Photoinduced accumulation of carotene in Phycomyces

Blue light stimulates the accumulation of beta-carotene (photocarotenogenesis) in the fungus Phycomyces blakesleeanus. To be effective, light must be given during a defined period of development, which immediately precedes the cessation of mycelial growth and the depletion of the glucose supply. The competence periods for photocarotenogenesis and photomorphogenesis in Phycomyces are the same when they are tested in the same mycelium. Photocarotenogenesis exhibits a two-step dependence on exposure, as if it resulted from the additon of two separate components with different thresholds and amplitudes. The low-exposure component produces a small beta-carotene accumulation, in comparison with that of dark-grown mycelia. The high-exposure component has a threshold of about 100 J· m^–2 blue light and produces a large beta-carotene accumulation, which is not saturated at 2·10⁶ J·m^–2. Exposure-response curves were obtained at 12 wavelengths from 347 to 567 nm. The action spectra of the two components share general similarities with one another and with those of other Phycomyces photoresponses. The small, but significant differences in the action spectra of the two components imply that the respective photosystems are not identical. Light stimulates the carotene pathway in the carB mutants, which contain the colourless precursor phytoene, but not beta-carotene. Carotenogenesis is not photoinducible in carA mutants, independently of their carotene content. This and other observations on various car mutants indicate that light prevents the normal inhibition of the pathway by the carA and carS gene products. The chromophore(s) for photocarotenogenesis are presumably flavins, and not carotenes.We thank Dr. A. Palit, C. Chmielewicz and D. Durant (same address as E.D.L.) and L.M. Corrochano, A. Fernández Estefane, and J. Córdoba López (same address as E.R.B.) for their help. This work was supported by grants from Comisión Asesora para Investigación Científica y Técnica and Comisión Interministerial para Ciencia y Tecnología to E.C.O.; from the National Institutes of Health and the National Science Foundation to E.D.L.; and from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation to E.C.O. and E.D.L.     

Investigation of the role of ubiquinone in rat liver subcellular compartments

The role of ubiquinone in the Golgi apparatus is still unknown, even if it might be considered as a lipid marker of the Golgi compartment because of its high content in these subcellular fractions. In vivo modulation of ubiquinone with ethanol and in vitro pentane extraction show that ubiquinone is not required either for NADH-ferricyanide reductase, acetaldehyde dehydrogenase activity, or Ca2+ and Mg2+ stimulated ATPases. Since ubiquinone does not seem to be involved in these enzymic activities in Golgi compartments, other possible functions are discussed, related to a role in membrane fluidity or as a barrier to the propagation of free radicals.     

High-and low-intensity photosystems in Phycomyces phototropism: Effects of mutations in genes madA,madB, and madC

Sporangiophores of Phycomyces blakesleeanus Burgeff that have been grown in darkness and are then suddenly exposed to unilateral light show a two-step bending response rather than a smooth, monotonic response found in light-adapted specimens (Galland and Lipson, 1987, Proc. Natl. Acad. Sci. USA 84, 104–108). The stepwise bending is controlled by two photosystems optimized for the low-and high-intensity ranges. These two photosystems have now been studied in phototropism mutants with defects in genes madA, madB, and madC. All three mutations raise the threshold of the low-intensity (low-fluence) photosystem by about 10⁶-fold and that of the high-intensity (high-fluence) system by about 10³-fold. Estimates for the light-adaptation time constants of the low-and high-intensity photosystems show that the mutants are affected in adaptation. In the mutants, the light-adaptation kinetics are only slightly affected in the low-intensity photosystem but, for the high-intensity photosystem, the kinetics are considerably slower than in the wild type.Abbreviations WT wild type     

Structure and function of the genes involved in the biosynthesis of carotenoids in the mucorales

Carotenoids are widely distributed natural pigments which are in an increasing demand by the market, due to their applications in the human food, animal feed, cosmetics, and pharmaceutical industries. Although more than 600 carotenoids have been identified in nature, only a few are industrially important (β-carotene, astaxanthin, lutein or lycopene). To date chemical processes manufacture most of the carotenoid production, but the interest for carotenoids of biological origin is growing since there is an increased public concern over the safety of artificial food colorants. Although much interest and effort has been devoted to the use of biological sources for industrially important carotenoids, only the production of biological β-carotene and astaxanthin has been reported. Among fungi, several Mucorales strains, particularlyBlakeslea trispora, have been used to develop fermentation process for the production of β-carotene on almost competitive cost-price levels. Similarly, the basidiomycetous yeastXanthophyllomyces dendrorhous (the perfect state ofPhaffia rhodozyma), has been proposed as a promising source of astaxanthin. This paper focuses on recent findings on the fungal pathways for carotenoid production, especially the structure and function of the genes involved in the biosynthesis of carotenoids in the Mucorales. An outlook of the possibilities of an increased industrial production of carotenoids, based on metabolic engineering of fungi for carotenoid content and composition, is also discussed.     

Variation in the amount of triacylglycerols and steryl esters in the outer sapwood of Pinus sylvestris L.

Variation in the amount of triacylglycerols and steryl esters was analysed enzymatically in the outer sapwood of Scots pine (Pinus sylvestris L.). Increment borings were taken at breast height from 40 stems of different diameter. Wood samples from outer sapwood (10 mm from the cambium) were extracted with acetone. Triacylglycerols and steryl esters were separated on TLC and the levels of glycerol and sterol were analysed enzymatically. The average amount of triacylglycerols and steryl esters was approximately 25 and 0.83 mg/g dry weight, respectively. However, variation between the stems studied was fairly large. The sapwood of young and small-diameter stems was not found to store larger amounts of either triacylglycerols or steryl esters than the sapwood of old stems. Neither was there any relationship between the amount of triacylglycerols and steryl esters in the outer sapwood. The possible role of these compounds in heartwood formation is discussed.     

Poly-cis carotene pathway in the Scenedesmus mutant C-6D

The mutation of Scenedesmus obliquus strain C-6D is expressed in the dark only. Under these conditions, this strain synthesizes acyclic poly-cis carotenes. Cyclic carotenoids like -carotene or xanthophylls are absent. In the light the normal cyclic carotenes and xanthophylls are synthesized in the all-trans configuration. The poly-cis carotencs present in dark cultures have been analysed and quantitated. It could be shown that these poly-cis carotenes are identical with the poly-cis carotenes synthesized by the tomato mutant Lycopersicon esculentum var. Tangella. The poly-cis pathways, however, are different regulated in the two organisms. The tomato mutant accumulates prolycopene as the major carotene, whereas the mutant C-6D accumulates mainly pro--carotene. Furthermore, the mutation in Tangella is constitutive in light in contrast to Scenedesmus C-6D. Besides that, Scenedesmus C-6D synthesizes a further cis-carotene isomer of phytofluene as well as of -carotene. The configuration of these carotenes still have to be elucidated. The occurrence of this poly-cis carotene biosynthetic pathway by a mutation of only one enzyme, the phytoene desaturase which, however, is only expressed in darkness under heterotrophic conditions, is discussed.     

Lipids and sterols of Pinus sylvestris L. sapwood and heartwood

Summary The lipid and sterol content and composition of three lipid fractions (free fatty acids/ sterols, triacylglycerols and sterol/triterpenoid esters) extracted from three stem discs of Pinus sylvestris were assessed to investigate metabolic changes related to heartwood formation. The wood was separated into (1) cambial zone, (2) outer sapwood, (3) inner sapwood, (4) transition zone, (5) outer heartwood and 6) inner heart-wood. The fractions were separated by thin-layer chromatography (TLC) and analysed by gas-liquid chromatography (GLC). The amount of fatty acids of sapwood triacylglycerols was about 1.5% (dry wt.) but a large reduction occurred in the transition zone. In contrast, noticeable amounts of free fatty acids were present only in the heart-wood. The most important fatty acids in the sapwood fractions were 16:0, 18:0, 18:1, 18:2 (the dominant fatty acid in all fractions), 18:3 and 20:3. Together 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids. Of the sterol/ triterpenoid esters, 18:2 and 18:3 were predominant. The fatty acid composition of all fractions changed in the transition zone. The sterols found were sitosterol, stigmastanol, campesterol and campestanol. The amount of sterol esters increased towards the heartwood, and the amount of free sterols was lowest in the inner sapwood. Sitosterol was the dominant sterol in both free sterols and sterol esters.     

Relationships among the generaAshbya,Eremothecium, Holleya andNematospora determined from rDNA sequence divergence

Summary Species of the generaAshbya, Eremothecium, Holleya, andNematospora were compared from extent of divergence in a 580-nucleotide region near the 5 end of the large subunit (26S) ribosomal DNA gene. The four genera are closely related and comprise a subclade of the hemiascomycetes. Because the taxa show little divergence, it is proposed that all be placed in the genusEremothecium. The family Eremotheciaceae, fam. nov., is proposed.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Ramicandelaber, a new genus of the Kickxellales, Zygomycetes

Ramicandelaber, a new genus of Zygomycetes is erected to accommodateRamicandelaber longisporus sp. nov. The fungus has hyphal septa with median plugs and forms homologous structures to sporocladia and pseudophialides. These characteristics suggest that it belongs to the Kickxellales, Zygomycetes.     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

Evidence that energy conserving electron transport pathways to nitrate and cytochrome o branch at ubiquinone in Paracoccus denitrificans

The organisation and function of electron transport pathways in Paracoccus denitrificans has been studied with both inhibitors and electrode probes for O₂ or N₂O respiration and membrane potential. Myxothiazol completely inhibits electron flow through the cytochrome bc₁ region of the electron transport chain, as judged by its effect on nitrous oxide respiration. Electron flow to oxygen via the cytochrome o oxidase was shown to be insensitive to myxothiazol in a mutant, HUUG 25, that lacks cytochrome c and in which the aa₃ oxidase is therefore inactive. Myxothiazol did not inhibit nitrate reduction. It is concluded that myxothiazol is a specific inhibitor of electron flow through the cytochrome bc₁ region and more potent than antimycin which does not give complete inhibition.As neither antimycin nor myxothiazol, nor a combination of the two, inhibits electron transport to either nitrate reductase or cytochrome o it is concluded that electron transport pathways to these enzymes do not involve the cytochrome bc₁ region but rather branch at the level of ubiquinone. Although the cytochrome o pathway branches at ubiquinone, it is associated with the generation of a protonmotive force; this is shown by measurements of membrane potential in vesicle preparations from the mutant HUUG 25.In contrast to antimycin and myxothiazol, the ubiquinone analogues 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-undecyl-3-hydroxy-1,4-naphthoquinone (UHNQ) inhibit electron flow through both the cytochrome bc₁ complex and the cytochrome o pathway, although a higher titre is required in the latter case. These two inhibitors were without effect on the nitrate reductase pathway. Thus myxothiazol is the inhibitor of choice for selective and complete inhibition of cytochrome bc₁.Recently published schemes for electron transport in P. denitrificans are analysed.Non standard abbreviations S-13 2,5-dichloro-3-t-butyl-4-nitrososalicylanilide - UHNQ 2-n-undecyl-3-hydroxy-1,4-naphthoquinone - UHDBT 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole     

Relationships among filamentous microorganisms in rotating biological contactors

The existing relationships were studied among the different types of filamentous microorganisms that appear in the biofilm of a biological contactor system. Using the hierarchical cluster analysis it was observed that, in all the stages, Beggiatoa sp. and the Eikelboom’s types 0803 and 1863 always appeared associated, while Sphaerotilus natans was always associated with the morphological type 021N. The remaining microorganisms were associated in variable forms in the plants. In addition, different association models were obtained according to the season of the year and the stage-season interaction. It has also been observed that a significant correlation exists among the filamentous microorganisms we have studied and the different physical-chemical parameters.     

An immunofluorescence study of mitosis in a mite-pathogen,Neozygites sp. (Zygomycetes: Entomophthorales)

Summary Mitosis in a mite-pathogenic species ofNeozygites (Zygomycetes: Entomophthorales) was investigated by indirect immunofluorescence microscopy using an antibody against -tubulin for visualization of microtubules (MTs). DAPI and rhodamine-conjugated phalloidin were used to stain chromatin and actin, respectively. Salient features of mitosis inNeozygites sp. are (1) a strong tendency for mitotic synchrony in any given cell, (2) conical protrusions at the poles of metaphase and anaphase nuclei revealed by actin staining, (3) absence of astral and other cytoplasmic MTs, (4) a spindle that occupies most of the nuclear volume at metaphase, (5) a spindle that remains symmetrical throughout most of mitosis, (6) kinetochore MTs that shorten during anaphase A, (7) a central spindle that elongates during anaphase B, pushing the daughter nuclei into the cell apices, and (8) interpolar MTs that continue to elongate even after separation of the daughter nuclei. Cortical cytoplasmic MTs are present in a few interphasic and post-cytokinetic cells. The data presented show thatNeozygites possesses features unique to this genus and support the erection of theNeozygitaceae as a separate family in theEntomophthorales.Abbreviations DAPI 4,6-diamidino-2-phenylindole - MT microtubule - SPB spindle pole body     

Negative phototropism in the piloboloid mutants of Phycomyces blakesleeanus Bgff.

The sporangiophore (spph) of a piloboloid mutant, genotype pil, of Phycomyces ceases elongation and expands radially in the growth zone shortly after reaching the developmental stage IV b. The pil spph is always negatively phototropic to unilateral visible light when its diameter exceeds 210 m. Photoinduction of spph initiation, light-growth response, threshold of light energy fluence rate for the negative phototropism, avoidance and gravitropism in the pil mutant are all normal. In liquid paraffin, the pil spph shows negative phototropism as does the wild-type spph. Genetic analyses indicate that the negative phototropism of the pil mutant is governed by the phenotypic characteristics of pil but not by specific gene(s) responsible for negative phototropism. These facts imply that the reverse phototropism of the pil mutant results from a loss of the convergent lens effect of the cell because of the increase in cell diameter.Abbreviations spph(s) sporangiophore(s) - wt(s) wild type(s)     

Main sterols from the ophiuroids Ophiocoma echinata, Ophiocoma wendtii, Ophioplocus januarii and Ophionotus victoriae

Sterol compositions of the cold water ophiuroids Ophioplocus januarii and Ophionotus victoriae and of the tropical ophiuroids Ophiocoma echinata and Ophiocoma wendtii are reported. The four sterol mixtures contain Δ⁵ mono- and di-unsaturated common 3β-hydroxy-sterols. Ophioplocus januarii and O. victoriae contain 24-methylcholesta-5,24(28)-dien-3β-ol and 24ζ-ethylcholesta-5,24(28)-dien-3β-ol in higher abundance than in O. echinata. These sterols were not found in O. wendtii. An interesting finding is the presence of Δ^5,24(28)-24-n-propylidenecholesterol in 7.6% in Ophionotus victoriae.     

Relationships among Cichorium species and related genera as determined by analysis of mitochondrial RFLPs

Mitochondrial DNA polymorphism was employed to assess cytoplasmic diversity among cytoypes of the genus Cichorium and related genera of the tribe Lactuceae (Asteraceae). Hybridization patterns of total DNA using six restriction enzymes and five heterologous mtDNA probes were examined. From estimates of mtDNA diversity, Cichorium spinosum appeared as an ecotype of C. intybus rather than a separate species. Interspecific mtDNA polymorphism in the genus Cichorium was higher than that observed in Cicerbita Crepis, Lactuca and Tragopogon. Molecular data seemed to indicate that Catananche is very distant from the other genera examined. Intergeneric comparisons allowed the clustering of Cicerbita, Lactuca and Cichorium, genera which belong to different subtribes. However, further molecular investigations on a larger number of genera are needed to clarify the relationships among genera within and between subtribes of the tribe Lactuceae.     

Water potential,glycerol synthesis,and water content of germinating Phycomyces spores

During early germination, the sporangiospores of Phycomyces blakesleeanus synthesized large amounts of glycerol. Glycerol started leaking out of the spores after some 20 min germination. Simultaneously the water content of the spores greatly increased. Water uptake was accompanied by disapperance of the phase contrast halo and an increase in spore cross-sectional area which all occurred during the same period between 10 and 30 min germination. When spores were incubated in 0.5 or 1 M sucrose, glycerol accumulated in the spores to much higher concentrations and the increase in cellular water content was greatly reduced and retarded. Glycerol synthesis and the concomitant lowering of spore osmotic potential was not the only mediator of spore swelling since equally important glycerol concentrations loaded into dormant spores did not cause spore water uptake or swelling. Also the swelling of the spores was less affected than water uptake by decreases in ambient water potential. Apparently also cell wall loosening was involved in the swelling phenomenon which might have important implications for cellular metabolism.     

Chemotaxonomic survey of sterols and fatty acids in six marine raphidophyte algae

Fatty acid and sterol profiles allowed for clear discrimination betweentheraphidophyte genera Chattonella,Heterosigma, Fibrocapsa andOlisthodiscus, but exhibited little differentiation forindividual Chattonella species(C.marina, C. antiqua and C.subsalsa). Sterol and fatty acid profiles do not support theseparation of Chattonella antiqua and C.marina as distinct species. Ecophenotypic variations in lipidprofiles were also observed between Chattonella strainsfromdifferent geographic locations. Sterol signatures which may be useful aschemotaxonomic markers were: the absence of C₂₇ sterols (cholesteroland 24-dihydrozymosterol) in Heterosigma akashiwo; thepresence of isofucosterol in Chattonella; and theoccurrence of brassicasterol, poriferasterol and fucosterol inOlisthodiscus luteus. High levels of eicosapentaenoic acid(EPA; 17-27% of fatty acids) were present in all raphidophyte species. Lipidcomposition correlated more closely to recent molecular classification ofraphidophytes than carotenoid pigments.     

Relationships among Eucheuma denticulatum,Eucheuma isiforme and Kappaphycus alvarezii (Gigartinales,Rhodophyta) based on nuclear ssu-rRNA gene sequences

The nuclear genes encoding small-subunit ribosomal RNAs (ssu-rDNAs) of the carrageenophytes Eucheuma denticulatum, E. isiforme and Kappaphycus alvarezii were amplified by the polymerase chain reaction, cloned and sequenced. The sequences range from 1767 (K. alvarezii) to 1781 (E. isiforme) nucleotides in length, and have guanine+cytosine contents between 51.2% (E. isiforme) and 51.5% (E. denticulatum). Pairwise sequence identities among these sequences ranged from 97.6% to 98.5%, levels comparable to some intergeneric identities within Gracilariales. In phylogenetic analyses, the two Eucheuma ssu-rDNAs group stably together vis-a-vis the ssu-rDNA of K. alvarezii, and these three ssu-rDNAs form a monophyletic group within a larger grouping of other carrageenophytes. The results demonstrate quantitatively that analysis of nuclear-encoded ssu-rDNA sequences is likely to be useful in resolving taxonomic, phylogenetic and biogeographic questions among tribe Eucheumatoideae Doty.