THE STRUCTURAL RELATIONSHIPS OF THE INTERNAL MEMBRANE SYSTEMS OF IN SITU AND ISOLATED CHLOROPLASTS OF HORDEUM VULGARE

Healthy chloroplasts of Hordeum vulgare are compared with chloroplasts subjected to abnormal stresses such as in situ disruption, isolation, isolation plus washing in 0.5 m sucrose, and isolation plus washing in 0.5 m sucrose and distilled H₂O. Normal chloroplasts resemble those of Nicotiana rustica and Phaseolus vulgaris in being composed of compartmented grana connected by an anastomosing fretwork system. They differ in having a somewhat greater incidence of parallel frets and double partitions. Under conditions of stress both grana and fretwork undergo varying degrees of swelling, and the double partition maintains its structural integrity. Grana are more resistant to abnormal stresses than the fretwork. Fret connections with more than 3 grana do not generally occur, but in some micrographs a single pathway may be traced through several grana. Washing isolated chloroplasts in distilled water results in an enlargement involving compartments of 2 or more grana together with the associated fretwork membranes. These results indicate that the grana in mature chloroplasts of Hordeum vulgare, like those of Nicotiana rustica and Phaseolus vulgaris, are compartmented structural units and not a series of localized aligned thickenings in regular extensive discs. These enlargements are complex structures comprising the membranes and spaces of both grana and frets. The swelling indicates an increase of locular and fret channel substance and possibly an enlargement of membrane surfaces. Dried down on grids, the compartments and frets appear as flat discs with radial appendages.     

THE GRANA OF STARCH-FREE CHLOROPLASTS OF NICOTIANA RUSTICA

The grana of chloroplasts of starch-free leaves of Nicotiana rustica are described in detail. Leaf sections were fixed in 2.5 per cent KMnO₄ and embedded in mixtures of butyl and ethyl methacrylate. Chain length of the polymer was modified by use of a transfer agent. The grana are composed of compartments consisting of electron-scattering partitions and electron-transparent loculi. Compartments are not open to the stroma so that the grana are distinct subplastid organelles. Adjacent grana are connected by an anastomosing fretwork system composed of flexuous channels bordered by electron-scattering membranes. Ten different kinds of granum margins are described. These marginal variations depend upon grana-fretwork connections and internal marginal connections between adjacent loculi. A study of serial sections suggests, at least in some plastids, the occurrence of a possible orderly spiral arrangement of compartment-fretwork connections. Adjacent grana may have common compartments. Grana may branch. Variations in shape may depend upon the angle the section bears to the axis of the cylinder. This should also influence the relative thickness and sharpness of the partitions. Since all shapes and variations in partition thickness and sharpness cannot be accounted for on the basis of the orientation of the grana, such variations probably occur naturally. Grana vary in size, ranging from those few which have a single partition to those having 50 or more compartments which extend completely through the width of a plastid. Relationships between grana of different sizes and between compartments and frets indicate the possibility of growth of grana from union or extension of compartments and formation of compartments from the union of frets.     

THE STRUCTURE OF GRANA IN FLOWERING PLANTS

A model of granum structure, consisting of multiple helical frets wound around each cylindrical granum, is proposed as a representation of granum structure for flowering plants in general. This model was originally formulated for grana in the mesophyll plastids of Zea mays and was subsequently extended to grana of Phaseolus vulgaris by additional studies. The model is now shown to be applicable also to grana in the plastids of Cannabis sativa, Elodea canadensis, Nicotiana rustica, Pisum sativum, and Spinacia oleracea. This sample of seven angiosperms includes genera commonly studied by other workers in their ultrastructural investigations of plastid structure.     

Continuity of the Chloroplast Membrane Systems in Zea mays L

Ultrastructural studies of the chloroplasts of the normal, yellow-green, and pale green phenotypes of Zea mays L. indicate that the internal membrane system is continuous with the plastid envelop. The intramembraneous spaces, loculi, and fret channels are also continuous with inner component of the plastid envelop. High energy compounds or other photosynthates, formed in the grana or frets are thus separated from both stroma and cytoplasm by a single membrane, either the fret membrane or the outer component of the plastid envelop. Since this type of plastid ultrastructure is apparently found only in plants exhibiting the Hatch and Slack pathways of photosynthesis there may be a relation between plastid ultrastructure and the pathways of photosynthetic carbon fixation.     

RECONSTRUCTIONS OF THE GRANA-FRETWORK SYSTEM OF A CHLOROPLAST

The grana-fretwork system was reconstructed from serial sections representing 0.5 μm slice, in profile view, from the midregion of a chloroplast of Nicotiana tabacum. Reconstructions show grana in multiple strata. The fretwork integrates the grana in all three dimensions. Large grana are visualized as complex structures consisting of two or more eccentrically stacked solids that vary in height and diameter. The close spacing and overlapping positions of grana in the plastid explain why phase microscopy cannot be used to demonstrate adequately the numerous strata of grana within a chloroplast. The grana are not far enough apart to allow for successful optical sectioning of a plastid in face view. In profile view no distinct grana can be resolved because the grana-fretwork system has the aspect of a “honeycomb.” Thus, observations with a light microscope are not adequate to determine the arrangement of grana in a chloroplast, and the recently-proposed model showing all grana of a plastid to be arranged in a single spiral ribbon must be rejected as incompatible with properties of real plastids.     

THE ULTRAMICRO STRUCTURE OF STARCH-FREE CHLOROPLASTS OF FULLY EXPANDED LEAVES OF NICOTIANA RUSTICA

Weier , T. Elliot . (U. California, Davis.) The ultramicro structure of starch-free chloroplasts of fully expanded leaves of Nicotiana rustica. Amer. Jour. Bot. 48(7): 615–630. Illus. 1961.—The grana of starch-free chloroplasts of fully expanded leaves of Nicotiana rustica are distinct, compartmented, subplastid entities. They vary in size, shape, orientation and in the distinctness with which their compartments are delineated. It has not been possible to equate accurately their micro and ultramicro appearances. At the ultramicro level, the grana are connected with each other at irregular intervals by a system of anastomosing channels. The partitions forming the compartments of the grana may be coarse or very fine but are constant in appearance in any given chloroplast. The loculi enclosed by the partitions may vary in size with a granum, depending upon their location or upon the physiological activity of the chloroplast. The stroma does not penetrate the grana; it may be relatively fluid and the grana-fretwork system may move within it. A double envelope, which may have pores connecting stroma and hyaloplasm, surrounds the chloroplasts. Materials may collect between the surfaces of the envelope. There is considerable variation in the ultramicro details of chloroplast structure of Nicotiana rustica. It is not yet possible to distinguish accurately between those variations which may be of physiological significance and those which may be induced by processing.     

Ultrastructural studies of isolatedZea mays L. Chloroplasts

Summary The ultrastructure of isolated mesophyll chloroplasts ofZea mays L. was studied using the shadow casting technique. Grana and interconnecting fret membranes were observed in the same basic arrangement as they appear in thin sections. Quantasome units, as previously described by other authors for other species, were detected. Portions of the peripheral reticulum and envelope were also observed. Swollen membranes, comparable to those observed when isolated plastids are placed in water, were frequent in the preparations. It can be assumed, then, that in any preparation of this nature the hydrophillic spaces will absorb a large amount of water, swelling the intramembranous spaces and giving the membranes the appearance of large vesicles which will appear as flattened sacs when dried on the electron microscope grids. As there is no indication of any quantasome pattern in these membranes it is assumed that the particle arrangement of these membranes was altered during the procedure. It was not possible to determine whether the membranes observed in the swollen vesicles belonged to the grana or frets. A change in the morphology of the membranes during the processing may be observed with the light microscope. Recognition of this change is frequently difficult in electron micrographs, consequently, membranes with similar appearance under the electron microscope may actually have arisen from different portions of the plastid membrane systems.     

Protein diffusion and macromolecular crowding in thylakoid membranes

The photosynthetic light reactions of green plants are mediated by chlorophyll-binding protein complexes located in the thylakoid membranes within the chloroplasts. Thylakoid membranes have a complex structure, with lateral segregation of protein complexes into distinct membrane regions known as the grana and the stroma lamellae. It has long been clear that some protein complexes can diffuse between the grana and the stroma lamellae, and that this movement is important for processes including membrane biogenesis, regulation of light harvesting, and turnover and repair of the photosynthetic complexes. In the grana membranes, diffusion may be problematic because the protein complexes are very densely packed (approximately 75% area occupation) and semicrystalline protein arrays are often observed. To date, direct measurements of protein diffusion in green plant thylakoids have been lacking. We have developed a form of fluorescence recovery after photobleaching that allows direct measurement of the diffusion of chlorophyll-protein complexes in isolated grana membranes from Spinacia oleracea. We show that about 75% of fluorophores are immobile within our measuring period of a few minutes. We suggest that this immobility is due to a protein network covering a whole grana disc. However, the remaining fraction is surprisingly mobile (diffusion coefficient 4.6 +/- 0.4 x 10(-11) cm(2) s(-1)), which suggests that it is associated with mobile proteins that exchange between the grana and stroma lamellae within a few seconds. Manipulation of the protein-lipid ratio and the ionic strength of the buffer reveals the roles of macromolecular crowding and protein-protein interactions in restricting the mobility of grana proteins.     

Three-dimensional architecture of grana and stroma thylakoids of higher plants as determined by electron tomography

We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by electron tomography of high-pressure-frozen/freeze-substituted intact chloroplasts. Higher-plant thylakoids are differentiated into two interconnected and functionally distinct domains, the photosystem II/light-harvesting complex II-enriched stacked grana thylakoids and the photosystem I/ATP synthase-enriched, nonstacked stroma thylakoids. The grana thylakoids are organized in the form of cylindrical stacks and are connected to the stroma thylakoids via tubular junctions. Our data confirm that the stroma thylakoids are wound around the grana stacks in the form of multiple, right-handed helices at an angle of 20° to 25° as postulated by a helical thylakoid model. The junctional connections between the grana and stroma thylakoids all have a slit-like architecture, but their size varies tremendously from approximately 15 × 30 nm to approximately 15 × 435 nm, which is approximately 5 times larger than seen in chemically fixed thylakoids. The variable slit length results in less periodicity in grana/stroma thylakoid organization than proposed in the original helical model. The stroma thylakoids also exhibit considerable architectural variability, which is dependent, in part, on the number and the orientation of adjacent grana stacks to which they are connected. Whereas some stroma thylakoids form solid, sheet-like bridges between adjacent grana, others exhibit a branching geometry with small, more tubular sheet domains also connecting adjacent, parallel stroma thylakoids. We postulate that the tremendous variability in size of the junctional slits may reflect a novel, active role of junctional slits in the regulation of photosynthetic function. In particular, by controlling the size of junctional slits, plants could regulate the flow of ions and membrane molecules between grana and stroma thylakoid membrane domains.     

Comparative Studies of the Thylakoid Proteins of Mesophyll and Bundle Sheath Plastids of Zea mays

The proteins from both grana and stroma lamellae of maize (Zea mays) mesophyll plastids and from maize bundle sheath plastid membranes have been compared by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels using a discontinuous buffer system. Peptide differences between grana and stroma lamellae were essentially quantitative and not qualitative. Bundle sheath plastid membrane peptides more closely resembled those of the ultrastructurally similar stroma lamellae. However, bundle sheath membranes contained several peptides not apparent in the stroma lamellae.     

Ultrastructural Development of Chloroplasts in Sacred Lotus Embryo Bud under Invisible Light

The present paper reports that the development ultrastructural observations of chloroplasts from sacred lotus (Nelumbo nucifera) embryo buds under invisible light. Embryo bud of sacred lotus is enclosed by three layers of thick integument (pericap, seed coat and thick fleshy cotyledons). During the period of the formation of embryo bud, it remained in dark condition, but turned from pale yellow to bluish-green. It was noteworthy that chloroplasts of the embryo bud had well developed giant grana under invisible light. Their developmental pathway in sacred lotus, however, was different from those of other higher plants grown under sunlight, intermittent light, or even in dark conditions (Fig. 1). The chloroplast development of embryo buds in Sacred lotus seeds in invisible light underwent only in the following three stages: (1) In the first stage the development was similar to that from other higher plants, the inner envelope membranes of the proplastids were invaginating. (2) In the second stage, a proplastid centre composed of prolamellar bodies (PLB)with semicrystalline structure was formed, and was accompanied by one or two huge starch grains in almost each proplastid. In the meantime, prothylakoid membranes extended parallelly from the plastid centre in three forms: (a) One plastid centre extending parallelly prothylakoid membranes from itself in one direction; (b) The same to (a), but extending in two directions; (c) Two plastid centres extending parallelly prothylakoid membranes between the centres. (3) In the third stage, grana and stroma thylakoid membranes of chloroplasts were formed. It is to be noted that most of chloroplasts had only one or two giant grana which often extended across the entire chloroplast body, and the length of the grana thylakoid membranes of the chloroplasts from embryo bud in Sacred lotus is 3 to 5 times as many as that in other higher plants. However, their stromatic thylakoid membranes were rather rare and very short. The giant grana were squeezed to the margin of the chloroplast envelope by one or two huge starch grains.     

THE ULTRASTRUCTURE OF GRANA IN MESOPHYLL PLASTIDS OF ZEA MAYS

The compartments of the grana of mesophyll plastids of Zea mays are extensively interconnected. In median sections of grana, 16.5% of compartment margins were involved in a Y-shaped conjunction of the loculi of two consecutive compartments with the loculus of an attached fret. Various configurations accounted for the remainder of the compartment margins seen in median sections of grana. Tangential views of grana and serial sections revealed an oblique relationship between the frets and the grana. Analysis of serial sections allows the conclusion that the components of the fretwork are helically wound around a granum.     

PLASTID ORGANIZATION IN PHENOTYPICALLY GREEN LEAF TISSUE OF A GENETIC ALBINO STRAIN OF NICOTIANA (SOLANACEAE)

Plastid organization within phenotypically green leaf tissue that forms in albino plants of a genetic albino strain of Nicotiana has been examined with the transmission electron microscope. Studies revealed the presence of plastids with and without thylakoids. When present, thylakoids were loosely and irregularly scattered in the stroma or organized either into several large spindle-shaped grana or into a single compound granum with deeply indented margins. Plastids without thylakoids were vesiculated and resembled the typical genetic albino type. Plastid types were not segregrated into individual cells and no plastid type appeared to be typical for the mutant tissue. Orientation of grana and thylakoid membrane associations were noted as well as the presence of osmiophilic globules, starch grains and DNA-like fibrillar areas.     

The Ultrastructure of Phaseolus vulgaris Chloroplasts

A comparison of bean chloroplasts after being fixed in potassiumpermanganate, osmium, and formaldehyde coupled with negativestaining shows that the general organization of the chloroplastis similar in all cases. However, the mature chloroplasts ofbean vary considerably in the extent and orientation of theinternal membranes—the grana and the interconnecting membranesbetween the grana. The interconnecting membranes are thin, branching,flexuous structures. This is illustrated by serial sectionsand by cross- and face-view sections of osmium- fixed chloroplastsand the best model, which allows for a considerable flexibilityin the orientation of the grana and also describes the thininterconnecting membranes between the grana, is the grana-fretworksystem proposed by Weier (1961). Furthermore, the comparative studies show that the internalregions of grana are separated from the stroma and that thegrana-fretwork systems appear to be a continuous membrane system.This membrane is single along the frets, end compartments, andgranal margins. It is double in the partitions of the granabut separated by a component, possibly a cementing material,which does not stain. It is suggested that this membrane isstructurally similar in the chloroplasts of higher plants, butthat its overall organization may vary from one plant to another.     

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.     

Light regulation of photosynthetic membrane structure,organization, and function

The light environment during plant growth determines the structural and functional properties of higher plant chloroplasts, thus revealing a dynamically regulated developmental system. Pisum sativum plants growing under intermittent illumination showed chloroplasts with fully functional photosystem (PS) II and PSI reaction centers that lacked the peripheral chlorophyll (Chi) a/b and Chl a light-harvesting complexes (LHC), respectively. The results suggest a light flux differential threshold regulation in the biosynthesis of the photosystem core and peripheral antenna complexes. Sun-adapted species and plants growing under far-red-depleted illumination showed grana stacks composed of few (3–5) thylakoids connected with long intergrana (stroma) thylakoids. They had a PSII/PSI reaction center ratio in the range 1.3–1.9. Shade-adapted species and plants growing under far-red-enrichcd illumination showed large grana stacks composed of several thylakoids, often extending across the entire chloroplast body, and short intergrana stroma thylakoids. They had a higher PSII/PSI reaction center ratio, in the range of 2.2–4.0. Thus, the relative extent of grana and stroma thylakoid formation corresponds with the relative amounts of PSII and PSI in the chloroplast, respectively. The structural and functional adaptation of the photosynthetic membrane system in response to the quality of illumination involves mainly a control on the rate of PSII and PSI complex biosynthesis.     

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.     

EPR characterization of photosystem II from different domains of the thylakoid membrane

We report electron paramagnetic resonance (EPR) studies on photosystem II (PSII) from higher plants in five different domains of the thylakoid membrane prepared by sonication and two-phase partitioning. The domains studied were the grana core, the entire grana stack, the grana margins, the stroma lamellae and the purified stromal fraction, Y100. The electron transport properties of both donor and acceptor sides of PSII such as oxygen evolution, cofactors Y D, Q A, the CaMn 4-cluster, and Cytb 559 were investigated. The PSII content was estimated on the basis of oxidized Y D and Q A (-) Fe (2+) signal from the acceptor side vs Chl content (100% in the grana core fraction). It was found to be about 82% in the grana, 59% in the margins, 35% in the stroma and 15% in the Y100 fraction. The most active PSII centers were found in the granal fractions as was estimated from the rates of electron transfer and the S 2 state multiline EPR signal. In the margin and stroma fractions the multiline signal was smaller (40 and 33%, respectively). The S 2 state multiline could not be induced in the Y100 fraction. In addition, the oxidized LP Cytb 559 prevailed in the stromal fractions while the HP form dominated in the grana core. The margins and entire grana fractions have Cytb 559 in both potential forms. These data together with previous analyses indicate that the sequence of activation of the PSII properties can be represented as: PSII content > oxygen evolution > reduced Cytb 559 > dimerization of PSII centers in all fractions of the thylakoid membrane with the gradual increase from stromal fractions via margin to the grana core fraction. The results further support the existence of a PSII activity gradient which reflects lateral movement and photoactivation of PSII centers in the thylakoid membrane. The possible role of the PSII redox components in this process is discussed.     

The structural and functional domains of plant thylakoid membranes

In plants, the stacking of part of the photosynthetic thylakoid membrane generates two main subcompartments: the stacked grana core and unstacked stroma lamellae. However, a third distinct domain, the grana margin, has been postulated but its structural and functional identity remains elusive. Here, an optimized thylakoid fragmentation procedure combined with detailed ultrastructural, biochemical, and functional analyses reveals the distinct composition of grana margins. It is enriched with lipids, cytochrome b₆f complex, and ATPase while depleted in photosystems and light‐harvesting complexes. A quantitative method is introduced that is based on Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) and dot immunoblotting for quantifying various photosystem II (PSII) assembly forms in different thylakoid subcompartments. The results indicate that the grana margin functions as a degradation and disassembly zone for photodamaged PSII. In contrast, the stacked grana core region contains fully assembled and functional PSII holocomplexes. The stroma lamellae, finally, contain monomeric PSII as well as a significant fraction of dimeric holocomplexes that identify this membrane area as the PSII repair zone. This structural organization and the heterogeneous PSII distribution support the idea that the stacking of thylakoid membranes leads to a division of labor that establishes distinct membrane areas with specific functions.     

Impact of the stringency of cell selection on plastid segregation in protoplast fusion-derived Nicotiana regenerates

Summary Vegetative segregation of a mixed plastid population in protoplast fusion-derived cell lines can be directed by a selection favouring the multiplication of one of the parental plastid types. This report defines some of the critical conditions leading to a homogeneous plastid population in cybrid plants generated by protoplast fusion between Nicotiana plumbaginifolia and an albino and streptomycin-resistant N. tabacum plastid mutant. Light (1,500 lx) conferred a strong selective advantage to chloroplasts versus albino plastids, while the lack of this effect in dim light (300 lx) indicated that a sufficient light intensity is essential to the phenomenon. Selection on streptomycin-containing medium in the dark, however, led to the preferential multiplication of resistant plastids. Streptomycin selection of resistant chloroplasts in the light, consequently, results in a plastid selection of doubled stringency. In another experiment a definite, but leaky, selection for chloroplast recombination (selection for greening on streptomycin-containing medium in dim light) was used to reveal various recombination products. Protoplast fusion in fact resulted in cybrid plants showing only simple chimeric segregation of unchanged parental plastids. These results demonstrate the essential requirement for stringent plastid selection, as defined by cell culture conditions, to precede the formation of shoots expected to possess the desired plastid genetic composition.