Grana Membranes Research Articles

Cytochemical study of diaminobenzidine oxidation by isolated Vicia chloroplasts under illumination.

A concurrent microchemical and topochemical study of DAB* oxidation by isolated chloroplasts of Vicia faba under illumination was carried out. A new method for the quantification of the oxidation product of DAB was explored. The microchemical analysis by this method revealed that the photosystem responsible for the DAB oxidation was the PS-I. It also proved that endogenous active oxygen in the chloroplasts played only a little role in the oxidation. The topochemical analysis revealed that both the grana and the stroma membrane were positive in DAB photooxidation. The densitometric traces across the grana membranes in both surface and side views proved effective in locating the precise reaction sites. The partition layer showed a high degree of positive reaction. The thylakoid membrane proper also contained particles of positive reaction. The positive site extended to the thylakoid loculus to form protrusions. These results are in harmony with the localization of the PS-I complex particles on the PF face side of the thylakoid membrane as revealed by the freeze fracture technique.

An ion-exchange model for thylakoid stacking in chloroplasts

1. Introduction Chloroplasts of higher plants contain stacks of internal membranes (thylakoids). These stacks (grana) can be reversibly unstacked in vitro by changing the salt composition ln the chloroplast suspension [I]. Attempts have been made to explain the mechanism of stacking in terms of the classical theory of colloidal particle interactions which involve the interplay of electrostatic and van der Waals’ forces [2]. In a [3] a quantitative analysis of these forces led to the conclu- sion that an additional short range force [4] was required to explain stacking. It was also estimated that for the stacking to occur the net surface charge density on the grana membranes should be <l electronic charge (1 e)/3000 A’. Such a density is low when compared with the density expected from the full dissociation of surface acidic groups of proteins and lipids [3]. The required low net surface charge density in the stacked regions of the membranes can arise from one of two possible mechanisms [3]: (a) cation binding [S--8]; or (b) redistribution of charges between grana (stacked) and stroma (unstacked) portions of the thylakoid mem- brane [9-l 11. Here, we present a model involving binding of cations which explains the mechanism of stacking. The model is inspired by recent studies on the nature of short range repulsive forces between lipid bilayers [4,12,13] and mica surfaces [13-161. 2. Balance of forces For phospholipid or lecithin bilayers in water an additional repulsive force arises at distances below -30 A [4,12]. This so called ‘hydration’ force domi- nates the interactions between the bilayers at the short distances. The origin of this additional force between mica surfaces has been studied in detail

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 chlorophyll a chlorophyll 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.

Freeze-fracture studies on barley plastid membranes. VI. Location of the P700-chlorophyll a-protein 1.

The photosystem I mutant of barley, viridis-zb63, which totally lacks the P700-chlorophyll a-protein 1 was characterised by rotary shadowed, freeze-fracture electron microscopy. Objective measurements were made of particle density and size distribution for all four fracture faces, and compared with values for wild-type. A highly significant reduction in the size of the PFu particles was found, which could be attributed to the loss of a population of large particles from the mutant PFu face. A corresponding loss of EFu pits was also observed. It is concluded that the photosystem I reaction centre and two or three ancillary polypeptides are located in the large PFu particles which account for two-thirds of the total, and that these particles span the membrane. Since no differences were seen on the PFs and EFs faces, there was no evidence for the localisation of any photosystem I in appressed granal membranes, supporting the concept of extreme lateral heterogeneity. A model is presented of the localisation of different functional polypeptide units to different freeze-fracture particles within the membrane. A peculiar feature of viridis-zb63 thylakoids was the presence of EFs particle arrays in vivo.

Influence of FeEDDHA on Growth and Manganese Accumulation in Flax

AbstractA lower leaf‐scorch abnormality, resembling Mn toxicity, was observed in flax (Linum usitatissium L.) plants growing on certain calcareous soils. The cause of this problem was studied in greenhouse experiments using a naturally occurring, slightly calcareous (pH 8.1) soil. Above‐ground parts, but not roots, of afflicted plants contained relatively high levels of Mn. Addition of 2 ppm FeEDDHA‐Fe eliminated the appearance of brownish spots on the distal portions of older leaves and subsequent leaf necrosis, decreased Mn accumulation in above‐ground plant parts, and increased yields. Ammonium molybdate (0.05 and 0.5 ppm Mo) had no effect on the severity of the abnormality or on Mn accumulation. Cytological examination of brown spots showed distortion of chloroplast membranes, absence of starch, decrease in grana membranes, absence of plastid ribosomes, and increase in plastoglobuli. It is hypothesized that the lower leaf scorch was caused by Mn toxicity. Flax, in the absence of FeEDDHA, was a Mn‐accumulator plant even though the soil in question was calcareous. Available soil Fe level appeared to be a dominant factor influencing Mn accumulation by flax.

Ethylene-induced changes of chloroplast structure in Satsuma mandarin (&lt;italic&gt;Citrus unshiu&lt;/italic&gt; Marc.)

Ethylene-induced changes of chloroplast structure in Satsuma mandarin (Citrus unshiu Marc.) were examined using light and electron microscopy. In ethylene-treated fruits, the number of chloroplasts decreased; this was especially remarkable in cells distant from the epidermis. Rapid reduction in chloroplast size was a characteristic feature. The inner membrane system of the chloroplasts of ethylene-treated fruits disintegrated prior to the disintegration of other cell structures. The disintegration of the membranes within the chloroplasts was expressed by the word “melt”. The double-layered structure of lamellar and granal membranes was degraded and the membrane layers became separated. Another interesting feature was the appearance of finger-like protuberances and peripheral reticula in the chloroplasts of the ethylene-treated fruits.

Relative speed of fixation of glutaraldehyde and osmic acid in plant cells measured by grana appearance in chloroplasts

Brief fixation in a mixture of glutaraldehyde and OsO4 caused stacked chloroplast grana membranes in leaf cells of wheat, barley, tobacco, maize, cowpea, pigweed or bean plants to distend and vesiculate. Fixation with glutaraldehyde followed by OsO4 prevented this fixation artifact. In a fixative mixture, OsO4 apparently reacted with cell contents before glutaraldehyde.

Chlorophyll, carotenoid, and lipid content in Triticum sativum L. plastid envelopes, prolamellar bodies, stroma lamellae, and grana

The pigment and lipid content, expressed on a protein basis, is compared in wheat etioplast and chloroplast membrane fractions. Chloroplast envelopes contain less carotenoid and 1/3 more lipid than etioplast envelopes. The minute amount of chlorophyll and carotenoid found in chloroplast envelopes could be due to thylakoid contamination. Prolamellar bodies and grana have nearly the same amount of total lipid and total carotenoid per mg of protein although their respective compositions differ. On a protein basis, the lipid, chlorophyll, and carotenoid contents are lower (2.3, 10, and 20 times, respectively) in stroma lamellae than in grana membranes, but the latter contains a higher proportion of β-carotene, chlorophyll a, and sulfolipid.

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.The unappressed membranes (stroma lamellae and bundle sheath plastid membranes) were enriched in heavy (60-40 kilodaltons) peptides and depleted in light (31-20 kilodaltons) peptides as compared to stacked grana membranes. The heavier peptides were tentatively identified as subunits of chloroplast coupling factor. These peptides in unappressed membranes were much more resistant to removal by washing with ethylenediaminetetraacetate (under conditions of low ionic strength) than they were in granal membranes.Ribulose-1,5-diphosphate carboxylase was identified on the gels and was localized exclusively in the bundle sheath cells. It is suggested that sodium dodecyl sulfate electrophoresis is a simple method to test for the localization of carboxylase in various C(4) plastid fractions.

The localization of (3H) thymidine incorporation in the DNA of replicating spinach chloroplasts by electron-microscope autoradiography.

Electron-microscope autoradiography has been used to obtain information on the localization of DNA labelled with [3H]thymidine in chloroplasts known to be replicating and concomitantly synthesizing and segregating DNA, in cultured leaf disks. The studies were made using both Microdol-X developer and a 'compact' developer which gave a smaller grain size. About 80% of the grains were associated with the granal membranes and with presumptive DNA regions (3-nm fibril material in clear areas). Few grains occurred in association with the chloroplast envelope. We suggest that the DNA of chloroplasts is associated with the grana lamellae and extends into the stroma. Some light-microscope autoradiographs of whole chloroplasts show spiral or helical-like labelling patterns. We interpret these patterns as demonstration of the possibility that DNA occurs along the length of a continuous lamellar membrane system. Chloroplast fractionation experiments provided data consistent with the electron-microscope autoradiographic studies as most of the label was associated with chlorophyll-containing lamellae. We consider an association of chloroplast DNA molecules along the length of a continuous lamellar system would ensure an orderly segregation of DNA to daughter chloroplasts, during the binary fission of spinach chloroplasts by constriction division.

A test of the binary chloroplast membrane hypothesis by using a nonpenetrating chemical probe,p-(diazonium)-benzenesulfonic acid

Spinach chloroplasts were exposed to35S-labeledp-(diazonium)-benzenesulfonic acid (DABS), a water soluble compound which does not penetrate lipophilie regions of membranes, and which is highly reactive toward amino acid functionagroups such as e-amino, sulfhydryl, histidine, and tyrosine groups. Amino groups inl lipids can also form similar, stable covalent bonds by diazo coupling. Both chloroplast lipids and proteins were labeled with DABS, the total binding being about 1 DABS per 10 chlorophylls, depending on the reaction conditions. After diazo coupling and subsequent digitonin fractionation into photosystems I and II enriched fractions, it was observed that PS-I was more highly labeled than PS-III usually by a factor of 10 to 24 times (on a per chlorophyll basis). After digitonin isolation, however, the PS-II portion bound an amount of DABS similar to the PS-I binding, We interpret these data as consistent with the binary membrane hypothesis (Arntzen. Dilley and Crane (1969),J. Cell Biol. 43:16), which visualizes PS-I on the externa, “half” of a 90 A grana membrane, and PS-II occurring on the interior “half” of thel membrane. The alternative explanation that PS-II and PS-I are arranged as a mosaic, and that the low DABS binding in PS-II is caused by burial of the diazo reactive groups in the interior of the proteins (and only exposed through the denaturing effect of digitonin) is not directly ruled out. However, this alternative is not consistent with the facts that: (a) most of the membrane proteins in PS-I and PS-II are identical in electrophoretic properties and therefore probably have similar overall structures; and (b) digitonin does not lead to appreciable denaturation of proteins, evidenced by the retention of PS-II electron transport activity.

Chloroplast Ultrastructure Preserved by a Water-Containing Glutaraldehyde-Urea Embedment

Conventional techniques for preparing aldehyde-fixed chloroplasts for electron microscopy result not only in the loss of their lipids, but also their chlorophyll so that they become obviously bleached. This extraction is prevented by embedding in glutaraldehyde polymerized with urea in the presence of substantial quantities of water. Glutaraldehyde-fixed tissue then retains its green color into the final sections. As a consequence of these factors, ultrastructural arrangements and details of the grana membranes are thought to be preserved with unusual precision.The thylakoids appear in precisely ordered arrays, individually separated by uniform translucent gaps 15-20Å wide, particularly well seen in sections stained with OsO4, vapor (Fig. 1).

Isolation of chloroplast envelope membranes.

LITTLE is known of the nature of the chloroplast envelope: investigations have been concerned chiefly with its role in granal membrane development (for review see ref. 1) and in the transport of metabolites between the chloroplast and the cytoplasm2–13. The lack of knowledge of the physical and chemical properties of the plastid envelope limits our understanding of its function in photosynthesizing chloroplasts and of its precise role in plastid development. As relatively few methods are available for the investigation of the envelope in vivo, it seemed valuable to devise a method for the isolation of envelope membranes from other chloroplast components and then to study the properties of the isolated envelopes. In this paper we give details of a protocol for the isolation of a membrane fraction containing substantially pure chloroplast envelope membranes.

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.

Lipid fixation during preparation of chloroplasts for electron microscopy

Reaction of osmium tetroxide with isolated spinach chloroplasts fixed completely the glycolipids, phosphatidyl glycerol, and phosphatidyl choline. Under the same reaction conditions only 30% of the chlorophyll was fixed. Reaction of potassium permanganate with isolated spinach chloroplasts fixed more than 90% of the glycolipids, phosphatidyl glycerol, and phosphatidyl choline, provided the reaction period was long enough. Potassium permanganate also fixed the chlorophyll. Reaction of osmium tetroxide and potassium permanganate with isolated (14)C-lipids from Chlorella pyrenoidosa fixed 59% and 66% of the radioactivity, respectively. The lipids that were not fixed included sterols and pigments. Electron micrographs show that chloroplasts extracted with chloroform-methanol after fixation in osmium tetroxide or potassium permanganate differ from those dehydrated with acetone mainly in that in the former, osmiophilic globules have been removed and there seems to be some fusion of the boundary membranes and grana membranes. These effects may be due to the extraction of unfixed, neutral lipids such as sterols and quinones.

Ultrastructural Development of Chromoplasts in Valencia Oranges

The development of chromoplasts from chloroplasts during ripening of Valencia oranges was followed with the electron microscope. The chloroplasts in the young, green fruit of Valencia were similar in structure to those in leaves of other plants. During ripening, there was a breakdown of the internal membrane system, the grana-fretwork system, and large, osmiophilic globules were formed. These globules apparently formed from breakdown products of the granal membranes as well as from carotenoids synthesized during ripening. During ripening, membranes developed from invagination of the inner part of the limiting membrane of the plastids. These membranes may be associated with chlorophyll that is synthesized during ripening. The breakdown of the grana-fretwork system seemed to be independent and not influenced by the limiting membrane of the plastid.

Subunits in chloroplast membranes of Scenedesmus quadricauda

The chloroplast membranes of Scenedesmus quadricauda consist, in Gibbs' (5) terms, of bands or grana formed by the following sequence of components: a thin dark lamella, a light space, a thick dark lamella, a light space, a thick dark lamella, a light space, and a thin dark lamella. High resolution studies show that the thin dark lamellae consist of a single row of approximately spherical subunits having light cores and dark rims. The thick dark lamellae are composed of two rows of such subunits. The width of the thin dark lamellae average 110 A, and the average width of the thick dark lamellae is 168 A. These lamellae are superficially divided into dark and light components by rows of dark rims and of light cores. The rows of light cores average 23 A. Thus, high resolution studies of the bands of Scenedesmus quadricauda confirms Gibb's viewpoint that they are comparable to the grana of higher plants. The thin dark lamellae are end partitions or end granal membranes, and they are composed of a single layer of subunits. The thick dark lamellae are partitions, and they are composed of a double layer of subunits; margins in the grana of Scenedesmus and the higher plants are formed of a single layer of subunits. Fret connections in Scenedesmus are not common, but when present they are composed of a single row of subunits. A comparison of models of chloroplast structure is presented.