Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides II. Role of carbamylphosphate in chloroplast regeneration

Levels of the activities of glutamine-dependent carbamylphosphatesynthetase, ornithine-and aspartate-transcabamylase and phosphoenolpyruvatecarboxylase were followed in greening cells of Chlorella prolothecoides.Among the enzymes examined the activity of carbamylphosphatesynthetase was extremely low, especially at the early phaseof greening. Arginine (but not ornithine or aspartate), when administeredto algal cells at the 24th hour of greening, stimulated thesyntheses of RNA, protein and chlorophyll in the subsequentperiod. It also affected the metabolic pathway of the ¹⁴CO₂supplied simultaneously with arginine in the presence of CMU.Arginine produced a decreased incorporation of ¹⁴C into proteinand an increased incorporation into nucleic acid. The mechanismof the action of CO₂ on chloroplast regeneration is discussed.We concluded that chloroplast regeneration in glucose-bleachedcells is limited by the synthesis of carbamylphosphate, especiallyin the early phase of greening. (Received August 19, 1975; )     

Spectral effectiveness in chlorophyll and 5-aminolevulinic acid formation during regreening of glucose-bleached cells of Chlorella protothecoides

Regreening of glucose-bleached cells of Chlorella protothecoidesis stimulated by light. Spectral effectiveness in the processshowed maxima around 370, 440 and 480 nm, suggesting a flavoproteinas primary photoreceptor. Action spectra of ALA synthesis provedto be similar to those of chlorophyll formation, indicatingthat light stimulation of greening in this alga is regulatedat the first step of chlorophyll biosynthesis. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo 113, Japan. (Received March 27, 1978; )     

Effects of disalicylidenepropandiamine and near far-red light on 14CO2-fixation in Chlorella cells

1) With Chlorella ellipsoidea cells, in the presence of 5x10^–6M DSPD, or in its absence, the amounts of ¹⁴CO₂ incorporatedin P-esters, serine-plus-glycine and alanine were larger underred light than under blue light, whereas blue light specificallyincreased ¹⁴CO₂-incorporation in aspartate, glutamate, malateand fumarate (blue light effect). The amount of total ¹⁴C fixedunder blue or red light was greatly decreased by the additionof DSPD. When the concentration of DSPD was raised to 5x10^–4M, practically no radioactivity was found, under blue or redlight, in aspartate, glutamate and fumarate. Radioactivity inalanine was greatly increased. Effects of higher concentrationof DSPD are explained as due to the inhibition of PEP carboxylaseactivity in Chlorella cells. 2) The percentage incorporation of ¹⁴C into aspartate and theother compounds mentioned above, under near infra-red illuminationwas significantly smaller than that under blue light and wasalmost equal to that under red light. These results along withthe effect of 5x10^–6 M DSPD, exclude the possibility thatcyclic photophosphorylation is involved in the "blue light effect"mechanism. (Received December 12, 1969; )     

Regulation of CO2-fixation in Chlorella by light of varied wavelengths and intensities

1) The wavelength effects on ¹⁴CO₂-fixation by Chlorella cellswere studied, using monochromatic light of different light intensities. 2) Blue light (453 mµ) stimulated the incorporation of¹⁴C into aspartate, glutamate and malate. Red light (679 mµ),on the other hand, stimulated its incorporation into P-esters,free sugars and insoluble material. 3) The blue light effect was observed in the presence of CMUat concentrations completely suppressing ordinary photosyntheticCO₂-fixation. 4) The blue light effect in the presence of CMU was inducedat very low intensities. At 453 mµ, 300 erg cm^–2sec^–1 was sufficient for complete saturation. 5) Time courses of ¹⁴C-incorporation into individual compoundswere investigated. Irrespective of the wavelength of the illuminatinglight, the first stable CO₂-fixation product formed under weaklight (400–500 erg cm^–2 sec^–1) was citrulline.At higher light intensities (4,000–7,000 erg cm^–2sec^–1), PGA was the first stable CO₂-fixation product.The incorporation of ¹⁴C into citrulline was not inhibited byCMU. 6) Experimental results indicate that both blue light-inducedincorporation of ¹⁴C into amino and organic acids and the incorporationof ¹⁴C into citrulline induced by low intensity light are operatedby a mechanism(s) independent of ordinary photosynthetic CO₂-fixation.Possible effects of light regulating the carbon metabolism inalgal cells are discussed. (Received July 24, 1969; )     

Studies on chlorophyll formation in Chlorella protothecoides II. Enhancing effects of light and suppressive effects of glucose on the development of porphyrin-synthesizing activity during light-induced greening of etiolated algal cells

Frozen and thawed cells, as well as sonicated cell preparationsof Chlorella protothecoides, were assayed for activity to synthesizeporphyrins from added ALA or PBG. Activity was very low in etiolatedcells, and markedly developed during the process of light-inducedgreening. The development of activity was strongly suppressedby glucose. Activity for the synthesis of URO(gen) from ALAwas initially developed, then the formation of COPRO(gen)-synthesizingactivity ensued. In glucose-suppressed cells as in cells incubatedin continuous darkness, URO was the main porphyrin producedand COPRO was virtually missing in the reaction products withadded ALA, indicating that development of activity for the conversionof URO(gen) to COPRO(gen) is greatly enhanced by light and isrepressed by added glucose. Suppressive effects of CP and CHon the development of porphyrin-synthesizing activity were alsostudied. From these and other results, a tentative scheme ispresented for the enhancing effects of light and the suppressiveeffects of glucose on the development of porphyrin-synthesizingactivity in etiolated algal cells, in correlation with the effectson other processes of chlorophyll biosynthesis. ¹ Present address: National Food Research Institute, Ministryof Agriculture and Forestry, Koto-ku, Tokyo 135, Japan. (Received April 6, 1972; )     

Studies on chlorophyll formation in Chlorella protothecoides I. Enhancing effects of light and added {delta}-aminoIevulinic acid, and suppressive effect of glucose on chlorophyll formation

Light-induced formation of chlorophyll in "etiolated" cellsof Chlorella protothecoides was studied under various experimentalconditions, (i) Two different types of enhancing effect of lightwere demonstrated: a "long-term" effect lasting for many hoursafter a relatively short illumination of etiolated cells anda "short-term" effect disappearing in a few hours after illumination,(ii) Addition of ALA caused enhancement of chlorophyll synthesisin etiolated cells in darkness as well as in light; the ALA-enhancedrate of dark chlorophyll synthesis, however, was much lowerthan the rate in light without added ALA. ALA was replaceablewith succinic acid plus glycine in light, but not in the dark,for enhancement of chlorophyll formation, (iii) Adding glucose,fructose, galactose, glycerol or acetate—at concentrationsmuch lower than those previously shown to induce "bleaching"of green algal cells-caused a more or less marked suppressionof light-induced greening in etiolated cells, (iv) Added glucosealmost instantaneously and completely stopped chlorophyll synthesisin light as well as in darkness either with or without addedALA. On the basis of these and other results, a tentative schemeis presented for the enhancing effects of light and the suppressiveeffects of glucose on chlorophyll formation in algal cells. (Received April 1, 1970; )     

Syntheses of {delta}-aminolevulinic acid and chlorophyll during chloroplast formation in Chlorella protothecoides

Greening cells of Chlorella prolothecoides were assayed foractivity of the in vivo synthesis of ALA, which was markedlydeveloped during light-induced greening. Effects of CH on thesyntheses of ALA and chlorophyll were also examined. The resultsstrongly suggested that a labile enzyme is involved in ALA synthesis,and that continuous formation of the enzyme is required forthe greening of cells. However, the prompt suppression of chlorophyllsynthesis when CH was added to rapidly greening cells was foundto be attributable not to the blockage of ALA synthesis butto the suppression of some later process(es) in the course ofchlorophyll synthesis, under the conditions used. The valueof the Hill coefficient for the CH inhibition of chlorophyllsynthesis as well as the CH concentration which caused 50% inhibitionremained unaltered whether it was measured when the ALA synthesisactivity was greatly inhibited by CH or when the activity wasonly slightly suppressed. (Received November 11, 1974; )     

Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )     

Studies on chlorophyllase of Chlorella protothecoides III. Purification and catalytic properties

Chlorophyllase was extracted from green cells of Chlorella protothecoidesby n-butanol treatment and purified 600-fold, as measured byenzyme activity in chlorophyll a hydrolysis, by ammonium sulfateprecipitation, chromatography on TEAE-cellulose column and gelfiltration with Sephadex G-200. At each purification step the following activities were compared:hydrolyses of chlorophyll a and methyl chlorophyllide a, methanolysisof chlorophyll a and transphythylation of methyl chlorophyllidea to chlorophyll a. The ratio of activities of chlorophyll a hydrolysis to chlorophylla methanolysis changed on purification and partial inactivationby heat, PCMB and phytol, as well as by varying the reactiontemperature, thus suggesting that the two reactions are notcatalyzed by a single enzyme. In contrast, the activity ratio of chlorophyll a methanolysisto transphytylation of methyl chlorophyllide a remained unaltered,indicating that these reactions can be forward and backwardreactions catalyzed by one enzyme. Results of kinetic studies also indicated that the chlorophyllaseof Chlorella protothecoides consists of at least two enzymes.One enzyme catalyzes chlorophyll a hydrolysis and the other,chlorophyll a methanolysis and the reverse reaction, transphytylationof methyl chlorophyllide a. (Received May 24, 1973; )     

The Respiratory Chain of Chlorella protothecoides: I. Inhibitor Responses and Cytochrome Components of Whole Cells

The respiration and cytochrome properties of “glucose-bleached” Chlorella protothecoides Krüger, Indiana strain 25, were studied. This organism, when grown heterotrophically with high glucose and a low organic nitrogen source, has no chlorophyll, little carotenoid, and diminished chloroplast structure—factors which make it suitable for respiration studies.     

Photosynthetic metabolism of 14CO2 in the process of the "glucose-bleaching" of Chlorella protothecoides

Changes in photosynthetic carbon metabolism during the glucosebleaching of Chlorella protothecoides cells were investigatedusing NaH¹⁴CO₃ as tracer. Several hours after incubating thegreen algal cells in the glucose medium in the dark, the ratesof ¹⁴C-incorporation into glucose polymers and sucrose decreasedand the incorporation into the lipid fraction (fatty acids)greatly increased. At this stage, the rate of photosynthetic¹⁴CO₂ fixation and the chlorophyll content were practicallythe same as in the starting green cells. Afterwards, the photosyntheticcapacity and chlorophyll content continued to decrease throughoutthe experimental period. In contrast, when photosynthetic ¹⁴CO₂fixation of green cells was carried out in the medium containingglucose, the rate of ¹⁴C-incorporation into glucose polymersincreased, though there was no change in the incorporationsinto sucrose and the lipid fraction. ¹Part of this investigation was reported at the Conference ComparativeBiochemistry and Biophysics of Photosynthesis (Japan-U.S. CooperativeScience Program) held at Hakone, Japan in 1967.²Present address: Faculty of Agriculture, Tamagawa University,Machida-shi, Tokyo, Japan. (Received June 10, 1974; )     

CO2-fixation Sites in Leaves of Maize and Oats

The primary site of photosynthetic carbon dioxide fixation wasdetermined in the leaves of two monocot species by means ofmicroautoradiographic techniques. Carbon fixation occurred primarilyin the vascular-bundle sheath cells in maize whereas fixationin oat occurred uniformly throughout the leaf. The evidencepresented suggests that the bundle sheath cells of maize fixcarbon via the C.4 dicarboxylic acid pathway.     

Studies on chlorophyllase of Chlorella protothecoides IV. Some properties of the purified enzyme

Some properties of chlorophyllase (chlorophyll-chlorophyllido-hydrolase,EC 3.1.1. 14) from Chlorella protothecoides are described. Themolecular weight estimated by sodium lauryl sulfate (SDS)-polyacrylamidegel electrophoresis was 38,000, and the isoelectric point determinedby the isoelectric focusing technique was 4.5. The enzyme hada constant high activity over a wide pH range from 6.0 to 8.5,and was stable from pH 4.0 to 9.2. (Received May 12, 1979; )     

Changes of ribulose 1,5-diphosphate carboxylase level during the processes of degeneration and regeneration of chloroplasts in Chlorella protothecoides

RuDP carboxylase was active mainly in chloroplasts and PEP carboxylaseactive principally outside of chloroplasts in Chlorella protothecoides. During the process of chloroplast degeneration in algal cellsinduced by addition of glucose, the activity of RuDP carboxylasesignificantly decreased, whereas the activities of PEP-carboxylaseand -carboxykinase markedly increased. During the process of chloroplast regeneration in "glucose-bleached"algal cells, which contained no detectable amounts of FractionI protein and showed only traces of RuDP carboxylase activity,a light-dependent development of RuDP carboxylase proceededalmost in parallel with the light-induced formation of chlorophyll.The activities of PEP-carboxylase and -carboxykinase, whichwere negligibly low in glucose-bleached cells, developed independentlyof light. Both chloramphenicol and cycloheximide severely inhibited thedevelopment of RuDP carboxylase activity. A relatively low concentrationof glucose also caused a significant suppression. Under theseconditions, chlorophyll formation was inhibited only slightlyby chloramphenicol and very strongly by cycloheximide and glucose. ¹ Deceased, 11 June, 1972. (Received April 25, 1972; )     

Effects of dark preincubation and chloramphenicol on blue light-induced CO2 incorporation in Chlorella cells

Chlorella cells incubated in the dark longer than 12 hr showedpronounced blue light-induced ¹⁴CO₂ fixation into aspartate,glutamate, malate and fumarate (blue light effect), whereasthose kept under continuous light showed only a slight bluelight effect, if any. 2) During dark incubation of Chlorellacells, phosphoenolpyruvate carboxylase activity and the capacityfor dark ¹⁴CO₂ fixation decreased significantly, whereas ribulose-1,5-diphosphatecarboxylase activity and the capacity for photosynthetic ¹⁴CO₂fixation (measured under illumination of white light at a highlight intensity) did not decrease. 3) In cells preincubatedin the dark, intracellular levels of phosphoenolpyruvate and3-phosphoglycerate determined during illumination with bluelight were practically equal to levels determined during illuminationwith red light. 4) The blue light effect was not observed incells incubated widi chloramphenicol, indicating that blue light-inducedprotein synthesis is involved in the mechanism of the effect. (Received April 9, 1971; )     

Role of chloroplast photosystems II and I in apoptosis of pea guard cells

We investigated the CN^–-induced apoptosis of guard cells in epidermal peels isolated from pea (Pisum sativum L.) leaves. This process was considerably stimulated by illumination and suppressed by the herbicides DCMU (an inhibitor of the electron transfer between quinones Q_A and Q_B in PS II) and methyl viologen (an electron acceptor from PS I). These data favor the conclusion drawn by us earlier that chloroplasts are involved in the apoptosis of guard cells. Pea mutants with impaired PS I (Chl-5), PS II (Chl-I), and PS II + PS I (Xa-17) were tested. Their lesions were confirmed by the ESR spectra of Signal I (oxidized PS I reaction centers) and Signal II (oxidized tyrosine residue Y_D in PS II). Destruction of nuclei (a symptom of apoptosis) and their consecutive disappearance in guard cells were brought about by CN^– in all the three mutants and in the normal pea plants. These results indicate that the light-induced enhancement of apoptosis of guard cells and its removal by DCMU are associated with PS II function. The effect of methyl viologen preventing CN^–-induced apoptosis in wild-type plants was removed or considerably decreased upon the impairment of the PS II and/or PS I activity.