Photosynthetic Properties of ac-31, a Mutant Strain of Chlamydomonas reinhardi Devoid of Chloroplast Membrane Stacking

A pale-green mutant strain of Chlamydomonas reinhardi, ac-31, is characterized by the absence of any stacking of its chloroplast membranes. The capacity for photosynthetic electron transport, phosphorylation, and CO₂ fixation in ac-31 is substantial, and it is concluded that these photosynthetic activities occur within the single membrane. The photosynthetic capacities of wild type and ac-31 as a function of increasing light intensity are compared. Saturation is attained at higher light intensities in ac-31, and the kinetics of the 2 sets of curves are distinctly different. The possibility that energy transfer is enhanced by membrane stacking is suggested by these results. The repeatedly-observed correlation between reduced stacking and disfunctional Photosystem II activities is discussed in view of the observation that ac-31 has no stacking but retains a functional Photosystem II.     

Chloroplast Ultrastructure in Mutant Strains of Chlamydomonas reinhardi Lacking Components of the Photosynthetic Apparatus

The fine structure of the chloroplast of wild-type and 9 photosynthetic mutant strains of Chlamydomonas reinhardi is described. The chloroplast phenotypes of the mutant strains are clearly distinct from the wild type in all but 2 cases. Moreover, strains with similar photosynthetic disabilities have structurally similar chloroplasts. These differences are apparently not the result of altered chlorophyll content, nor of photosynthetic inactivity. It is therefore proposed that the structural alterations are in some way related to the mutant strains' inability to synthesize active components of the photosynthetic electron transport chain.     

Polypeptide Composition of Photosynthetic Membranes from Chlamydomonas reinhardi and Anabaena variabilis

Anabaena variabilis, a blue-green alga lacking chlorophyll b, shows an absence of the major 22 and 24 kilodalton polypeptides which are present in the photosynthetic membranes of Chlamydomonas reinhardi and higher plants. These data are consistent with other investigations which have shown that these polypeptides are associated with chlorophyll b in the chloroplasts of higher plants, and indicate the presence of a light harvesting chlorophyll-protein complex in higher plants which contains the chlorophyll b of the photosynthetic membrane.     

The Effect of Manganese on Chloroplast Structure and Photosynthetic Ability of Chlamydomonas reinhardi

Manganese deficiency was induced in mixotrophically-grown cultures of the green alga Chlamydomonas reinhardi and the effects of this deficiency on photosynthetic activity and cell structure were investigated. Manganese-deficient cells are unable to carry out, at normal rates, photosynthetic reactions involving system II of the photosynthetic electron transport chain. Reactions requiring only system I are not inhibited. Normal system II activity returns within 2 hr in the light following addition of manganous ions to deficient cells.Significant structural alterations for deficient cells were observed in the chloroplast, in the arrangement of discs into stacks. Stacking distributions for normal, deficient, and recovered cells were quantitatively characterized and compared by the introduction of an experimentally-defined distribution function and its attendant parameters. The results provide evidence for a role for manganese in maintaining chloroplast structure.     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi: VIII. The 520 nm Light-induced Absorbance Change in the Wild-type and Mutant Strains 1

The 520 nm light-induced absorbance change in wild-type and 4 mutant strains of Chlamydomonas reinhardi was investigated. In the wild-type strain the absorbance change is composed of at least 2 components, P520 I and P520 II, sensitized by Systems I and II respectively. Some of the properties of these components can be studied by using the appropriate photosynthetic mutant strain. A group of mutant strains modified in the photochemical complex of System II shows only the P520 I absorbance change, whereas a mutant strain deficient in active P700 exhibits only the P520 II absorbance change. The possible relationship between these absorbance changes and the photosynthetic electron transport pathway is discussed.     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. III. Light-Induced Absorbance Changes in Chloroplast Fragments of the Wild Type and Mutant Strains

Light-induced absorbance changes were investigated in chloroplast fragments of wild type Chlamydomonas reinhardi and 5 different mutant strains having impaired photosynthesis. Two absorbance changes were detected, 1 having a maximum at 553 nm and the other at 559 nm. The component exhibiting the 553 nm change is a cytochrome similar to cytochrome f from higher plant chloroplasts. The component exhibiting the 559 nm change has the properties of a cytochrome similar to cytochrome b(3). Two of the mutant strains (ac-115 and ac-141) were found to lack the 559 cytochrome and light induced only the oxidation of the 553 cytochrome. A third mutant strain (ac-206), previously shown to lack the 553 cytochrome, exhibited only the light-induced reduction of the 559 cytochrome. A fourth mutant strain (ac-208), shown to lack plastocyanin, exhibited absorbance changes attributable to both cytochromes. However, light was capable of inducing the reduction of the 559 cytochrome but not its oxidation. On the other hand, light induced the oxidation of the 553 cytochrome but not its reduction.These observations are discussed in terms of the series formulation for photosynthetic electron transport in which the 559 cytochrome is reduced by system II and transfers electrons via the component affected in ac-21 to the 553 cytochrome. Accordingly, system I sensitizes the oxidation of the 3 components of the electron transport chain.     

Fluorescence Properties of Wild-Type Chlamydomonas reinhardi and Three Mutant Strains Having Impaired Photosynthesis

The wild-type strain of Chlamydomonas reinhardi and 3 mutant strains ac-21, ac-141, and ac-115 have been compared for their fluorescence (and luminescence) properties. The different fluorescence levels, the rapid and slow photochemical responses affecting fluorescence, and the intensity of luminescence have been studied under various conditions: air, nitrogen, 3(p-chlorophenyl)-1,1-dimethylurea. The strain ac-21 exhibits fluorescence properties only quantitatively different from those of the wild-type strain, and it is believed to be affected in some component of the electron transport chain between the 2 light reactions. Both ac-141 and ac-115 have an abnormally high initial fluorescence level; ac-115 does not show the normal photochemical response associated with System II and has a very low luminescence. Mutant strains ac-141 and ac-115 both seem to be modified in the System II photochemical center. These conclusions are compared with a previous analysis based on absorbance changes of cytochrome 559.     

Chloroplast Development in the Chloroplast Ribosome Deficient Mutant (y-1 ac-20) of Chlamydomonas reinhardtii

To study the participation of chloroplast protein synthesisduring the three phases [Matsuda (1974) Biochim. Biophys. Acta366:45] of the greening process in Chlamydomonas reinhardtiiy-1, the greening characteristics in the low-chloroplast ribosomemutant y-1 ac-20 were compared with those in the y-1. In thedouble mutant cells Chl synthesis proceeded with an extendedlag and without a second transition point. The development ofpotential for rapid Chl synthesis (P-factor formation) was alsodelayed. Furthermore, PS I activity increased significantly,whereas PS II activity developed very little during greeningof the double mutant cells. The results indicate that greeningin double mutant cells occurs with no apparent late phase. (Received November 26, 1984; Accepted February 25, 1985)     

Synthesis and Turnover of the Chloroplast Coupling Factor 1 in Chlamydomonas reinhardi

Studies on the biosynthesis of the chloroplast coupling factor 1 (CF₁) in Chlamydomonas reinhardi have been initiated. The ratio of CF₁ to chlorophyll in the cell was shown to be independent of the density of the culture. No turnover of assembled CF₁ could be detected, thus suggesting that CF₁ was synthesized at a rate equivalent to that of net chlorophyll synthesis. A lag of between 5 to 7 minutes in the incorporation of radioactive precursor sulfate into assembled CF₁ was measureable. This puts an upper limit on the pool size of any precursor to the assembled CF₁ complex. The pool size is estimated to be equivalent to 1% of the total CF₁ in the cell.     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. IV. Purification and Properties of Plastocyanin

The copper protein plastocyanin has been found to be an essential component of the photosynthetic electron transport chain of Chlamydomonas reinhardi, and in this paper we describe a method for its isolation and purification from the wild-type strain. In addition, we describe some of its properties and compare them with those reported for spinach plastocyanin.     

Resistance and Sensitivity of Chloroplast Ribosomes to Streptomycin in Mutants of Chlamydomonas reinhardi

• 1 In a mendelian (sr₃) and an uniparental (sr₃₅) streptomycin resistant mutant of Chlamydomonas reinhardi the influence of streptomycin on protein synthesis on the chloroplast and cytoplasmic ribosomes was investigated in vitro. Hetero-, mixo- and phototrophic agar cultures and heterotrophic liquid cultures were used.
• 2 Protein synthesis on the cytoplasmic ribosomes, measured by the activity of glyceraldehyde-3-phosphate: NADP dehydrogenase (EC 1.2.1.9), was not inhibited, but rather stimulated by streptomycin.
• 3 Protein synthesis on the chloroplast ribosomes of sr₃, measured by the activity of ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39), was greatly inhibited by streptomycin, especially in hetero- and mixotrophic cultures. In sr₃₅ the chloroplast ribosomes were resistant to streptomycin.
• 4 Heterotrophically grown cultures of sr₃ and of a streptomycin-sensitive strain are yellow in the presence of streptomycin and form no or only reduced thylakoids on solid media. But 70-S organelle-ribosomes are present in a normal amount.
• 5 The relationship between chloroplast protein synthesis and thylakoid formation is discussed.

     

The Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. VII. Photosynthetic Phosphorylation by a Mutant Strain of Chlamydomonas reinhardi Deficient in Active P700

Electron transport activity and absorbance changes associated with P700 were investigated in a mutant strain of Chlamydomonas reinhardi with impaired photosynthesis. This mutant strain, ac-8oa, cannot reduce NADP with electrons from either water or dye and ascorbate, but it has considerable Hill activity. The mutant strain shows none of the absorbance changes characteristic of P700. Although unable to carry out cyclic photosynthetic phosphorylation, ac-8oa is able to synthesize ATP when ferricyanide is provided as an electron acceptor.

These observations lead to the conclusion that a site for the coupling of photosynthetic phosphorylation with electron transport must exist between the 2 photochemical systems.

     

Photosynthetic Properties of Chloroplasts from Chlamydomonas reinhardii

Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (−), fix CO₂ at rates between 25 and 50 micromoles per milligram chlorophyll per hour. The upper value is approximately half of the rate of the intact cell.

During storage in the dark on ice, the chloroplast preparation loses 30 to 50% of its CO₂ fixing capability per hour. Under reducing conditions (+ 1 millimolar dithiothreitol), this loss of activity is about twice as fast. The same reducing conditions stimulate CO₂ fixation in the light.

High concentrations of inorganic phosphate (>2 millimolar) inhibit CO₂ fixation. This inhibition is overcome by the addition of glycerate 3-phosphate. It is concluded that chloroplasts from C. reinhardii possess a higher plant type phosphate translocator. With respect to dependency upon light intensity, pH and Mg²⁺ concentration, the results were similar to that reported for chloroplasts from higher plants. However, in contrast to higher plant chloroplasts, maximum CO₂ fixation is observed at the relatively low osmotic concentration of 0.12 molar mannitol in the reaction buffer.

     

Synthetic Requirements of Chloroplast Development in Chlamydomonas reinhardi*†

SYNOPSIS. The synthesis of chlorophyll by chlorotic cultures of Chlamydomonas reinhardi is stimulated by a utilizable carbon source. This synthesis is almost completely inhibited by cycloheximide in concentrations as low as 0.50 μg/ml. The period of chloroplast development during which stimulation by carbon source and inhibition by cylcoheximide are most effective corresponds to a period of rapid formation of chloroplast lamellae.     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. V. Purification and Properties of Cytochrome 553 and Ferredoxin

Cytochrome 553 and ferredoxin were isolated and purified from acetone powders prepared from intact cells of the wild-type strain of Chlamydomonas reinhardi. Purification was achieved by ion exchange chromatography on DEAE cellulose and gel filtration on Sephadex G-75.     

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi VI. Electron Transport in Mutant Strains Lacking Either Cytochrome 553 or Plastocyanin

A mutant strain of Chlamydomonas reinhardi, ac-206, lacks cytochrome 553, at least in an active and detectable form. Chloroplast fragments of this mutant strain are inactive in the photoreduction of NADP when the source of electrons is water, but they are active when the electron source is 2,6-dichlorophenolindophenol and ascorbate. The addition of either cytochrome 553 or plastocyanin, obtained from the wild-type strain, has no effect upon the photosynthetic activities of the mutant strain. Cells of the mutant strain lack both the soluble and insoluble forms of cytochrome 553, but they possess the mitochondrial type cytochrome c. Thus, the loss of cytochrome 553 appears to be specific.