Measurement of the transmembrane electrical potential of Dunaliella acidophila by microelectrodes

A method was developed to determine electrical potential differences across the plasma membrane of the microalga Dunaliella by means of potential-sensitive microelectrodes. Special emphasis was put on the measurement of the membrane potential in the acidophilic Dunaliella acidophila (optimal growth at pH 1.0), but neutrophilic, halotolerant Dunaliella species were used as reference systems. For Dunaliella acidophila positive membrane potentials (cytoplasma relative to the medium), ranging from +30 to +65mV were measured. Illumination caused a decrease of the positive potential by about 10 mV. The ATPase inhibitor omeprazole caused an increase of the positive membrane potential ranging from +60 to +100 mV, whereas the ionophore gramicidin caused a decrease of the MP to +10 to +30 mV. The salt tolerant, neutrophilic Dunaliella parva and Dunaliella bardawil exhibited negative membrane potentials in the order of -40 to -60mV, and light caused a hyperpolarization of about 10 mV. A negative membrane potential was measured also in D. acidophila cells transferred to pH 7.0. The physiological significance of a positive membrane potential for acidophilic algae is discussed.Abbreviations E _m membrane potential - PM plasma membrane - TPB^– tetraphenylborone anion - TPP⁺ tetraphenyl-phosphonium cation - SCN^– isothiocyanate     

Uptake and Effect of Abscisic Acid during Induction and Progress of Radicle Growth in Seeds of Chenopodium album

In the seeds of Chenopodium album L. visible phenomena preceding the final protrusion of the radicle enable a clear distinction between the induction and the progress of growth inside the covering structures. The light-dependent induction of radicle growth is not inhibited by exogenously applied abscisic acid (ABA). Experiments with 1-¹⁴C-ABA ruled out a lack of penetration of the hormone. However, ABA does inhibit the growth of the radicle before final protrusion. This inhibition and the uptake of 1-¹⁴C-ABA are enhanced at lower pH values, indicating absorption of the undissociated molecule. The uptake of labeled hormone strongly increases during the growth of the radicle. This increase is not merely a reflection of extra water uptake. Seeds of different degrees of dormancy contain equallly low levels of endogenous ABA. Much higher levels of ABA in the seeds were obtained by exogenous application of the hormone but these levels stills do not prevent the breaking the dormancy by light. It is concluded that ABA has no function in the regulation of dormancy in C. album seeds.     

Phylogenetic position of Dunaliella acidophila (Chlorophyceae) based on ITS and rbcL sequences

Dunaliella acidophila is one of the most extreme acidophiles on earth and is able to survive in highly acidic habitats. This characteristic has made this organism the universal model for the study of abiotic stress. Although D. acidophila is currently circumscribed to the subgenus Pascheria within Dunaliella Teodoresco (Chlorophyceae), its taxonomic position has stirred controversy. The comparison of D. acidophila CCAP19/35 internal transcribed spacers (including ITS2 secondary structure analysis) and RuBisCo large subunit (rbcL) sequences with other Dunaliella species confirms that D. acidophila should maintain its phylogenetic position within the genus Dunaliella, suggesting its inclusion within the subgenus Dunaliella. Furthermore, the ITS1 and ITS2 data revealed that D. acidophila was highly divergent from the other freshwater species assessed, D. lateralis, with which it barely shares a 56.8% similarity.     

Possible explanation of IAA-stimulated transport of14C-ABA in long pea (Pisum sativum L.) epicotyl segments

Effects of 2,3,5-triiodobenzoic acid (TIBA) and age of etiolated pea epicotyl segments on the indol-3-ylacetic acid (IAA) stimulated transport of¹⁴C-abscisic acid (ABA) was studied. In spite of a slight decrease of IAA transport after the application of TIBA, the IAA stimulation of¹⁴C-ABA transport did not change. In segments excised from epicotyls of different age,³H-IAA transport was identical and the induction of prolongation growth by IAA in segments from the upper part of the epicotyl was observed. The IAA ap{ie226-01}ation to the growing segments was connected with intensive attraction of¹⁴C-ABA to the site {ie226-02}AA application, while the application of IAA to the older segments was growth ineffective ana no stimulation of¹⁴C-ABA transport by IAA was observed.     

Metabolism of14C-abscisic acid during its IAA-promoted transport in etiolated segments of pea (Pisum sativum L.) epicotyls†

The metabolism of exogenously supplied abscisic acid (ABA) during translocation attracted under the influence of indolyl-3-acetic acid (IAA) was studied in etiolated segments of pea (Pisum sativum L.). After 8 and 24 h 90% and 60% of the ABA, respectively, were found in the segments in unchanged form. Phaseic acid, dihydrophaseic acid and the glucose ester of ABA were found as ABA metabolites. Results indicated that the growth processes initiated by the application of IAA were associated neither with an increased immobilization nor increased metabolization of this growth regulator. † Part II. Influence of Auxin-like Substances upon the Transport of¹⁴C-ABA in Long Pea Epicotyl Segments.     

Translocation of14C-abscisic acid from roots into the aboveground part of pea (pisum sativum L.) seedlings

The translocation of¹⁴C-ABA from roots into other parts of the plant was followed in intact and decapitated pea seedlings. In intact plants ABA from roots was translocated above all into the apical part of epicotyl. In decapitated plants the regulative ability of intact apex can be partly simulated by exogenous IAA. The growth of lateral buds occurring after decapitation was associated with an intensive flow of¹⁴C-ABA from roots into released lateral buds as late as 72 h after decapitation,i.e. in the stage of intensive elongation growth of buds.     

Movement of Abscisic Acid in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Plants

THE movement of abscisic acid (ABA) in plants seems to have been studied only in isolated segments of tissue^1–4. We have used ¹⁴C-labelled ABA of relatively high specific activity to investigate its movement in a number of plant species, in both isolated tissue segments and whole plants. The movement of 2-¹⁴C-ABA in intact seedlings of Phaseolus vulgaris is described here.     

Dunaliella acidophila: an algae with a positive zeta potential at its optimal pH for growth*

Abstract. The zeta potential (which approximates the surface potential) of the acid resistant green alga Dunaliella acidophila (optimal growth at pH 1.0) and the salt resistant D. parva (grown at pH 7.6) were calculated from the electrophoretic mobility of cells as determined by means of free-flow electrophoresis. Dunaliella acidophila cells exhibit a positive zeta potential (+5 to +20mV) at acidic external pH values, whereas negative zeta potentials (-30 mV) were measured at neutral pH values. Negative zeta potentials of the same order of magnitude were also measured for D. parva cells (pH 7.6). Low concentrations of La³⁺ and A1³⁺ did not affect the positive zeta potential of D. acidophila at acidic pH values, whereas higher concentrations caused a shift to more positive potentials. However, at neutral pH these cations caused a significant decrease of the negative zeta potential. The impermeant polycation poly-L-lysine acted in a similar manner to A1³⁺ or La³⁺. The effect of Impermeant cations and anions on various physiological reactions also supports the existence of a positive zeta potential for D. acidophila and of a negative zeta potential for D. parva: polycations such as DEAE-dextran and poly-L-lysine strongly inhibitied photosynthesis and mobility of D. parva, but did not affect these reactions in D. acidophila. Comparable differential inhibitions were also observed for A1³⁺ and La³⁺. Impermeant anions such as Dextran-sulfate exhibited effects in the opposite direction: inhibition was stronger with D. acidophila and weaker with D. parva. However, the differential inhibition by impermeant anions was much less pronounced in comparison with impermeant cations due to the relatively high pK_a values of anionic solutes and consequently relatively high protonation at pH 1.0. The physiological consequences of an asymmetrically charged plasma membrane (excess of positive charges outside, excess of negative charges on the cytoplasmic side) of D. acidophila are discussed in regard to the extreme acid resistance of this alga and its resistance to cationic toxic solutes in industrial wastes.     

Effect of abscisic acid and ontogenic phases of the host alga on the infection process in the pathosystem Scenedesmus acutus — Phlyctidium scenenedesmi

The parasite contamination make difficult laboratory cultivation of green algae and decrease significantly the production of microalgal biomass during industrial cultivation. In the present study the influence of the endogenous abscisic acid content (determinate by gas chromatography) on the host-parasite relationship in different ontogenetic phases of the host Scenedesmus as well as resistance induction after treatment of synchronous algal culture with ABA were studied. Synchronization of algae was carried out by alteration of light and dark periods. The age groups under study were: autospores (at the beginning of the light period), growing cells, mature cells (belonging to in the end of the light period) and mature cells, starting to release autospore at the beginning of dark phases. The higher levels of endogenous ABA during the algal autospore formation as well as exogenous ABA supply of (10⁻⁵ M) inhibited the infection process in the pathosystem green microalga Scenedesmus acutus and unicellular fungal parasite Phlyctidium scenedesmi. The treatment with fluridone 10⁻⁷ M (an inhibitor of ABA biosynthesis) increased the host susceptibility during all ontogenetic phases. The susceptibility of S. acutus to the chytridial infection depended on the endogenous ABA level during different ontogenetic stages of the alga.     

Distribution and Diversity of Cyanobacteria and Eukaryotic Algae in Qinghai–Tibetan Lakes

Cyanobacteria and eukaryotic algae are important primary producers in a variety of environments, yet their distribution and response to environmental change in saline lakes are poorly understood. In this study, the community structure of cyanobacteria and eukaryotic algae in the water and surface sediments of six lakes and one river on the Qinghai–Tibetan Plateau were investigated with the 23S rRNA gene pyrosequencing approach. Our results showed that salinity was the major factor controlling the algal community composition in these aquatic water bodies and the community structures of water and surface sediment samples grouped according to salinity. In subsaline–mesosaline lakes (salinity: 0.5–50 g L^?1), Cyanobacteria (Cyanobium, Synechococcus) were highly abundant, while in hypersaline lakes (salinity: >50 g L^?1) eukaryotic algae including Chlorophyta (Chlorella, Dunaliella), Bacillariophyta (Fistulifera), Streptophyta (Chara), and Dinophyceae (Kryptoperidinium foliaceum) were the major members of the community. The relative abundance ratio of cyanobacteria to eukaryotic algae was significantly correlated with salinity. The algae detected in Qinghai–Tibetan lakes exhibited a broader salinity range than previously known, which may be a result of a gradual adaptation to the slow evolution of these lakes. In addition, the algal community structure was similar between water and surface sediment of the same lake, suggesting that sediment algal community was derived from water column.     

Distribution of IAA and ABA in gravistimulated primary roots ofZea mays. L.

The transport of¹⁴C-IAA and¹⁴C-ABA applied exogenously to root cap toward the elongation zone was investigated in gravi- and light-stimulated primary roots ofZea mays L. cv. Golden Cross Bantam 70. No significant difference of either IAA or ABA in radioactivities was observed between upper and lower halves of elongation zones during the latent period (0–60 min after the stimulation) of gravitropic response. When quantitative analysis of endogenous IAA and ABA by an internal standard method was carried out 60 min after gravi- and/or light-stimulation, no asymmetric redistribution of either IAA or ABA was observed between upper and lower halves of elongation zones. Light irradiation increased by 20% the contents of ABA in elongation zones. These results suggest that although both IAA and ABA are basipetally transportable and can transmit their information to the elongation zone during a latent period we cannot explain the gravitropic curvature by their redistributions between the two (upper and lower) halves of primary roots ofZea. On the basis of results from the present work and previous papers, the distribution of IAA and ABA in gravistimulatedZea roots is discussed. A part of this study was reported at the Eighth Annual Meeting of the IUPS Commission on Gravitational Physiology at Tokyo 1986.     

The Uptake and Degradation of 1-14C-Abscisic Acid by Apple Seeds during Stratification

The distribution and degradation of I-¹⁴C-ABA in apple seedsduring stratification was investigated. It was found that totalradioactivity of labelled ABA present in the apple seed after4⁸ h of soaking in I-¹⁴C-ABA solution decreased gradually duringthe stratification period. The decrease of radioactivity wasobserved in seed coats and endosperm with the simultaneous considerableincrease in the cotyledon and embryo axis. Decarboxylation ofI-¹⁴C-ABA was demonstrated and it is postulated as a mode ofABA degradation in apple seed.     

Algal biotechnology products and processes — matching science and economics

Several microalgae, such as species ofChlorella, Spirulina andDunaliella, are grown commercially and algal products such as -carotene and phycocyanin are available. The main focus of algal biotechnology continues to be on high value fine chemicals and on algae for use as aquaculture feeds. This paper provides the outline for a rational approach in evaluating which algae and which algal products are the most likely to be commercially viable. This approach involves some simple market analysis followed by economic modelling of the whole production process. It also permits an evaluation of which steps in the production process have the greatest effect on the final production cost of the alga or algal product, thus providing a guide as to what area the research and development effort should be directed to. An example of this approach is presented and compared with other models. The base model used here gives a production cost of microalgal biomass at about AS 14 to 15 kg^–1, excluding the costs of further processing, packaging and marketing. The model also shows that some of the key factors in microalgal production are productivity, labor costs and harvesting costs. Given the existing technology, high value products such as carotenoids and algal biomass for aquaculture feeds have the greatest commercial potential in the short term.This paper was presented at the Symposium on Applied Phycology at the Fourth International Phycological Congress, Duke University.     

The effect of ampicillin plus streptomycin on growth and photosynthesis of two halotolerant chlorophyte algae

Streptomycin and ampicillin are antibiotics commonly used to eliminate prokaryotes from the cultures of eukaryotic algae. We studied the effects of 25 mg l⁻¹ streptomycin plus 50 mg l⁻¹ ampicillin on the growth and photosynthesis of two broadly halotolerant algae, Picochlorum oklahomensis and Dunaliella sp. (Chlorophyceae). We measured growth rate, oxygen evolution, chlorophyll fluorescence kinetics, and pigment content in low (150 μmol photons m⁻² s⁻¹) and high (600 μmol photons m⁻² s⁻¹) light grown batch cultures. Our results show only a minor effect of the antibiotics on P. oklahomensis, and none on Dunaliella sp., so this combination of antibiotics is suitable for maintenance of stock cultures for physiological experiments. We also show that these antibiotics can be used in turbidostat cultures of P. oklahomensis, which otherwise tend to succumb to bacteria.     

ADAPTATION OF THE UNICELLULAR ALGA DUNALIELLA PARVA TO A SALINE ENVIRONMENT1

The holophilic alga Dunaliella parva produces glycerol as a major product of photosynthetic ¹⁴CO₂ incorporation and accumulates very large amounts of intracellular glycerol. A method was adopted for the determination of the cell water space based on the distribution of ¹⁴C sorbitol and ³H₂O between the cells and the medium. Using these measurements the internal concentration of glycerol was found to be isoomotic with that of the medium over a broad range of 0.6 to 2.1 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular water content immediately varied so as to keep an osmotic equilibrium between the cells and the medium. During the following 90 min under metabolic conditions, glycerol content changed until a new level was reached. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. Determination of the glycerol sensitivity of enzymic and photosynthetic reactions of cell-free preparations from D. parva showed a broad range of tolerance to high concentrations of glycerol. These results indicate that osmoregulation in Dunaliella depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration. A proposed scheme of glycerol synthesis in Dunaliella is suggested.     

Resistance of the alga Spermatozopsis acidophila Kalina (Chlorophyta,Volvocales) to heavy metals

Abstract

The toxic effects of 14 chemical elements (prevalently heavy metals) on the alga Spermatozopsis acidophila Kalina (Volvocales, Polyblepharidaceae) have been investigated.

The results obtained show a high resistance to heavy metals, particularly to Cu, in comparison with data found in literature on other algae.

This phenomenon might be interpreted as a consequence of evolutionary adaptation to highly acid environments.     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

Nitrogen limitation and amino-acid metabolism of Chlorella symbiotic with green hydra

Chlorella algae symbiotic in the digestive cells of Hydra viridissima Pallas (green hydra) were found to contain less amino-N and smaller pools of free amino acids than their cultured counterparts, indicating that growth in symbiosis was nitrogen-limiting. This difference was reflected in uptake of amino acids and subsequent incorporation into protein; symbiotic algae incorporated a greater proportion of sequestered radioactivity, supplied as ¹⁴C-labelled alanine, glycine or arginine, than algae from nitrogen-sufficient culture, presumably because smaller internal pools diluted sequestered amino acids to a lesser extent. Further experiments with symbiotic algae showed that metabolism of the neutral amino acid alanine differed from that of the basic amino acid arginine. Alanine but not arginine continued to be incorporated into protein after uptake ceased, and while internal pools of alanine were exchangeable with alanine in the medium, those of arginine were not exchangeable with external arginine. Thin-layer chromatography of ethanol-soluble extracts of algae incubated with [¹⁴C]alanine or [¹⁴C]arginine showed that both were precursors of other amino acids. The significance of nitrogen-limiting growth of symbiotic algae is discussed in terms of host-cell regulation of algal cell growth and division.