Occurrence of protochlorophyll and its phototransformation to chlorophyll in mutant C-2A' of Scenedesmus obliquus

The pigment mutant C-2A' of the unicellular green alga Scenedesmus obliquus accumulates protochlorophyllide and small amounts of protochlorophyll in darkness. Protochlororphyll was isolated and characterized by thin layer chromatography and absorption and fluorescence spectroscopy. The protochlorophyll was photoreduced by light to chlorophyll both in vitro and in vivo.     

Photoreduction of monovinyl- and divinyl-protochlorophyllide in vitro and in vivo in the light-dependent greening mutant C-2A' of Scenedesmus obliquus

Dark-grown cells of mutant C-2A' of Scenedesmus obliquus accumulate monovinyl-and mainly divinyl-protochlorophyllide (PChlide). Both PChlide-forms are equally well photoconverted in vitro by the NADPH-protochlorophyllide oxidoreductase of the light-dependent greening mutant C-2A'of Scenedesmus obliquus. The chlorophyllide (Chlide) resulting from this photoconversion in vivo has a predominantly monovinyl character. Only small traces of a transient Chlide-form with divinyl fluorescence could be detected.     

Biosynthesis of chlorophyll b in pigment mutant C-2A' of Scenedesmus obliquus

It is demonstrated that chlorophyll b does not only derive from chlorophyll a , but is also formed separately from an in vivo-occurring chlorophyllide b . The branching point for the latter synthesis is at the level of chlorophyllide, since no protochlorophyllide b was detectable. We have indications that the enzyme oxidizing chlorophyll a to chlorophyll b accepts also non-phytylated 17,18 dihydroporphyrins and is not restricted to chlorophylls. Preparations of chlorophyllide a and chlorophyll a could both be transferred with the same enzyme fraction to chlorophyllide b and chlorophyll b , respectively. Preliminary experiments show this enzyme to be membrane bound and light independent. An updated scheme for chlorophyll b biosynthesis is presented.     

Enantioselective toxicological response of the green alga Scenedesmus obliquus to isocarbophos

Chiral compounds usually behave enantioselectively in phyto‐biochemical processes. Isocarbophos (ICP) is a chiral pesticide that is widely used. To evaluate the toxicological response of ICP and its enantiomers to Scenedesmus obliquus, algal growth, total chlorophyll, total soluble protein, and the superoxide anion radicals (O₂^?‐) were investigated. The microalgae were treated with ICP and its enantiomers at 0.01–10 mg/l for 96 h. The growth of S. obliquus was stimulated at low levels of ICP and its enantiomers (0.01–1 mg/l), but all were inhibited at high concentrations (10 mg/l). The total soluble protein content and total chlorophyll content of the tested green alga S. obliquus gradually increased, depending on the growth of algal cells in the medium. Meanwhile, the content of O₂^?‐ was decreased. Interestingly, the cell number and content of the chlorophylls and protein decreased with increasing levels of concentration, whereas O₂^?‐ increased. Our results indicated that enantioselectivity was observed in the dose–response of ICP and its enantiomers in S. obliquus. The high O₂^?‐ level might lead to the death of S. obliquus. The stimulation of growth suggests a regulatory mechanism that is related to the capability of the algae to adapt to the O₂^?‐. Chirality 24:481–485, 2012. © 2012 Wiley Periodicals, Inc.     

Solubilization and hydrophobicity test by Triton X-114-partitioning of NADPH-protochlorophyllide oxidoreductase from the unicellular alga Scenedesmus obliquus, mutant C-2A'

NADPH-protochlorophyllide oxidoreductase (PChilde reductase, EC 1.3.1.33), a key enzyme in light-dependent greening and the conversion of etioplasts into chloroplasts was investigated in the the greening mutant C-2A' of the unicellular green alga Scenedesmus obliquus. In the absence of detergent, the solubilization of the enzyme increased with high glycerol concentrations in the buffer. Solubilization capacities of 4 non-ionic or zwitterionic detergents, Triton X-100, CHAPS, octylglucoside and decyl-maltopyranoside, were compared. Due to the addition of these detergents, the enzyme activity in the soluble fraction was increased severalfold. Hydrophobicity of the enzyme was analyzed by Triton X-114 phase partitioning. The protein had a preference for the aqueous phase, but its distribution was strongly influenced by the glycerol concentration of the buffer. These results indicate that the PChlide reductase of the green alga Scenedesmus obliquus is a hydrophobic, membrane-associated enzyme, but not an integral membrane protein.     

Accumulation of uranium at low concentration by the green alga Scenedesmus obliquus 34

Accumulation of UO ₂ ^{2 +} by Scenedesmus obliquus 34 was rapid and energy-independent and the biosorption of UO ₂ ^{2 +} could be described by the Freundlich adsorption isotherm below the maximum adsorption capacity (75 mg g-¹ dry wt). The optimum pH for uranium uptake was between 5.0_8.5.0.1_2.0 M NaCl enhanced uranyl, while Cu²⁺, Ni²⁺, Zn²⁺, Cd²⁺ and Mn²⁺ competed slightly with uranyl. Pretreatment had an unexpected effect on biosorption. After being killed by 0.1 M HCl, S. Obliquus 34 showed 45% of the uptake capacity of the control in which fresh cells were suspended directly in uranyl solution, while the pretreatment of cells by 0.1 M NaOH, 2.0 M NaCl, ethanol or heating decreased uptake slightly. Fresh S. obliquus 34 at 1.2_2.4 mg dry wt mL-1 was able to decrease U from 5.0 to 0.05 mg L-1 after 4_6 equilibrium stages with batch adsorption. Deposited U could be desorbed by pH 4.0 buffer. It is suggested that U was captured by effective groups or by capillary action in the cell wall in the form of [UO2OH]+. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biotransformation of 1-naphthalenesulfonic acid by the green alga Scenedesmus obliquus

Under sulfate limitation, axenic batch cultures of the green alga Scenedesmus obliquus metabolized 1-naphthalenesulfonic acid and partially used the sulfonate as a source of sulfur. The main metabolite, 1-hydroxy-2-naphthalenesulfonic acid, which was not metabolized further in the algal culture, was formed by hydroxylation of the substrate in position 1 and by migration of the sulfonic acid group to position 2 of the naphthalene ring (NIH shift). A smaller amount of 1-naphthalenesulfonic acid was desulfonated. The resulting 1-naphthol was mostly transformed into 1-naphthyl β-d-glucopyranoside. Received: 27 March 1996 / Revision received: 18 October 1996 / Accepted: 30 October 1996     

Temperature dependent reduction of protochlorophyllide in darkness followed by the assembly of active photosystems in pigment mutant C-2A' of Scenedesmus obliquus

In the wild type of Scenedesmus obliquus strain D3 grown heterotrophically, the chlorophyll biosynthesis and thus the reduction of protochlorophyllide to chlorophyllide takes place in darkness. However, in pigment mutant C-2A' of Scenedesmus obliquus only traces of protochlorophyllide are reduced under optimal growth conditions in darkness. By lowering the growth temperature from 33° to 15–25°C, protochlorophyllide can be reduced in darkness. At 20°C this process is about 10 times more active than at 33°C, but reaches only about 13% of the light-dependent chlorophyll biosynthesis. The chlorophylls synthesized at the lower temperatures are inserted into the pigment-protein complexes and photosystem I as well as photosys-tem II capacities are developed. The rate of light-independent protochlorophyllide reduction at lower temperatures is not limited by the enzyme PChlide-oxidoreductase itself, but rather by its substrate, being in turn limited by the amount of 5-amino levulinic acid (ALA) available.     

Fast and reversible response of thylakoid-associated polyamines during and after UV-B stress: a comparative study of the wild type and a mutant lacking chlorophyll b of unicellular green alga Scenedesmus obliquus

The functional and biochemical aspects of the photosynthetic apparatus in response to UV-B radiation were examined in unicellular oxygenic algae Scenedesmus obliquus. The wild type (Wt) and a chlorophyll b-less mutant (Wt-lhc) were used as a specific tool for the understanding of antenna role. Photosynthesis was monitored during and after UV-B stress by time resolved fluorescence spectroscopy and polarography. Carotenoids, such as neoxanthin, loroxanthin, lutein, violaxanthin, antheraxanthin, zeaxanthin, alpha- and beta-carotene, cellular and thylakoid-associated putrescine, spermidine, spermine and subcomplexes of light-harvesting complex (LHCII) of photosystem II (PSII) were investigated to assess their possible involvement in response to UV-B. Oxygen evolution depression by UV-B was higher in the Wt-lhc mutant than in the Wt. Photosynthesis recovery occurred in the Wt, but not in the mutant. The dissipation of excess excitation energy during UV-B stress was accompanied by changes in the thylakoid-associated polyamines which were much higher than changes in xanthophylls. We conclude that, at least in the unicellular green alga S. obliquus, mutants lacking chlorophyll b have significant lower capacity for recovery after UV-B stress. In addition, the comparison of xanthophylls and thylakoid-associated polyamines reveals that the latter are more responsive to UV-B stress and in a reversible manner.     

The carotenoid-deficient mutant, C-6E, of Scenedesmus obliquus is blocked at the site of phytoene synthase

In contrast to the wild type strain of Scenedesmus , mutant C-6E synthesized only trace amounts of the carotenoids violaxanthin and lutein during prolonged heterotrophic growth. All other carotenoids and carotenoid precursors, such as phytoene, were undetectable. Additionally, only reduced levels of chlorophyll a and no chlorophyll b were formed. To evaluate the potential site of inhibition in the pathway for carotenoid biosynthesis the enzymatic activities of geranylgeranyl pyrophosphate synthase and phytoene synthase were assayed in cell-free extracts. Both enzymes were highly active in extracts of the wild type but only geranylgeranyl pyrophosphate synthase was active in comparable extracts from mutant C-6E . This observation strongly indicates that the phenotype of C-6E results from either a mutation of the phytoene synthase structural gene or of a regulatory gene involved in expression of this enzyme. Other phenotypic effects on composition and structure of the photosynthetic apparatus are discussed as a secondary consequence of the carotenoid deficiency in the thylakoid membranes.     

Purification and kinetic studies on a porphobilinogen deaminase from the unicellular green alga Scenedesmus obliquus

Porphobilinogen deaminase, the enzyme condensing four molecules of porphobilinogen, was isolated and purified from light grown Scenedesmus obliquus (wild type). The purification procedure included heat treatment, ammonium sulphate fractionation, gel filtration, high-resolution anion-exchange chromatography and hydrophobic interaction chromatography. The enzyme was purified 1368-fold, compared to the initial crude extract. Its final specific activity was 6812 units · (mg · protein)^?1 at pH 7.4 with a recovery of 44%. The relative molecular mass was 33000, as determined by Sephadex G-100 gel filtration, and 35900 by lithium dodecyl sulfate-polyacrylamide-gel electrophoresis, indicating that the enzyme is a monomer. Studies of initial reaction velocities showed a linear progress curve for hydroxymethylbilane formation and a hyperbolic dependence of the initial reaction rate on substrate concentration, consistent with a sequential displacement mechanism. Apparent kinetic constants (K _m and V _max) for the conversion of porphobilinogen to hydroxymethylbilane at 37 ° C, pH 7.4, were 79 μM and 176 pmol · min^?1, respectively. Variation of both V _max and K _max with pH indicated the presence of ionizable groups in the enzyme-substrate complex(es), showing a single ionization (pK 7.15) in V _max/K _m plots. A sharp pH-profile for V _max was interpreted as a positive cooperative proton dissociation. In spite of the two pathways existing for 5-aminolevulinate biosynthesis in Scenedesmus, currently there is no indication of the existence of two porphobilinogen deaminases or even of isoenzymes.     

Physiological and structural effects of phosphorus starvation on the unicellular green alga Scenedesmus

The effects of phosphorus starvation on morphology and intracellular structure and on reactions related to the energy metabolism of the unicellular green alga Scenedesmus obtusiusculus (Chod.) were studied over a period of 96 h by employing transmission electron microscopy and various methods for measurement of physiological reactions. Increase in cell size and shape and in cell wall thickness are dominating features of phosphorus starvation. There is also an increase in number and size of starch granules and lipid globules and the internal structure of the cells appears successively disorganized. Shortage of phosphorus in the medium initially induces an increase of the adenylate pool whereas the energy charge value remains the same as for the controls. The photosynthetic and respiratory activities are high during incipient phosphorus starvation. After 24 h, as shortage of phosphorus becomes critical, the internal phosphorus reaches a low steady-state value, and this is also true for the adenylate energy charge. The total content of adenylates, however, peaks after 24 h of starvation and then decreases with increasing length of phosphorus starvation. Light-induced oxygen evolution appears not to be as much inhibited by a low phosphorus content in the cells as by the concomitant starch accumulation. The data indicate that the strategy for survival of the cells in a phosphorus-poor environment includes morphological and physiological changes that facilitate the transfer and adaption of the cells to environments with a more favourable supply of phosphorus, such as the often oxygen-poor but phosphorus-rich bottom zones.     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

斜生栅藻中虾青素的积累过程及其光合活性变化

分析了斜生栅藻（Scenedesmus obliquus）在光温（30℃,180 μmol/m2·s）胁迫条件下积累虾青素的过程,观察了该过程中细胞形态及细胞光合生理的变化。胁迫条件下,细胞在48h内生成并积累了包括海胆酮、角黄素、金盏花黄素和金盏花红素在内的多种次生类胡萝卜素,并合成了虾青素及其酯。该过程中,细胞形态由两端尖细变得不规则、膨大,原来由4、8个细胞组成的定形群体变为游离的单个细胞或2个细胞组成的群体。藻细胞光合速率在24h内先下降后上升,而后又呈现下降趋势,从34.29 μmol O2/mg Chla/h迅速下降为5.21 μmol O2/mg Chla/h;呼吸速率在前24h内升高至60.37 μmol O2/mg Chla/h,而后缓慢下降到38.40 μmol O2/mg Chla/h;光合系统Ⅱ的活性随着胁迫时间的延续而逐步下降,较初始值降低了63.9％。结果表明,斜生栅藻细胞在高光照条件下可以合成虾青素,并通过调节光合速率、呼吸速率以及光合系统Ⅱ的效率来应对胁迫。     

The effect of arsenate and arsenite on photosynthesis in Scenedesmus obliquus. A Potentiometric study in a closed CO2-system

Abstract

A Potentiometric titration method was used to study the adverse effect of arsenate (As(V)) and arsenite (As(III)) on inorganic carbon uptake in suspensions of the green alga Scenedesmus obliquus. The measurements were performed in a closed CO₂-system with diluted synthetic seawater (1‰ salinity) as ionic medium. Usually, the algal chlorophyll concentration was 0.4 mg dm^?3, while the arsenate- and arsenite-concentrations were varied within the limits 0.1 to 200 μmol dm^?3. In some experiments arsenate toxicity was studied in the presence of 1 to 100 μmol dm^?3 of phosphate (P(V)).

With concentrations of arsenate or arsenite less than 0.1 μmol dm^?3 no toxic effects were observed. However, at As-concentrations of 200 μmol dm^?3, the algal carbon uptake was reduced by 41% with arsenate and 29% with arsenite, i.e., arsenate is more toxic to Scenedesmus obliquus than arsenite. The toxicity of arsenate was negligible in the presence of a ten fold excess of phosphate. This is probably due to chemical similarities between arsenate and phosphate causing competition between the ions for the binding sites.

The importance of taking the speciation as well as the buffer capacity of the algal system into account, when calculating the carbon uptake, is also discussed.     

Aggregation effects due to aluminum adsorption to cell walls of the unicellular green alga Scenedesmus obtusiusculus

Cells of the unicellular green alga Scenedesmus obtusiusculus Chod. were cultivated for 2–24 h in nutrient media with low (60 ?mol/L) or high (1000 ?mol/L) phosphorus (P) concentration, and in the presence or absence of 222 ?mol/L aluminum chloride (Al). Cell aggregation was studied by using light microscope, sedimentation and centrifugation. After 2 h, Al was adsorbed to the cell surface and cell aggregates were formed by the attraction of the cells to each other. Aluminum is bound by the negative charges of the cell walls, and studies at different pH showed that a high proportion of positively charged Al forms promote cell aggregation. This effect was most pronounced in low phosphorus cultures as phosphate reduces the effect of Al on cell aggregation by forming aluminum‐phosphate. Algae cultivated in the absence of Al did not show any cell aggregation tendencies.     

Characterisation of two forms of phosphoribulokinase isolated from the green alga, Scenedesmus obliquus

Two forms of phosphoribulokinase from the alga, Scenedesmus obliquus, have been purified to homogeneity by DEAE-cellulose, Ultrogel AcA34 and hydroxyapatite chromatography. An active form of the enzyme is a dimer of identical 42,000-Mr subunits. A latent form of phosphoribulokinase, requiring incubation with dithiothreitol for activity, is of Mr 470,000 and apparent subunit composition X8Y4. The subunits X and Y are of Mr 39,000 and 42,000 respectively. The latent form of phosphoribulokinase is lost during DEAE-cellulose chromatography but this is prevented by NAD. Depolymerisation of the latent phosphoribulokinase to give the low-Mr form of the enzyme accompanied its activation by dithiothreitol. An algal protein with all the properties of thioredoxin stimulates activation of the latent phosphoribulokinase when incubated with low concentrations of dithiothreitol. The latent form of phosphoribulokinase predominates in the heterotrophically grown algae whilst under photoheterotrophic conditions equal amounts of both enzyme forms are present in algal extracts. This is consistent with the suggestion that light activation of phosphoribulokinase in vivo is also due to depolymerisation of the large-Mr latent form of the enzyme.     

Evidence for the Presence of Chlorophyllide b in the Green Alga Scenedesmus obliquus in vivo

Chlorophyllide b could be extracted from the wild type of Scenedesmus obliquus and its pigment mutant C-2A'. Its identity was proved by absorption and fluorescence spectroscopy and by a positive hydroxylamine test. Chlorophyllide b could be transformed into pheophorbide b and methylpheophorbide b. The formation of chlorophyllide b from chlorophyll b by dephytylation with chlorophyllase could be ruled out. The stimulation of chlorophyllide b biosynthesis with o-phenanthroline, as described in the literature, could not be confirmed under physiological conditions.     

Effect of acetate on growth and ammonium uptake in the microalga Scenedesmus obliquus

The effect of acetate on growth and rate of ammonium uptake in Scenedesmus obliquus (UTEX 78) was investigated under light-limiting conditions. Addition of acetate to autotrophic cells with a growth constant of 0.71 day⁻¹ resulted in an increase in the growth rate (mixotrophy, k = 1.3 day⁻¹), and in the presence of acetate, growth occurred in the dark (heterophy, k = 0.44 day⁻¹). The rate of ammonium uptake in autotrophy (17.8 amol cell⁻¹ min⁻¹) was similar to that in heterotrophy (17.4 amol cell⁻¹ min⁻¹) but was 3.7 times lower than that in mixotrophy (65.9 amol cell⁻¹ min⁻¹). In general, mixotrophic cells showed optimum ammonium uptake at the acetate concentration at which they were grown. In autotrophy, uptake of ammonium leveled off at about 12.5 μ M while no saturation was observed in mixotrophic cells. An increase in the rate of uptake of ammonium was observed in autotrophic cells within 1 h after the addition of acetate. The activity of isocitrate lyase (EC 4.1.3.1), a key enzyme for the regulation of the glyoxylate cycle responsible for acetate catabolism, showed a 3.9-fold increase in activity after 24 h in the dark in the presence of acetate. The level of isocitrate lyase activity in cells grown for 24 h in the dark in the presence of 0–20 m M acetate also increased as a function of acetate concentration.