Chlorophyll Formation in the YG-6 Mutant of Chlorella regularis: Accumulation of Protochlorophyllide and Protochlorophyll Esterified with Geranylgeraniol

A chlorophyll-less mutant, YG-6, was produced by UV treatmentof the wild strain of Chlorella regularis (S-50). Cells grownfor 3 days in darkness showed a red absorption maximum at 634nm and a shoulder near 650 nm, indicative of the accumulationof at least two spectral forms of protochlorophyll(ide). Protochlorophyllide(Pchlide), and one species only of Pchlide ester, protochlorophyllesterified with geranylgeraniol (Pchl GG) were separated, thelatter with absorption maxima in diethyl ether at 438, 574 and624 nm. Spectroscopically, Pchl GG was identical with divinyl-protochlorophyll.The content of Pchlide was 10 to 13 times that of Pchl GG. Bothpaper and high-performance liquid chromatography showed thephototransformation of Pchlide, but no Pchl GG was present.This suggests that Pchl GG is not a direct precursor of chlorophylla esterified with geranylgeraniol. (Received June 4, 1983; Accepted November 11, 1983)     

Bisulphite-induced Inactivation of Growth and Chlorophyll Formation in Chlorella pyrenoidosa

Treatment of S-sufficient or S-deficient Chlorella pyrenoidosacells with NaHSO₃, during an 8-h period in the light, significantlydecreased their chlorophyll and dry matter contents when thecells were incubated in the presence or absence of SO₄²⁺. Incontrols lacking HSO₃^–, when the starting pH was 7.5,dry matter and chlorophyll contents increased slightly, whereasno significant changes in either occurred at a starting pH of3.0 when the cells and medium contained SO₄^–. In the dark,at both pH 3.0 and 7.5, dry matter and chlorophyll contentsdecreased slightly. Bisulphite treatment in the dark causedlittle decrease of either dry matter or chlorophyll when cellsand medium contained SO₄^2–. However, in its absence, drymatter decreased markedly, but there was little change of chlorophyllcontent in the dark. The interactions between HSO₃^– asa source of S and as an inhibitor of growth and chlorophyllformation are discussed in the context of the changes inducedby light and alternative sources of S. Overall, the harmfuleffects of HSO₃^– outweigh any role it has as a sourceof S, since its effects are ameliorated by SO₄^2–.     

Purification and Characterization of 4,5-Dioxovalerate Reductase (NADPH) from Chlorella regularis

Two types of 4,5-dioxovalerate reductases (NADPH) were partiallypurified and characterized from green alga, Chlorella regularis.The enzyme was separated by DEAE-Sephacel chromatography intotwo peaks: type I (first peak) and type II (second peak). Theactivity ratio of the type II to type I enzyme varied between5 to 7 with a starting cell material. Both enzymes had the samepH optimum at 6.0 and pI value of 4.9. The molecular weightestimated by gel filtration was 33,000 for type I and 99,000for type II enzyme. Both enzymes used only NADPH, but were notspecific for 4,5-dioxovaleric acid (DOVA). Type I enzyme reducedglyoxylate 68-fold faster than DOVA, whereas type II enzymeacted more specifically on a variety of aldehydes than DOVA.It is suggested that these enzymes may not function primarilyas NADPH-DOVA reductases in the metabolic pathway of DOVA. (Received June 15, 1985; Accepted October 14, 1985)     

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)     

The Spectral Response of Light Dependent Chlorophyll b Formation

Dark grown seedlings of barley will obtain a high ratio chlorophyll a/chlorophyll b when exposed to intermittent light (1 min of incandescent light or one electronic flash every hour). Such material has been exposed to monochromatic light of different wavelengths for 1 h. The ratio a/b gets a minimum in light of 670 nm, indicating the highest rate of chlorophyll b formation at this wavelength. The possibility is discussed that the light absorber (and also precursor) could be a short-lived chlorophyll a form, existing prior to the forms in the Shibata-shift. Chlorophyll b formation in darkness is discussed from the findings that the rate of formation of chlorophyll b is higher in intermittent light than it should be, calculated from the rate in continuous light, where the saturation intensity is rather low.     

Purification and properties of L-alanine:4,5-dioxovalerate aminotransferase from Chlorella regularis

The enzyme L-alanine:4,5-dioxovalerate aminotransferase (EC 2.6.1.43), which catalyzes the synthesis of 5-aminolevulinic acid, was purified 161-fold from Chlorella regularis. The enzyme also showed L-alanine:glyoxylate aminotransferase activity (EC 2.6.1.44). The activity of glyoxylate aminotransferase was 56-fold greater than that of 4,5-dioxovalerate aminotransferase. The ratio of the two activities remained nearly constant during purification, and when the enzyme was subjected to a variety of treatments. 4,5-Dioxovalerate aminotransferase activity was competitively inhibited by glyoxylate, with a Ki value of 0.5 mM. Double-reciprocal plots of velocity versus 4,5-dioxovalerate with varying L-alanine concentrations indicate a ping-pong reaction mechanism. The apparent Km values for 4,5-dioxovalerate and L-alanine were 0.12 and 3.5 mM, respectively. The enzyme is an acidic protein having an isoelectric point of 4.8. The molecular weight of the enzyme was estimated to be 126,000, with two identical subunits. These results suggest that, in Chlorella, as in bovine liver mitochondria and Euglena, both 4,5-dioxovalerate and glyoxylate aminotransferase activities are associated with the same protein. From the activity ratio of transamination and catalytic properties, it is concluded that this enzyme does not function primarily as a part of the 5-carbon pathway to 5-aminolevulinic acid synthesis.     

Photoconversion of protochlorophyll to chlorophyll a in a mutant of Chlorella regularis

Dark-grown cells of a mutant strain of Chlorella regularis containedchlorophyll a and protochlorophyll, phytyl ester of protochlorophyllide.Under illumination, protochlorophyll was quantitatively anddirectly converted into chlorophyll a. The photoconversion wasdependent on light intensity and temperature and proceeded ina cell-free preparation. The pathway of chlorophyll formation found in the mutant cellsis entirely different from that from protochlorophyllide byway of chlorophyllide a, which is generally observed in greenplants. ¹Present address: Division of Biology, Medical College of Miyazaki,Miyazaki 889-16, Japan. ²Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Ibaragi 300-21, Japan. (Received October 24, 1975; )     

The Regulation of Chlorophyll Biosynthesis by the Action of Protochlorophyllide on glut-RNA-Ligase

The pigment mutant C-2A' of the green alga Scenedesmus obliquus accumulates considerable amounts of protochlorophyllide (PChlide), when grown in darkness. In this paper it is demonstrated that the accumulated PChlide directly acts on ^glut-RNA-ligase and thereby blocks further biosynthesis of ALA and chlorophyll. By increasing the amount of ligase at constant concentrations of PChlide and ^glut-RNA it could clearly be demonstrated that PChlide directly inhibits ligase activity and does not act on the t-RNA. The inhibitory effect of other tetrapyrroles like chlorophyll a, pheophytin a and protoporphyrin IX was much less effective even at oversaturating concentrations.     

Esterification and Spectral Shifts of Chlorophyll(ide) in Wildtype and Mutant Seedlings Developed in Darkness

Spectral changes and esterification (presumably with phytol) of newly formed chlorophyllide a in dark-grown leaves of wildtype bean (Phaseolus vulgaris) and barley (Hordeum vulgare) and a number of chloroplast mutants in barley, were studied by spectrofluorimetry on leaves and on solvent extracts. The shift of the fluorescence emission maximum from 692–694 to 678 nm (excitation shift: 682–684 to 672 nm) and esterification of chlorophyllide a have a similar time course, and both processes are temperature dependent in a similar manner. After completion of the spectral shift and esterification, the fluorescence efficiency of chlorophyll a increases with a subsequent reaccumulation of protochlorophyllide. In leaves of mutants where the shift of fluorescence from 692 to 678 nm is lacking, esterification and the subsequent processes are also blocked. In leaves of mutants with a rapid shift of the fluorescence from 692 to 678 nm, or with direct photoconversion to chlorophyllide a with the fluorescence at 678 nm, esterification is also rapid. The results are interpreted as a sequence of molecular events involving a conformational relaxation of the chlorophyllide holochrome and a translocation of chlorophyll a to reaction centers of the photosystems.     

Presence of the Photoactive Reaction-Center Chlorophyll of PSI (P700) in Dark-Grown Cells of a Chlorophyll-Deficient Mutant of Chlorella kessleri

Compositions of pigments and polypeptides of pale green membranesthat had been isolated from dark-grown cells of a chlorophyll-deficientmutant of Chlorella kessleri were investigated. They containedChl a in a level corresponding to about 1% of that present inthe thylakoid membranes isolated from autotrophically grownwild-type cells and a trace amount of chlorophyllide a, butneither Chl b nor carotenoids. The polypeptide profile of themutant membranes was similar to that of membranes isolated fromwild-type cells that were grown in the dark. Neither the chlorophyll-bindingsubunits of PSI nor the apoproteins of LHCP were detected bySDS-PAGE and immunoblot analysis. However, the light-minus-darkdifference spectrum of the mutant membranes revealed the presenceof the reaction-center chlorophyll of PSI (P700) at a molarratio of 190 chlorophyll (Chl a plus Chlide a) per P700. P700was more stable than Chl a and Chlide a in the light so thatprolonged illumination led to a decline in the Chl/P700 ratioto 24. The initial rate of P700 photooxidation in the mutantmembranes was comparable to that in CP1 isolated from the dark-grownwild-type cells. Under illumination with strong light, the initialrate was decreased in parallel to the decrease in Chl/P700 ratio.The results suggest that most of Chi present in the mutant membranescan transfer excitation energy to P700. (Received March 13, 1998; Accepted August 7, 1998)     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells I. Chlorophyll Accumulation and Phototransformation of Protochlorophyll(ide) in Callus Cells under Blue and Red light

A marked accumulation of chlorophyll was observed in calluscells of Nicotiana glutinosa when they were grown under bluelight, while under strong red light no chlorophyll accumulated.This blue light effect saturated at an intensity of about 500mW.m^–2. The effects of white, blue and red light on the transformationof protochlorophyll (ide) (Pchl) accumulated in dark-grown calluscells were studied by following the changes in the intensityof fluorescence emitted by Pchl and different forms of chlorophyll(ide) (Chi). Pchl with a fluorescence maximum at 633 nm (absorptionmaximum: 630 nm) decreased slowly, concomitant with an increasein Chl having a fluorescence maximum at 677 nm (absorption maximum:675 nm), which was subsequently transformed, independently oflight, to Chi with a fluorescence maximum at 683 nm (absorptionmaximum: 680 nm). Both blue and red light of low intensitieswere effective for the phototransformation, while red light,but not blue light, of high intensities caused significant destructionof Pchl. An action spectrum for this photodestruction showedthat the maximum destruction took place at 630 nm. White lightof high intensities was effective for the photoreduction withonly slight destruction of Pchl, suggesting that blue lightcounteracts the destructive effect of red light. At low temperatures,however, blue light as well as red light of low intensitiescaused photodestruction of Pchl. It was inferred that blue lightenhances a certain step or steps involved in the productionof a reductant required for the photoreduction of Pchl to Chl. (Received July 3, 1981; Accepted November 11, 1981)     

Chlorophyll Formation in the Dark I. Chlorophyll in Pine Seedlings

In black pine (Pinus nigra) chlorophyll was first detected two days after planting of the soaked seeds. There was a slight change in the chlorophyll content between two and five days, then a faster chlorophyll synthesis started. The maximum chlorophyll content was found after 15 days of germination. Thereafter the content showed a gradual decrease. The chlorophyll a/b ratio decreased from 10 at two days to 3 after five days. Protochlorophyllide did not accumulate after 6 days of germination.     

Further Chemical and Morphological Characterization of Chloroplast Membranes from a Chlorophyll b-less Mutant of Hordeum vulgare

A comparative study of peptide composition and freeze-fracture morphology of chloroplast membranes from a chlorophyll b-less mutant and a normal barley plant (Hordeum vulgare L.) is reported in this work. Using a high resolution, discontinuous sodium dodecyl sulfate—acrylamide gel electrophoretic system, we show that the mutant chloroplast membranes not only completely lack the 25-kilodalton peak, which accounts for about 18% of the chloroplast membrane protein in the normal plant, but also exhibit gross reduction in other components at 27.5- and 20-kilodalton regions. Despite such extensive deletions in the peptide composition of the mutant chloroplast lamellae, no alteration could be detected in either density or size of the intramembranous particles, visualized by freeze-fracturing.     

Characterization of Growth and Acid Formation in a Bacillus subtilis Pyruvate Kinase Mutant

Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO₂ production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.     

Serological Characterization of the Glycolate-oxidizing Enzymes from Tobacco, Euglena gracilis, and a Yellow Mutant of Chlorella vulgaris

An antiserum to tobacco glycolate oxidase has been prepared by injection of the purified enzyme into rabbits. Double gel diffusion tests between the antiserum and purified antigen and also with a crude tobacco preparation gave a single immunoprecipitation band. Crude extracts of Euglena gracilis Z Klebs, containing glycolate dehydrogenase, and of Chlorella vulgaris 211-11h/20, containing glycolate oxidase, also formed single bands with the tobacco antiserum. The algal bands were identical and showed partial identity with the tobacco band. The antiserum inhibited the glycolate oxidase activities of the tobacco and Chlorella extracts but did not affect Euglena glycolate dehydrogenase activity.     

Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes

Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM–N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites as did the addition to BM–N of cycloheximide or a chelator, 2,2-bipyridyl. In contrast, chloramphenicol or the protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase. During growth in BM–N, protease activity increased and proteins immunologically detectable with an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM–N and BM+N cells were used for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expression (dee) in C. protothecoides. Several different dees were identified with different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM–N cultures. Analysis of the dee sequences showed that they encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca²⁺-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.