Roy Powls

A series of mutant strains of Scenedesmus obliquus with abnormal carotenoid compositions

Several mutant strains of Scenedesmus obliquus (Chlorophyta) have been isolated which, when cultured heterotrophically, are pale green or yellow, in contrast to the dark green of the wild type. On the basis of their carotenoid compositions, four groups of pale-green strains have been delineated. These accumulate, respectively, no carotenoid, phytoene, mainly ζ-carotene and mainly ζ-carotene together with some neurosporene and lycopene. All these strains synthesized no chlorophyll b and only small amounts of chlorophyll a. A further group of yellow strains produced the normal Scenedesmus obliquus range of cyclic carotenes and xanthophylls, but no chlorophyll. Most of the pale-green strains were killed by exposure to light, but two strains, PG1 and 1E, which accumulated predominantly ζ-carotene when grown in the dark, survived exposure to the light and developed photosynthetically active chloroplasts containing the normal pigments.The possible biosynthetic implications of the carotenoid composition of these mutant strains, and the relationship between the carotenoid composition and protection of the cells from photooxidative destruction are discussed.

The biosynthesis of quinones from p-hydroxybenzoic acid in Euglena gracilis var. bacillaris☆

Abstract Etiolated cultures of E. gracilis readily incorporated p -[U- 14 C]hydroxybenzoic acid into ubiquinone-9. The evidence suggests that the ubiquinone-9 was then metabolized to rhodoquinone-9. Autotrophic cultures failed to incorporate p -[U- 14 C]hydroxybenzoic acid into plastoquinone and α-tocopherol quinone although both ubiquinone and rhodoquinone were again labelled. Experiments with both sonicated and whole chloroplasts gave similar negative incorporation into the plastidic quinones. Whether this was the result of the chloroplast membrane being impermeable to p -hydroxybenzoic acid or due to this compound not being on the biosynthetic pathway of plastidic benzoquinones was not resolved.

The roles of isomers of phytoene, phytofluene and ζ-carotene in carotenoid biosynthesis by a mutant strain of Scenedesmus obliquus

Considerable changes in pigment composition accur during a period of 10 h when dark-grown cultures of PG1, a ζ-carotenic strain of Scenedesmus obliquus, are illuminated. These changes are consistent with a biosynthetic pathway in which 15-cis-phytoene is converted via 15-cis-phytofluene and 15-cis-ζ-carotene into all-trans-gz-carotene and trans-bicyclic carotenoids.The findings also support the view that the xanthophylls lutein and zeaxanthin are formed from the corresponding carotenes and are especially important in the development of a normal chloroplast structure.

Algal glyceraldehyde-3-phosphate dehydrogenases conversion of the NADH-linked enzyme of Scenedesmus obliquus into a form which preferentially uses NADPH as coenzyme

Scenedesmus obliquus contains two glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.-) one of which uses NADH as its preferred coenzyme (D-enzyme) and the other NADPH (T-enzyme). On incubation of the D-enzyme with cysteine and a 1,3-diphosphoglycerate-generating system the specific activity with NADH as coenzyme decreased whilst that with NADPH increased by a factor of 10. The components of the generating system had no effect on the D-enzyme individually and it is concluded that 1,3-diphosphoglycerate was probably responsible for the change in nucleotide specificity. The coenzyme specificity of the T-enzyme was not affected by such treatment. A similar type of activation occurred to a lesser extent on incubation of the D-enzyme with 2,3-diphosphoglycerate. The NADPH-dependent activity of the D-enzyme could also be promoted by incubation with NADPH. However, in this case the activation was less than that seen with either 1,3- or 2,3-diphosphoglycerate. The change in coenzyme specificity of the D-enzyme occurred in parallel with changes in sedimentation behaviour. Initially, a single boundary of S20,w equals 14.5 S was present, but on conversion to NADPH-dependent activity by incubation with the 1,3-diphosphoglycerate-generating system, new boundaries of 7.5 S and 5.5 S appeared. The first of these corresponds in sedimentation coefficient to the native T-enzyme. On removal of 1,3-diphosphoglycerate the 7.5 S boundary disappeared accompanied by an increase in that of 14.5 S, whilst the 5.5 S boundary persisted. These changes are consistent with the reversible conversion of the D-enzyme into a form similar to the native T-enzyme in response to cysteine and 1,3-diphosphoglycerate. These effects may be explained if acylation of the active site of the D-enzyme by 1,3-diphosphoglycerate results in displacement of the bound nucleotide, thus promoting nucleotide exchange. These findings are consistent with the kinetic mechanism established for other glyceraldehyde-3-phosphate dehydrogenases. Similar activation was seen in extracts of other species of the Chlorophyta but not in other photosynthetic organisms. The significance of this type of activation of enzyme activity to the metabolism of these species of algae is discussed.

A carotenoprotein, containing violaxanthin, isolated from Scenedesmus obliquus D3.

Abstract A yellow water-soluble carotenoprotein, which does not contain any chlorophyll has been isolated from the unicellular alga, Scenedesmus obliquus. The protein of approximate molecular weight 140 000 has been isolated using methods which did not require the use of detergents or organic solvents. Complexing of the carotenoid with the protein results in a marked red shift in its absorption spectrum. The carotenoprotein has absorption maxima in the visible region of 424, 449 and 479 nm, whereas the carotenoid component, which can be obtained by precipitation of the protein with acetone, has absorption maxima at 420, 441 and 470 nm in ethanol solution. The carotenoid component of the chromoprotein was identified by thin layer chromatography as predominantly violaxanthin, although neoxanthin was also present. No lipid other than carotenoid was present and the carotenoid to protein ratio was approximately 1:1.

Carotenoid transformations during chloroplast development in Scenedesmus obliquus PG1 demonstrated by deuterium labelling

THE unicellular green alga Scenedesmus obliquus (Chlorophyta), when grown photosynthetically, contains pigments (chlorophylls and carotenoids) similar to those of the chloroplasts of higher plants1. Several mutant strains1 have abnormal pigmentation1. One of them, PG1, when grown in the dark accumulates the acyclic carotenoids phytoene (7,8,11,12,7′,8′,11′, 12′-octahydro-ψ,ψ-carotene), phytofluene (7,8,11,12,7′,8′-hexahydro-ψ,ψ-carotene) and ζ-carotene (7,8,7′,8′-tetrahydro-ψ,ψ-carotene) in place of cyclic carotenes and xanthophylls and cannot make more than trace amounts of chlorophylls. The pigmentation of light-grown PG1, however, is virtually identical to that of the wild type. When dark-grown PG1 is illuminated, the content of acyclic carotenoids decreases and that of cyclic carotenoids increases2. Time-course studies3 agree with the hypothesis that the cyclic carotenoids produced on illumination are formed from the acyclic precursors that accumulate in dark-grown cultures. It has not been possible to confirm this by radioisotopic labelling, but we have now done so using deuterium labelling. Our procedure also provides a general method for following the kinetics of formation of important chloroplast constituents during chloroplast development in this and other organisms.

The properties and significance of rhodoquinone-9 in autotrophic and etiolated cultures of Euglena gracilis var. bacillaris☆

Abstract A new quinone isolated from Euglena gracilis was characterized, using principally spectrometric techniques, as rhodoquinone-9. The level of this compound was similar in both autotrophic and etiolated cultures suggesting that it did not function in photosynthesis. The evidence suggests that rhodoquinone is probably of mitochondrial origin. Also the absence of rhodoquinone and ubiquinone and the presence of “plastidic quinones” in Anabaena variabilis is contrasted with the reverse situation in another procaryotic, photosynthetic micro-organism Rhodospirillum rubrum.

Phytoene, phytofluene and ζ-carotene isomers from a Scenedesmus obliquus mutant

Abstract A number of mutant strains of the green alga, Scenedesmus obliquus , when grown in the dark, accumulated ζ-carotene as their major carotenoid together with appreciable concentrations of phytoene and phytofluene. The phytoene was almost entirely the 15- cis isomer, and phytofluene was also present mainly as the 15- cis form, whereas the ζ-carotene could be separated into three isomers, predominantly all-trans ζ-carotene accompanied by the 15- cis and an unidentified cis isomer. All the ζ-carotene isomers, when illuminated in the presence of iodine, gave the same equilibrium mixture of stereo-isomers, including a product with unusual spectroscopic and chromatographic properties, which may be a cyclic compound. The pathway of carotenoid biosynthesis in S. obliquus is discussed. On illumination, most of the ζ-carotenic strains were killed, but PGI strain survived, due to the production of cyclic carotenoids with chromophores long enough to protect chlorophyll from photosensitized oxidation.

Glyceraldehyde-3-phosphate dehydrogenase of Scenedesmus obliquus effects of dithiothreitol and nucleotide on coenzyme specificity

NADH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.--) of the photosynthetic alga Scenedesmus obliquus is converted to an NADPH specific form by incubation with dithiothreitol. The change in nucleotide specificity is accompanied by a reduction in the molecular weight of the enzyme from 550 000 to 140 000. Prolonged incubation with dithiothreitol results in the further dissociation of the enzyme to an inactive 70 000 dalton species. The 140 000 dalton, NADPH-specific enzyme is stabilized against dissociation and inactivation by the presence of NAD(H) or NADP(H). Optimum stimulation of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase activity is achieved on incubation of the NADH-specific enzyme with dithiothreitol and NADPH, or dithiothreitol and a 1,3-diphosphoglycerate generating system. The relevance of these observations to in vivo light-induced changes in the nucleotide specificity of the enzyme is discussed.

The effects of excess exogenous mevalonic acid on sterol and steryl ester biosynthesis in celery (Apium graveolens) cell suspension cultures

Abstract Cell suspension cultures of celery ( Apium graveolens ) have been used to investigate the fate of excess added mevalonic acid and the effects on the amounts of free sterols and steryl esters in the cells. Radiolabelling studies using [2- 14 C] MVA showed that the main effect of excess MVA (5 mM) was an increase in the synthesis of steryl esters. Further analysis revealed that esterified 4,4-dimethylsterols were more highly labelled than 4-mono- and 4-desmethylsterols in MVA-treated cells, suggesting a restriction in the flow of carbon downstream from cycloartenol and 24-methylenecycloartanol, and thus indicating the presence of a post-mevalonic acid control point in the sterol biosynthesis pathway. The total fatty acids recovered from the celery cell lipids showed a very low incorporation of radioactivity from the [2- 14 C] mevalonate, indicating that a limited breakdown of excess mevalonate to acetate may occur in these cells, possibly by the operation of the mevalonate shunt. However, this does not appear to be a major route for the disposal of excess mevalonate. Analysis of the free sterols and steryl esters showed that the administration of excess exogenous MVA (5 mM) to Apium graveolens cells caused a two-fold increase in the steryl ester content when compared with control cells, although there was little effect on the free sterol content or composition. When excess MVA was administered in conjunction with the sterol biosynthesis inhibitor paclobutrazol (50 μM), the steryl ester content increased almost three-fold. It is possible that, as well as providing a means of storage of excess sterols, the esterification of sterols may have a regulatory role in sterol biosynthesis, by controlling the rate of C-4 demethylation of precursor 4,4-dimethylsterols.