Characterization of a Photosynthetic Mutant Strain of Chlamydomonas reinhardi Deficient in Phosphoribulokinase Activity

A mutant strain of the unicellular green alga, Chlamydomonas reinhardi, is unable to fix carbon dioxide by photosynthesis because it is deficient in phosphoribulokinase activity. The absence of light-dependent carbon dioxide fixation in cells of the mutant strain supports the operation of the Calvin-Benson scheme of photosynthetic carbon dioxide fixation in this organism. No deficiency other than low phosphoribulokinase activity was found which would account for the inability of cells of the mutant strain to fix carbon dioxide by photosynthesis. Activities comparable to those in the wild-type strain were found for eight other enzymes of the Calvin cycle and two enzymes associated with the C₄ dicarboxylic acid pathway. The normal rates of nicotinamide adenine dinucleotide phosphate photoreduction and of photosynthetic phosphorylation observed in chloroplast fragments prepared from cells of the mutant strain indicated that the photosynthetic electron transport chain in the mutant is intact.     

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 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.     

Low Energy Effects of Light on Growth and Pigment Content in a Yellow-in-the-Dark Mutant of Chlamydomonas reinhardi

The y-2 mutant of Chlamydomonas reinhardi differs from the wild type in being unable to synthesize chlorophyll in the dark and in a requirement for catalytic amounts of light for organotrophic growth. Light-grown y-2 cells given acetate are capable of the equivalent of 9 to 10 divisions when placed in darkness. Cultures adapt gradually to dim white or monochromatic light and after 8 to 10 generations assume a steady state with respect to growth and pigment content.

Two energetically distinct light reactions promote the growth of y-2 on acetate. A low energy requirement is satisfied at about 0.1 μw/cm² of white light which results in a growth rate of 0.5 log unit per day. A high energy response, which saturates at 2000 μw/cm² and a growth rate of 0.9 log unit per day, is probably attributable to net photosynthesis. An action spectrum for the low energy growth response contains a broad major peak in the blue between 462 and 502 nm and a minor peak in the far-red between 700 and 736 nm. All intermediate wavelengths have low but positive activity. The action spectrum was investigated with y-2 cultures that were grown for many generations under steady-state conditions in growth-limiting monochromatic light. Many wavelengths resulted in a selection pressure that strongly favored a strain of green-in-the dark cells that usually appeared after 5 to 8 generations of light-limited growth. Under the low light intensity of these experiments (0.15 ± 0.05 μw/cm²) the green strain was much richer in chlorophyll than y-2 and divided more rapidly with the consequence that y-2 was generally replaced in the course of a few generations. Consideration of the results led to the conclusion that both chlorophyll and carotenoids act as photoreceptors in the low energy growth response of y-2.

     

BIOGENESIS OF CHLOROPLAST MEMBRANES : V. A Radioautographic Study of Membrane Growth in a Mutant of Chlamydomonas reinhardi y-1

The development of photosynthetic lamellae during greening of dark-grown Chlamydomonas y-1 cells was investigated by radioautography. Acetate-³H was used as a marker for membrane lipids. In short pulse-labeling experiments, about 50–60% of the radioactivity incorporated was found in the lipid fraction and about 25–50% in starch granules present in the chloroplast of these algae. The relative specificity of acetate-³H used as a marker for membranes was artificially increased through quantitative removal of the starch granules from fixed cells by amylase treatment. Analysis of turnover coefficients of different membrane constituents and of the contribution of turnover and net synthesis to the total label incorporated in pulse experiments indicated that the incorporation of acetate into specific lipids was mainly due to net synthesis. The distribution of radioactivity in the different lipid constituents at the end of a short pulse and after 30- and 60-min chases indicated that transacylation is minimal and may be disregarded as a possible cause of randomization of the label. Statistical analysis of radioautographic grain distribution and measurements of different structural parameters indicate that (a) the chloroplast volume and surface remain constant during the process, whereas the growth of the photosynthetic lamellae parallels the increase in chlorophyll; (b) the lamellae do not develop from the chloroplast envelope or from the tubular system of the pyrenoid; (c) all the lamellae grow by incorporation of new material within preexisting structures; (d) different types of lamellae grow at different rates. The pyrenoid tubular system develops faster than the thylakoids, and single thylakoids develop about twice as fast as those which are paired or fused to grana. It is concluded that growth of the membranes occurs by a mechanism of random intussusception of molecular complexes within different types of preexisting membranes.     

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.     

Responses of Chlamydomonas reinhardi to Specific Nutritional Limitation in Continuous Culture*†

SYNOPSIS. Responses of wild type and mutant strains of Chlamydomonas reinhardi were studied in a simple but stable chemostat. No chlorosis was observed under conditions of phosphate, arginine or Na acetate limitation. Competition between wild type and the mutant strain y-2 was studied in continuous culture with phosphate as the limiting nutrient in continuous light, continuous dark and alternating periods of light and darkness. In all cases, the mutant strain y-2 overgrew the wild type.     

Colchicine-resistant mutants of Chlamydomonas reinhardi

Five colchicine-resistant mutant strains of Chlamydomonas reinhardi have been isolated. In colchicine-free medium they all have abnormally long cell doubling times and tend to occur within palmella envelopes, rather than as free-swimming cells. Zygote germination of all the mutants is abnormal, but crosses with wild type suggest that resistance is in each case due to a Mendelian mutation. It is suggested, though not proved, that the mutations may affect microtubular structures.     

Amide metabolism of Chlamydomonas reinhardi

Chlamydomonas reinhardi can utilise the lower aliphatic amides (C₁–C₄) as nitrogen sources. Of these only acetamide can serve as a sole carbon source. The acetamide analogue F-acetamide kills cells after conversion to F-acetate and F-citrate. This conversion is controlled by exogenous ammonia and, in part, acetate levels. Only one enzyme and one active site are involved in acetamidase function. Enzymatic analysis indicates an increased substrate range as compared to the growth — supported range, indicating uptake, toxicity or metabolic control restrictions.Abbreviations TCA trichloroacetic acid - TAP tris-acetate-phosphate medium - MIC mimmum inhibitory concentration - BSA bovine serum albumin     

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.     

An Electron Spin Resonance Study of Manganese in Wild-Type and Mutant Strains of Chlamydomonas reinhardi

Changes in the intensity of the electron spin resonance signal of divalent manganese were found to occur in suspensions of wild-type Chlamydomonas reinhardi. The observed manganese signal decreased in the light and increased in the dark. Through the use of a continuous-flow system it was possible to determine that the manganous ions responsible for the observed signal were localized solely in the medium. Changes in the signal intensity associated with wild-type cells were independent of the ability of fragments prepared from these cells to perform the Hill reaction with 2,6-dichlorophenol-indophenol (DPIP) as the oxidant.

The manganese signal changes were still evident, though smaller, in cell suspensions of wild-type cells treated with 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, and in mutant strains unable to carry out the Hill reaction, ac-115 and ac-141.

From these data it is concluded that the changes in intensity of the manganese resonance are not related to the function of manganese in photosynthesis but may reflect the capacity of cells for ion uptake in the light.

     

Effect of ethylmethanesulfonate on Chlamydomonas reinhardi

Summary Ethylmethane sulfonate (EMS) is known to cause a considerably high mutation rate in higher plants. In our experiments with Chlamydomonas reinhardi however, the mutagenic effect was unexpectedly low, whereas the toxic effect was quite remarkable. It is supposed that the reason for the low rate of mutants is the high toxicity, since non-toxic EMS concentrations induce no mutants. The toxic effect on Chlamydomonas cells is caused not only by the products of hydrolysis of the EMS, but also by the EMS itself. The damaged cells begin to bleach, furthermore they are not able to deliver their daughter cells. To a certain degree both effects are reversible. Finally it was found that the sensivity to EMS was higher in cells of the mating type — than it was in those of the+mating type.     

莱茵衣藻Nfr-4突变株中类胡萝卜素含量的变化及其对藻生长的影响

文章对相同条件下培养的莱茵衣藻野生型CC-137和八氢番茄红素脱氢酶(phytoene desaturase,PDS)基因突变株Nfr-4的生长进行分析;并用反相高效液相色谱分析总有色类胡萝卜素以及叶绿素含量变化,结果表明两者生长的差异明显;Nfr-4突变株的单细胞叶绿素和总有色类胡萝卜素含量高于野生型CC-137的。