Copper tolerance in the green alga, Chlorella vulgaris

Abstract. The effect of sub-lethal concentrations of copper upon tolerant and non-tolerant strains of Chlorella vulgaris was investigated. Copper concentrations of 0.2 and 0.4 mg dm⁻³ increased the lag phase of both strains, the effect being greater in the non-tolerant strain. No difference was observed in the toxicity of copper to the photosynthetic rates of the isolated chloroplasts of either strain. However, significant differences were shown at the whole cell level.
Lower copper uptake was shown by the tolerant cells. In both strains initial uptake of copper was followed by a phase of desorption before cell division occurred. In cultures of both strains the concentration of ionic copper was decreased by complexation with extracellular organic material. Over a 14 day growth period more organic material was produced by the tolerant cells. The organic material produced by the tolerant cell formed organo-copper complexes which had a higher conditional stability constant.
It is proposed that the cell wall acts as a barrier to copper in the tolerant cells and prevents copper from affecting cell metabolism. Organo-copper complexation occurs at this barrier and this complex is then released into the medium.     

A new inducible expression system in a transformed green alga, Chlorella vulgaris

Genetic transformation is useful for basic research and applied biotechnology. However, genetic transformation of microalgae is usually quite difficult due to the technical limitations of existing methods. We cloned the promoter and terminator of the nitrate reductase gene from the microalga Phaeodactylum tricornutum and used them for optimization of a transformation system of the microalga Chlorella vulgaris. This species has been used for food production and is a promising candidate as a bioreactor for large-scale production of value-added proteins. A construct was made containing the CAT (chloramphenicol acetyltransferase) reporter gene driven by the nitrate reductase promoter. This construct was transferred into the C. vulgaris genome by electroporation. Expression of CAT in transgenic Chlorella conferred resistance to the antibiotic chloramphenicol and enabled growth in selective media. Overall efficiency for the transformation was estimated to be approximately 0.03%, which is relatively high compared with other available Chlorella transformation systems. Expression of CAT was induced in the presence of nitrate and inhibited in the presence of ammonium as a sole nitrogen source. This study presented an inducible recombinant gene expression system, also providing more gene regulation elements with potential for biotechnological applications.     

Nitrogen,phosphorus and high CO2 modulate photosynthesis,biomass and lipid production in the green alga Chlorella vulgaris

Photosynthesis Research - Climate change could impact nutrient bioavailability in aquatic environment. To understand the interaction of nutrient bioavailability and elevated CO2, Chlorella vulgaris...     

Mechanistic characterization of omega-3 desaturation in the green alga Chlorella vulgaris

alpha-Linolenic acid (ALA, 9(Z),12(Z),15(Z)-octadecatrienoic acid) derivatives are important plant lipids which play a critical key role in cold tolerance. The final steps of ALA biosynthesis feature a series of regio- and stereoselective dehydrogenation reactions which are catalyzed by a set of enzymes known as fatty acid desaturases. In conjunction with ongoing research into the structural biology of these remarkable catalysts, we have examined the mechanism of double bond introduction at C15,16 as it occurs in a model photosynthetic organism, Chlorella vulgaris. The individual deuterium kinetic isotope effects associated with the C-H bond cleavages at C-15 and C-16 of a thialinoleoyl analogue were measured via competition experiments using appropriately deuterium-labelled 7-thia substrates. A large kinetic isotope effect (KIE) (k(H)/k(D)=10.2+/-2.8) was observed for the C-H bond-breaking step at C-15 while the C-H bond cleavage at C-16 was found to be relatively insensitive to deuterium substitution (k(H)/k(D)=0.8+/-0.2). These results point to C-15 as the site of initial oxidation in omega-3 desaturation and imply that the Chlorella and corresponding plant systems share a common active site architecture.     

Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris.

The hypothesis that the Delta9 desaturase of Chlorella vulgaris might operate by a synchronous mechanism has been tested using a kinetic isotope effect (KIE) approach. Thus the intermolecular primary deuterium KIE on the individual C-H bond cleavage steps involved in Delta9 desaturation have been determined by incubating growing cultures of C. vulgaris (strain 211/8K) with mixtures of the appropriate regiospecifically deuterated fatty acid analogues. Our analysis shows that the introduction of a double bond between C-9 and C-10 occurs in two discrete steps as the cleavage of the C9-H bond is very sensitive to isotopic substitution (kH/kD = 6.6 +/- 0.3) whereas a negligible isotope effect (kH/kD = 1.05 +/- 0.05) was observed for the C10-H bond-breaking step. Similar results were obtained for linoleic acid biosynthesis (Delta12 desaturation). These data clearly rule out a synchronous mechanism for these reactions.     

Uptake and metabolism of taurine in the green alga Chlorella fusca

Taurine entered the alga Chlorella fusca Shihira et Krauss strain 21l-8b via a pH and energy-dependent system ("permease"). Transport followed triphasic kinetics from 10⁻⁶ to 10⁻² M with K_m values for taurine of 5.4 × 10⁻⁵, 4.1 × l0⁻⁴ and l.5 × 10⁻³ M. This uptake system was specific for sulfonic acids and showed no affinity for α- and β -amino acids or Na⁺; thus the permease of C. fusca is different from all known taurine transport systems with respect to structural specificity and lack of Na⁺ -dependence. Uptake was not observed in sulfate-grown algae but developed as a response to sulfate limitation within 2 h. Sulfate addition caused a rapid decline in taurine transport capacity. Labeled taurine was rapidly metabolized in C. fusca to sulfate and ethanolamine, suggesting oxidative hydrolysis as the mechanism of C-S bond cleavage. Further incorporation of these catabolic products in C - and S -metabolism was demonstrated. Taurine catabolism was also detected in other green algae and some cyanobacteria.     

Enzymatic conversion of glutamate to delta-aminolevulinate in soluble extracts of the unicellular green alga, Chlorella vulgaris

Cell-free preparations from the unicellular green alga, Chlorella vulgaris, catalyze the conversion of glutamate to delta-aminolevulinate, which is the first committed step in heme and chlorophyll biosynthesis. Most activity remains in the supernatant fraction after centrifugation at 264,000g. Additional activity can be solubilized from the high-speed pellet by treatment with 0.5 M NaCl. After gel filtration through Sephadex G-25, the reaction catalyzed by the high-speed supernatant requires glutamate, ATP, Mg2+, and NADPH. Boiled extract is inactive. The pH optimum is between 7.8 and 7.9 and the temperature optimum is 30 degrees C. Concentrations required for half-maximal activity are 0.05 mM glutamate, 0.4 mM ATP, 6 mM MgCl2, and 0.4 mM NADPH or 0.7 mM NADH. The reaction requires no additional amino donor. Involvement of pyridoxal phosphate in the catalytic mechanism is suggested by sensitivity to pyridoxal antagonists; 50% inhibition is achieved with 5 microM gabaculine or 0.4 mM aminooxyacetate. Involvement of two or more enzymes is suggested by the nonlinear reaction rate dependence on protein concentration. Evidence for the involvement of an activated glutamate intermediate was obtained by product formation after sequential addition and removal of substrates, and by inhibition (80%) with 1 mM hydroxylamine. Protoheme inhibits the activity by 50% at 1.2 microM. Preincubation of the extract with ATP causes stimulation and/or stabilization of the activity compared to preincubation without ATP or no preincubation. In preparations obtained from C. vulgaris strain C-10, which requires light for greening, dark-grown cells yield one-third as much activity as 4-h-greened cells.     

Kinetics of uptake and intracellular location of cobalt,manganese and zinc in the estuarine green alga Chlorella salina

Summary The uptake kinetics and intracellular location of cobalt (⁶⁰Co), manganese (⁵⁴Mn) and zinc (⁶⁵Zn) have been characterized in Chlorella salina. Uptake of all three metals was biphasic. The initial rapid phase was independent of light, temperature or the presence of metabolic inhibitors. This first phase of metabolism-independent biosorption was followed by a slower phase of uptake that was apparently dependent on metabolism and decreased by incubation in the dark, or in the light at low temperature or in the presence of metabolic inhibitors. This latter phase of metal accumulation followed Michaelis-Menten kinetics. However, when expressed in the form of a Lineweaver-Burk plot two distinct phases were apparent for each metal with the following K_m values (M); Co²⁺, 19 and 266; Mn²⁺, 2 and 760; Zn²⁺, 4 and 635. For all three metals cellular compartmentation analysis showed that large amounts of metal were bound to intracellular components and to the cell wall. There was also a higher concentration of each metal in the vacuole than in the cytosol, indicating transport of the metals across the tonoplast which may, in part, account for the multi-phasic uptake systems detected. The influence of competing divalent ions on the active uptake of Co²⁺ and Mn²⁺ was also studied. When the concentration of divalent ion was the same as that of Co²⁺ the uptake of the latter was not affected, indicating a specific system for the uptake of Co²⁺. However, Mn²⁺ uptake inhibited by Mg²⁺, Zn²⁺ and Cd²⁺, but not by Co²⁺, which indicated that Mn²⁺, Mg²⁺ and Cd²⁺ may enter the cells via a common system with different affinities for each metal.     

The chloroplast chlL gene of the green alga Chlorella vulgaris C-27 contains a self-splicing group I intron

The chlL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome of Chlorella vulgaris strain C-27 contains the first example of a split chlL gene, which is interrupted by a 951?bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5′ and 3′ splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522?nt), separating the catalytic core from the 3′ splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3′ splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

A freshwater green alga under cadmium stress: Ameliorating calcium effects on ultrastructure and photosynthesis in the unicellular model Micrasterias

Cadmium is a highly toxic heavy metal pollutant arising mainly from increasing industrial disposal of electronic components. Due to its high solubility it easily enters soil and aquatic environments. Via its similarity to calcium it may interfere with different kinds of Ca dependent metabolic or developmental processes in biological systems. In the present study we investigate primary cell physiological, morphological and ultrastructural responses of Cd on the unicellular freshwater green alga Micrasterias which has served as a cell biological model system since many years and has proved to be highly sensitive to any kind of abiotic stress. Our results provide evidence that the severe Cd effects in Micrasterias such as unidirectional disintegration of dictyosomes, occurrence of autophagy, decline in photosystem II activity and oxygen production as well as marked structural damage of the chloroplast are based on a disturbance of Ca homeostasis probably by displacement of Ca by Cd. This is indicated by the fact that physiological and structural cadmium effects could be prevented in Micrasterias by pre-treatment with Ca. Additionally, thapsigargin an inhibitor of animal and plant Ca(2+)-ATPase mimicked the adverse Cd induced morphological and functional effects on dictyosomes. Recovery experiments indicated rapid repair mechanisms after Cd stress.     

Characterization and amino acid sequences of cytochromes c6 from two strains of the green alga Chlorella vulgaris

Cytochromes c6 from the green algae Chlorella vulgaris CK-5 (CK5cyc6) and C. vulgaris CK-22 (CK22cyc6) were characterized and their amino acid sequences were analyzed. CK5cyc6 had a molecular mass of 9.3 kDa, isoelectric points of 3.0 (reduced) and 3.6 (oxidized), and a redox potential of +362 mV at pH 7.0. CK22cyc6 had a molecular mass of 9.5 kDa, isoelectric points of 2.9 (reduced) and 3.5 (oxidized), and a redox potential of +355 mV at pH 7.0. The absorption spectra of both cytochromes c6 showed 4 maxima in reduced form, and 2 maxima and a weak peak at 695 nm in oxidized form. The pyridine ferrohemochrome spectra indicated that their prosthetic group was heme c. These physicochemical properties were similar to those of other algal cytochromes c6. The amino acids (88 residues) of CK5cyc6 and CK22cyc6 were sequenced and the sequence motif -CXXCH-, which is typical of the heme-binding site of c-type cytochrome, was clearly confirmed in both cytochromes. Twenty-six amino acid residues were substituted, and the similarity score of each of them was 70.45%.     

Effects of high light and ozone fumigation on photosynthesis in Phaseolus vulgaris

In the present work, the aim was to study the combined effects of high irradiance (about 1 000 μmol·m^–2·s^–1) on the effects of O₃ (150 nL·L^–1 for 2 h) on the photosynthetic process of primary pinto (Phaseolus vulgaris L.) leaves. The CO₂ assimilation rate significantly decreased in high light and/or O₃-treated leaves, as did the stomatal conductance. The present work shows that changes occur in photosynthesis-related parameters due to light stress. Photoinhibition was detected as a decrease in the F_v/F_m ratio. This indicates that the light treatment considerably exceeded the photoprotective capacity and resulted in significant photon damage. Moreover O₃ fumigation alone determined an impairment of the photosynthetic process, that in this case is related to the dark reactions of photosynthesis as also showed by the strong increase in (1-q_P). The data presented in this paper illustrate the complexity of photosynthetic response to high light intensities and ozone, which are two stress factors that often occur simultaneously under natural conditions. High light can modify the reaction of leaves to ozone treatment by reducing the chloroplastic electron transport rate and the quantum yield for PSII photochemistry. Thus, high light seems to enhance the detrimental effects of ozone on photosynthesis.     

Interactive effects of UV-B irradiation and triadimefon on nodulation and nitrogen metabolism in Vigna radiata plants

Supply of aqueous solution of triadimefon (20 mg dm⁻³) to unstressed green gram plants increased the contents of soluble proteins, amino acids, nitrate and nitrite, and the activity of nitrate reductase in the leaves and nitrate reductase in nodules. The nitrogenase activity in nodules and roots was also increased. Number and fresh mass of nodules and their nitrate and nitrite contents were also higher than those of the controls. In contrast, the UV-B stress (12.2 kJ m⁻² d⁻¹) suppressed nodulation and nitrogen metabolism in leaves and roots compared to plants under natural UV-B (10 kJ m⁻² d⁻¹). Triadimefon-treated plants did not show such severe inhibitions after exposure to elevated UV-B. Thus triadimefon increased their tolerance to UV-B stress.     

Early-season effects of supplemented solar UV-B radiation on seedling emergence, canopy structure, simulated stand photosynthesis and competition for light

Mixtures and monocultures of wheat (Triticum aestivum) and wild oat (Avena fatua), a common weedy competitor of wheat, were exposed to enhanced solar UV-B radiation simulating a 20% reduction in stratospheric ozone to assess the timing and seasonal development of the UV-B effects on light competition in these species. Results from two years of field study revealed that UV-B enhancement had no detectable effect on the magnitude or timing of seedling emergence in either species. End-of-season measurements showed significant UV-B inhibition of leaf insertion height in wild oat in mixture and monoculture in the second year (irrigated year) but not in the first year (drought year). Leaf insertion height of wheat was not affected by UV-B in either year. The UV-B treatment had no detectable effect on monoculture or total (combined species) mixture LAI but did significantly increase (5–7%) the fractional contribution of wheat to the mixture LAI after four weeks of growth in both years. In addition, the UV-B treatment had subtle effects on LAI height profiles with early season mixtures showing significant reductions in wild oat LAI in lower canopy layers in both years while midseason Year 2 mixtures showed significant reductions in wild oat LAI in upper canopy layers. The changes in canopy structure were found to significantly increase (6–7%) the proportional simulated clear sky canopy photosynthesis and light interception of wheat in mixture. These findings, and others, indicate that the effects of UV-B enhancement on competition are realized very early in canopy development and provide additional support for the hypothesis that UV-B enhancement may shift the balance of competition between these species indirectly by altering competitive interactions for light.     

The effect of natural and synthetic auxins on the growth,metabolite content and antioxidant response of green alga Chlorella vulgaris (Trebouxiophyceae)

The effect of exogenously applied natural [indole-3-acetic acid (IAA), phenylacetic acid (PAA), indole-3-butyric acid (IBA)] and synthetic [1-naphthaleneacetic acid (NAA)] auxins on the growth and metabolism of green microalga Chlorella vulgaris was examined. Exogenous auxins acted in a concentration-dependent manner on algal growth. Phytohormones at concentration of 100 μM inhibited algal growth expressed as the number of cells. IAA and IBA displayed the highest biological activity at 0.1 μM, whereas PAA and NAA were characterized by the greatest stimulatory effect on the number of cells at 1 μM. Treatment with IAA and IBA at 0.1 μM or NAA and PAA at 1 μM increased the concentration of photosynthetic pigments, monosaccharides and soluble proteins in C. vulgaris. Moreover, all auxins stimulated enzymatic (ascorbate peroxidase, catalase, superoxide dismutase) and non-enzymatic antioxidant (ascorbate, glutathione) systems in C. vulgaris, and therefore, suppressed lipid peroxidation and hydrogen peroxide accumulation. The data supports the hypothesis that auxins play a central role in the regulation of C. vulgaris growth and metabolism and the components of cellular redox systems that are thought to have a prominent role in the regulation of auxin-dependent processes.