Study of amino acid composition and biosynthesis of protein components of the membrane system of corn plastids during biogenesis of chloroplasts]

The biosynthesis of membrane proteins in maize plastids at different stages of differentiation of the chloroplast lamellar system was studied. Prolamellar and lamellar system preparations were isolated from maize plastids, disintegrated by osmotic shock under hypotonic conditions. Changes in the amino acid composition of 14C membrane proteins were observed at all stages of chloroplast ultrastructure formation. The maximal level of the apolar amino acids was observed in the membrane fraction of chloroplasts. Washed membranes from maize proplastids and chloroplasts can be resolved into at least 14 protein bands on formic acid--urea polyacrylamide gel. It is pointed out that biogenesis process leads to the increase of lipophylic protein content in the chloroplast lamellae fraction.     

A POSSIBLE MECHANISM FOR THE MORPHOGENESIS OF LAMELLAR SYSTEMS IN PLANT CELLS

A mechanism for the formation of lamellar systems in the plant cell has been proposed as a result of electron microscope observations of young and mature cells of Nitella cristata and the plastids of Zea mays in normal plants, developing plants, and certain mutant types. The results are compatible with the concept that lamellar structures arise by the fusion or coalescence of small vesicular elements, giving rise initially to closed double membrane Structures (cisternae). In the chloroplasts of Zea, the cisternae subsequently undergo structural transformations to give rise to a compound layer structure already described for the individual chloroplast lamellae. During normal development, the minute vesicles in the young chloroplast are aggregated into one or more dense granular bodies (prolamellar bodies) which often appear crystalline. Lamellae grow out from these bodies. In fully etiolated leaves lamellae are absent and the prolamellar bodies become quite large, presumably because of inhibition of the fusion step which appears to require chlorophyll. Lamellae develop rapidly on exposure of the plant to light, and subsequent development closely parallels that seen under normal conditions. The plastids of white and very pale green mutants of Zea similarly lack lamellae and contain only vesicular elements. A specialized peripheral zone immediately below the double limiting membrane in Zea chloroplasts appears to be responsible for the production of vesicles. These may be immediately converted to lamellae under normal conditions, but accumulate to form a prolamellar body if lamellar formation is prevented, as in the case of etiolation and chlorophyll-deficient mutation, or when the rate of lamellar formation is slower than that of the production of precursor material (as appears to be the case in the early stages of normal development).     

Effect of Chloramphenicol on Formation of Chloroplast Structure and Protein During Greening of Etiolated Leaves of Phaseolus vulgaris

Greening of leaves of Phaseolus vulgaris in the presence of chloramphenicol inhibits formation of A) total chloroplast protein, B) an easily extractable fraction removed during isolation of chloroplasts in isotonic media by differential centrifugation, and C) the insoluble lamellar fraction which remains after extracting osmotically shocked freeze-dried plastids. The inhibition of insoluble chloroplast protein formation is correlated with decreased formation of lamellae and increased formation of vesicular structures. In contrast, chloramphenicol increases the formation of a fraction not removed during differential centrifugation, but removed by water extraction after osmotic shock and freeze-drying of chloroplasts. Analysis of this fraction by electrophoresis and column chromatography, indicates that the increased accumulation of this protein fraction is largely due to accumulation of a protein which is normally present in this fraction in small quantities. It was suggested that this protein may be a precursor which is normally incorporated into the lamellae. The protein extracted from freeze-dried chloroplasts of chloramphenicol treated chloroplasts contains a smaller proportion of one or more proteins than a similar extract of untreated plastids. However, per plastid, no such difference exists.     

FINE STRUCTURE OF PROTEIN-STORING PLASTIDS IN BEAN ROOT TIPS

The fine structure of leucoplasts in root tip cells of Phaseolus vulgaris L. has been studied in material fixed in glutaraldehyde followed by osmium tetroxide and poststained in uranyl acetate and lead citrate. Plastid development has been followed from the young stages in and near the meristematic region, through an ameboid stage, to the larger forms with more abundant storage products in the outermost cells. The plastids contain a dense stroma penetrated by tubules and cisternae arising from the inner membrane of the plastid envelope. Also located in the stroma are lamellae, ribosome-like particles, phytoferritin granules, and fine fibrils in less dense regions. In some elongate plastids microfilaments run lengthwise in the stroma near the surface. The same plastids store both starch and protein, but in a strikingly different manner. The starch is deposited in the stroma, while the protein always is accumulated within membrane-bounded sacs. These sacs arise as outgrowths from a complex of interconnected tubules which in turn appears to originate by coalescence and proliferation of tubules and cisternae arising from the inner plastid membrane. This "tubular complex" bears a strong resemblance to the prolamellar body of etiolated chloroplasts, but is smaller and ordinarily less regularly organized, and is apparently light-insensitive. Crystallization of the protein commonly occurs in the sacs and occasionally takes place within the tubules of the complex as well. The fine structure of the leucoplasts is discussed in relation to that of etiolated chloroplasts. Suggestions are made concerning the function of the tubular complex, role of the ameboid plastid forms, and manner of accumulation of the storage protein in the plastids.     

Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells

The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope. The presence of catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.     

ULTRASTRUCTURAL CHANGES OF CHLOROPLASTS IN ATTACHED AND DETACHED,AGING PRIMARY WHEAT LEAVES

Degradation of chloroplasts is shown in mesophyll cells of primary leaves of wheat. The sequence of ultrastructural changes in chloroplasts of naturally senescing leaves is compared with that of detached, aging leaves. In chloroplasts of naturally senescing leaves, the first indications of aging are the appearance of osmiophilic globuli and reorientation of the thylakoidal system. The membranes of the grana and intergrana lamellae then become distended and later dissociate into distinct vesicles. Concurrent with these membrane changes, osmiophilic globuli increase in size and number, and the stroma breaks down. Finally, the chloroplast envelope ruptures and plastid contents disperse throughout the cell's interior. In chloroplasts of mesophyll cells in detached, aging leaves, initial changes also include appearance of osmiophilic globuli, but later stages of chloroplast degradation are different. The chloroplast envelope ruptures before the lamellae break down. Swelling of grana and intergrana lamellae is not pronounced and, additionally, the thylakoidal system degenerates without forming vesicles or numerous osmiophilic globuli. These differences in the sequence of chloroplast degradation indicate that naturally senescing leaves rather than detached, aging leaves should be used in studies of chloroplast senescence.     

甜菊组织培养物中叶绿体的超微结构与脱分代

含有叶绿体的甜菊（Ｓｔｅｖｉａｒｅｂａｕｄｉａｎａ）愈伤组织细胞转移至新鲜培养基后,导致光合片层的逐渐减少或消失,最后叶绿体脱分化形成原质体样的结构。超微结构观察表明,光合片层的减少或消失与降解及叶绿体分裂特别是不均等缢缩分裂而致基质组分和类囊体膜稀释有关。这一过程并不完全同步,一些质体含有少量正常的片展而另一些质体含有退化的片层甚至片展结构完全消失。细胞的一个明显特点是细胞器大多聚集在细胞核附近,细胞质增加并向细胞中央伸出细胞质丝。同时可观察到原质体。培养７ｄ后,许多细胞呈分生状态,细胞质富含细胞器,充满了细胞的大部分空间。此时细胞中的质体大多呈原质体状态。在细胞生长的稳定期,质体内膜组织成基质基粒片层,同时质体核糖体增加。文中讨论了高度液泡化细胞脱分化与细胞中叶绿体脱分化的关系。     

Multiple fates of non‐mature lumenal proteins in thylakoids

Most proteins found in the thylakoid lumen are synthesized in the cytosol with an N–terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid‐transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling‐lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1‐null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid‐transfer signal removal on protein targeting and accumulation by examining the localization of non‐mature lumenal proteins in the Arabidopsis plsp1‐null mutant and performing a protein import assay using pea chloroplasts. In plsp1‐null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non‐mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid‐transfer signal removal did not disrupt cpSEC‐ and cpTAT‐dependent translocation, but prevented release of proteins from the membrane. Interestingly, non‐mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440‐kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1‐null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.     

Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells

Summary The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope.The presence of catalase (E.C. 1.11. 1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.Abbreviations used CAT catalase - CYT-OX cytochrome oxidase - DAB diaminobenzidine - ER endoplasmic reticulum - f filaments - HVEM high voltage electron microscopy - M mitochondrion - MT microtubule - P peroxisome - PB protein body - PER peroxidase - Pl plastid - Pg plastoglobuli - RER rough endoplasmic reticulum - RuBPcase ribulose-1,5-bisphosphate carboxylase - S starch - T tubule - V vacuole Scientific Article No. A3997, Contribution No. 6981, of the Maryland Agricultural Experiment StationThe scale bar on each micrograph is 0.1 , unless indicated otherwise     

ULTRASTRUCTURE OF THE LAMELLAE AND GRANA IN THE CHLOROPLASTS OF ZEA MAYS L

The fine structure of the chloroplasts of maize (Zea mays L.) has been investigated by electron microscopic examination of ultrathin sections of leaves fixed in buffered osmium tetroxide solutions. Both the parenchyma sheath and mesophyll chloroplasts contain a system of densely staining lamellae about 125 A thick immersed in a finely granular matrix material (the stroma), and are bounded by a thin limiting membrane which often appears as a double structure. In the parenchyma sheath chloroplasts, the lamellae usually extend the full width of the disc-shaped plastids, and grana are absent. The mesophyll chloroplasts, however, contain numerous grana of a fairly regular cylindrical form. These consist of highly ordered stacks of dense lamellae, the interlamellar spacing being ca. 125 A. The grana are interlinked by a system of lamellae (intergrana lamellae) which are on the average about one-half as numerous as the lamellae within the grana. In general, this appears to be due to a bifurcation of the lamellae at the periphery of the granum, but more complex interrelationships have been observed. The lamellae of the parenchyma sheath chloroplasts and those of both the grana and intergrana regions of the mesophyll chloroplasts exhibit a compound structure when oriented normally to the plane of the section. A central exceptionally dense line (ca. 35 A thick) designated the P zone is interposed between two less dense layers (the L zones, ca. 45 A thick), the outer borders of which are defined by thin dense lines (the C zones). Within the grana, the C zones, by virtue of their close apposition, give rise to thin dense intermediate lines (I zones) situated midway between adjacent P zones. A model of the lamellar structure is proposed in which mixed lipide layers (L zones) are linked to a protein layer (P zone) by non-polar interaction. Chlorophyll is distributed over the entire lamellar surface and held in the structure by van der Waals interaction of the phytol "tail" with the hydrocarbon moieties of the mixed lipide layers. The evidence in favour of the model is briefly discussed.     

Electron microscopic evidence for the reversible transformation ofEuonymus plastids

Contradictory concepts on whether the differentiation of plastids is monotropically directed or reversibly transformable with one another have been argued for a long time. In the present report, the evidence to support the latter concept, i.e. the reversible transformation, will be presented. The seasonal yellowing and regreening ofEuonymus leaves were observed by means of electron microscopic study. In the yellowing of chloroplasts during winter, plastoglobules appeared in the plastid stroma and increased in number according to the disintegration of lamellae; then the degenerated chloroplasts (chromoplasts) were filled up with these plastoglobules. In the next spring, however, regreening of the yellowed leaves took place; the lamellae were regenerated in the chromoplasts to again restore the normal chloroplast structure. Infolding of the inner membrane was never observed in these regreening plastids. The number of plastoglobules in the plastids decreased as the lamellae regenerated, and the chlorophyll content increased. These observations suggest that the plastoglobules in chromoplasts (plastids in yellowed leaves) are made of material of the disintegrating lamellae and are re-used as the source of supply for the reformation of lamellae in the spring reversal.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

DEVELOPMENT OF CHLOROPLAST FINE STRUCTURE IN ASPEN TISSUE CULTURE

Chloroplast ontogeny has been examined in 42-day etiolated triploid aspen callus (Populus tremuloides Michx.) subjected to two different light conditions. White and low-intensity red illumination showed little differences in their stimulatory effects on plastid development, the red light-irradiated plastids developing only slightly more slowly. Asynchronous plastid development was noted in both lighting systems. Etioplasts contained an interconnected tubular net, phytoferritin aggregates, electron-transparent vesicles which seem to invaginate from the inner plastid membrane, membrane-bound homogeneous spheroids and starch grains. Irradiation caused various morphological changes within the proplastids; the tubular complex became transformed into the more ordered prolamellar body-like structure from which radiated membrane-bound sacs filled with electron-dense material. These sacs, characterized as thylakoid precursors, were transformed into a thylakoidal system typical of mature chloroplasts. This ontogenetic scheme represents an additional pathway for the development of photosynthetic lamellae. Other light-induced changes in the developing plastid include disappearance of phytoferritin particles and homogeneous spheroids, decrease in starch content, and appearance of osmiophilic droplets.     

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.

     

Transport of proteins into chloroplasts. Organization, orientation, and lateral distribution of the plastocyanin processing peptidase in the thylakoid network

Plastocyanin is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen of the chloroplast. Maturation of preplastocyanin involves successive cleavages by a stromal peptidase and a distinct thylakoidal peptidase. In this report we have analyzed the precise location and orientation of the thylakoidal peptidase with respect to the thylakoid membrane. Experiments involving differential centrifugation of thylakoid extracts and sonication of isolated vesicles indicate that the peptidase is tightly bound to the thylakoid membrane but not intimately associated with any of the major thylakoid protein complexes. Analysis of the lateral distribution of the peptidase has shown that the enzyme is exclusively located in the non-appressed lamellae of the thylakoid network. The active site of the peptidase is on the lumenal face of the thylakoid membrane.     

Osservazioni Citologiche Su Peschi Clorotici E Rinverditi in Seguito A Trattamenti Con Soluzioni Minerali

Abstract

A Cytological study of peach leaves, chlorotic or regreening after treatment with salt solutions. — Whitening and regreening (for treatment with solutions containing Fe) leaves of chlorotic (Fe deficient) peaches were examined both at the optical and the electron microscope. The nuclei, as seen at the optical microscope, and the plastids infrastructures of chlorotic leaves sharply differ from the same structure of the leaves of virused plants. The differentiation of plastids of peach chlorotic leaves is arrested at a very early stage comparable to that of proplastids in etiolated plants before a vescicolar body is formed. In peach plastids a prolamellar body is never formed, not even during the greening of plastids. This is a further confirmation that such a structure, although normal in etiolated and genetically variegated plants, does not represent a fixed stage during differentiation of the lamellar system.

The present observations put into evidence that, even when the formation of lamellae is not immediate, the formation of the prolamellar body is not a necessary condition for the further development of the lamellar apparatus.     

Identification of chloroplast signal recognition particle RNA genes

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the ER membrane in eukaryotes, the plasma membrane in bacteria and the thylakoid membrane in chloroplasts. In higher plants two different SRP-dependent mechanisms have been identified: one post-translational for proteins imported to the chloroplast and one co-translational for proteins encoded by the plastid genome. The post-translational chloroplast SRP (cpSRP) consists of the protein subunits cpSRP54 and cpSRP43. An RNA component has not been identified and does not seem to be required for the post-translational cpSRP. The co-translational mechanism is known to involve cpSRP54, but an RNA component has not yet been identified. Several chloroplast genomes have been sequenced recently, making a phylogenetically broad computational search for cpSRP RNA possible. We have analysed chloroplast genomes from 27 organisms. In higher plant chloroplasts, no SRP RNA genes were identified. However, eight plastids from red algae and Chlorophyta were found to contain an SRP RNA gene. These results suggest that SRP RNA forms a complex in these plastids with cpSRP54, reminiscent of the eubacterial SRP.     

Absorption Microscopy of Enzymatically Treated Cell-Free Chloroplasts

Monochromatic light microscopy at 435 mµ shows in Euglena gracilis, the distribution of chlorophyll and the general orientation and geometry of chloroplasts in vivo. In addition it discloses, in swelling chloroplasts, a lamellar pigmented structure. Changes in this structure are observed in extruded swollen chloroplasts treated with lipolytic or proteolytic enzymes. Lipolytic enzymes produce an increase in the number of visible lamellae while proteolytic enzymes disrupt the lamellar array. Correlation of chloroplast swelling behavior and the effects of enzymatic degradation with current electron microscope observations support the following: (1) the pigment lamellae observed in vivo consist of component laminae; (2) the lamellae are separated by sites of swelling; and (3) the integrity of the lamellar structure is primarily dependent upon the intact state of the protein.     

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.