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; )     

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.     

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.     

Protochlorophyll(ide) accumulation and degradation in the dark and photoconversion to chlorophyll in the light in pigment mutant C-2A' of Scenedesmus obliquus

Pigment mutant C-2A' of Scenedesmus obliquus accumulates only traces of chlorophyll, when grown heterotrophically in the dark. Immediately upon transfer of cells into fresh medium protochlorophyllide and protochlorophyll are formed, which accumulate to their maximum concentrations within 8 to 12 h. Subsequently, this protochlorophyll(ide) is degraded in the dark, but not transformed into chlorophyll. After 6–8 days of dark growth no protochlorophyll(ide) can be detected any more. The protochlorophyll(ide) pool of cultures, which contain reduced concentrations, can be reestablished either by addition of glucose or illumination with blue light; both increase the rate of respiration.
By low temperature spectroscopy in vivo and by absorption and fluorescence emission spectroscopy of pigment extracts it is shown that the protochlorophyllide accumulated in freshly inoculated cultures can be converted to chlorophyll in light.
From the action spectrum for chlorophyll formation after addition of glucose it can be seen that protochlorophyllide 636 and 649 are present and are photoconvertible in this mutant.     

Influence of Calcium on Formation and Reduction of Protochlorophyllide in the Pigment Mutant C-2A' of Scenedesmus obliquus

The levels of protochlorophyllide and protochlorophyll of pigmentmutant C-2A' of Scenedesmus obliquus grown in darkness dependupon the calcium concentration in the growth medium. In thepresence of calcium both the protochlorophyllide and protochlorophylllevels decrease upon irradiation whereas the amount of photoreducedchlorophyllide increases. In contrast to light-dependent protochlorophyllide reduction,the activity of light-independent protochlorophyllide reductionis higher in calcium free cultures compared to those grown inthe presence of calcium. It is discussed whether calcium actsdirectly on the activity of the protochlorophyllide oxidoreductaseor stabilizes the newly formed chlorophyllide. (Received September 1, 1989; Accepted February 19, 1990)     

Formation of 5-aminolevulinate via glutamate-1-semialdehyde and 4,5-dioxovalerate with participation of an RNA component in Scenedesmus obliquus mutant C-2A'

In the yellow mutant C-2A' of the unicellular green alga Scenedesmus obliquus the participation of an RNA species in the conversion of glutamate to 5-aminolevulinate is clearly demonstrated by the fact that RNAase treatment of a soluble enzyme preparation drastically decreases the formation of 5-aminolevulinate. The involvement of 4,5-dioxovalerate in the C5 pathway is demonstrated by the decrease of label in enzymatically formed 5-aminolevulinate from [14C]glutamate by providing an increased unlabelled pool of 4,5-dioxovalerate. Evidence supporting the role of glutamate-1-semialdehyde as an additional intermediate in the reaction sequence is also presented. We propose a new reaction scheme, consistent with the results reported here, for the formation of 5-aminolevulinate via the C5 pathway.     

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.     

Role of Light in 5-Aminolevulinic Acid Formation in Wild Strain and Mutant C-2A' Cells of Scenedesmus obliquus

In dark-grown wild strain cells of Scenedesmus obliquus, 5-aminolevulinicacid (ALA) formation was induced by irradiation with a weakblue light, as in its mutant C-2A' cells. The induction wasinhibited by distamycin A, 6-methylpurine, cycloheximide andchloramphenicol. After the light induction, the ALA formationcould proceed in the dark as well as in the light, in such heterotrophicallygrown wild type cells, but not in the greening mutant C-2A'cells. In the latter, ALA formation was dependent on red light,as well as on blue light, in the presence of CMU. The amountsof protochlorophyll in the mutant cells increased upon cessationof illumination and decreased with subsequent irradiation withblue and red light. The possible role of protochlorophyll asa photoreceptor in regulation of ALA formation in the mutantcells is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otuka, Hachioji, Tokyo 192-03, Japan. (Received January 17, 1981; Accepted April 30, 1981)     

Monovinyl and divinyl protochlorophyll in different stages of esterification isolated from mutant C-2A'of the unicellular green alga Scenedesmus obliquus

The mutant C-2A'of the unicellular green alga Scenedesmus obliquus accumulates the chlorophyll-precursors protocbloropbyllide and the already eslerified protochlorophyll when grown heterotrophically in the dark. Two derivatives of protochlorophyll. monovinyl protochlorophyll (MV-PChl) and divinyl protochlorophyll (DV-PChl), were isolated from dark-grown cells of mutani C-2A'and characterized by absorption and fluorescence spectroscopy. Their molecular masses were determined by plasma desorption mass spectrometry. Both MV- and DV-PChl were mainly esterified with geranylgeraniol. However, some esterification with the more saturated alcohols dihydrogeranylgeraniol. tetrahydrogeranylgeraniol and phytol could also be detected.     

Changes in Hydrogen Metabolism during Greening of Pigment Mutant C-2A' from Scenedesmus obliquus

Capacities of H₂-photoproduction and photoreduction were determinedin the greening pigment mutant C-2A' of Scenedesmus obliquus. In the dark grown culture the capacity for H₂-photoproductionis low and for photoreduction not detectable. Both processesincrease with chloroplast development in the light. Photoreductionincreases in parallel to the photosynthetic capacity using H₂instead of water as electron donor. H₂ reaches optimal valuesduring the stage of highest quantum efficiency of photosyntheticoxygen evolution, but does not increase further. Hydrogenase of adapted cells is most active in dark grown culturesand declines during greening. This indicates that hydrogenaseactivity is not the limiting factor in the hydrogen metabolismstudied in this investigation. Studies with cells in which the formation of the reaction centerswere reversibly suppressed by chloramphenicol demonstrate thathydrogen metabolism depends on intact reaction centers and cannot be mediated by light harvesting chlorophylls. (Received March 2, 1985; Accepted June 11, 1985)     

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.     

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.     

Photoisomerization of lycopene during carotenogenesis in mutant C-6D of Scenedesmus obliquus

Mutant C-6D of the unicellular green alga Scenedesmus obliquus has lost the ability to form cyclic carotenoids during heterotrophic growth in the dark. In the dark it accumulates acyclic intermediates, i.e. lycopene, neurosporene and ζ-carotene. The lycopene and two neurosporene forms were identified to be cis-isomers. Upon transfer to light, intermediates decrease and a normal set of carotenoids is synthesized. Inhibition of the cyclization reaction by nicotine reveals a lightinduced isomerization of cis-lycopene to trans-lycopene. Since the spectral characteristics of these two isomers differ drastically the isomerization can be followed in vivo by measuring a light-induced absorbance change. This absorbance change has its maximum at 520 nm and shows fast kinetics under high light intensities reaching a saturation level after about 2 min. Fluence-response curves for this absorbance change were performed for different wavelengths of actinic light. From the linear parts of these curves an action spectrum was caculated showing maxima at 670, 630 and 440 nm originating from chlorophyll and a maximum at shorter wavelengths (400–510 nm) which is interpreted to derive from ζ-carotene. A model for the light regulation of carotenogenesis in mutant C-6D is presented and the relation to the so-called 520-change observed in many plants is discussed.     

Estimation of protochlorophyll(ide) contents in plant extracts; re-evaluation of the molar absorption coefficient of protochlorophyll(ide)

In an attempt to solve the controversy about the evaluation of the molar absorption coefficient of PChl(ide), this coefficient is estimated in this work by using an original experimental approach. The calculated molar absorption coefficient of PChl(ide) is 30.4.10³ 1 mole^–1 cm^–1 at 626 nm in acetone 80%; it is close to that derived from the specific absorption coefficient of Koski and Smith when assuming that the pigment extracted by these authors was the esterified pigment: PChl. Sets of equations for the quantification of Chl(ide) a, Chl b and PChl(ide) in 80% acetone extracts are derived.Abbreviations PChl(ide) protochlorophyll(ide) - Chl(ide) chlorophyll(ide)     

Estimation of protochlorophyll(ide) contents in plant extracts; re-evaluation of the molar absorption coefficient of protochlorophyll(ide)

In an attempt to solve the controversy about the evaluation of the molar absorption coefficient of PChl(ide), this coeffecient is estimated in this work by using an original experimental approach. The calculated molar absorption coefficient of PChl(ide) is 30.4.10³ l mole^-1 cm^-1 at 626 nm in acetone 80%; it is close to that derived from the specific absorption coefficient of Koski and Smith when assurning that the pigment extracted by these authors was the esterified pigment: PChl. Sets of equations for the quantification of Chl(ide) a, Chl b and PChl(ide) in 80% acetone extracts are derived.     

Phosphorescence of protochlorophyll(ide) and chlorophyll(ide) in etiolated and greening bean leaves

The assignment is presented for the principal phosphorescence bands of protochlorophyll(ide), chlorophyllide and chlorophyll in etiolated and greening bean leaves measured at -196°C using a mechanical phosphoroscope. Protochlorophyll(ide) phosophorescence spectra in etiolated leaves consist of three bands with maxima at 870, 920 and 970 nm. Excitation spectra show that the 870 nm band belongs to the short wavelength protochlorophyll(ide), P627. The latter two bands correspond to the protochlorophyll(ide) forms, P637 and P650. The overall quantum yield for P650 phosphorescence in etiolated leaves is near to that in solutions of monomeric protochlorophyll, indicating a rather high efficiency of the protochlorophyll(ide) triplet state formation in frozen plant material. Short-term (2–20 min) illumination of etiolated leaves at the temperature range from -30 to 20°C leads to the appearance of new phosphorescence bands at about 990–1000 and 940 nm. Judging from excitation and emission spectra, the former band belongs to aggregated chlorophyllide, the latter one, to monomeric chlorophyll or chlorophyllide. This indicates that both monomeric and aggregated pigments are formed at this stage of leaf greening. After preillumination for 1 h at room temperature, chlorophyll phosphorescence predominates. The spectral maximum of this phosphorescence is at 955–960 nm, the lifetime is about 2 ms, and the maximum of the excitation spectrum lies at 668 nm. Further greening leads to a sharp drop of the chlorophyll phosphorescence intensity and to a shift of the phosphorescence maximum to 980 nm, while the phosphorescence lifetime and a maximum of the phosphorescence excitation spectrum remains unaltered. The data suggest that chlorophyll phosphorescence belongs to the short wavelength, newly synthesized chlorophyll, not bound to chloroplast carotenoids. Thus, the phosphorescence measurement can be efficiently used to study newly formed chlorophyll and its precursors in etiolated and greening leaves and to address various problems arising in the analysis of chlorophyll biosynthesis.Abbreviations Pchl protochlorophyll and protochlorophyllide - Chld chlorophyllide - Chl chlorophyll     

Studies on nucleic acids in plastids of the pigment mutant C-2A{acute} of Scenedesmus obliquus

Pigment mutant C-2A{acute} of Scenedesmus obliquus whose chlorophyllformation and chloroplast development are light dependent, wasstudied for the nucleic acid content of its plastids. The ribosomalRNA of plastids of the achlorophyllous or greened mutant C-2A{acute},did not show any difference from that of the wild type. Incorporationof [5-³H] uridine into mutant cells was partially inhibitedby rifampicin, indicating this part as being plastidial incorporation.Since there were no significant differences in the ribosomalRNA of plastids between the mutant and the wild type of Scenedesmus,the ribosomal system in the plastids of mutant C-2A' seemednot to be affected by the mutation. C_sCl gradient patterns ofScenedesmus mutant and wild-type DNA were almost identical withthose of Chlorella DNA. A peak at a buoyant density of 1.69g/cm³, the same as that of Chlorella chloroplast DNA, couldbe identified in Scenedesmus also as plastid DNA because itdisappeared after prolonged treatment with myxin and hybridizedwith rifampicin-sensitive pulse-labelled RNA. This peak waspresent to nearly the same degree in the mutant and the wildtype, indicating that a larger deficit of plastid DNA did notoccur in the mutant. Whether or not the mutation might be localizedin the plastid genome is discussed. (Received March 19, 1976; )     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm