Chloroplast movement in Mougeotia induced by blue light pulses

The profile-to-face chloroplast movement in the green alga Mougeotia has been induced by strong blue and near-ultraviolet light pulses (6 J m^-2). Simultaneously, strong red or far-red light (10 W m^-2) was applied perpendicularly to the inducing beam. The response was measured photometrically. Against the far-red background the reciprocity law was found to hold for pulse durations varying two orders of magnitude. The action spectrum exhibited a maximum near 450 nm and a distinct increase in near-ultraviolet. The time-course and the spectral dependence of pulse responses of chloroplasts in Mougeotia were similar to those recorded for other plants which are sensitive only to blue. This points to an alternative sensor system active in the short-wavelength region in addition to the phytochrome system.Abbreviations FR far-red light - Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome - R red light This paper is dedicated to the memory of Professor Jan Zurzycki     

Characterization of the isolated calcium-binding vesicles from the green alga Mougeotia scalaris,and their relevance to chloroplast movement

The calcium-binding vesicles from the green alga Mougeotia scalaris were isolated and characterized after staining in vivo by neutral red or rhodamine B. They were found to possess, a protonated group with a pK_a-9.9, typifying phenolic hydroxyl groups; upon titration, both, phenolic compound(s) and vital dye were concomitantly released from the vesicular matrix. A shift in peak absorbance from 450 nm to 540 nm of the vitally stained vesicles indicated that the neutral form of neutral red was bound to the vesicular, matrix as an intermediate form, stabilized via intermolecular hydrogen bonds to the phenolic compound(s). Up to 8.5.10⁹ dye molecules were calculated to be adsorbed to a mean-size vesicle. Analysis of Langmuir adsorption isotherms, indicated that there were two binding sites each for both neutral red and rhodamine B. The isolated vesicles were devoid of calcium, probably because vesicular calcium, bound to the vesicle matrix, was displaced upon dye binding. Dye adsorption to the vesicles in vivo results in substantial inhibition of the reorientational movement of the Mougeotia chloroplast and is explained by dye-mediated disorder of the cellular calcium homoeostasis.Abbreviations NR neutral red - RB rhodamine B - SDS sodium dodecyl sulfate This paper is part of the Ph.D. thesis of F. Grolig at Justus-Liebig-Universität Giessen, FRG     

X-ray microanalysis and chlorotetracycline staining of calcium vesicles in the green alga Mougeotia

Calcium ions have been proposed to play a key role in the sensory transduction of phytochrome-governed chloroplast movement in the green alga Mougeotia. To test this hypothesis, the intracellular pattern of calcium distribution was studied in this alga by two independent techniques, namely, X-ray microanalysis of fixed and of unfixed frozen-hydrated cells, as well as in vivo fluorescence by chlorotetracycline. Both methods of detection reveal a significant compartmentation of calcium in vesicles close to the chloroplast edge and, less frequently, in the cortical cytoplasm. Microfilaments, presumably actin, which could function in driving chloroplast movement, have been observed running between the chloroplast edge and the cortical cytoplasm (Wagner, G., Klein, K. (1978) Photochem. Photobiol. 27, 137). The vesicular calcium concentration is stable and decays only slowly in the absence of extracellular calcium much in the same way as the ability of the chloroplast to perform movements decreases. A functional relationship between vesicular calcium compartmentation and phytochrome-governed chloroplast movement in the green alga Mougeotia seems indicated.Abbreviation CTC chlorotetracycline     

Blue-light-induced chloroplast orientation in Mougeotia. Evidence for a separate sensor pigment besides phytochrome

Chloroplast orientation in the green alga Mougeotia has been induced by unidirectional red or blue light, given continuously during one hour. In addition, part of the preparations obtained scattered strong far-red light simultaneously with the orienting light. This far-red light completely abolished the response to red light, consistent with phytochrome as the sensor pigment for orientation in Mougeotia. In blue light, however, the response was completely insensitive to far-red light, thus pointing to a different sensor pigment in the shortwavelength region.Abbreviation P_fr far-red-absorbing form of phytochrome     

Mechanism of chloroplast movement inMougeotia

Summary The single, ribbon-shaped chloroplast in the filamentous green algaMougeotia performs orientational movements with respect to light. The chain of reaction involves phytochrome as the photoreceptor pigment to perceive the light signal differentiated by wavelength and direction, calcium probably to convert the light signal into a chemical message and actomyosin to respond to this message and to move the chloroplast accordingly.Precise reorientation of the chloroplast is proposed to be brought about by a dual function of phytochrome: regulation of the cellular level of calciumand regulation of membrane anchorage sites to actin.The Institute where ProfessorKamiya did early research together with ProfessorKüster.     

Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-buffered media (-log [Ca²⁺] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca²⁺]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca²⁺] in M. scalaris that was nearly independent of the external [Ca²⁺] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca²⁺ from intracellular stores. Increased cytoplasmic [Ca²⁺] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca²⁺-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.Abbreviations EGTA ethylene glycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - indo-1 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,Ntetraacetic acid - pCa log [Ca²⁺] - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - x_G geometric mean Dedicated to Professor Wolfgang Haupt on the occasion of his 70th birthdayThis paper is part of the Ph.D. thesis of U. Russ at the Justus-Liebig-Universitat Giessen (FRG). Part of this work has been presented at a meeting on Calcium and intracellular signalling in plants in Plymouth, UK, Dec. 1990We are indebted to Dr. G. Seibold and Dipl. Phys. H. Weintraut for their advice on the technique of microspectrofluorometry and for allowing access to the microspectrophotometric facilities in the Strahlenzentrum der Justus-Liebig-Universität, Giessen, FRG. We thank Mrs. A. Quanz for reliable culture of the algae and evaluation of the videotapes. Bay-K8644 was a generous gift of Bayer AG, Wuppertal, FRG. U. russ was supported by a scholarship according to the Hessisches Graduierten Förderungsgesetz. This work was supported by the Deutsche Forschungsgemeinschaft.     

Phytochrome-mediated uptake of calcium in Mougeotia cells

Ca²⁺ is proposed to function as a messenger in such phytochrome-mediated responses as localized cell growth, intracellular movements, and control of plasma membrane properties. To test this hypothesis, the uptake of Ca²⁺ in irradiated and non-irradiated regions of individual threads of the green alga Mougeotia was studied with the aid of ⁴⁵Ca²⁺ and low temperature autoradiography: 10–20 cells within 40–60 cell-long threads were irradiated for up to 1 min, transferred to darkness for 3 to 10 min, submersed in a radioactive medium for 1 min, washed in an unlabelled medium for 30 min, and then autoradiographed at-80° C for several days.The autoradiographs show that those cells which had been pre-irradiated with red light did take up 2–10 times more Ca²⁺ than the adjacent non-irradiated cells of the same thread. Cells pre-irradiated with farred light or red light followed by far-red light showed no enhanced uptake of Ca²⁺. These results might be interpreted to indicate, firstly, that phytochrome-Pfr is involved in the enhanced uptake of Ca²⁺ and secondly, that the accumulation of radioactive Ca²⁺ in red light irradiated cells is an expression of an increased intracellular concentration of Ca²⁺. This interpretation is based on the data that (i) the dark interval between irradiation and labelling precluded the involvement of photosynthesis, (ii) the effect of red light was reversible with far-red light, and (iii) the accumulation of Ca²⁺ persisted during the long wash-out period. We speculate, that the red light-enhanced accumulation of Ca²⁺ in Mougeotia cells is caused by a Pfr-mediated increase of the Ca-permeability of the plasma membrane, and perhaps by a Pfr-impeding of an active Ca²⁺-extrusion.Abbreviations APW artificial pond water - EGTA ethylene glycol-bis-(-amino ethyle ether) N,N-tetraacetic acid - R red irradiation - D darkness - FR far-red irradiation - Pfr physiologicallyactive form of phytochrome - Pr physiologically inactive form of phytochrome This paper is part of a Ph. D. Thesis submitted to the University of Erlangen-Nürnberg by E.M. Dreyer     

Vital staining permits isolation of calcium vesicles from the green alga Mougeotia

The calcium vesicles of the green alga Mougeotia (G. Wagner and R. Rossbacher, 1980, Planta 149, 298–305) were isolated for characterization in vitro by fractionation of algal homogenate on sucrose density gradients. A new technique, based on vital staining by neutral red or rhodamine B, permitted isolation. Minimum dye binding to the calcium vesicles prevented desintegration, and for isolation a single, thoroughly defined centrifugation step sufficed, facilitated by the exceptionally high vesicular density of 1.3 g· cm^-3. Neutral red in particular seems to be accumulated by the vesicles via hydrogen bonds to abundant phenolic hydroxyl groups which, reversibly bound to an as yet undefined vesicle core, may well provide coordination sites for the observed calcium binding.Dedicated to Professor Wilhelm Nultsch on the occasion of his 60th birthdayA preliminary version of this paper has been presented at Tagung der Deutschen Gesellschaft für Zellbiologie (Grolig and Wagner 1985). This paper is part of the Ph. D. thesis of F. Grolig at Justus-Liebig-Universität Giessen     

Heavy-meromyosin-decoration of microfilaments from Mougeotia protoplasts

The cell wall of the green alga Mougeotia was enzymatically digested by macerase and cellulysin. Released protoplasts were spread on poly-L-ornithine, formvar-carbon-coated grids, and cell fragments were collected for structural characterization. Large numbers of 5–7 nm filaments are seen which may be decorated with heavy meromyosin (HMM), a digest product of muscle myosin that binds specifically to actin, supporting the hypothesis that the phytochrome-mediated chloroplast movements in these algae are driven by a contractile complex of actomyosin.Abbreviation HMM heavy meromyosin Dedicated to Professor Wolfgang Haupt on the occasion of his 60th birthday     

Phytochrome of the green alga Mougeotia: cDNA sequence, autoregulation and phylogenetic position

A cDNA clone encoding phytochrome (apoprotein) of the zygnematophycean green alga Mougeotia scalaris has been isolated and sequenced. The clone consisted of 3372 bp, encoded 1124 amino acids, and showed strain-specific nucleotide exchanges for M. scalaris, originating from different habitats. No indication was found of multiple phytochrome genes in Mougeotia. The 5 non-coding region of the Mougeotia PHY cDNA harbours a striking stem-loop structure. Homologies with higher-plant phytochromes were 52–53% for PHYA and 57–59% for PHYB. Highest homology scores were found with lower-plant phytochromes, for example 67% for Selaginella (Lycopodiopsida), 64% for Physcomitrella (Bryopsida) and 73% for Mesotaenium (Zygnematophyceae). In an unrooted phylogenetic tree, the position of Mougeotia PHY appeared most distant to all other known PHYs. The amino acids Gly-Val in the chromophore-binding domain (-Arg-Gly-Val-His-Gly-Cys-) were characteristic of the zygnematophycean PHYs known to date. There was no indication of a transmembrane region in Mougeotia phytochrome in particular, but a carboxyl-terminal 16-mer three-fold repeat in both, Mougeotia and Mesotaenium PHYs may represent a microtubule-binding domain. Unexpected for a non-angiosperm phytochrome, its expression was autoregulated in Mougeotia in a red/far-red reversible manner: under P_r conditions, phytochrome mRNA levels were tenfold higher than under P_fr conditions.     

Regulation of the orientation movement of chloroplasts in epidermal cells of Vallisneria: cooperation of phytochrome with photosynthetic pigment under low-fluence-rate light

In epidermal cells of the leaves of the aquatic angiosperm Vallisneria gigantea Graebner, the chloroplasts accumulate in the outer periclinal layer of cytoplasm (P side) under light at low fluence rates. The nature of such intracellular orientation of chloroplasts was investigated in a semiquantitative manner. Time-lapse video microscopy revealed that, while irradiation with red light (650 nm, 0.41 W · m^–2) rapidly accelerated the migration of chloroplasts, not only from the anticlinal layers of cytoplasm (A sides) to the P side but also from the P side to the A sides, the increased rate of migration in both directions returned to the control rate upon subsequent irradiation with far-red light (746nm, 0.14W · m^–2). These effects of red and far-red light could be observed repeatedly, both in the presence and in the absence of inhibitors of photosynthesis, suggesting the involvement of phytochrome as the photoreceptor. After saturating irradiation with red light, the increased rate of migration of chloroplasts from the P side to the A sides declined more rapidly than the increased rate of migration in the opposite direction. This imbalance in the migration of chloroplasts between the two opposing directions resulted in the accumulation of chloroplasts on the P side. The more rapid decline in the rate of migration of chloroplasts from the P side to the A sides than in the opposite direction was not observed in the presence of an inhibitor of photosynthesis. It appears, therefore, that phytochrome and photosynthetic pigment cooperatively regulate the accumulation of chloroplasts on the P side through modulation of the nature of the movement of the chloroplasts.Abbreviations A side cytoplasmic layer that faces the anticlinal wall - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - P side cytoplasmic layer that faces the outer periclinal wall This work was supported in part by Grants-in-Aid from the Japanese Ministry of Education, Science and Culture to S.T. and R.N. The authors are indebted to the Osaka branch of Kashimura Inc. for their kind cooperation in preparing the GREEN software.     

Photoregulation systems for light-oriented chloroplast movement

Movements of the chloroplasts induced by a directional stimulus of light are found to occur in various plant materials ranging from algae to terrestrial angiosperms. Depending on the fluence rate of light, chloroplasts move toward the area of the maximum light absorption and escape from it, which are named as low- and high-fluence-rate responses, respectively. In most materials the effective wavelengths are exclusively found in the blue-UV region of spectrum, (a) flavin pigment(s) being considered as the photoreceptor, while in a few species of plants phytochrome is involved. The arrangement of chloroplasts as a result of the movement depends on the orientation of the electrical vector of light, which reflects the dichroic orientation of the photoreceptor for perception of light direction. Photosystems involved in these responses, however, are not only for perception of light direction, but also for realization of the movement and for holding of the chloroplasts in the reached site. Possible interactions and dual roles of photosystems in regulation of chloroplast movement are discussed.     

Influence of calcium on blue-light-induced chloroplast movement in Lemna trisulca L.

The presence of calcium is essential for chloroplast movement induced by blue light in Lemna trisulca L. The regulatory role of calcium was confirmed by the inhibition of chloroplast movement by cytochalasin B and trifluoperazine. The calcium concentration in tissues was modified by ethylene glycol-bis(2-aminoethylether)-N,N,N, N-tetraacetic acid (EGTA), the calcium ionophore A23187 and La³⁺. Only a long period of incubation (12h) in EGTA or La³⁺ caused distrubances in chloroplast movement. This indicates that calcium influx is not essential for chloroplast movement. Those conditions that dramatically changed the internal calcium concentration, either applications of calcium ionophore A23187 and EGTA, or ionophore and La³⁺, markedly decreased the amplitude of response to blue-light pulses. This demonstrates that disturbances of chloroplast movement are observable only when internal stores of calcium are affected by Ca²⁺-antagonists. We suggest that the calcium involved in blue-light-induced chloroplast movement is derived from intracellular stores. The addition of Mg²⁺ to EGTA buffer counteracted its effect, indicating that Mg²⁺, as well as Ca²⁺, might possibly be involved in chloroplast movement.Abbreviations EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes 4(2-hydroxyethyl-1-piperazine) ethanesulfonic acid - A23187 calcium ionophore We express our gratitude to Professor W. Korohoda for valuable critical comments on this paper and stimulating discussion. We also thank Mr. P. Malec for help in preparing the experiment with trifluoperazine and Mr. A. Waloszek for taking the photographs. We are indebted to Mr. Tim Kline (International House, Krakow, Poland) for improving the English style. This research was supported by grant No. 1042/P2/92/03 from the State Committe for Scientific Research.     

Calcium-binding and its effect on circular dichroism of plant calmodulin

The Ca²⁺-binding properties of calmodulin purified from zucchini (Cucurbita pepo L.) has been determined. A value of 3.3 mol Ca²⁺ per mol of zucchini calmodulin was measured at pH 7.5 by equilibrium chromatography. The far-and near-UV circular-dichroic spectra of the Ca²⁺-and Mg²⁺-saturated as well as from the metal-free forms of zucchini calmodulin reveal that upon Ca²⁺-binding the -helix content increases. A comparison with the spectra of vertebrate calmodulin indicates that both calmodulin have a similar secondary structure, similar Ca²⁺-induced conformational changes and the same number of Ca²⁺-binding sites.Abbreviations CAPP 10-(3-aminopropyl)-2-chloro-phenothiazine - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - EDTA ethylenediaminetetraacetic acid Dedicated to Prof. Dr. Karl Decker on the occasion of his 60th birthday     

Blue- and red-light action in photoorientation of chloroplasts in Adiantum protonemata

An action spectrum for the low-fluencerate response of chloroplast movement in protonemata of the fern Adiantum capillus-veneris L. was determined using polarized light vibrating perpendicularly to the protonema axis. The spectrum had several peaks in the blue region around 450 nm and one in the red region at 680 nm, the blue peaks being higher than the red one. The red-light action was suppressed by nonpolarized far-red light given simultaneously or alternately, whereas the bluelight action was not. Chloroplast movement was also induced by a local irradiation with a narrow beam of monochromatic light. A beam of blue light at low energy fluence rates (7.3·10^-3-1.0 W m^-2) caused movement of the chloroplasts to the beam area (positive response), while one at high fluence rates (10 W m^-2 and higher) caused movement to outside of the beam area (negative response). A red beam caused a positive response at fluence rates up to 100 W m^-2, but a negative response at very high fluence rates (230 and 470 W m^-2). When a far-red beam was combined with total background irradiation with red light at fluence rates causing a low-fluence-rate response in whole cells, chloroplasts moved out of the beam area. When blue light was used as background irradiation, however, a narrow far-red beam had no effect on chloroplast distribution. These results indicate that the light-oriented movement of Adiantum chloroplasts is caused by red and blue light, mediated by phytochrome and another, unidentified photoreceptor(s), respectively. This movement depends on a local gradient of the far-red-absorbing form of phytochrome or of a photoexcited blue-light photoreceptor, and it includes positive and negative responses for both red and blue light.Abbreviations BL blue light - FR far-red light - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - UV ultraviolet     

Nuclear genes encoding chloroplast hemoglobins in the unicellular green alga Chlamydomonas eugametos

When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclear genes are periodically activated in response to the changes in light conditions. These genetic responses are dependent upon the activation of genes associated with photosynthesis (LI616 and LI637), nonphotosynthetic photoreceptors (LI410 and LI818) and the biological clock (LI818). We report here that the LI410 and LI637 genes are part of a small gene family encoding hemoglobins (Hbs) related to those from two unicellular eukaryotes, the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis, and from the cyanobacterium Nostoc commune. Investigations of the intracellular localization of C. eugametos Hbs by means of immunogold electron microscopy indicate that these proteins are predominantly located in the chloroplast, particularly in the pyrenoid and the thylakoid region. To our knowledge, this constitutes the first evidence for the presence of Hbs in chloroplasts. Alignment of the LI637 cDNA nucleotide sequence with its corresponding genomic sequence indicates that the L1637 gene contains three introns, the positions of which are compared with those in the Hb genes of plants, animals and the ciliate P. caudatum. Although the LI637 gene possesses a three-intron/four-exon pattern similar to that of plant leghemoglobin genes, introns are inserted at different positions. Similarly the position of the single intron in the P. caudatum gene differs from the intron sites in the LI637 gene. The latter observations argue against the current view that all eukaryotic Hbs have evolved from a common ancestor having a gene structure identical to that of plant or animal Hbs.     

Phylogenetic relationship of the green alga Nanochlorum eukaryotum deduced from its chloroplast rRNA sequences

The marine green coccoidal alga Nanochlorum eukaryotum (N.e.) is of small size with an average diameter of 1.5 m. It is characterized by primitive-appearing biochemical and morphological properties, which are considerably different from those of other green algae. Thus, it has been proposed that N.e. may be an early developed algal form. To prove this hypothesis, DNA of N.e. was isolated by a phenol extraction procedure, and the chloroplast DNA separated by preparative CsCl density-gradient centrifugation. The kinetic complexity of the nuclear and of the chloroplast DNA was evaluated by reassociation kinetics to 3 × 10⁷ by and 9 × 10⁴ bp, respectively. Several chloroplast genes, including the rRNA genes, were cloned on distinct fragments. The order of the rRNA genes corresponds to the common prokaryotic pattern. The 16S rRNA gene comprises 1,548 bases and is separated from the 23S rRNA gene with its 2,920 bases by a short spacer of 460 bases, which also includes the tRNA^Ile and tRNA^Ala genes. The 5S rRNA gene has not been found; it must start further than 500 bases downstream from the 3-end of the 23S rRNA gene. From the chloroplast rRNA sequences, we have deduced secondary structures of the 16S and 23S rRNAs, which are in agreement with standard models. The rRNA sequences were aligned with corresponding chloroplast sequences; phylogenetic relationships were calculated by several methods. From these calculations, we conclude that N.e. is most closely related to Chlorella vulgaris. Therefore, N.e. does not represent an early developed algal species; the primitive-appearing morphological and biochemical characteristics of N.e. must rather be explained by secondary losses. Correspondence to: D. Weinblum     

Kinetics of the dichroic reorientation of phytochrome during photoconversion inMougeotia

In the green algaMougeotia, the dichroic orientation of the red-absorbing form of phytochrome (P_r) is parallel of the cell surface, whereas the far-red-absorbing form (P_fr) is oriented normal to it. The time course of the change from parallel to normal was investigated by double-flash irradiation with polarized red and far-red light. The results obtained by two different methods indicate that most of the phytochrome intermediates existing in the first 5 ms after the inducing red flash are still oriented parallel to the cell surface, similar to P_r. At increasing intervals between the red and the far-red flashes, more and more phytochrome molecules turn their transition moments to the P_fr orientation. This reaction is finished after approximately 30 ms. We conclude that the change in dichroic orientation of the phytochrome molecules inMougeotia occurs during the last relaxation steps of the intermediates on the way from P_r to P_fr. It cannot be decided yet, whether the first surface-normal phytochrome species is an intermediate or P_fr itself.Abbreviations P_r red-absorbing form of phytochrome - P_fr far-red-absorbing form of phytochrome A preliminary report of this work was presented at the European Symposium on Photomorphogenesis, University of Reading, UK (Kraml et al. 1982)     

Ultrastructural organization of chloroplast membranes in mutants of Pisum sativum L. with impaired activity in the photosystems

The ultrastructural organization and the photosynthesis reactions of chloroplast membranes were studied in three lethal mutants of Pisum sativum, Chl-1, Chl-19 and Chl-5, all lacking the capacity to evolve oxygen. The rates of 2,6-dichloroindophenol reduction, delayed fluorescence and electron-spin-resonance signal 1 indicate that Chl-1 and Chl-19 have an impaired activity in photosystem II (PS II), while in Chl-5 the electron transport is blocked between PS I and the reactions of CO₂ fixation. Ultrathin sectioning demonstrates the presence of giant grana in the chloroplasts of Chl-1 and Chl-19, while the chloroplast structure of the Chl-5 is very similar to that of the wild-type. The grana of the Chl-19 mutant contain large multilamellar regions of tightly packed membranes. When the chloroplast membranes were studied by freeze-fracture, the exoplasmic and protoplasmic fracture faces (EF and PF, respectively) in both stacked and unstacked membranes were found to show large differences in particle concentrations and relative population area (per m²), and also in particle size distribution, between all mutant chloroplast membranes and the wild-type. A close correlation between increasing k_mt (ratio of particle concentrations on PF/EF) and PS II activity was observed. The differences in particle concentrations on both fracture faces in different regions of the intact chloroplast membranes of the wild-type are the consequence of a rearrangement of existing membrane components by lateral particle movements since quantitative measurements demonstrate almost complete conservation of intramembrane particles in number and size during the stacking of stroma thylakoid membranes. The results indicating particle movements strongly support the concept that the chloroplast membranes have a highly dynamic structure.Abbreviations DPIP 2,6-dichloroindophenol - EF and PF exoplasmic and protoplasmic fracture faces, respectively - PS I and PS II photosystems I and II, respectively