Further studies of the relationship between cation-induced chlorophyll fluorescence and thylakoid membrane stacking changes

Salt-induced changes in thylakoid stacking and chlorophyll fluorescence do not occur with granal membranes obtained by treatment of stacked thylakoids with digitonin. In contrast to normal untreated thylakoids, digitonin prepared granal membranes remain stacked under all ionic conditions and exhibit a constant high level of chlorophyll fluorescence. However, unstacking of these granal membranes is possible if they are pretreated with either acetic anhydride or linolenic acid.Trypsin treatment of the thylakoids inhibits the salt induced chlorophyll fluorescence and stacking changes but stacking of these treated membranes does occur when the pH is lowered, with the optimum being at about pH 4.5. This type of stacking is due to charge neutralization and does not require the presence of the 2000 dalton fragment of the polypeptide associated with the $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b}$ light harvesting complex and known to be lost during treatment with trypsin (Mullet, J.E. and Arntzen, C.J. (1980) Biochim. Biophys. Acta 589, 100–117).Using the method of 9-aminoacridine fluorescence quenching it is argued that the surface charge density, on a chlorophyll basis, of unstacked thylakoid membranes is intermediate between digitonin derived granal and stromal membranes, with granal having the lowest value.The results are discussed in terms of the importance of surface negative charges in controlling salt induced chlorophyll fluorescence and thylakoid stacking changes. In particular, emphasis is placed on a model involving lateral diffusion of different types of chlorophyll protein complex within the thylakoid lipid matrix.     

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.     

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.     

Dynamic flexibility in the structure and function of photosystem II in higher plant thylakoid membranes: the grana enigma

Grana are not essential for photosynthesis, yet they are ubiquitous in higher plants and in the recently evolved Charaphyta algae; hence grana role and its need is still an intriguing enigma. This article discusses how the grana provide integrated and multifaceted functional advantages, by facilitating mechanisms that fine-tune the dynamics of the photosynthetic apparatus, with particular implications for photosystem II (PSII). This dynamic flexibility of photosynthetic membranes is advantageous in plants responding to ever-changing environmental conditions, from darkness or limiting light to saturating light and sustained or intermittent high light. The thylakoid dynamics are brought about by structural and organizational changes at the level of the overall height and number of granal stacks per chloroplast, molecular dynamics within the membrane itself, the partition gap between appressed membranes within stacks, the aqueous lumen encased by the continuous thylakoid membrane network, and even the stroma bathing the thylakoids. The structural and organizational changes of grana stacks in turn are driven by physicochemical forces, including entropy, at work in the chloroplast. In response to light, attractive van der Waals interactions and screening of electrostatic repulsion between appressed grana thylakoids across the partition gap and most probably direct protein interactions across the granal lumen (PSII extrinsic proteins OEEp-OEEp, particularly PsbQ-PsbQ) contribute to the integrity of grana stacks. We propose that both the light-induced contraction of the partition gap and the granal lumen elicit maximisation of entropy in the chloroplast stroma, thereby enhancing carbon fixation and chloroplast protein synthesizing capacity. This spatiotemporal dynamic flexibility in the structure and function of active and inactive PSIIs within grana stacks in higher plant chloroplasts is vital for the optimization of photosynthesis under a wide range of environmental and developmental conditions.     

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.     

Changes in the Cationic Regulation of Structure and Function in Thylakoids Isolated from Wheat Seedlings Treated with BASF 13.338

When wheat seedlings (Triticum vulgare cf HD 2189) were grown in the presence of BASF 13.338 (4-chloro-5-[dimethylamino]-2-phenyl-3[2H]-pyridazinone), there was a decrease in the ratio of linolenic acid to linoleic acid in the thylakoid membrane lipids (JB St John 1976 Plant Physiol 57: 38) and an increase in the ratio of photosystem II to photosystem I (RM Mannan, S Bose 1984 Photochem Photobiol 41: 63). Accompanying these gross structural changes were alterations in the cationic regulation of structure and functioning of the thylakoid membranes: (a) Mg²⁺-induced increase in the room temperature fluorescence was totally absent; (b) Mg²⁺-induced increase in absorbance at 560 nm, indicative of granal stacking, was slightly higher in thylakoids isolated from the BASF 13.338 treated plants suggesting an increased degree of stacking; and (c) absorption changes in the red and Soret regions of the absorption spectrum, normally resulting from the addition of divalent cation or alkyl anion, or from osmotic shrinkage were almost totally absent in thylakoid membranes isolated from BASF 13.338 treated plants. These observations have been interpreted in terms of: (a) significant alterations in the lipid matrix of the thylakoids from treated plants, (b) absence of cation-induced reorganization of the pigment-protein complexes in the horizontal plane of the treated thylakoid membranes suspended in low salt medium, and (c) absence of dynamic changes even within the individual pigment-protein complexes of treated thylakoids.     

The importance of grana stacking for xanthophyll cycle-dependent NPQ in the thylakoid membranes of higher plants

In the present study we have examined the effects of grana stacking on the rate of violaxanthin (Vx) de-epoxidation and the extent of non-photochemical quenching of chlorophyll a fluorescence (NPQ) in isolated thylakoid membranes of spinach. Our results show that partial and complete unstacking of thylakoids in reaction media devoid of sorbitol and MgCl₂ did not significantly affect the efficiency of Vx de-epoxidation. Under high light (HL) illumination we found slightly higher values of Vx conversion in stacked membranes, whereas in thylakoids incubated at pH 5.2 in the dark, representing the pH-optimum of Vx de-epoxidase, de-epoxidation was slightly increased in the unstacked membranes. Partial and complete unstacking of grana membranes, however, had a dramatic effect on the HL-induced NPQ. High NPQ values could only be achieved in stacked thylakoid membranes in the presence of MgCl₂ and sorbitol. In unstacked membranes NPQ was drastically decreased. The effects of grana stacking on the xanthophyll cycle-dependent component of NPQ were even more pronounced, and complete unstacking of thylakoid membranes led to a total loss of this quenching component. Our data imply that grana stacking in the thylakoid membranes of higher plants is of high importance for the process of overall NPQ. For the xanthophyll cycle-dependent component of NPQ it may even be essential. Possible effects of grana stacking on the mechanism of zeaxanthin-dependent quenching are discussed.     

Granal stacking of thylakoid membranes in higher plant chloroplasts: the physicochemical forces at work and the functional consequences that ensue.

The formation of grana in chloroplasts of higher plants is examined in terms of the subtle interplay of physicochemical forces of attraction and repulsion. The attractive forces between two adjacent membranes comprise (1) van der Waals attraction that depends on the abundance and type of atoms in each membrane, on the distance between the membranes and on the dielectric constant, (2) depletion attraction that generates local order by granal stacking at the expense of greater disorder (i.e. entropy) in the stroma, and (3) an electrostatic attraction of opposite charges located on adjacent membranes. The repulsive forces comprise (1) electrostatic repulsion due to the net negative charge on the outer surface of thylakoid membranes, (2) hydration repulsion that operates at small separations between thylakoid membranes due to layers of bound water molecules, and (3) steric hindrance due to bulky protrusions of Photosystem I (PSI) and ATP synthase into the stroma. In addition, specific interactions may occur, but they await experimental demonstration. Although grana are not essential for photosynthesis, they are ubiquitous in higher plants. Grana may have been selected during evolution for the functional advantages that they confer on higher plants. The functional consequences of grana stacking include (1) enhancement of light capture through a vastly increased area-to-volume ratio and connectivity of several PSIIs with large functional antenna size, (2) the ability to control the lateral separation of PSI from PSII and, therefore, the balanced distribution of excitation energy between two photosystems working in series, (3) the reversible fine-tuning of energy distribution between the photosystems by State 1-State 2 transitions, (4) the ability to regulate light-harvesting via controlled thermal dissipation of excess excitation energy, detected as non-photochemical quenching, (5) dynamic flexibility in the light reactions mediated by a granal structure in response to regulation by a trans-thylakoid pH gradient, (6) delaying the premature degradation of D1 and D2 reaction-centre protein(s) in PSII by harbouring photoinactived PSIIs in appressed granal domains, (7) enhancement of the rate of non-cyclic synthesis of adenosine triphosphate (ATP) as well as the regulation of non-cyclic vs. cyclic ATP synthesis, and (8) the potential increase of photosynthetic capacity for a given composition of chloroplast constituents in full sunlight, concomitantly with enhancement of photochemical efficiency in canopy shade. Hence chloroplast ultrastructure and function are intimately intertwined.     

Thylakoid membrane remodeling during state transitions in Arabidopsis

Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.     

EFFECT OF SHORT-TERM SHADING ON THE CYTOLOGY OF PALISADE TISSUE IN MATURE LEAVES OF THE SUNFLOWER HELIANTHUS ANNUUS

Mature sunflower leaves were exposed to partial shading (35 or 14% of normal sun) or darkness (0% of normal sun) for approximately 8 hr. During this period one-half of each test leaf was shaded; the other half was used as a normal sun control. Palisade cell structure from both halves of each leaf was compared. Shading of leaves had little effect on organelle percent volume values (V_v) with exception of the starch compartment which decreased as shading increased. The surface to volume ratio (S_v) of the chloroplast thylakoids increased while the S_v of the mitochondrial membranes decreased as shading increased. Palisade cell volume did not change in shaded portions of the leaf, except in the fully shaded (dark) tissues where cell volume decreased. Changes in the actual volume of organelle compartments were strongly correlated with changes in cell volume. Thus a general osmotic response may account for some of the volume changes associated with differences in light intensity. Shading increased thylakoid surface areas 10–30% over the full sun controls. The ratio of stromal to granal thylakoid surface area remained constant in both the control and partially shaded samples. However, in darkened samples this ratio decreased as stromal membranes increased more than granal membranes. Changes observed in thylakoid surface areas associated with shading did not support thylakoid models which propose the interconversion of granal membranes to stromal membranes and vice versa.     

Structure and Function of Chloroplast Membrane III. The Effects of Mg++ and K+ Ions Toward the Ultrastructure of Two Kinds of Chloroplast Thylakoid Membranes

Based upon our previous work on the relationship between structure and function of chloroplast of wheat in connection with PSⅡ reaction, we studied the effects of MgCl2 and KC1 toward two kinds of thylakoid membranes. After exposing etiolated wheat seedlings to intermittent light (cycle of 2 min. light, 118 min. dark) for 24 hr, we obtain ed an incompletely developed chleroplast membrane. Completely developed chloroplast membrane was obtained from wheat seedlings grown under normal light-dark regime. Thylakoid membranes of plants grown under intermittent light failed to form grana stacks they remained as single lamellae in the suspension containing Mg++ or K+ of high concentration although simple stackings not more than two thylakoids c.ould be found. However, thylakoids grown under normal light-dark regime showed well developed grand stacks. Isolated chloroplast samples from two kinds of seedlings were suspended in 5 mM MgCl2 and 100 mM KC1 solutions for a definite time, portions of each samples were processed for electron microscopic observations and their photosynthetic activities were measured at the same time (It will be dealt with in another article). When these two kinds of isolated plastids were suspended either in MgCl2 (5 mM) or KC1 (100), the normally developed grana thylakoids stacked closely but the incompletely developed thylakoid- membranes did not stack. The incompletely developed chloroplast thylakoid membranes,, in either Mg++ or K+ ions could not induce stacking of the scattered thylakoid membranes to form grana. Therefore, we presume that light- harvesting chlorophyll a/b protein complex is on internal factor to induce thylakoid- membranes stacking and a definite concentration of caionions is an important factor in maintaining the stacking of thylakoid membranes. These results further prove the close association between structure and function in our previous studies on the mesophyll cell of the winter wheat.     

Thylakoid and grana formation during the development of pea chloroplasts,illuminated by white,red, and blue low intensity light

Summary We analyzed the formation of thylakoids and grana during the development of pea chloroplasts, illuminated by white, red and blue low intensity light. The total length of granal and intergranal thylakoids, and the length of granal thylakoids per unit area of plastid section were measured. Initially the greatest increase in length of granal thylakoids and the highest incidence of grana with large thylakoid content occurred in red light. On the other hand, with illumination times of over 12 hours blue light appeared to be more efficient in stimulating grana formation and thylakoid growth.     

Plants under Climatic Stress: II. Low Temperature, High Light Effects on Chloroplast Ultrastructure

Mesophyll chloroplasts of the C₄-pathway grasses Sorghum and Paspalum and of the C₃-pathway legume soybean undergo ultrastructural changes under moderate light intensities (170 w·m⁻², 400-700 nanometers) at a tme when photosynthesis is much reduced by low temperature (10 C). The pattern of ultrastructural change was similar in these species, despite some differences in the initial sites of low temperature action on photosynthesis and differences in their mechanisms of CO₂ fixation. Starch grains in the chloroplasts rapidly reduce in size when chilling stress is applied. At or before the time starch grains completely disappear the membranes of the individual stromal thylakoids close together, reducing the intraspace between them while the chloroplast as a whole begins to swell. Extensive granal stacking appears to hold the thylakoids in position for some time, causing initial swelling to occur in the zone of the peripheral reticulum, when present. At more advanced stages of swelling the thylakoid system unravels while the thylakoid intraspaces dilate markedly. Initial thylakoid intraspace contraction is tentatively ascribed to an increase in the transmembrane hydrogen ion gradient causing movement of cations and undissociated organic acids from the thylakoid intraspace to the stroma. Chloroplast swelling may be caused by a hold-up of some osmotically active photosynthetic product in the chloroplast stroma. After granal unraveling and redilation of the thylakoid intraspaces, chloroplasts appear similar to those isolated in low salt hypotonic media. At the initial stages of stress-induced ultrastructural change, a marked gradient in degree of chloroplast swelling is seen within and between cells, being most pronounced near the surface of the leaf directly exposed to light.     

Light-induced structural changes of thylakoids in normal and carotenoid deficient chloroplasts of maize

Summary Changes of membrane thickness and loculi were studied after red (650 nm) and far-red (707 nm) light in thylakoids of maize with different stacking and pigment compositions.The most intensive shrinkage of thylakoid membranes occurred in grana and under red light. Membranes of stroma thylakoids responded more to far-red light. Bundle sheath thylakoid membranes did not change in thickness. Loculi decreased in all types of thylakoids under both, red and far-red light. Thylakoids obtained from a -carotenic mutant exhibited a contrasting response: swelling under red light followed by photodestruction. Changes under far-red light were similar to that of normal stroma thylakoids.The data on normal chloroplasts show that the light induced shrinkage of membranes and the decrease of loculi are coupled to a different degree in various kinds of thylakoids; that the thylakoid flattening can be correlated with the Photosystem content of the membranes; and that two kinds of single thylakoids (stroma lamellae and bundle sheath lamellae) are different in molecular structure and function.Data on carotenoid deficient chloroplasts indicate a photooxidative destruction of the thylakoids by Photosystem 2 that occurs in the absence of normal carotenoids.     

A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg⁺⁺) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg⁺⁺. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg⁺⁺-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5±0.1 or 1.4±0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes.     

Effect of Light Quality on the Composition, Function, and Structure of Photosynthetic Thylakoid Membranes of Asplenium australasicum (Sm.) Hook

The effect of light quality on the composition, function and structure of the thylakoid membranes, as well as on the photosynthetic rates of intact fronds from Asplenium australasicum, a shade plant, grown in blue, white, or red light of equal intensity (50 microeinsteins per square meter per second) was investigated. When compared with those isolated from plants grown in white and blue light, thylakoids from plants grown in red light have higher chlorophyll a/chlorophyll b ratios and lower amounts of light-harvesting chlorophyll a/b-protein complexes than those grown in blue light. On a chlorophyll basis, there were higher levels of PSII reaction centers, cytochrome f and coupling factor activity in thylakoids from red light-grown ferns, but lower levels of PSI reaction centers and plastoquinone. The red light-grown ferns had a higher PSII/PSI reaction center ratio of 4.1 compared to 2.1 in blue light-grown ferns, and a larger apparent PSI unit size and a lower PSII unit size. The CO₂ assimilation rates in fronds from red light-grown ferns were lower on a unit area or fresh weight basis, but higher on a chlorophyll basis, reflecting the higher levels of electron carriers and electron transport in the thylakoids.

The structure of thylakoids isolated from plants grown under the three light treatments was similar, with no significant differences in the number of thylakoids per granal stack or the ratio of appressed membrane length/nonappressed membrane length. The large freeze-fracture particles had the same size in the red-, blue-, and white-grown ferns, but there were some differences in their density. Light quality is an important factor in the regulation of the composition and function of thylakoid membranes, but the effects depend upon the plant species.

     

Desiccation‐induced alterations in surface topography of thylakoids from resurrection plant Haberlea rhodopensis studied by atomic force microscopy,electrokinetic and optical measurements

With their ability to survive complete desiccation, resurrection plants are a suitable model system for studying the mechanisms of drought tolerance. In the present study, we investigated desiccation‐induced alterations in surface topography of thylakoids isolated from well‐hydrated, moderately dehydrated, severely desiccated and rehydrated Haberlea rhodopensis plants by means of atomic force microscopy (AFM), electrokinetic and optical measurements. According to our knowledge, so far, there were no reports on the characterization of surface topography and polydispersity of thylakoid membranes from resurrection plants using AFM and dynamic light scattering. To study the physicochemical properties of thylakoids from well‐hydrated H. rhodopensis plants, we used spinach thylakoids for comparison as a classical model from higher plants. The thylakoids from well‐hydrated H. rhodopensis had a grainy surface, significantly different from the well‐structured spinach thylakoids with distinct grana and lamella, they had twice smaller cross‐sectional area and were 1.5 times less voluminous than that of spinach. Significant differences in their physicochemical properties were observed. The dehydration and subsequent rehydration of plants affected the size, shape, morphology, roughness and therefore the structure of the studied thylakoids. Drought resulted in significant enhancement of negative charges on the outer surface of thylakoid membranes which correlated with the increased roughness of thylakoid surface. This enhancement in surface charge density could be due to the partial unstacking of thylakoids exposing more negatively charged groups from protein complexes on the membrane surface that prevent from possible aggregation upon drought stress.     

ULTRASTRUCTURE OF CHLOROPLASTS AND CHROMOPLASTS IN CAPSICUM ANNUUM I. THYLAKOID MEMBRANE CHANGES DURING FRUIT RIPENING

Developing chromoplasts in the fruit of Capsicum annuum were examined by electron microscopy. Special attention was given to changes in the thylakoid system. All grana and some intergranal thylakoids in the mature chromoplasts of the seven cultivars studied underwent lysis. The particulate nature of the granal membranes disappeared during lysis before the relationship between the partitions and locules was obscured. The changes during lysis support the globular concept of membrane structure. The selective lysis of the synaptic membranes of the granal partitions may be attributed to their distinctive composition and structure. Lipid globules (osmio-philic) did not accumulate in the immediate region of granal lysis, indicating that they are not directly derived from membranes undergoing degradation. During and following granal lysis a profuse development of intergranal thylakoid membranes occurred in several cultivars. In some instances a thylakoid plexus (prolamellar body) was formed. This specialized structure of the thylakoid system occurs in the chromoplasts of other species as well as in other types of plastids. Extensive, concentrically arranged thylakoid sheets with specific interspaced membrane relationships were frequently associated with the plexus. Several types of membrane associations and interrelationships in the plastid are described. An analysis of one type of membrane configuration, the thylakoid sheets, indicated that one method of growth may be through intussusception into the original membrane. The development of thylakoid plexes and of extensive thylakoid sheets during or after granal lysis indicates that dynamic synthetic activities occur in the chromoplasts of some cultivars of pepper during fruit ripening.     

A Comparison of the Effects of Chilling on Thylakoid Electron Transfer in Pea (Pisum sativum L.) and Cucumber (Cucumis sativus L.)

Experiments comparing the photosynthetic responses of a chilling-resistant species (Pisum sativum L. cv Alaska) and a chilling-sensitive species (Cucumis sativus L. cv Ashley) have shown that cucumber photosynthesis is adversely affected by chilling temperatures in the light, while pea photosynthesis is not inhibited by chilling in the light. To further investigate the site of the differential response of these two species to chilling stress, thylakoid membranes were isolated under various conditions and rates of photosynthetic electron transfer were determined. Preliminary experiments revealed that the integrity of cucumber thylakoids from 25°C-grown plants was affected by the isolation temperature; cucumber thylakoids isolated at 5°C in 400 millimolar NaCl were uncoupled, while thylakoids isolated at room temperature in 400 millimolar NaCl were coupled, as determined by addition of gramicidin. The concentration of NaCl in the homogenization buffer was found to be a critical factor in the uncoupling of cucumber thylakoids at 5°C. In contrast, pea thylakoid membranes were not influenced by isolation temperatures or NaCl concentrations. In a second set of experiments, thylakoid membranes were isolated from pea and cucumber plants at successive intervals during a whole-plant light period chilling stress (5°C). During wholeplant chilling, thylakoids isolated from cucumber plants chilled in the light were uncoupled even when the membranes were isolated at warm temperatures. Pea thylakoids were not uncoupled by the whole-plant chilling treatment. The difference in integrity of thylakoid membrane coupling following chilling in the light demonstrates a fundamental difference in photosynthetic function between these two species that may have some bearing on why pea is a chilling-resistant plant and cucumber is a chilling-sensitive plant.     

Electron and proton transport in chloroplasts taking into account lateral heterogeneity of thylakoids. Mathematical model]

A mathematical model of a chloroplast was constructed, which takes into account the inhomogeneous distribution of complexes of photosystems I and II between granal and intergranal thylakoids. The structural and functional complexes of photosystems I and II, which are localized in intergranal and granal thylakoids, respectively, and the b/f complex, which is uniformly distributed in thylakoid membranes, are assumed to be immobile. The interactions between spatially distant electron transport complexes are provided by plastoquinone and plastocyanine, which diffuse in the thylakoid membrane and intrathylakoid space, respectively. The main stages of proton transport associated with the functioning of photosystem II and oxidation-reduction transformations of plastoquinone are considered. The model takes into account the interactions of protons with membrane-bound buffer groups, the lateral diffusion of hydrogen ions in the intrathylakoid space and in the lumen between adjacent granal thylakoids, and the transmembrane proton transport associated with the function of ATP synthase and passive leakage of protons from thylakoids outside. The numerical integration of two systems of differential equations describing the behavior of some variables in two different regions: granal and intergranal thylakoids was performed. The model describes adequately the kinetics of processes being studied and predicts the occurrence of inhomogeneous lateral profiles of proton potentials and redox state of electron carriers. Modeling the electron and proton transport with allowance for the topological features of chloroplasts (lateral heterogeneity of thylakoids) is important for correct interpretation of "power-flux" interactions and the experimentally measured kinetic parameters averaged over the entire spatially inhomogeneous thylakoid system.