Regulation of photosynthetic carbon metabolism in synchronously growing Chlorella pyrenoidosa

The physical properties and photosynthetic metabolism of synchronizedcells of Chlorella pyrenoidosa are described. Cells, synchronizedby successive periods of light and dark, photosynthesized understeady-state conditions for 30 min with ¹⁴CO₂. Pool sizes ofmetabolic intermediate compounds, and rates of flow of carbonthrough these pools, were determined. Cell properties and metabolismwere studied for cells just divided, at three periods duringthe growing stage, at the time of maximum DNA synthesis, justprior to division (after continuous light, and after 5 hr darkness),and following division after continuous light for 37 hr. Changes in pool sizes and flow rates are correlated with relativeshifts between amino acid and protein synthesis, which is greatestduring the growing period and DNA-synthesizing stage, and sucrosesynthesis, which is greatest in the divided cells (after darkness)and pre-division cells (after darkness). The effects of thestage of cell growth and of a prior period of darkness can beseparated to some extent by these studies, and in some respectsare additive. Specific sites of metabolic regulation discerned in these experimentsinclude the following: 1) Ribulose diphosphate carboxylase,2) fructose diphosphatase and sedoheptulose diphosphatase, 3)the synthesis of sucrose, probably at the reaction between fructose-6-phosphateand uridine diphosphoglucose to give sucrose phosphate and uridinediphosphate, 4) amino acid synthesis, at the level of nitratereduction, and 5) amino acid synthesis, at the level of carbonflow from the photosynthetic carbon reduction cycle to aminoacid carbon skeletons. (Received October 31, 1969; )     

Effect of cadmium on photosynthetic pigments in synchronously growing Chlorella cells

Cadmium ions introduced at concentration of 30 ppm to the cultivation medium of synchronously growing Chlorella vulgaris decreased concentration of chlorophylls a and b, carotenes alpha and beta and lutein at various stages of the cell cycle, while at concentration of 1 ppm synthesis of the photosynthetic pigments was stimulated. The pigment content in the cadmium treated cells was related to the morphometrically determined changes in the size and shape of the cells.     

Sequential synthesis of membrane polypeptides in chloroplast thylakoids of synchronized Chlorella pyrenoidosa cells

Synchronized Chlorella pyrenoidosa cells (strain 211-8b) were incubated for 15 min with [³H]leucine at different times after the start of the light period. Chloroplast membranes were isolated and the polypeptides of the thylakoid membranes separated by polyacrylamide gel electrophoresis. The distribution of the radioactive label incorporated in polypeptides at different times indicates a sequential synthesis of protein-chlorophyll complexes (CPC) of the chloroplast membrane proteins. Synthesis of CPC of photochemical systems I and II is promoted by light, whereas the synthesis of the polypeptide “K” falls down with the beginning of the light period. The experiments suggest for different polypeptides during the differentiation process an independent variation in the rates of synthesis or in the incorporation into the membrane.     

RNA synthesis in synchronously growing populations of HeLa S3 cells. I. Rate of total RNA synthesis and its relationship to DNA synthesis

The rate of RNA synthesis in synchronously growing HeLa S3 cells was determined as a function of position in the cell generation cycle. Measurements throughout the cycle of both the rate of incorporation of radioactively-labeled uridine and of the total amount of RNA indicate that (1) the rate of RNA synthesis is constant (or increases only slightly) during G₁, approximately doubles during the first half of S, and then remains constant during the remainder of S and G₂, and (2) cells attain the average G₁ rate of RNA synthesis very early in G¹, and maintain the average G₂ rate until mitosis. If the initiation of DNA synthesis is blocked, the acceleration of RNA synthesis is markedly reduced or eliminated. Further experiments in which DNA synthesis was inhibited at different times in S, or to varying degrees from the beginning of S, suggest that the extent to which RNA synthesis is accelerated depends on the amount of DNA duplicated. These data also indicate that duplication of the first half, and in particular the first few per cent, of the DNA complement results in a disproportionate acceleration of RNA synthesis. The possibility that fluctuations in the sizes of precursor pools may lead to misinterpretation of labeled-uridine incorporation data was examined. Experiments indicate that in this system pool fluctuations do not cause invalid measures of RNA synthesis. It is concluded that RNA synthesis occurs throughout interphase, but undergoes a two-fold increase in rate which is dependent on the duplication of DNA.     

Adaptations in Pigment Composition and Photosynthesis by Far Red Radiation in Chlorella pyrenoidosa

Chlorella pyrenoidosa has been cultivated in radiation of wavelengths between 690–975 nm for several months. Absorption spectra and action spectra of photo-synthesis have been determined for far red and “white” light brown cultures, In vivo spectrophotometric analyses and action spectra showed that fur red growth Chlorella adapted to the extreme light conditions by an increase both in absorption and photosynthesis above 700 nm. It is proposed that som of the in vivo normal chlorophyll a forms were converted to a far red absorbing chlorophyll a form, giving the far red exposed suspension an increased photosynthetic activity between 700–740 nm. The analyses of far red grown Chlorella have also shown an increased photosynthesis in the blue part of the spectrum, presumably due to a decrease in photosynthetically inactive carotenoid content. By culturing Chlorella in a “white” light gradient between 0.5 × 10⁴ and 3.7 × 10⁴ erg cm^?2 s^?1, it has been demonstrated that light intensity did not influence pigment ratios between 500–750 nm. In the blue part, however, high light levels caused increased absorption because of increased carotenoid content. Some ecological aspects of this far red effect have also been discussed.     

Transport changes in synchronously growing CHO and L cells

A reduced rate of transport of 2-amino-isobutyric acid (AIB), was found to be associated with M and early G₁ periods in synchronized populations of CHO and L cells. A doubling of the transport rate occurred with progress of CHO cells further into G₁, and the specific transport activity remained constant during the rest of the cycle, The rate of uridine and thymidine transport also doubled at this point in the cell cycle. There was no change in Km values. The rise in transport rate was blocked by cycloheximide, or when the cells were prevented from leaving metaphase by colcemid.     

Photoproduction of hydrogen by adapted cells of Chlorella pyrenoidosa

Photoproduction of hydrogen gas by the green alga Chlorella pyrenoidosa was studied in a large scale culture of 2.1. Hydrogen was produced by adding sodium hydrosulfite directly to an algal suspension after anaerobiosis in darkness for activation of hydrogenase. The hydrogen production rate showed a characteristic course of an initial burst of gas then steady production, and this course appeared most clearly at cell concentrations around 0.6–0.7 kg/m³. In the final third phase, the hydrogen production rate gradually decreased until evolution ceased. The steady hydrogen evolution was inhibited 75% by a herbicide, DCMU, which blocks electron flow through photosystem II, indicating that the electron donor for hydrogen production was mainly water. The average light intensity within the culture vessel was measured with a diffusing sphere photoprobe. The rate of hydrogen evolution increased hyperbolically with the average light intensity. The duration of hydrogen photoproduction was shorter at higher light intensity due to the inhibition of hydrogenase by concomitantly released oxygen. The duration was shorter also at higher concentrations of algal suspension. It was foudd that the optimum concentration of algae, about 0.7 kg/m³ in this system, must be selected to maximize the yield of hydrogen.     

Protoplast induction in Chlorella pyrenoidosa

Chlorella pyrenoidosa (UTEX 1230) cells in late log phase of growth were induced to form viable protoplasts by enzymatic digestion only when incubated in 2-deoxy-d-glucose (2DG) for 24 h. The combination of hemicellulase (4% w/v), Cellulysin (4% w/v), and glucuronidase (5% v/v) with 0.8 M mannitol and 8 mM CaCl₂ in modified Bristol's solution, was most effective for obtaining viable protoplasts as determined by light and electron microscopy, and vital staining with primuline (0.01% w/v). Resistance of cell walls to extensive extraction (acetolysis), and infrared analysis indicated that sporopollenin is a component of the cell wall. Transmission electron miscroscopy of acetolysed cell walls also allowed visualization of the laminate nature of the wall. This is the first report of successful induction of protoplasts from algae which contain sporopollenin in their cell walls.     

Terminal Oxidases of Chlorella pyrenoidosa

In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O₂) of 2.1 ± 0.3 μm, while the second oxidase has a Km (O₂) of 6.7 ± 0.5 μm, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8-hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake.     

Disappearance of isocitrate lyase enzyme from cells of Chlorella pyrenoidosa

1. Mitochondria isolated from livers of fed adult, starved adult, and embryonic rats can be separated into three distinct bands by isopycnic density centrifugation on a sucrose density gradient. The least dense band (B1) has a mean buoyant density of 1.162 and consists mainly of disrupted mitochondria. The middle band (B2) has a mean buoyant density of 1.184. The most dense band (B3) has a mean buoyant density of 1.216. B2 and B3 consist of intact mitochondria. 2. The mitochondria in B2 and B3 have very similar protein/phospholipid ratios, virtually identical phospholipid and fatty acid compositions and similar specific activities for cytochrome oxidase, malate dehydrogenase and monoamine oxidase. Both fractions have very low glucose 6-phosphatase and acid phosphatase activities. 3. As isolated, adult rat liver mitochondria have electron-dense matrices (condensed forms); some embryonic liver mitochondria are condensed, but a significant proportion have dilated matrices. All B2 mitochondria are in the condensed form. B3 mitochondria from adult rats are condensed if fixed in their equilibrium-density sucrose, but when this is diluted rapidly to 0.25m they become swollen. Some B3 mitochondria from embryonic rats are condensed, the others have dilated matrices. They all swell if rapidly diluted to 0.25m-sucrose. B2 mitochondria retain their condensed form on dilution of the sucrose. 4. It is concluded that the matrix space of B2 mitochondria is almost totally inaccessible to sucrose, but that of B3 mitochondria is readily accessible to sucrose. 5. In liver from normally fed adult rats the B2 mitochondria predominate, whereas in starved rats B2 and B3 are present in approximately equal proportions. Mitochondrial preparations from embryonic liver consist predominantly of B3 mitochondria, but the proportion of these decreases progressively as development proceeds. 6. The B2 mitochondria from livers of fed adult rats can be converted into B3 mitochondria by incubation with 10mm-succinate and 10mm-phosphate. 7. Some B2 mitochondria are converted into B3 mitochondria by exposure to high concentrations of sucrose.     

Specific deficit in the synthesis of 6-sulfoquinovsyl diglyceride in Chlorella pyrenoidosa.

It was found that when Chlorella pyrenoidosa was grown on cysteine as the sole sulfur source, it lost the ability to grow photoautotrophically. When grown in the presence of glucose, cysteine-grown cells displayed a doubling time in the light or dark of 45 h, which is identical to that of cells grown on glucose and SO4 in the dark. This suggests that cells grown on cysteine as sole sulfur source can only grow heterotrophically. In support of this hypothesis, it was found that cysteine-grown cells were defective both in vivo and in vitro in CO2 fixation, although O2 evolution in such cells was normal. Assays of the enzymes of the Calvin cycle indicated that the deficit in CO2 fixation could be ascribed to a lowered phosphoribulokinase activity. A total lipid analysis of Chlorella grown on cysteine revealed that such cells showed a 100-fold deficiency in the purportedly chloroplast-associated 6-sulfoquinovsyl diglyceride. This agrees with earlier reports that cysteine could not serve as a precursor of sulfolipid in Chlorella. No other polar lipid was affected. Large amounts of triglyceride, however, were found in cysteine-grown cells. The biosynthesis of triglyceride provides a means of utilizing reduced nicotinamide adenine dinucleotide reducing equivalents not being used for CO2 fixation.     

Synthesis of Citrulline and Arginine in Chlorella pyrenoidosa

The distribution of radioactivity in Chlorella during dark ¹⁴CO₂fixation was investigated either (a) in normal cells with andwithout added ammonium chloride, or (b) in nitrogen-starvedcells supplied with intermediates of the Krebs-Henseleit ureacycle. In the control experiments almost all the activity was presentin compounds of or associated with, the tricarboxylic acid cycle. The amino-acids citrulline and arginine became radioactive onlyin the presence of ammonia or ornithine where initially theycomprised 40–60 per cent. of the total activity, reactionsof the Krebs–Henseleit urea cycle being implicated intheir formation. No evidence could be found for a complete ureacycle. Unidentified compounds deriving their radioactivity fromthe C₄ carbon of citrulline and/or arginine were detected andformed up to 40 per cent. of the total ¹⁴CO₂ incorporated after25 min.