A procedure for purifying the chloroplast envelope subfractionates it into two membrane fractions ofcomparable quantities. This procedure differs from previous ones in that the chloroplasts are ruptured by freezing and thawing in hypertonic medium rather than by osmotic shock. The two membrane fractions have qualitatively similar polar lipid compositions but differ in their content of individual lipids, specifically monogalactosyldiacylglycerol and phosphatidylcholine. The two fractions also differ in their constituent polypeptides and in their appearance when examined by electron microscopy. The light (density = 1.08 g/ ml) and heavy (density = 1.13 g/ml) membrane fractions have been tentatively identified as the outer and inner envelope membranes, respectively.The chloroplasts ofhigher plants are enclosed by a pair ofclosely spaced membranes, the envelope, consisting of an outer membrane in contact with the cytoplasm of the cell and an inner membrane bounding the matrix or stroma of the organelle. The chloroplast envelope mediates the complex interactions between the chloroplast and the cell cytoplasm. For example, both reactants and products of photosynthesis must be transported across the envelope (1). In addition, those chloroplast proteins that are synthesized on cytoplasmic ribosomes must cross the envelope to reach their correct location (2). The envelope is also the site of various biosynthetic reactions, including those responsible for the formation of the galactolipids, major components of both envelope and thylakoid membranes (1).The two membranes of the envelope have major differences in both structure and function, as shown by studies of isolated intact chloroplasts. It has been shown by freeze-fracture electron microscopy that the density of intramembranous particles is much lower in the outer membrane than in the inner, suggesting that the protein content ofthe outer membrane is lower (3). Also, experimental evidence indicates that the outer membrane is nonspecifically permeable to low molecular weight compounds although the inner is impermeable to such compounds and contains several translocator systems for the transport of metabolites (4).In previously published procedures for the purification of envelopes from intact chloroplasts, the plastids are first broken by hypotonic lysis and the envelopes are then isolated by centrifugation (1). Unfortunately, during isolation, the inner and outer membranes presumably become an inseparable mixture. This makes it impossible to use these preparations to investigate the reported differences between the two membranes or to determine which one contains which biosynthetic enzymes.We report here a new procedure for preparing the chloroplast envelope that subfractionates the envelope into two distinct fractions tentatively identified as the inner and outer membranes.MATERIALS AND METHODS Percoll, uridine-5'-diphosphogalactose, and trypsin inhibitor (Type I-P from beef pancreas) were from Sigma. Trypsin and chymotrypsin were from Boehringer Mannheim an...
Segments of mature tobacco leaves were fixed in glutaraldehyde, incubated in medium containing 3,3'-diaminobenzidine (DAB) and hydrogen peroxide, and postfixed in osmium tetroxide . Electron microscopic observation of treated tissues revealed pronounced deposition of a highly electron-opaque material in microbodies but not in other organelles . The coarsely granular reaction product is presumably osmium black formed by reaction of oxidized DAB with osmium tetroxide . Reaction of the microbodies with DAB was completely inhibited by 0 .02 M 3-amino-1 ,2,4-triazole and was considerably reduced by 0 .01 M potassium cyanide . These results, when considered in light of recent biochemical studies, strongly suggest that catalase is responsible for the reaction . Sharp localization of this enzyme in microbodies establishes that they are identical to the catalase-rich "peroxisomes" recently isolated from leaf cell homogenates . A browning reaction that occurred in leaves during the incubation step was inhibited by cyanide but not by aminotriazole and therefore could not have been caused by the same enzyme . This reaction and a slight deposition of dense material within primary and secondary walls are ascribed to oxidation of DAB by soluble and wall-localized peroxidases .
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.
Morphology and distribution of the relatively less well known organelles of plants have been studied with the electron microscope in tissues fixed in glutaraldehyde and postfixed in osmium tetroxide. An organelle comparable morphologically to the animal microbody and similar to the plant microbody isolated by MOLLENHAUER et al. (1966) has been encountered in a variety of plant species and tissues, and has been studied particularly in bean and radish roots, oat coleoptiles, and tobacco roots, stems and callus. The organelle has variable shape and is 0.5 to 1.5 μ in the greatest diameter. It has a single bounding membrane, a granular to fibrillar matrix of variable electron density, and an intimate association with one or two cisternae of rough endoplasmic reticulum (ER). Microbodies are easily the most common and generally distributed of the less well characterized organelles of plant cells. It seems very probable that they contain the enzymes characteristic of animal lysosomes (containing hydrolases) or animal microbodies (containing catalase and certain oxidases). Spherosomes are also possible sites of enzyme activity but are not as common or as widely distributed as microbodies. For this reason it appears likely that the particles designated as "plant lysosomes", "spherosomes", "peroxisomes", etc., in some of the cytochemical and biochemical studies on enzyme localization will prove to be microbodies.Variations in the morphology and ER associations of microbodies in tissues of bean and radish are described and discussed. "Crystal-containing bodies" (CCBs) are interpreted as a specialized type of microbody characteristic of metabolically less active cells. Stages in the formation of CCBs from microbodies of typical appearance are illustrated for Avena.The general occurrence of microbodies in meristematic and differentiating cells and their close association with the ER suggest that they may play active roles in cellular metabolism. The alterations in their morphology and numbers that are observed in certain differentiating cells suggest further that the enzyme complements and metabolic roles of microbodies might change during cellular differentiation. If so, microbodies could be the functional equivalent of both microbodies and lysosomes of animal cells.
The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of micro.
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