Plastids with densely staining thylakoid contents inNymphoides indica

Summary Most chloroplasts of the lower epidermal and sub-epidermal cells in mature floating leaves ofNymphoides indica have densely staining thylakoid loculi which contrast markedly with electron translucent loculi of chloroplasts elsewhere in the leaves. Examination of leaf primordia (< 1 mm long) showed that granal membranes arise within the plastid stroma and already at that stage the stainable substance can be seen associated with the lamellae. In slightly enlarged primordia (5 mm long), nearly all plastids of the young leaf contain thylakoids with stained loculi, but by the time the leaf reaches 25 mm in length, the staining is restricted to the lower epidermal layers.     

Changes of Thylakoid Membrane Stacks and Chl a/b Ratio of Chloroplast from Sacred Lotus (Nelumbo nucifera) Seeds During Their Germination Under Light

Changes of chloroplast thylakoid membrane stacks and Chl a/b ratio in the plumule of sacred lotus (Nelumbo nucifera Gaertn) seeds during their germination under light were as follows: Before germination there were giant grana and very low Chi a/b ratio (0.9) in the chloroplasts. Two days after germination, the thylakoid membranes of the giant grana gradually loosened and even destacked (disintegrated), the Chl a/b ratio was 1.06. Four clays after germination, the newly formed grana thylakoid membranes were 3–5 times shorter than those of the supergrana thylakoid membranes before germination and less grana stacks were seen; the Chl a/b ratio was 1.42. Six days after germination, the stacked thylakoi membranes became more orderly arranged. In addition the grana increased in number, the stroma thylakoid membranes were scarce, the Chl a/b ratio was 2.16. Eiglt days after germination, the thylakoid membranes in each granum decreased, but the total number of grana increased only slightly. In the meantime, some large starch grains and more stroma thylakoid membranes appeared; the Chl a/b ratio was 2.77. Ten days after germination normal thylakoid membrane structure was formed both in grana and stroma lamellae. They were arranged orderly as in the chloroplasts of other higher plants; the Chl a/b ratio was 2.80. The following conclusions could be drawn from the above mentioned results: 1) There was a negative correlation between the degree of stacking of the grana thylakoid membranes and the Chl a/b ratio. This statement further proved that the membranes stacking might mainly be induced by LHCII. 2) Development of the grana thylakoid membranes within chloroplasts from sacred lotus plumule followed that of the stroma thylakoid membranes, and the tendency of changes of their Chl 2/b ratio being from the lowest to the highest and then to normal were quite different from those of other higher plants. The chloroplasts iri the latter plants contain long parallel stacks of nonappressed primary thylakoids at second step, and the changes of their ratio of Chl a/b tend to be from the highest to the lowest and then to normal. There are indications that sacred lotus plumule might employ a distinctive developing pathway. This provides an important basis for Nelumbo to possess an unique position in phylogeny of Angiospermae.     

Heat sensitivity of chloroplasts and leaves: Leakage of protons from thylakoids and reversible activation of cyclic electron transport

In illuminated intact spinach chloroplasts, warming to and beyond 40 °C increased the proton permeability of thylakoids before linear electron transport through Photosystem II was inhibited. Simultaneously, antimycin A-sensitive cyclic electron transport around Photosystem II was activated with oxygen or CO2, but not with nitrite as electron acceptors. Between 40 to 42 °C, activation of cyclic electron transport balanced the loss of protons so that a sizeable transthylakoid proton gradient was maintained. When the temperature of darkened spinach leaves was slowly increased to 40°C, reduction of the quinone acceptor of Photosystem II, Q_A, increased particularly when respiratory CO2 production and autoxidation of plastoquinones was inhibited by decreasing the oxygen content of the atmosphere from 21 to 1%. Simultaneously, Photosystem II activity was partially lost. The enhanced dark Q_A reduction disappeared after the leaf temperature was decreased to 20 °C. No membrane energization was detected by light-scattering measurements during heating the leaf in the dark. In illuminated spinach leaves, light scattering and nonphotochemical quenching of chlorophyll fluorescence increased during warming to about 40 °C while Photosystem II activity was lost, suggesting extra energization of thylakoid membranes that is unrelated to Photosystem II functioning. After P700 was oxidized by far-red light, its reduction in the dark was biphasic. It was accelerated by factors of up to 10 (fast component) or even 25 (slow component) after short heat exposure of the leaves. Similar acceleration was observed at 20 °C when anaerobiosis or KCN were used to inhibit respiratory oxidation of reductants. Methyl viologen, which accepts electrons from reducing side of Photosystem II, completely abolished heat-induced acceleration of P700+ reduction after far-red light. The data show that increasing the temperature of isolated chloroplasts or intact spinach leaves to about 40 °C not only inhibits linear electron flow through Photosystem II but also activates Photosystem I-driven cyclic electron transport pathways capable of contributing to the transthylakoid proton gradient. Heterogeneity of the kinetics of P700+ reduction after far-red oxidation is discussed in terms of Photosystem I-dependent cyclic electron transport in stroma lamellae and grana margins.     

Thylakoid-bound proteolytic activity against LHC II apoprotein in bean

Triton X-100 solubilized thylakoids, isolated from Phaseolus vulgaris chloroplasts, degrade endogenous or exogenously added LHC II. The degradation, as monitored by immunodetection of the remaining LHC II after incubation at 37°C, is activated by Mg⁺⁺ and inhibited by pCMB, EDTA, PMSF and benzamidine; the activity under high light conditions parallels chlorophyll photooxidation. The thylakoid-bound proteolytic activity is under phytochrome control. Etiolated plants pretreated by a white light pulse, and kept in the dark thereafter, show enhanced proteolytic activity, which follows rhythmical oscillations. On the other hand, chloramphenicol pretreatment of etiolated plants, prior to their transfer to continuous light, reduces the proteolytic activity against LHC II. The results suggest that the degradation involves a serine type protease, which depends on SH group(s), coded by the plastid genome; the protease action on LHC II is regulated by Mg⁺⁺, phytochrome, the biological clock and chlorophyll accumulation in the thylakoid. The stroma lamellar fraction, separated from French press disrupted chloroplasts, exhibits higher activity towards exogenous LHC II than the grana fraction. The stroma of intact chloroplasts exhibits also high proteolytic activity, which is drastically reduced when the lysis medium is supplemented with cations. This suggests that the protease is bound mainly on stroma lamellae and peripheral granal membranes, its association to the membranes being possibly under cation control.Abbreviations CAP chloramphenicol - CL continuous light - LHC II light harvesting complex of Photosystem II     

Insights into the complex 3-D architecture of thylakoid membranes in the unicellular cyanobacterium Cyanothece sp. ATCC 51142

In cyanobacteria and chloroplasts, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. In plant chloroplasts, thylakoids are differentiated into a highly interconnected system of stacked grana and unstacked stroma membranes. In contrast, in cyanobacteria, the evolutionary progenitors of chloroplasts, thylakoids do not routinely form stacked and unstacked regions, and the architecture of the thylakoid membrane systems is only now being described in detail in these organisms. We used electron tomography to examine the thylakoid membrane systems in one cyanobacterium, Cyanothece sp. ATCC 51142. Our data showed that thylakoids form a complicated branched network with a rudimentary quasi-helical architecture in this organism. A well accepted helical model of grana-stroma architecture of plant thylakoids describes an organization in which stroma thylakoids wind around stacked granum in right-handed spirals. Here we present data showing that the simplified helical architecture in Cyanothece 51142 is lefthanded in nature. We propose a model comparing the thylakoid membranes in plants and this cyanobacterium in which the system in Cyanothece 51142 is composed of non-stacked membranes linked by fret-like connections to other membrane components of the system in a limited left-handed arrangement.Key words: cyanobacteria, Cyanothece 51142, thylakoid membrane, electron tomography, chloroplast     

Significance of structural variation in thylakoid membranes in maintaining functional photosystems during reproductive growth

Structural variation in the stroma‐grana (SG) arrangement of the thylakoid membranes, such as changes in the thickness of the grana stacks and in the ratio between grana and inter‐grana thylakoid, is often observed. Broadly, such alterations are considered acclimation to changes in growth and the environment. However, the relation of thylakoid morphology to plant growth and photosynthesis remains obscure. Here, we report changes in the thylakoid during leaf development under a fixed light condition. Histological studies on the chloroplasts of fresh green Arabidopsis leaves have shown that characteristically shaped thylakoid membranes lacking the inter‐grana region, referred to hereafter as isolated‐grana (IG), occurred adjacent to highly ordered, large grana layers. This morphology was restored to conventional SG thylakoid membranes with the removal of bolting stems from reproductive plants. Statistical analysis showed a negative correlation between the incidences of IG‐type chloroplasts in mesophyll cells and the rates of leaf growth. Fluorescence parameters calculated from pulse‐amplitude modulated fluorometry measurements and CO₂ assimilation data showed that the IG thylakoids had a photosynthetic ability that was equivalent to that of the SG thylakoids under moderate light. However, clear differences were observed in the chlorophyll a/b ratio. The IG thylakoids were apparently an acclimated phenotype to the internal condition of source leaves. The idea is supported by the fact that the life span of the IG thylakoids increased significantly in the later developing leaves. In conclusion, the heterogeneous state of thylakoid membranes is likely important in maintaining photosynthesis during the reproductive phase of growth.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material.The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic cross-point (pH 4.1) was observed for the fraction enriched in inside-out grana thylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

Light-induced Changes in the Ultrastructure of Pea Chloroplasts in Vivo: Relationship to Development and Photosynthesis

Light-induced structural changes of chloroplasts and their lamellae were studied in leaves of Pisum sativum L., cv. Blue Bantam, using electron microscopy. Upon illumination of 14-day-old plants with 2000 lux, the chloroplasts decreased in thickness by about 23% with an accompanying increase in electron scattering by the stroma. Concomitantly, the average thickness of granal lamellae (thylakoids) decreased from 195 ± 4 angstroms in the dark to 152 ± 4 angstroms in the light, and this change was half-saturated at only 50 lux. Lamellar flattening at 50 lux and its reversal in the dark both had half-times of a minute or less. The thickness of a partition (a pair of apposed lamellar membranes) was 140 ± 9 angstroms in both the light and the dark, indicating that the observed light-induced change was in the volume enclosed within the thylakoid. The effect of illumination could be inhibited by various uncouplers of photophosphorylation but not by 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea, suggesting that it depended on ATP (or its precursor). In the presence of 0.5 micromolar nigericin, the thickness of the granal lamellae increased in the light to 213 ± 3 angstroms; this may reflect an uptake of K⁺ into an osmotically responding space within the thylakoids.     

Comparative ultrastructural properties of pallisade tissue and spongy tissue chloroplasts from normal and mutant leaves of Pisum sativum L.*

Abstract. The ultrastructure of chloroplasts from palisade and spongy tissue was studied in order to analyse the adaptation of chloroplasts to the light gradient within the bifacial leaves of pea. Chloroplasts of two nuclear gene mutants of Pisum sativum (chlorotica-29 and chlorophyll b-less 130A), grown under normal light conditions, were compared with the wild type (WT) garden-pea cv. ‘Dippes Gelbe Viktoria’. The differentiation of the thylakoid membrane system of plastids from normal pea leaves exhibited nearly the same degree of grana formation in palisade and in spongy tissue. Using morphometrical measurements, only a slight increase in grana stacking capacity was found in chloroplasts of spongy tissue. In contrast, chloroplasts of mutant leaves differed in grana development in palisade and spongy tissue, respectively. Their thylakoid systems appeared to be disorganized and not developed as much as in chloroplasts from normal pea leaves. Grana contained fewer lamellae per granum, the number of grana per chloroplast section was reduced and the length of appressed thylakoid regions was decreased. Nevertheless, chloroplasts of the mutants were always differentiated into grana and stroma thylakoids. The structural changes observed and the reduction of the total chlorophyll content correlated with alterations in the polypeptide composition of thylakoid membrane preparations from mutant chloroplasts. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), polypeptide bands with a relative molecular mass of 27 and 26 kilodalton (kD) were markedly reduced in mutant chloroplasts. These two polypeptides represented the major apoproteins of the light harvesting chlorophyll a/b complex from photosystem II (LHC-II) as inferred from a comparison with the electrophoretic mobility of polypeptides isolated from the LHC-II.     

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes

ABSTRACT: BACKGROUND: The thylakoid system in plant chloroplasts is organized into two distinct domains: granaarranged in stacks of appressed membranes and non-appressed membranes consisting ofstroma thylakoids and margins of granal stacks. It is argued that the reason for thedevelopment of appressed membranes in plants is that their photosynthetic apparatus need tocope with and survive ever-changing environmental conditions. It is not known however,why different plant species have different arrangements of grana within their chloroplasts. Itis important to elucidate whether a different arrangement and distribution of appressed andnon-appressed thylakoids in chloroplasts are linked with different qualitative and/orquantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranesand whether this arrangement influences the photosynthetic efficiency. RESULTS: Our results from TEM and in situ CLSM strongly indicate the existence of differentarrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids areregularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, whileirregular appressed thylakoid membranes within bean chloroplasts correspond to smaller andless distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show adistinct spatial separation of stacked thylakoids from stromal spaces whereas spatial divisionof stroma and thylakoid areas in bean chloroplasts are more complex. Structural differencesinfluenced the PSII photochemistry, however without significant changes in photosyntheticefficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well asspectroscopic investigations indicated a similar proportion between PSI and PSII corecomplexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones.Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSIsupercomplexes between species are suggested. CONCLUSIONS: Based on proteomic and spectroscopic investigations we postulate that the differences in thechloroplast structure between the analyzed species are a consequence of quantitativeproportions between the individual CP complexes and its arrangement inside membranes.Such a structure of membranes induced the formation of large stacked domains in pea, orsmaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with eachother and not always parallel to each other.     

Effect of Doubled-CO2 Concentration on the Ultrastructure of Chloroplasts from Medicago sativa and Setaria italica

It has been reported in quite a number of literatures that doubled CO2 concentration increased the photosynthetic rate and dry matter production of C3 plants, but substantially affected C4 plants little. However, why may CO2 enrichment promote growth and either no change or decrease reproductive allocation of the C3 species, but havinag no effects on growth characteristics of the C4 plants? So far, there has been no satisfactory explanation on that mentioned above, except the differences in their CO2 compensatory points. In the past, although some studies on ultrastructure of the chloroplasts under doubled CO2 concentration were limitedly conducted. Almost all the relevant experimental materials were only from C3 plants not from C4 plants, and even though the results were of inconsistancy. Thereby, it needs to verify whether the differences in photosynthesis of C3 and C4 plants at doubled CO2 level is caused by the difference in their chloroplast deterioration. Experiments to this subject were conducted at the Botanical Garden of Institute of Botany, Academia Sinica in 1993 and 1994. Both experimental materials from C3 plant alfalfa (Medicago sativa) and C4 plant foxtail millet (Setaria italica) were cultivated in the cylindrical open-top chambers (2.2 m in diameter × 2.4 m in height) with aluminum frames covered by polyethylene film. Natural air or air with 350× 10-6 CO2 were blown from the bottom of the chamber space with constant temperature between inside and outside of the chamber 〈0.2℃〉. Electron microscopic observation revealed that the ultrastructure of the chloroplasts from C3 plant Medicago sativa and C4 plant Seteria italica growing under the same doubled CO2 concentration were quite different from each other. The differential characteristics in ultrastructure of chloro plasts displayed mainly in the configuration of thylakoid membrances and the accumulation of starch grains. They were as follows: 1. The most striking feature was the building up of starch grains in the chloroplasts of the bundle sheath cells (BSCs) and the mesophyll cells (MCs) at doubled CO2 concentra tion. The starch grains appeared centrifugally first in the BSCs and then in the chloroplast of the other MCs. It was worthy to note that the starch grains in the chloroplasts of C4 plant Setaria ira/ica were much more than those of the C3 plant Medicago sativa . The decline of photosynthesis in the doubled CO2-grown C4 plants might be caused by an over accumulation of starch grains, that deformed the chloroplast even demaged the stroma thylakoids and grana. There might exsist a correlation between the comformation of thylakoid system and starch grain accumulation, namely conversion and transfer of starch need energy from ATP, and coupling factor (CF) for ATP formation distributed mainly on protoplastic surface (PSu) of stroma thylakoid membranes, as well as end and margin membranes of grana thylakoids. Thereby, these results could provide a conclusive evidence for the reason of non effectiveness on growth characteristics of C4 plant. 2. Under normal condition , the mature chlolroplats of higher plants usually develop complete and regularly arranged photosynthetic membrane systems . Chloroplasts from the C4 plant Setaria italica, however, exerted significant changes on stacking degree, grana width and stroma thylakoid length under doubled CO2 concentration; In these changes, the grana stacks were smaller and more numerous, and the number of thylakoids per granum was greatly increased, and the stroma thylakoid was greatly lengthened as compared to those of the control chloroplasts. But the grana were mutually intertwined by stroma thylakoid. The integrity of some of the grana were damaged due to the augmentation of the intrathylakoid space . Similarly, the stroma thylakoids were also expanded. In case. the plant was seriously effected by doubled CO2 concentration as observed in C4 plant Setaria italica , its chloroplasts contained merely the stroma (matrix) with abundant starch grains, while grana and stroma thylakoid membranes were unrecognizable, or occasionally a few residuous pieces of thylakoid membranes could be visualized, leaving a situation which appeared likely to be chloroplast deterioration. However, under the same condition the C3 plant Medicago sativa possessed normally developed chloroplasts, with intact grana and stroma thylakoid membranes. Its chloroplasts contained grana intertwined with stroma thylakoid membranes, and increased in stacking degree and granum width, in spite of more accumulated starch grains within the chloroplasts. These configuration changes of the thylakoid system were in consistant with the results of the authors another study on chloroplast function, viz. the increased capacity of chloroplasts for light absorption and efficiency of PSⅡ.     

The constant proportion of grana and stroma lamellae in plant chloroplasts

The relative proportion of stroma lamellae and grana end membranes was determined from electron micrographs of 58 chloroplasts from 21 different plant species. The percentage of grana end membranes varied between 1 and 21% of the total thylakoid membrane indicating a large variation in the size of grana stacks. By contrast the stroma lamellae account for 20.3 ± 2.5 ( sd )% of the total thylakoid membrane. A plot of percentage stroma lamellae against percentage of grana end membranes fits a straight line with a slope of zero showing that the proportion of stroma lamellae is independent of the size of the grana stacks. That stroma lamellae account for about 20% of the thylakoid membrane is in agreement with fragmentation and separation analysis (Gadjieva et al . Biochim. Biophys. Acta 144: 92–100, 1999). Chloroplasts from spinach, grown under high or low light, were fragmented by sonication and separated by countercurrent distribution into two vesicle populations originating from grana and stroma lamellae plus end membranes, respectively. The separation diagrams were very similar lending independent support for the notion that the proportion of stroma lamellae is constant. The results are discussed in relation to the composition and function of the chloroplast in plants grown under different environmental conditions, and in relation to a recent quantitative model for the thylakoid (Albertsson, Trends Plant Sci. 6: 349–354, 2001).     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

Energization and ultrastructural pattern of thylakoids formed under periodic illumination followed by continuous light

Bean leaves grown under periodic illumination (56 cycles of 2 min light and 98 min darkness) were subsequently exposed to continuous illumination, and in connection with granum formation and accumulation of the light-harvesting pigment-protein complex thermoluminescence and light-induced shrinkage of thylakoid membranes were studied. Juvenile chloroplasts with large double sheets of thylakoids obtained under periodic light exhibited low temperature spectra of polarized fluorescence yielding fluorescence polarization (FP) values < 1 at 695 nm, characteristic for pheophytin emission. In the course of maturation under continuous light when normal grana appeared and the chlorophyll a/b light-harvesting photosystem II complex was incorporated into the membrane, at 695 nm the relative intensity of fluorescence dropped and FP changed to a value of > 1, suggesting an overlap between the emission of pheophytin and that of the chlorophyll a/b light-harvesting photosystem II complex. Thermoluminescence glow curves recorded with juvenile thylakoids displayed a relatively high proportion of emission at low temperatures (around -10°C) while with mature chloroplasts, more thermoluminescence originated from energetically deeper traps (discharged around 28°C). This means that during thylakoid development the capacity of the membrane to stabilize the separated charges increases, which might be favourable for the ultimate conservation of energy. The more extensive energization of mature thylakoids was also indicated by a light-induced decrease in the thickness of the membranes upon illumination; a change which could not be detected in juvenile thylakoids.Abbreviations EDTA ethylenediamine tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

Functional role of aminoacyl phosphatidylglycerols in the lamellar system of chloroplasts]

The role of 14C-aminoacyl-tRNAs in the formation of aminoacyl phosphatidyl glycerols in isolated chloroplasts of haricot bean leaves was studied. The formation of 14C-aminoacyl-tRNAs was more intensive in the case when 14C-aminoacyl phosphatidyl glycerols were the source of amino acids. On incubation of lamellae with 14C-aminoacyl phosphatidyl glycerols, 14C-amino acids proved to be incorporated intensively in protein of the lamellae. Membrane-bound chloroplast ribosome-like particles were observed on the outermost thylakoid membranes of the grana stacks as well as on the stroma thylakoids. It is concluded that aminoacyl phosphatidyl glycerols play an important role in lateral transport of amino acids within the chloroplasts lamellar system.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition.

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material. The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic crosspoint (pH 4.1) was observed for the fraction enriched in inside-out granathylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

A mechanism for the formation of inside-out membrane vesicles. Preparation of inside-out vesicles from membrane-paired randomized chloroplast lamellae

Inside-out thylakoid membrane vesicles can be isolated by aqueous polymer two-phase partition of Yeda press-fragmented spinach chloroplasts (Andersson, B. and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472). The mechanism for their formation has been investigated by studying the yield of inside-out vesicles after various treatments of the chloroplasts prior to fragmentation. No inside-out vesicles were isolated during phase partitioning if the chloroplasts had been destacked in a low-salt medium prior to the fragmentation. Only in those cases where the chloroplast lamellae had been stacked by cations or membrane-paired by acidic treatment did we get any yield of inside-out vesicles. Thus, the intrinsic properties of chloroplast thylakoids seem to be such that they seal into right-side out vesicles after disruption unless they are in an appressed state. This favours the following mechanism for the formation of inside-out thylakoids. After press treatment, a ruptured membrane still remains appressed with an adjacent membrane. Resealing of such an appressed membrane pair would result in an inside-out vesicle.If the compartmentation of chloroplast lamellae into appressed grana and unappressed stroma lamellae is preserved by cations before fragmentation, the inside-out vesicles are highly enriched in photosystem II. This indicates a granal origin which is consistent with the proposed model outlined. Inside-out vesicles possessing photosystem I and II properties in approximately equal proportions could be obtained by acid-induced membrane-pairing of chloroplasts which had been destacked and randomized prior to fragmentation. Since this new preparation of inside-out thylakoid vesicles also exposes components derived from the stroma lamellae it complements the previous preparation.It is suggested that fragmentation of paired membranes followed by phase partitioning should be a general method of obtaining inside-out vesicles from membranes of various biological sources.     

Chloroplast structure: from chlorophyll granules to supra-molecular architecture of thylakoid membranes

This review provides a brief historical account of how microscopical studies of chloroplasts have contributed to our current knowledge of the structural and functional organization of thylakoid membranes. It starts by tracing the origins of the terms plastid, grana, stroma and chloroplasts to light microscopic studies of 19th century German botanists, and then describes how different types of electron microscopical techniques have added to this field. The most notable contributions of thin section electron microscopy include the elucidation of the 3-D organization of thylakoid membranes, the discovery of prolamellar bodies in etioplasts, and the structural changes in thylakoid architecture that accompany the light-dependent transformation of etioplasts into chloroplasts. Attention is then focused on the roles that freeze-fracture and freeze-etch electron microscopy and immuno electron microscopy have played in defining the extent to which the functional complexes of thylakoids are non-randomly distributed between appressed, grana and non-appressed stroma thylakoids. Studies reporting on how this lateral differentiation can be altered experimentally, and how the spatial organization of functional complexes is affected by alterations in the light environment of plants are also included in this discussion. Finally, the review points to the possible uses of electron microscope tomography techniques in future structural studies of thylakoid membranes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.