Probing the organization of photosystem II in photosynthetic membranes by atomic force microscopy

Efficient photosynthetic energy transduction and its regulation depend on a precise supramolecular arrangement of the plant photosystem II (PSII) complex in grana membranes of chloroplasts. The topography of isolated photosystem II supercomplexes and the supramolecular organization of this complex in grana membrane preparations are visualized by high-resolution atomic force microscopy (AFM) in air in tapping mode with an active feedback control to minimize tip-sample interactions. Systematic comparison between topographic characteristics of the protrusions in atomic force microscopic images and well-established high-resolution and freeze-fracture electron microscopic data shows that the photosystem II organization can be properly imaged by AFM in air. Taking the protruding water-splitting apparatus as a topographic marker for PSII, its distribution and orientation in isolated grana membrane were analyzed. For the latter a new mathematical procedure was established, which revealed a preference for a parallel alignment of PSII that resembles the organization in highly ordered semicrystalline arrays. Furthermore, by analyzing the height of grana membrane stacks, we conclude that lumenal protrusions of adjacent photosystem II complexes in opposing membranes are displaced relative to each other. The functional consequences for lateral migration processes are discussed.     

Effect of Salts and Electron Transport on the Conformation of Isolated Chloroplasts. II. Electron Microscopy

Spinach chloroplasts isolated in media containing salts and the rare chloroplasts which are still within their envelopes alike retain grana similar to those seen in chloroplasts in situ.

Chloroplasts isolated in low-salt media lose their grana without losing any chlorophyll. These grana-free chloroplasts are considerably swollen and consist almost entirely of continuous sheets of paired-membrane structures. These double structures, the lamellae, are only loosely held together, primarily at the edges, by tenuous material which does not react with permanganate.

Addition of salts (methylamine hydrochloride, NaCl, MgCl₂) to the grana-free low-salt chloroplasts provide strong interlamellar attractions. These attractions result in a stacking of the lamellae which is sometimes almost random but sometimes results in regular structures indistinguishable from the original grana.

The phosphorylation-uncoupler atebrin causes further swelling of the chloroplasts in the absence of electron transport by increasing the space between the paired membranes of the lamellae.

The rapid electron transport (Hill reaction) made possible by atebrin-uncoupling is associated with a great decrease in chloroplast volume. This decrease results from a collapsing together of the widely separated lamellar membrane pairs. The pairs approach each other so closely that they usually appear as a single membrane when viewed with the electron microscope. The much slower electron transport which occurs in the absence of uncouplers is associated with a similar but smaller decrease in the space between the lamellar membrane pairs.

Chloroplasts swell during the rapid electron transport made possible by the phosphorylation-uncoupler methylamine. This swelling is accompanied by a degree of membrane distortion which precludes an interpretation of the mechanism. As with atebrin-faciliated electron transport, obviously paired membranes disappear but it is not yet clear whether this is by association or dissociation of the pairs.

     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

Function and evolution of grana

Chloroplasts are descended from cyanobacteria, and they retain many features of the cyanobacterial photosynthetic apparatus. However, land-plant chloroplasts have a strikingly different thylakoid membrane organization to that of cyanobacteria. Usually the two photosystems are laterally segregated; Photosystem II is concentrated in complex stacked-membrane structures known as grana. The function of grana has long been debated. Recent studies on membrane organization in chloroplasts, cyanobacteria and purple bacteria now offer a new perspective. I argue that grana allow the presence of a large light-harvesting antenna for Photosystem II, without excessively restricting electron transport. Other organisms solve this problem in different ways. Land plants evolved from macroalgae that were adapted to high light conditions; they evolved grana as a new solution to the problem of efficient photosynthesis in shade.     

Photosynthetic activity and membrane polypeptide composition of supergranal chloroplasts from plant tissue cultures containing a viruslike particle

Tissue culture cells of Streptanthus tortuosus (Kell.) var. orbiculatus (Greene) Hall (Cruciferae), having a viruslike particle in their nucleoli, the STV cell line, contain “supergranal” chloroplasts. Freeze-fracture studies of chloroplasts of a control cell line, which lacks the viruslike particles, reveal two complementary faces similar to those observed in spinach chloroplasts. Replicas of freeze-fractured STV supergranal chloroplasts, however, show that one membrane face (B) contains widely spaced 80 Å particles and the other face (C) is essentially smooth. Isolated STV supergranal chloroplasts lack photosystem II activity as indicated by their inability to reduce dichlorophenolindophenol and are unable to reduce NADP with electrons from photosystem II or from ascorbate-reduced dichlorophenolindophenol. However, partial photosystem I activity is indicated by the reduction of methyl viologen with electrons from dichlorophenolindophenol-ascorbate. This supports the concept that there is not a direct correspondence between grana formation and photosystem II activity. Electrophoresis shows that all of the major polypeptide bands present in the STV supergranal chloroplasts are also present in the control chloroplast membranes. One band, molecular weight 33,000, is present in a greatly increased amount in the STV supergranal chloroplast membranes and may be associated with grana stacking.     

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.     

Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.     

Purification of structurally intact grana from plants thylakoids membranes

Thylakoid membranes in higher plant chloroplasts are composed by two distinct domains: stacked grana and stroma lamellae. We developed a procedure for biochemical isolation of grana membranes using mild detergent to maintain membrane structure. Pigment and polypeptide analyses of membrane preparation showed the preparations were indeed enriched in grana membranes. The method was shown to be effective in four different plant species, although with small changes in detergent concentration. Electron microscopy analyses also showed that the preparation consisted of large membrane patches with roughly round shape and diameter comparable with grana membranes in vivo. Furthermore, protein complexes distribution was shown to be maintained with respect to freeze fracture studies, demonstrating that the protocol was successful in isolating membranes close to their in vivo state.     

Fine structure of granal thylakoid membrane organization using cryo electron tomography

The architecture of grana membranes from spinach chloroplasts was studied by cryo electron tomography. Tomographic reconstructions of ice-embedded isolated grana stacks enabled to resolve features of photosystem II (PSII) in the native membrane and to assign the absolute orientation of individual membranes of granal thylakoid discs. Averaging of 3D sub-volumes containing PSII complexes provided a 3D structure of the PSII complex at 40 ? resolution. Comparison with a recently proposed pseudo-atomic model of the PSII supercomplex revealed the presence of unknown protein densities right on top of 4 light harvesting complex II (LHCII) trimers at the lumenal side of the membrane. The positions of individual dimeric PSII cores within an entire membrane layer indicates that about 23% supercomplexes must be of smaller size than full C(2)S(2)M(2) supercomplexes, to avoid overlap.     

Investigations of the role of the main light-harvesting chlorophyll- protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex

The functions of the light-harvesting complex of photosystem II (LHC- II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication- freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.     

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.     

Evidence for the role of surface-exposed segments of the light-harvesting complex in cation-mediated control of chloroplast structure and function.

Chloroplast membranes contain a light-harvesting pigment-protein complex (LHC) which binds chlorophylls a and b. A mild trypsin digestion of intact thylakoid membranes has been utilized to specifically alter the apparent molecular weights of polypeptides of this complex. The modified membrane preparations were analyzed for altered functional and structural properties. Cation-induced changes in room temperature fluorescence intensity and low temperature chlorophyll fluorescence emission spectra, and cation regulation of the quantum yield of photosystem I and II partial reactions at limiting light were lost following the trypsin-induced alteration of the LHC. Electron microscopy revealed that cations can neither maintain nor promote grana stacking in membranes which have been subjected to mild trypsin treatment. Freeze-fracture analysis of these membranes showed no significant differences in particle density or average particle size of membrane subunits on the EF fracture face; structural features of the modified lamellae were comparable to membranes which had been unstacked in a “low salt” buffer. Digitonin digestion of trypsin-treated membranes in the presence of cations followed by differential centrifugation resulted in a subchloroplast fractionation pattern similar to that observed when control chloroplasts were detergent treated in cation-free medium. We conclude that: (a) the initial action of trypsin at the thylakoid membrane surface of pea chloroplasts was the specific alteration of the LHC polypeptides, (b) the segment of the LHC polypeptides which was altered by trypsin is necessary for cation-mediated grana stacking and cation regulation of membrane subunit distribution, and (c) cation regulation of excitation energy distribution between photosystem I and II involves the participation of polypeptide segments of the LHC which are exposed at the membrane surface.     

AN ULTRASTRUCTURAL STUDY OF CHLOROPLAST STRUCTURE AND DEDIFFERENTIATION IN TISSUE CULTURES OF STREPTANTHUS TORTUOSUS (CRUCIFERAE)

Cells of Streptanthus tortuosus callus tissue contain chloroplasts when cultured in a liquid medium in the light. Similar cells grown in the dark contain proplastids that fail to develop prolamellar bodies but do contain a complex of loosely-associated membranes. When green, light-grown cultures are cut into small pieces and subcultured to a fresh culture medium, they become bleached even though maintained under the same illumination. The fine structure of the chloroplasts and the chlorophyll content of the cells indicate a dedifferentiation of the chloroplasts to a proplastid state during the early culture period. The changes in the ultrastructure of the plastids are paralleled by a dedifferentiation of the vacuolate cells to a less differentiated, meristematic state. Subsequent growth in the light results in a re-formation of chloroplasts and an increase in the chlorophyll content of the cells. The period of chloroplast redevelopment is associated with the re-formation of large central vacuoles in the cultured cells. Invaginations of the inner membrane of the plastid envelope occur at all stages of plastid development and are not lost during the period of grana degeneration. The proplastids formed from the dedifferentiation of the chloroplasts contain a large number of these invaginations and the redevelopment of grana is associated with a change in the electron density of the invaginating membranes. The degradation of the chlorophyll-containing membranes of the grana occurs during a period of rapid cytoplasmic synthesis induced by the fresh supply of nutrients in the culture medium. These results suggest that the high levels of nutrients may act directly on the chloroplasts and cause their dedifferentiation or that the rapid cell growth induced by the nutrients may cause a degradation of the membrane proteins in the grana of the chloroplasts and an incorporation of the released amino acids into non-plastid components of the cytoplasm.     

定位于类囊体膜的PPF1在转基因拟南芥中的表达影响叶绿体的发育

PPF1是一个与植物营养生长相关的基因。它编码的产物可能是一个膜蛋白并与拟南芥叶绿体中的类囊体蛋白ALB3有很高的同源性。免疫电镜分析表明PPF1蛋白同样主要定位于类囊体膜 ,而且在短日照G2豌豆开花两周后仍发育良好的叶绿体中有很高的表达 ,在长日照豌豆同时期非正常叶绿体中丰度非常低。对转基因拟南芥和野生型植株的叶片衰老进程比较发现 ,PPF1在拟南芥中的过量表达可以延缓叶片的衰老 ,而用PPF1反义mRNA抑制拟南芥中的同源基因ALB3则明显加快叶片衰老速度。对转基因拟南芥的超微结构分析显示 ,PPF1在拟南芥中过量表达时 ,转基因植株的叶绿体比野生型植株的叶绿体大并含有更多的基粒和基质类囊体膜 ;相反 ,反义PPF1表达抑制其在拟南芥中的同源物时 ,转基因植株的叶绿体比野生型植株的叶绿体小并含有较少的基粒和发育较差的类囊体膜系统。这些数据表明叶绿体的发育状况与PPF1或拟南芥同源物ALB3的表达水平呈正相关。我们的结果提示PPF1基因可能通过控制叶绿体的发育状况来调节植物的发育。