0.1 to 2.4 within 3 h. The rate of dark C fixation during N depletion was only 1.0 fmol·cell⁻¹·h⁻¹. The addition of NH₄⁺ strongly stimulated dark C fixation, leading to an assimilation rate of 4.0 fmol·cell⁻¹·h⁻¹, corresponding to a molar C/N uptake ratio of 0.33. Biochemical fractionation of organic ¹⁴C showed no significant ¹⁴C fixation into amino acids during N depletion, but during the first 1–2 h of NH₄⁺ assimilation, amino acids were rapidly radiolabeled, accounting for virtually all net ¹⁴C fixation. These results indicate that anaplerotic β-carboxylation is activated during NH₄⁺ assimilation to provide C₄ intermediates for amino acid biosynthesis. The level of cellular β-1,3-d-glucan was constant (16.5 pg·cell⁻¹) during N depletion, but NH₄⁺ assimilation activated a mobilization of 28% of the reserve glucan within 3 h. The results indicate that β-1,3-glucan in diatoms is the ultimate substrate for β-carboxylation, providing precursors for amino acid biosynthesis in addition to energy from respiration.