Luis Oliveira

Evidence of Nickel Ion Requirement for Autotrophic Growth of a Marine Diatom with Urea serving as Nitrogen Source

The diatom Cyclotella cryptica Reimann, Lewin & Guillard was unable to grow on urea added to seawater enriched with phosphate, silicate, vitamins (Bag, thiamine, biotin), trace metal ions (Fe, Mn, Zn, Mo, Cu, Co) and buffered at pH 7.5 8-0. On addition of minute Ni 2 + concentrations to this medium, the diatom showed good growth on urea compared to that on equivalent nitrate without Ni 2+ addition. The growth on urea was optimal at Ni 2+ concentrations in the range 0.1-1 gM, but good growth was also obtained at 0-01 and 10 gM N i 2 +. Higher Ni 2 + concentrations were inhibitory to growth on both urea and nitrate, while the lower Ni 2 + concentration limit for measurable urea growth was reached at 4-5 riM. In the simultaneous presence of a chelating agent, the diatom failed to grow on urea, indicating a growth requirement for free Ni 2+ ions. This Ni 2+ requirement could not be replaced by Cu 2 +, Mn 2 +, Zn 2 +, Pd 2 +, while Co z + could substitute for N i 2 + to the extent of about 20~o. At a Ni z+ concentration of 0.1 g~, the diatom yield increased to a saturation maximum with increasing urea concentration up to 5 mM and was little depressed at higher urea levels. While a Ni 2 + requirement could be universal to urease-produeing phytoplankters, it was inferred that this requirement may be greater and more stringent for C. cryptica so that other algae could meet their requirement, without Ni 2+ supplementation, from the trace metal concentrations normally present in seawater. Alternatively, they might more readily substitute Ni 2+ by a trace metal analogue (such as Co 2+) from the nutrient enrichments normally made in preparing culture media.

The use of an ultra‐low viscosity medium (VCD/HXSA) in the rapid embedding of plant cells for electron microscopy

The VCD/HXSA ultra‐low viscosity medium is characterized by a viscosity of only 20 cps at 298 K. This is extremely useful for rapid embedding schedules and facilitates cutting large sections even from difficult materials. The suitability of this medium for ultrastructural studies is tested with plant specimens ranging from soft, highly vacuolated parenchymatous tissue to hard thick‐walled cells impregnated with a variety of substances or covered with mucilage. The results, when compared with those from similar materials infiltrated with Spurrs and Epon embedding media, show that the general preservation of the cellular structure is comparable for all the three media tested. In addition, embeddment in VCA/HXSA medium results in better preservation of some vacuolar features and in the reduction of plasmolysis.

Ultrastructural, cytochemical, and enzymatic studies on the adhesive “plaques” of the brown algaeLaminaria saccharina (L.) lamour. andNereocystis luetkeana (nert.) post. et rupr. 1

SummaryNewly settled zoospores of bothLaminaria saccharina andNereocystis luetkeana are surrounded by adhesive plaques. At this stage cell wall deposition has not occurred and similar structures cannot be detected in the cytoplasm. In free swimming zoospores, however, plaques are observed exclusively in small vesicles within the cytoplasm. Enzymatic extractions and cytochemical tests indicate that plaques are glycoproteid in nature. Studies on the influence of several enzymes on the attachment of zoospores show that plaques are involved in the adhesion of zoospores to the substratum.

Aluminum toxicity studies in Vaucheria longicaulis var. macounii (Xanthophyta, Tribophyceae). II. Effects on the F-actin array.

In this study, we test the hypothesis that exposure to environmentally significant concentrations of aluminum (Al, 80 µM) causes the microfilament array of Vaucheria longicaulis var. macounii vegetative filaments to become fragmented and disorganized. Changes in F-actin organization following treatment of vegetative filaments by Al are examined using vital staining with fluorescein phalloidin. In the cortical cytoplasm of the apical zone of pH 7.5 and pH 4.5 control cells, axially aligned bundles of F-actin lead to a region of diffuse, brightly stained material. Dimly stained focal masses are noted deeper in the cytoplasm of the apical zone whereas they are absent from the zone of vacuolation. The F-actin array is visualized in the cortical cytoplasm of the region of the cell, distal to the apical tip, which exhibits vigorous cytoplasmic streaming (zone of vacuolation) as long, axially aligned bundles with which chloroplasts and mitochondria associate. Thirty minutes following treatment with aluminum, and for the next 8-16 h, the F-actin array is progressively disorganized. The longitudinally aligned F-actin array becomes fragmented. Aggregates of F-actin, such as short rods, amorphous and stellate F-actin focal masses, curved F-actin bundles and F-actin rings replace the control array. Each of these structures may occur in association with chloroplasts or independently with no apparent association with organelles. Images are recorded which indicate that F-actin rings not associated with organelles may self-assemble by successive bundling of F-actin fragments. The fragmentation and bundling of F-actin in cells of V. longicaulis upon treatment with aluminum resembles those reported after diverse forms of cell disturbance and supports the hypothesis that aluminum-induced changes in the F-actin array may be a calcium-mediated response to stress.

Organization of the cytoskeleton in the coenocytic algaVaucheria longicaulis var.macounii: an experimental study

SummaryThe organization of the cytoskeleton of vegetative filaments ofVaucheria longicaulis Hoppaugh var.macounii Blum is investigated by fluorescence microscopy using monoclonal anti β-tubulin antibodies and fluorescein (FITC)-labelled phalloidin. Confocal laser scanning microscopy observations give further information on the distribution of the cytoskeletal elements. Phalloidin labelling reveals F-actin bundles in the cortical cytoplasm of both fixed and unfixed vegetative filaments of this alga. In addition a more diffuse fluorescent component, seen at higher magnification to be made up of thinner F-actin bundles, can also be detected in unfixed cells. The distribution of the F-actin bundles resemble that of filamentous structures observed with differential interference contrast (DIC) microscopy in living cells. These structures seem to correspond to the “microtubule associated reticulum” (MAR) described in literature and overall the evidence suggests that actin and MAR elements are co-distributed. F-actin bundles are always found in association with focal masses (foci) of phalloidin-positive material. Foci are also observed by DIC microscopy associated with the cytoplasmic filamentous structures in living cells.Depolymerization of F-actin with cytochalasin D and the subsequent repolymerization that occurs on transfer ofVaucheria vegetative filaments to cytochalasin-free medium suggest that these foci are involved in the organization of the F-actin array. Immunofluorescence for β-tubulin reveals microtubule bundles that are shorter in length and straighter in configuration than microfilament bundles. Microtubule bundles are associated with spot-like focal structures that, in many instances, show a close relationship with respect to nuclei. Oryzalin and cold temperature cause the depolymerization of the microtubule bundles and suggest, in conjunction with repolymerization studies, that these fluorescent spots associated with the ends of the microtubule bundles are involved in their organization; hence, they represent microtubule organizing centres or MTOCs. The importance of both microfilament and microtubule bundle focal regions is discussed with respect to the apical growth exhibited by the vegetative filaments of this alga.

Aluminum toxicity studies in Vaucheria longicaulis var. macounii (Xanthophyta, Tribophyceae). I. Effects on cytoplasmic organization

Using differential interference contrast (DIC) and epifluorescence microscopy, we tested the hypothesis that exposure to environmentally significant levels of aluminum (Al) would cause rapid changes in cytoplasmic organization in vegetative filaments of the coenocytic alga, Vaucheria longicaulis Hoppaugh var. macounii Blum resulting in the loss of cytoplasmic streaming. In untreated cells, DIC microscopy revealed the presence of cortical cytoplasmic strands that were oriented longitudinally to the cell axis as well as sub-cortical cytoplasmic strands that exhibited a reticulate morphology. Organelles such as chloroplasts and mitochondria translocated throughout the cell in close association with the cortical longitudinal cytoplasmic strands. Staining with the lipophilic dye, 3,3-dihexyloloxacarbocyanine, revealed structures that appeared to be endoplasmic reticulum (ER). This organelle closely resembled, in location and appearance, the cytoplasmic strands visualized using DIC microscopy. The addition of Al (80 µM) resulted in the inhibition of cytoplasmic streaming as well as the dissipation of the putative cortical longitudinal ER within one minute. Subsequently, the DIC-visible cortical cytoplasmic strands exhibited progressive degrees of disorganization. Throughout these changes, chloroplasts and mitochondria remained visibly associated with the cortical cytoplasmic strands.

The effect of organic calcium channel modulators on the germination ofVaucheria longicaulis aplanospores

SummaryInVaucheria longicaulis var.macounii aplanospore germination and filament growth are severely inhibited by the Ca2+-channel antagonists (−)202–791, diltiazem, nifedipine and verapamil, whereas the agonists (+)202–791, Bay K-8644 and CGP-28392 stimulate those processes. Both antagonist and agonist actions suggest that voltage-controlled Ca2+ influx plays a major role in the regulation of the initial events of germination and filament growth. Increases in45Ca2+ influx are observed after pretreatment of the aplanospores with low temperature shocks of brief duration or FCCP. Both agents are known to depolarize the surface membrane.45Ca2+ influx is reduced in material treated with FC, an agent known to hyperpolarize cell membranes. The results indicate that Ca2+ influx takes place through voltage-sensitive Ca-channels.