Two Class I Aldolases in the Green Alga Chara foetida (Charophyceae)

Aldolase activity of Chara foetida (Braun) could be separated into a minor (peak I) and a major peak (peak II) by ion-exchange chromatography on DEAE-cellulose. Affinity chromatography on P-cellulose resulted in highly purified aldolase preparations with specific activities of 3.2 and 4.8 units per milligram protein and molecular subunit masses of 37 and 35 kilodalton, as shown by SDS-PAGE, for the aldolase of peak I and peak II, respectively. Both aldolases belong to class I aldolase since the activity is not inhibited by 1 millimolar EDTA. The K_m (fructose-1,6-bisphosphate) values were 0.64 and 13.4 micromolar, respectively. The aldolase of peak I showed a 6.7 times stronger crossreaction with a specific antiserum against the cytosol aldolase of spinach than with an antiserum against the chloroplast aldolase of spinach. On the other hand the aldolase of peak II showed a 5.1 times stronger cross-reaction with the α-plastidaldolase antiserum than with the α-cytosol-aldolase antiserum. For algae this is the first separation of two class I aldolases. They are similar to the cytosol and chloroplast aldolases in higher plants, but different from a reported class I (Me²⁺ independent) and class II (Me²⁺ dependent) aldolase in other algae.     

Rapid Biotransformation of Arsenate into Oxo-Arsenosugars by a Freshwater Unicellular Green Alga,Chlamydomonas reinhardtii

We examined the short-term metabolic processes of arsenate for 24 h in a freshwater unicellular green alga, Chlamydomonas reinhardtii wild-type strain CC-125. The arsenic species in the algal extracts were identified by high-performance liquid chromatography/inductively coupled plasma mass spectrometry after water extraction using a sonicator. Speciation analyses of arsenic showed that the levels of arsenite, arsenate, and methylarsonic acid in the cells rapidly increased for 30 min to 1 h, and those of dimethylarsinic acid and oxo-arsenosugar-glycerol also tended to increase continuously for 24 h, while that of oxo-arsenosugar-phosphate was quite low and fluctuated throughout the experiment. These results indicate that this alga can rapidly biotransform arsenate into oxo-arsenosugar-glycerol for at least 10 min and then oxo-arsenosugar-phosphate through both reduction of incorporated arsenate to arsenite and methylation of arsenite and/or arsenate retained in the cells to dimethylarsinic acid via methylarsonic acid as an possible intermediate.     

Fine filaments observed within the cytoplasm surrounding the leading edge of the septum in telophase cells of Spirogyra (Zygnematales, Chlorophyta)

Electron microscope observation revealed the presence of many fine filaments within the cytoplasm surrounding the leading edge of the septum in telophase cells of Spirogyra verruculosa Jao. These filaments, about 7 nm each in diameter, ran parallel to one another along the leading edge of the septum and, sometimes, they appeared to be gathered into at least two bundles. These filament distribution patterns coincided well with those of the fluorescence of rhodamine-labeled phalloidin in the vicinity of the septum in telophase cells. The present results suggest that the fine filaments observed within the cytoplasm surrounding the leading edge of the septum may be actin filaments.     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Chemical Composition and Biological Activities of Trans-Himalayan Alga Spirogyra porticalis (Muell.) Cleve

The freshwater alga Spirogyra porticalis (Muell.) Cleve, a filamentous charophyte, collected from the Indian trans-Himalayan cold desert, was identified on the basis of morpho-anatomical characters. Extracts of this alga were made using solvents of varying polarity viz. n-hexane, acetonitrile, methanol and water. The antioxidant capacities and phenolic profile of the extracts were estimated. The methanol extract showing highest antioxidant capacity and rich phenolic attributes was further investigated and phytochemical profiling was conducted by gas chromatography-mass spectrometry (GC/MS) hyphenated technique. The cytotoxic activity of methanol extract was evaluated on human hepatocellular carcinoma HepG2 and colon carcinoma RKO cell lines. The anti-hypoxic effect of methanol extract of the alga was tested on in vivo animal system to confirm its potential to ameliorate oxidative stress. The antioxidant assays viz. ferric reducing antioxidant power (FRAP), 2,2''-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging capacities, β-carotene-linoleic acid bleaching property and lipid peroxidation exhibited analogous results, wherein the algal extracts showed significantly high antioxidant potential. The extracts were also found to possess high content of total proanthocyanidin, flavonoid and polyphenol. GC/MS analysis revealed the presence of thirteen chemotypes in the methanol extract representing different phytochemical groups like fatty acid esters, sterols, unsaturated alcohols, alkynes etc. with substantial phyto-pharmaceutical importance. The methanol extract was observed to possess anticancer activity as revealed from studies on HepG2 and RKO cell lines. In the present study, S. porticalis methanol extract also provided protection from hypoxia-induced oxidative stress and accelerated the onset of adaptative changes in rats during exposure to hypobaric hypoxia. The bioactive phytochemicals present in this trans-Himalayan alga are of enormous interest and can be utilized sustainably for discovery of novel drugs against oxidative stress.     

Active Hydrocarbon Biosynthesis and Accumulation in a Green Alga,Botryococcus braunii (Race A)

Among oleaginous microalgae, the colonial green alga Botryococcus braunii accumulates especially large quantities of hydrocarbons. This accumulation may be achieved more by storage of lipids in the extracellular space rather than in the cytoplasm, as is the case for all other examined oleaginous microalgae. The stage of hydrocarbon synthesis during the cell cycle was determined by autoradiography. The cell cycle of B. braunii race A was synchronized by aminouracil treatment, and cells were taken at various stages in the cell cycle and cultured in a medium containing [¹⁴C]acetate. Incorporation of ¹⁴C into hydrocarbons was detected. The highest labeling occurred just after septum formation, when it was about 2.6 times the rate during interphase. Fluorescent and electron microscopy revealed that new lipid accumulation on the cell surface occurred during at least two different growth stages and sites of cells. Lipid bodies in the cytoplasm were not prominent in interphase cells. These lipid bodies then increased in number, size, and inclusions, reaching maximum values just before the first lipid accumulation on the cell surface at the cell apex. Most of them disappeared from the cytoplasm concomitant with the second new accumulation at the basolateral region, where extracellular lipids continuously accumulated. The rough endoplasmic reticulum near the plasma membrane is prominent in B. braunii, and the endoplasmic reticulum was often in contact with both a chloroplast and lipid bodies in cells with increasing numbers of lipid bodies. We discuss the transport pathway of precursors of extracellular hydrocarbons in race A.     

Photosynthetic Eukaryotes of Freshwater Wetland Biofilms: Adaptations and Structural Characteristics of the Extracellular Matrix in the Green Alga, Cosmarium reniforme (Zygnematophyceae, Streptophyta)

ABSTRACT. Cosmarium reniforme (Zygnematophyceae, Streptophyta) is a green alga that is commonly found in biofilms of wetlands of the Adirondack region, NY (USA). Two distinctive characteristics that are critical to this alga's survival in a benthic biofilm are its elaborate cell morphology and extracellular matrix (ECM). In this study, ultrastructural, immunocytochemical, and experimental methodologies were employed in order to elucidate the cellular characteristics that are critical for survival in a biofilm. The ECM consists of a thick, outwardly lobed cell wall (CW), which contains a patterned network of structurally complex pores. Each pore consists of a narrow channel, terminating internally at a bulb that invaginates localized regions of the plasma membrane. The outer region of the pore contains arabinogalactan protein-like and extensin epitopes that are likely involved in adhesion mechanisms of the cell. External to the CW is the extracellular polymeric substance that is employed in ensheathment of the cell to the substrate and in gliding motility. The architectural design/biochemical make-up of the CW and a secretory system that encompasses the coordinated activities of the endomembrane and cytomotile/cytoskeletal systems provide the organism with effective mechanisms to support life within the biofilm complex.     

The Role of a Green Alga in the Precipitation of Calcite and the Coprecipitation of Phosphate in Freshwater

The precipitation of calcite from a calcium bicarbonate solution, similar in ionic strength to natural hardwaters, was observed in a series of experiments utilizing an automated culture apparatus. Seeded growth experiments, using calcite seed crystals, were performed at a range of phosphate concentrations to observe inhibitory effects. These experiments demonstrated a linear relationship of increasing inhibition with increasing initial phosphate concentration. A further series of experiments was performed in which an actively photosynthesizing culture of a unicellular green alga (Chlorococcum sp.) was added to the culture vessel in order to initiate precipitation. Experiments to observe spontaneous precipitation, occurring in the absence of both seed and alga additions, were carried out to compare with precipitation rates in the algal experiments. A control experiment was also performed to investigate whether precipitation occurrred in algal cultures maintained in darkness. The carbonate site mechanistic model, developed for calcite precipitation in abiotic conditions, was used to analyse the results of the algal experiments and found to be applicable.     

Division of Chloroplasts in a Green Alga, Pediastrum duplex

The division of chloroplasts in a green alga, Pediastrum duplex,was studied by electron microscopy. Cells were treated for observationwith the freeze-substitution method. Fibrils, or fibrous belts,which we had observed previously at the dividing constrictionsof chloroplasts in Trebouxia potteri were not visible in Pediastrum,even though the method of preparation was the same for bothsets of samples. Microtubules (MTs) and the septum seem notto participate directly in the division of the single chloroplastin Pediastrum cells. Many thin fibrils, 7–20 nm in diameter,attached to, or protruding from, the surface of the dividingconstriction were seen. These fibres were less densely distributedat the constrictions of non-dividing chloroplasts. It is suggestedthat these fibrils are involved in the divison of chloroplastsin Pediastrum duplex. Cell wall, chloroplast division, freeze-substitution, intermediate fibres, Pediastrum duplex, transmission electron microscopy     

Division of Chloroplasts in a Green Alga, Trebouxia potteri

The division of chloroplasts in Trebouxia potteri was studiedby electron microscopy. At the beginning of chloroplast division,vesicles with fine fibrils (FVs) and ER attach to the isthmusof the chloroplast. Then, filaments appear around the isthmusparallel to the direction of constriction and seem to contractin order to decrease the diameter of the isthmus. It is suggestedthat the FVs are involved in the formation of the filamentsand that the ER is involved in the contraction of the filaments.At the final stages of the division of the chloroplast, thefilaments decompose. FVs are partially surrounded and decomposedby lysosomal sheets. For the next cycle of division of the chloroplast,the recovery of substances from decomposed filaments by functionalFVs seems a realistic possibility. chloroplast division, division apparatus, division cycle, transmission electron microscopy, Trebouxia potteri.     

Ultrastructure of mitosis and cytokinesis inZygnema sp. (Zygnematales,Chlorophyta)

Summary Mitosis and cytokinesis have been studied in the green algaZygnema C. A. Agardh using interference-contrast light and transmission electron microscopy. At prophase, the nucleolus disintegrates and numerous extranuclear microtubules near the nuclear periphery penetrate into the nucleoplasm. When aligned in the equatorial plane of the open metaphase spindle the chromosomes are coated with persistent nucleolar fragments. At anaphase, vacuoles intrude into the interzonal spindle region and seemingly contribute to the anaphase movement of the chromosomes. At telophase, the spindle is persistent and the reforming nuclei are separated by cytoplasmic strands containing microtubules, interspersed with vacuoles. Extensive bundles of microtubules, dictyosomes and parallel, slightly inflated ER-profiles extend from the poles of the telophase nucleus along the longitudinal side of the chloroplast. Conceivably, these microtubules guide the nucleus during its post-mitotic migration towards its central interphase position between the two halves of the dividing chloroplast. Throughout the mitotic cycle, ubiquitous dictyosomes, positioned near the chloroplast core, seem very active. Arrays of microtubules run towards these dictyosomes and may conduct the dictyosome-vesicles to the cleavage plane. At metaphase, septum growth becomes visible as an annular ingrowth of the plasmalemma. At late telophase or at entering interphase, an extensive clump of vesicles, associated with longitudinal bundles of microtubules, appears between the leading edges of the advanced furrow. Apparent fusion of these vesicles with the head of the centripetally-growing furrow results in its completion. The pattern of mitosis and cytokinesis inZygnema is compared with that of closely related green algae.     

Division of Chloroplasts in a Green Alga, Trebouxia potteri

The division of chloroplasts in Trebouxia potteri was studiedby electron microscopy. At the beginning of chloroplast division,vesicles with fine fibrils (FVs) and ER attach to the isthmusof the chloroplast. Then, filaments appear around the isthmusparallel to the direction of constriction and seem to contractin order to decrease the diameter of the isthmus. It is suggestedthat the FVs are involved in the formation of the filamentsand that the ER is involved in the contraction of the filaments.At the final stages of the division of the chloroplast, thefilaments decompose. FVs are partially surrounded and decomposedby lysosomal sheets. For the next cycle of division of the chloroplast,the recovery of substances from decomposed filaments by functionalFVs seems a realistic possibility. chloroplast division, division apparatus, division cycle, transmission electron microscopy, Trebouxia potteri     

Homeostasis in Nitrogenous Uptake/Assimilation by the Green Alga Platymonas (Tetraselmis) striata (Prasinophyceae)

When nitrate, ammonium, or urea were the sole nitrogen sourcesfor the growth of Platymonas striata there was a short initiallag period, after which uptake/assimilation occurred at a constantrate. Glycine ‘uptake’ was however non-linear withtime. Addition of ammonium or urea markedly inhibited nitrate‘uptake’ whereas although glycine was initiallytotally inhibitory, nitrate ‘uptake’ recommencedabout 1 h after addition, but at a greatly reduced rate. Ammoniumtotally inhibited urea ‘uptake’, but did not inhibitglycine ‘uptake’. When ammonium, urea, or glycinewere present at the same time as nitrate the total rate of nitrogen'uptake/assimilation was within 40% (or closer) of that of nitratenitrogen ‘uptake’ alone. In the first two casesthis reflected a total switching off of nitrate ‘uptake’whilst the new substrate was assimilated at a rate similar tothe initial nitrate rate, whereas in the case of glycine, bothsubstrates were used simultaneously. Once the concentrationof any of the inhibitory nitrogen sources had been reduced toa low level the ‘uptake’/assimilation of nitraterecommenced. It is suggested that the ability of P. striatato maintain a more or less constant rate total nitrogen ‘uptake’/assimilationirrespective of the mixture of utilizable nitrogen sources presentedto it, is not due to controls at the level of the entry of substrateinto the cell, but to intracellular actions at the enzyme level.The relative constancy of, and high rate of ‘uptake’,irrespective of nitrogen source and over a wide range of extracellularconcentrations, is tentatively considered to be due to saturationof the assimilatory mechanisms under the conditions employed;all the sources lead, either directly or indirectly, to theproduction of amino groupings for entry into nitrogenous metabolism.This homeostasis of nitrogen ‘uptake’/assimilationwould clearly be of considerable benefit to the alga in nature,enabling it to maximize growth in changing environmental conditions. Platymonas striata, nitrogen update, nitrogen assimilation     

Uptake of Neutral Red by the Vacuoles of a Green Alga, Micrasterias pinnatifida

Neutral red (NR) in the culture medium entered the vacuolesof a green alga, Micrasterias pinnatifida, at a higher rateat pH 8 than at pH 5. NR remained soluble in vacuoles of cellscultured at pH 5, while it precipitated and formed granulesin cells cultured at pH 8. The vacuoles of cells cultured atpH 8 contained fibrils, but those of cells cultured at pH 5did not. The amount of NR that entered the cells was markedlyreduced by the addition to the medium of nigericin at 10^-5M,monensin at 10^-5M, bafilo-mycin A₁ at 10^-5M, or ammonium chlorideat 50 mM. The formation of NR granules in vacuoles were stronglyinhibited and the disorganization of NR granules were acceleratedby the addition of nigericin at 10^-5M, or bafilomycin A₁ at10^-5M to the culture medium. The possibility is discussed thatNR which enters vacuoles might become positively charged (NRH⁺)by protons brought into vacuoles by proton pumps and that NRH⁺might combine with some negatively charged macromolecules toform aggregates or granules. (Received April 18, 1996; Accepted May 27, 1996)     

Red And far‐red regulation of filament movement correlates with the expression of phytochrome and FHY1 genes in Spirogyra varians (Zygnematales,Streptophyta)1

Spirogyra filaments show unique photomovement that differs in response to blue, red, and far‐red light. Phototropins involved in the blue‐light movement have been characterized together with downstream signaling components, but the photoreceptors and mechanical effectors of red‐ and far‐red light movement are not yet characterized. The filaments of Spirogyra varians slowly bent and aggregated to form a tangled mass in red light. In far‐red light, the filaments unbent, stretched rapidly, and separated from each other. Mannitol and/or sorbitol treatment significantly inhibited this far‐red light movement suggesting that turgor pressure is the driving force of this movement. The bending and aggregating movements of filaments in red light were not affected by osmotic change. Three phytochrome homologues isolated from S. varians showed unique phylogenetic characteristics. Two canonical phytochromes, named SvPHY1 and SvPHY2, and a noncanonical phytochrome named SvPHYX2. SvPHY1 is the first PHY1 family phytochrome reported in zygnematalean algae. The gene involved in the transport of phytochromes into the nucleus was characterized, and its expression in response to red and far‐red light was measured using quantitative PCR. Our results suggest that the phytochromes and the genes involved in the transport system into the nucleus are well conserved in S. varians.     

Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga,Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.     

Isolation,Growth, Ultrastructure,and Metal Tolerance of the Green Alga,Chlamydomonas acidophila (Chlorophyta)

An acidophilic volvocine flagellate, Chlamydomonas acidophila (Volvocales) that was isolated from an acid lake, Katanuma, in Miyagi prefecture, Japan was studied for growth, ultrastructural characterization, and metal tolerance.

Chlamydomonas acidophila is obligately photoautotrophic, and did not grow in the cultures containing acetate or citrate even in the light. The optimum pH for growth was 3.5-4.5. To characterize metal tolerance, the toxic effects of Cd, Co, Cu, and Zn on this alga were also studied. Effective metal concentrations, which limited the growth by 50%, EC₅₀ were measured, after 72h of static exposure. EC₅₀s were 14.4 μM Cd²⁺, 81.3 μM Co²⁺, 141μM Cu²⁺, and 1.16 mM Zn²⁺ for 72 h of exposure. Thus, this alga had stronger tolerance to these metals than other species in the genus Chlamydomonas.