Prolamellar body transformation with increasing cell age in the maize leaf

Summary The fine structure of the plastids in the leaf's basal meristem and in the leaf tissues 2 cm immediately above has been studied in maize leaves of different ages. In the young leaves the proplastids of the basal meristem differentiate, in the tissues within 2 cm above the meristem, into chloroplasts containing two or more prolamellar bodies, indipendently whether the tissues have been fixed 11 hours after a period of illumination or of darkness. In the oldest leaves, in the tissues immediately above the basal meristem, no prolamellar body is present in the plastids, and the proplastids differentiate directly into chloroplasts, without passing through an etio-chloroplast stage.Supported by a grant of C.N.R.     

Protein crystalloids in ribosome-deficient plastids of Aeonium domesticum cv. variegatum (Crassulaceae)

Protein crystalloids are typical constituents of Aeonium domesticum plastids. They are composed of hexagonally arranged tube-like elements situated in the stroma without a bordering membrane. The single tubule has an external diameter of about 20 nm and an internal one of about 10 nm. The green-white-green mesochimera Ae. domesticum cv. variegatum contains normal chloroplasts in the green tissue and colourless plastids in the pale tissue. The defective plastids have a double-layered envelope, scarce internal membrane structures and contain, in the mature stage, a large vacuole. Plastid ribosomes can be detected only rarely in proplastids. They lose their ribosome complement entirely in the course of development. Polyacrylamide gel electrophoresis of total nucleic acids extracted from white tissue revealed the absence of the 23S and 16S rRNA normally present in plastids. Despite the loss of ribosomes, the plastids contain large protein crystalloids, which are structurally identical with those of normal green chloroplasts. Consequences concerning problems of encoding and transport of crystalloid protein(s) are briefly discussed.Abbreviations CAM crassulacean acid metabolism - FIP fraction I protein - L I epidermis - L II subepidermal layer - L III leaf core - SPC succulent protein crystalloid This is the first part of a series on the crystalloid-forming succulent protein     

Activation of polyphenol oxidase of chloroplasts

Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or —18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.     

Purification and Properties of an NADPH-Aldose Reductase (Aldehyde Reductase) from Euonymus japonica Leaves

Intact chloroplasts isolated from Euglena gracilis exhibit high rates of light-driven protein synthesis, whereas protein synthesis by isolated proplastids is absolutely dependent upon the addition of an exogenous energy source in the form of equimolar ATP and Mg²⁺. ATP and Mg²⁺ also stimulate translation by chloroplasts. The greatly increased rates of protein synthesis obtained by supplementing proplastids with ATP and Mg²⁺ have allowed the first clear characterization of proplastid translation products. Two-dimensional polyacrylamide gel electrophoretic analysis of proteins synthesized in organello shows that, while many translation products are common to both plastid types, most are unique to either the proplastid or the chloroplast. Pulse-chase experiments using both proplastids and chloroplasts indicate similar rates of turnover of newly synthesized proteins in both types of plastids. Thus, the differences seen between proplastid and chloroplast translation products are apparently not due to turnover. Immunoprecipitation of large subunit of ribulose-1,5-bisphosphate carboxylase (LS) from pulse-chase experiments indicates that LS is made in both proplastids and in chloroplasts and that the rate of LS turnover is similar in both types of plastids.     

Concanavalin A-mediated agglutination of plant plastids

Cucumber (Cucumis sativus L.) and pear (Pyrus domestica Medik.) fruit proplastids, and pea (Pisum sativum L., cv. Meteor) leaf chloroplasts, extracted by osmotic rupture of protoplasts isolated after degradation of the cell walls by cellulase and pectinase, agglutinated in the presence of Con A. Agglutination of cucumber proplastids was inhibited by anti-Con A and by methyl D-gluco/manno pyranosides but not by methyl D-galactopyranoside. Fluorescein isothiocyanate-conjugated Con A (FITC-Con A) rendered agglutinated clumps fluorescent. If cellulase was omitted from the macerating medium, Con A-mediated agglutination did not occur even if proplatids were subsequently incubated with cellulase. Proplastids and chloroplasts extracted by conventional mechanical disruption methods were not agglutinated by Con A and did not acquire fluorescence with FITC-Con A. However, cucumber proplastids so extracted could be agglutinated by Con A if incubated with cellulase after preparation.Abbreviation Con A Concanavalin A (Jackbean phytohemagglutinin)     

Dynamics of localization and protein composition of plastid nucleoids in light-grown pea seedlings

Summary Localization and protein composition of plastid nucleoids was analyzed in light-grown pea seedlings at various stages of leaf development. In young plastids of unopened leaf buds, nucleoids were abundant and localized in the periphery of plastids, whereas, in mature leaves, chloroplasts contained nucleoids within narrow spaces restricted by thylakoids or grana. The migration of nucleoids into the interior of plastids preceded the formation of grana, and hence, the maturation of the photosynthetic apparatus. The protein composition of nucleoids was considerably different in young plastids and mature chloroplasts. Polypeptides with a molecular mass of 70–100 kDa predominated in the nucleoids of young plastids, whereas polypeptides with molecular mass of 20–30 kDa were abundant in the nucleoids of mature chloroplasts. Immuno-blot analysis with antibodies against the nucleoids of young plastids identified various polypeptides that were significantly more abundant in the nucleoids of young plastids than in the nucleoids of mature chloroplasts. These results demonstrate that plastid nucleoids are subject to dynamic changes in both localization and composition during the normal development of chloroplasts in the light.Abbreviations DAPI 4,6-diamidino-2-phenylindol - DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Intracellular localization of VDAC proteins in plants

Voltage-dependent anion channels (VDACs) are porin-type -barrel diffusion pores. They are prominent in the outer membrane of mitochondria and facilitate metabolite exchange between the organelle and the cytosol. Here we studied the subcellular distribution of a plant VDAC-like protein between plastids and mitochondria in green and non-green tissue. Using in vitro studies of dual-import into mitochondria and chloroplasts as well as transient expression of fluorescence-labeled polypeptides, it could be clearly demonstrated that this VDAC isoform targets exclusively to mitochondria and not to plastids. Our results support the idea that plastids evolved a concept of solute exchange with the cytosol different from that of mitochondria.Abbreviations AOX Alternative oxidase - p Precursor form - POM36 Putative outer mitochondrial membrane proteins of 36 kDa - SSU Small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) - VDAC Voltage-dependent anion channel     

Variation of plastid types in spinach

Summary During growth in the light the plastids of cultured leaf discs of spinach divide, increase in size, and differentiate in a similar manner to those in intact leaves. By contrast when l'eaf discs are grown in the dark prolamellar bodies begin to develop in partially differentiated chloroplasts within 2 hours. After 7 days growth in the dark the plastids contain many vesicles which appear to arise from swelling of thylakoids. These vesicles often contain large crystals. When dark grown discs are illuminated they regreen and fully differentiated chloroplasts are reformed.Proplastids are found in the stem apex of whole plants; these develop into the partially differentiated chloroplasts of young leaves, which divide and increase in size to form the fully differentiated chloroplasts of mature leaves. The cotyledons of dormant seeds contain proplastids; these develop into amyloplasts during germination and after exposure to white light differentiate into chloroplasts.     

Morphological and chemical studies on the crystalloid-forming ‘succulent protein’ from normal and ribosome-deficient Aeonium domesticum plastids

Aeonium domesticum cv. variegatum is a mesochimera of the constitution green/white/green with normal proplastids and chloroplasts in the unaffected tissues and ribosome-deficient colourless mutant plastids in the white leaf tissues. All the different plastid types contain succulent protein crystalloids (SPC). For more detailed characterization, the SPC elements were freed from the plastids and purified by gel filtration. Electron microscopy of different fractions revealed five levels of structural organization. Beginning with the most complex state, the levels are designated as succulent protein (SP) organizational state V (hexagonally arranged and closely packed tubules in the stroma of intact plastids) to I (globular protomers of 5 nm diameter as the basic structure of SPCs). Highly purified SP-fractions were shown by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to consist of two or three proteins of M_r 56 kdalton, 58 kdalton and 60 kdalton, depending on the buffer medium used for SP isolation and the duration of storage of leaves in the frozen state. In the urea/SDS-PAGE system, these proteins show similar mobilities to - and -tubulin, but no immunoreaction against antitubulin. The proteolytic cleavage pattern of tubulin subunits and SP proteins are different. Their locations on two-dimensional isoelectric focusing-SDS gels show some overlappings because of microheterogeneities in both proteins in the pH gradient from pH 4.5 to 6.5. Malatedehydrogenase activity could not be detected in the purified SP fractions.Abbreviations CAM Crassulacean acid metabolism - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SP succulent protein - SPC succulent protein crystalloid - SPOS succulent protein organizational state     

Double ring structure around the constricting neck of dividing plastids ofAvena sativa

Summary Ultrastructure of the constricting neck of dividing proplastids and young chloroplasts in the first leaves ofAvena sativa was examined by electron microscopy. An electron-dense, double ring structure (plastid-dividing ring doublet; PD ring doublet) with a width of 15–40 nm was revealed around the narrow neck of the constricted and dividing plastids by serial section technique. The inner and outer ring of the doublet coated the inside (stromal side) of the inner envelope membrane and the outside (cytoplasmic side) of the outer envelope membrane, respectively. However, electron-dense materials were not observed within the lumen between the outer and inner envelope membranes.Although the PD ring doublet was commonly observed in the constricted plastids with a 70–140 nm wide neck, they could be scarcely observed in the constricted plastids with a 160 or more nm wide neck. The components of the PD ring were assumed not to be concentrated enough to identify by electron microscopy in the early stage of constriction and the PD ring may be formed and recognized at the final stage.The significance of the formation of the PD ring and its role in plastokinesis (plastid kinesis) were discussed.     

Distinct intracellular vesicle transport mechanisms are selectively modified by spastin and spastin mutations

Spastin is a microtubule severing ATPase that regulates intracellular and axonal transport of vesicles. Intracellular vesicle trafficking was analyzed in differentiated SH‐SY5Y‐neuroblastoma cells, transfected with spastin wild‐type and three spastin mutations (ΔN, K388R, S44L) to investigate spastin‐mediated effects on the velocity of vesicles, stained with LysoTracker Red®. The vesicle velocity varied considerably between mutations and detailed analysis revealed up to five distinct velocity classes. Microtubule severing by overexpressed wild‐type spastin caused reduced vesicle velocity. S44L and ΔN mutations, which were functionally impaired, showed similar velocities as control cells. K388R‐transfected cells exhibited an intermediate velocity profile. The results support the idea that spastin mutations not only alter axonal transport, but in addition regulate intracellular trafficking in the cell soma as well. J. Cell. Physiol. 226: 362–368, 2011. © 2010 Wiley‐Liss, Inc.     

Ultrastructural localization of photosynthetic activity in thylakoids during chloroplast development in maize

The localization of photosynthetic activity in developing maize (Zea mays L.) chloroplasts was studied in situ by two electron-microscopic-cytochemical methods. The activity of photosystem I was detected by photooxidation of 3,3-diaminobenzidine (DAB) and the activity of the photosystem II by photoreduction of thiocarbamyl nitrotetrazolium blue (TCNBT). During the transformation of proplastids into chloroplasts, at the base of the leaf blade the DAB reaction appeared before the TCNBT reaction. A positive DAB reaction was observed in the single thylakoids of plastids in cells located only about 0.5 mm above the base. Dark, osmiophilic DAB polymers accumulated in the lumina of the thylakoids. Plastid envelopes and tubules of the prolamellar bodies in immature chloroplasts were DAB-negative. In fully differentiated leaf tissue the DAB reaction was intense in the thylakoids of bundle-sheath chloroplasts, as well as in the stroma thylakoids and the peripheral grana thylakoids of mesophyll chloroplats. The photoreduction of TCNBT started in leaf tissue about 1 mm above the base. Dark granular material of reduced TCNBT appeared mostly in the partitions of grana, i.e. interthylakoidally, but some granules were also attached to the stroma thylakoids. The membranes of plastid envelopes and the tubules of prolamellar bodies showed a negative TCNBT reaction. Young bundle-sheath chloroplasts contained some reduced TCNBT in their grana; these deposits largely disappeared in the course of further differentiation. In mature leaf tissue the photoreduction of TCNBT was conspicuous in the grana of mesophyll chloroplasts, but very weak in the single thylakoids and in the granal rudiments of bundle-sheath chloroplasts.Abbreviations DAB 3,3-diaminobenzidine·4 HCl - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS(I,II) photosystem (I,II) - TCNBT thiocarbamyl nitrotetrazolium blue chloride     

A novel strategy to visualize vesicle‐bound kinesins reveals the diversity of kinesin‐mediated transport

In mammals, 15 to 20 kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each kinesin or the functional significance of such diversity. To characterize the transport mediated by different kinesins, we developed a novel strategy to visualize vesicle‐bound kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin‐1, ‐2, and ‐3 families. We observed vesicle labeling with nearly all kinesins. Only six kinesins bound vesicles that undergo long‐range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle‐bound kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on‐vesicle regulation is crucial for kinesin targeting. Surprisingly, several kinesins were associated with populations of somatodendritic vesicles that underwent little long‐range transport. This assay should be broadly applicable for investigating kinesin function in many cell types.     

Proteomic analysis of Medicago truncatula root plastids

Despite the recognized importance of non‐photosynthetic plastids in a wide array of plant processes, the root plastid proteome of soil‐grown plants still remains to be explored. In this study, we used a protocol allowing the isolation of Medicago truncatula root plastids with sufficient protein recovery and purity for their subsequent in‐depth analysis by nanoscale capillary LC‐MS/MS. Besides providing the first picture of a root plastid proteome, the results obtained highlighted the identification of 266 protein candidates whose functional distribution mainly resembled that of wheat endosperm amyloplasts and tobacco proplastids together with displaying major differences to those reported for chloroplasts. Most of the identified proteins have a role in nucleic acid‐related processes (16%), carbohydrate (15%) and nitrogen/sulphur (12%) metabolisms together with stress response mechanisms (10%). It is noteworthy that BLAST searches performed against the proteins reported in different plastidomes allowed detecting 30 putative root plastid proteins for which homologues were previously unsuspected as plastid‐located, most of them displaying a common putative role in participating in the plant cell responses against abiotic and/or biotic stresses. Taken together, the data obtained provide new insights into the functioning of root plastids and reinforce the emerging idea for an important role of these organelles in sustaining plant defence reactions.     

DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus.

We have identified a mutation at the DAG locus of Antirrhinum majus which blocks the development of chloroplasts to give white leaves with green revertant sectors. The green areas contain normal chloroplasts whereas the white areas have small plastids that resemble proplastids. The cotyledons of dark-grown dag mutant seedlings have plastids which also resemble proplastids. The palisade cells in the white areas of dag mutant leaves also lack their characteristic columnar shape. The DAG locus was cloned by transposon tagging: DAG encodes a novel protein with a predicted Mr of 26k, which is targeted to the plastids. Cleavage of its predicted transit peptide gives a mature protein of Mr 20k. Screening of databases and analysis of Southern blots gave evidence that DAG belongs to a protein family with homology to several proteins of unknown function from plants. Expression of DAG is required for expression of nuclear genes affecting the chloroplasts, such as CAB and RBCS, and also for expression of the plastidial gene RPOB encoding the plastidial RNA polymerase beta subunit, indicating that it functions very early in chloroplast development.     

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.     

Ultrastructure of Carposporogenesis in the Red Alga Caulacanthus ustulatus (Gigartinales, Caulacanthaceae)

Carposporangium differentiation in Caulacanthus ustulatus (Turner)Ktzing proceeds through four developmental stages. The youngestcarposporangia are embedded within confluent mucilage and containa nucleus, a few small starch granules, concentric membranebodies and proplastids without a peripheral thylakoid. The intermediate-agedcarposporangia are characterized by the formation of fibrousvacuoles by a fibrous vacuole associated organelle (FVAO). Plastidsalso start to develop their internal thylakoid system. In nearlymature carposporangia, highly active, curved dictyosomes producecored vesicles, while fibrous vacuoles increase in number anddisplay a perinuclear arrangement. Abundant starch granulesare present, some of which exhibit a degenerating appearance.A carposporangium wall is formed and plastids complete theirinternal thylakoid system. Carposporangium maturation is signalledby the presence of adhesive vesicles. Fully developed and peripherallyarranged plastids, centrally located fibrous vacuoles, fewerstarch granules and a monolayered wall are the features of maturecarposporangia. Carposporogenesis, Caulacanthus ustulatus (Turner) Kützing, red algae, ultrastructure     

Pigment composition and plastid structure in leaves of carotenoid mutants of maize

Summary In monogenic, recessive chloroplast mutants of maize which contain chlorophylls, and lycopene or -carotene but no normal carotenoids, great variability in the size of plastids was associated with a number of ultrastructural abnormalities. In the mutant accumulating lycopene some plastids contain dense bundles of lamellae, whereas the chloroplasts of the -carotene mutant show poor thylakoid development. Neither of the mutants was able to form normal grana.A comparison of chlorophyll/carotenoid ratios in different chloroplast fractions of normal and mutant leaves showed that plastids of small size and delicate structure contain relatively less chlorophyll than fully differentiated chloroplasts.     

On vesicles and membrane compartments

Summary Two different mechanisms have been proposed to explain transport along the endocytic and biosynthetic transport routes in cells. The first involves stable compartments connected by vesicular traffic while the second argues that the key organelles (early endosomes or the cis Golgi) form de novo by fusion of vesicles and subsequently mature into later forms. In the first part of this article, I propose a classification that distinguishes between stable, preexisting membrane compartments and vesicles that are, by definition, transient organelles. In this scheme, compartments, but not vesicles, are capable of homotypic fusion while vesicles, but not compartments, are able to mature, a process defined as an irreversible set of biochemical events which lead to a physiologically distinct end-state of the vesicle prior to its vectorial fusion with a target compartment. In the second part, I summarize my current ideas about the ultrastructural organization of the ER-Golgi region. Finally, I review the cell biology of selected examples of different vesicle types in order to exemplify the fascinating diversity of functions that this class of membrane organelles has evolved.Abbreviations COP coatomer - ECV endosome carrier vesicle - ER endoplasmic reticulum - HRP horseradish peroxidase - IC intermediate compartment between ER and Golgi - MVB multivesicular body - NSF N-ethyl maleimide sensitive factor - SNAPS soluble NSF associated proteins - TGN trans Golgi network Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Ulfrastructure of the histological zones in growing vegetative buds of Salix spp.

Ultrastructural differentiation in the shoot apex of growing vegetative buds of Salix was studied, and some micrographs analysed morphometrically. The distribution of inorganic phospahte (P;) was analysed cytochemically. A distinct histological zona–tion was observed in the apex. The relative volumes of nuclei and plastids were significantly higher in the central tunica zone than in the peripheral one. The corpus differed from the central tunica zone by significantly lower volume density of nuclei and higher of vacuoles and mitochondria. During differentiation of the rib meristem vacuole volume increased significantly, while the relative volumes of nuclei, mitochondria, nucleoli, and heterochromatin decreased. It was not possible to decide whether the vacuoles originate from ER or GERL. Morphogenesis of chloroplasts with large starch grains and grana from proplastids was evident in the rib meristem; dedifferentiation to S–plastids was found in the protophloem. Prolamellar bodies were observed in the procambium plastids. The protophloem was characterized by P–protein and spiny vesicles. P_i was found in the nucleoli of most epidermis cells, several procambium cells, and a few chlorencyma cells, but never in the tunica of the growing apical and developing lateral buds. P_i also occurred in some plasmalemma–somes and occasionally in the walls in connection with plasmodesmata.