Biogenesis and cytochemistry of unspecialized peroxisomes in root cortical cells of Yucca torreyi L

Microbodies containing bipyramidal crystalline nucleoid inclusions occur within every cortical cell in roots of Yucca torreyi. Reaction product deposition attributable to catalase, glycolate oxidase, and urate oxidase activities are cytochemically localized to Yucca root microbodies and classifies them as unspecialized peroxisomes on the basis of their enzyme complement and tissue origin. Crystalline nucleoids do not stain for glycolate or urate oxidase activities, appearing as negatively-stained inclusions, but are apparently reactive for catalase activity. Development of unspecialized peroxisomes in Yucca roots is consistent with all evidence for glyoxysome and leaf-type peroxisome biogenesis from ER. Dilated ends of ER cisternae accumulate cytochemically detectable glycolate oxidase activity. After considerable dilation, paracrystalline precursors to nucleoids form within the bulge, and the inclusion enlarges to comprise the majority of peroxisomal volume. Peroxisomes that are not attached to ER are observed with high voltage electron microscopy and in serial thin sections, implying that eventually the budding peroxisomes are vesiculated. The functions of these unspecialized peroxisomes are suggested based upon cytochemical detection of their partial enzyme complement and their spatial and developmental timing relationships within developing Yucca root cortical parenchyma cells.     

Leaf peroxisomes are directly transformed to glyoxysomes during senescence of pumpkin cotyledons

Summary After the functional transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and against two glyoxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increased strikingly. Double labeling experiments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomes are directly transformed to glyoxysomes during senescence.     

Immunocytochemical Analysis Shows that Glyoxysomes Are Directly Transformed to Leaf Peroxisomes during Greening of Pumpkin Cotyledons

The functional transition of glyoxysomes to leaf peroxisomes occurs during greening of germinating pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). The immunocytochemical protein A-gold method was employed in the analysis of the transition using glyoxysomal specific citrate synthase immunoglobulin G and leaf peroxisomal specific glycolate oxidase immunoglobulin G. The labeling density of citrate synthase was decreased in the microbodies during the greening, whereas that of glycolate oxidase was dramatically increased. Double labeling experiments using different sizes of protein A-gold particles show that both the glyoxysomal and the leaf peroxisomal enzymes coexist in the microbody of the transitional stage indicating that glyoxysomes are directly transformed to leaf peroxisomes during greening.     

Isolation of microbodies from plant tissues

Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm³ in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues.     

CYTOCHEMICAL LOCALIZATION OF MALATE SYNTHASE IN GLYOXYSOMES

Cytochemical staining techniques for microbodies (peroxisomes) are limited at present to the enzymes catalase and α-hydroxy acid oxidase, and neither technique can distinguish glyoxysomes from other microbodies. Described here is a procedure using ferricyanide for the cytochemical demonstration by light and electron microscopy of malate synthase activity in glyoxysomes of cotyledons from fat-storing cucumber and sunflower seedlings. Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of acetyl CoA with glyoxylate to form malate and release free coenzyme A. Localization of the enzyme activity is based on the reduction by free CoA of ferricyanide to ferrocyanide, and the visualization of the latter as an insoluble, electron-opaque deposit of copper ferrocyanide (Hatchett's brown). The conditions and optimal concentrations for the cytochemical reaction mixture were determined in preliminary studies using a colorimetric assay developed to measure disappearance of ferricyanide at 420 nm. Ultrastructural observation of treated tissue reveals electron-opaque material deposited uniformly throughout the matrix portion of the glyoxysomes, with little background deposition elsewhere in the cell. The reaction product is easily visualized in plastic sections by phase microscopy without poststaining. Although the method has been applied thus far only to cotyledons of fat-storing seedlings, it is anticipated that the technique will be useful in localizing and studying glyoxylate cycle activity in a variety of tissues from both plants and animals.     

Development of enzymes in the cotyledons of watermelon seedlings

Changes in hypocotyl length, cotyledon weight, lipid content, chlorophyll content, and capacity for photosynthesis have been described in seedlings of Citrullus vulgaris, Schrad. (watermelon) growing at 30 C under various light treatments. Corresponding changes in the levels of 19 enzymes in the cotyledons are described, with particular emphasis on enzymes of microbodies, since during normal greening, enzymes of the glyoxysomes are lost and those of leaf peroxisomes appear. In complete darkness enzymes of the glyoxysomes reach a peak at 4 days and decline as the fat is depleted. Enzymes of mitochondria and of glycolytic pathways also peak at 4 to 5 days and either remain unchanged or decline to a lesser extent. Exposure to light at 4 days, when the cotyledons emerge, results in a selectively greater destruction of enzymes of the glyoxylate cycle; chlorophyll synthesis and capacity for photosynthesis increase in parallel, and there is a striking increase in the activities of chloroplast enzymes and in those of the leaf peroxisomes, hydroxypyruvate reductase and glycolate oxidase. The reciprocal changes in enzymes of the glyoxysomes and of leaf peroxisomes can be temporally dissociated, since even after 10 days in darkness, when malate synthetase and isocitrate lyase have reached very low levels, hydroxypyruvate reductase and glycolate oxidase increase strikingly on exposure to light and the cotyledons become photosynthetic. Furthermore, the parallel development of enzymes of leaf peroxisomes and functional chloroplasts is not immutable, since hydroxypyruvate reductase and glycolate oxidase activity can be elicited in darkness following a 5-minute exposure to light at day 4 while chlorophyll does not develop under these conditions.     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena.     

Cytochemical localization of glycolate oxidase in microbodies of Klebsormidium

The filamentous green alga Klebsormidium flaccidum A.Br. was fixed with glutaraldehyde, incubated in a cytochemical medium designed to detect glycolate-oxidase activity, and prepared for electron microscopy. Heavy deposits of stain were observed in microbodies following incubation with either glycolate or L-lactate as substrate, but not after incubation with D-lactate or H₂O. When Chlamydomanas reinhardi Dangeared cells were treated in the same way, their microbodies did not appear stained. The results establish that in Klebsormidium glycolate-oxidase occurs in microbodies (peroxisomes), as it does in angiosperms; also, they emphasize the dichotomy between those green algae which contain glycolate-oxidase and those, such as Chlamydomonas, which possess the mitochondrial enzyme glycolate dehydrogenase.     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.     

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds

Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.     

Cytochemical demonstration of malate synthase and glycolate oxidase in microbodies of cucumber cotyledons

The cytochemical localizations of malate synthase (glyoxysomal marker) and glycolate oxidase (peroxisomal marker) have been examined in cotyledon segments and sucrose-gradient fractions from germinated cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in the dark for 4 days, transferred to 4 hours of continuous light, then returned to the dark for 24 hours. Under these conditions, high specific activities for both glyoxysomal and peroxisomal enzymes are maintained in cotyledon homogenates and microbody-enriched fractions. Electron cytochemistry of the marker enzymes reveals that all or virtually all the microbodies observed in cotyledonary cells and sucrose-gradient fractions contain both enzymes. The staining in gradient fractions was determined from scoring a minimum of 600 photographed microbodies for each enzyme. After correcting for the number of particles stained for catalase reactivity (representing true microbodies), 94 and 97% of the microbodies were found stained for malate synthase and glycolate oxidase activity, respectively.     

Cytochemical localization of glycolate dehydrogenase in mitochondria of chlamydomonas

Mildly disrupted cells of Chlamydomonas reinhardi Dangeard were incubated in a reaction medium containing glycolate, ferricyanide, and cupric ions, and then processed for electron microscopy. As a result of the cytochemical treatment, an electron opaque product was deposited specifically in the outer compartment of mitochondria; other cellular components, including microbodies, did not accumulate stain. Incubation with d-lactate yielded similar results, while treatment with l-lactate produced only a weak reaction. Oxamate, which inhibits glycolate dehydrogenase activity in cell-free extracts, also inhibited the cytochemical reaction. These findings demonstrate in situ that glycolate dehydrogenase is localized in mitochondria, and thus corroborate similar conclusions reached on the basis of enzymic studies of isolated algal organelles.     

Enzymes of beta-Oxidation in Different Types of Algal Microbodies

The algae Mougeotia and Eremosphaera were used for isolation of microbodies with the characteristics of leaf peroxisomes and unspecialized peroxisomes, respectively. In both types of organelles, the following enzymes of the β-oxidation pathway were determined: acyl-CoA oxido-reductase, enoyl-CoA hydratase, and 3-hydroxyacyl-CoA dehydrogenase. There are indications that the peroxisomal oxidoreductase of both algae is a H₂O₂-forming oxidase rather than a dehydrogenase.

The enzymes enoyl-CoA hydratase and acyl-CoA oxidoreductase are located also in the mitochondria from Eremosphaera but not from Mougeotia. The mitochondrial acyl-CoA oxidizing enzyme was found to be a dehydrogenase. The specific activities of acyl-CoA oxidase and enoyl-CoA hydratase are lower than in spinach leaf peroxisomes. However, the activity of 3-hydroxyacyl-CoA dehydrogenase in the peroxisomes of both algae is almost 2-fold higher. The capability for degradation of fatty acids is a common feature of all different types of peroxisomes from algae.

     

Transport of chimeric proteins that contain a carboxy-terminal targeting signal into plant microbodies

Malate synthase is a glyoxysome-specific enzyme. The carboxy-terminal tripeptide of the enzyme is Ser—Arg—Leu (SRL), which is known to function as a peroxisomal targeting signal in mammalian cells. To analyze the function of the carboxy-terminal amino acids of pumpkin malate synthase in plant cells, a chimeric gene was constructed that encoded a fusion protein which consisted of β-glucuronidase and the carboxyl terminus of the enzyme. The fusion protein was expressed and accumulated in transgenic Arabidopsis that had been transformed with the chimeric gene. Immunocytochemical analysis of the transgenic plants revealed that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for transport of the fusion protein into glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL. The results suggest that tripeprides with S, A or P at the −3 position, K or R at the −2 position, and L or M at the carboxyl terminal position can function as a targeting signal for three kinds of plant microbody.     

Electron microscopic cytochemical localization ofα-hydroxyacid oxidase in rat kidney cortex

Summary The substrate specificity of-hydroxyacid oxidase in the rat kidney has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay applied to isolated renal peroxisomes. Rat kidneys were fixed by perfusion via the abdominal aorta with a low concentration (0.25%) of glutaraldehyde. Vibratome sections were incubated for 60 min at 37°C in a medium containing 3 mM CeCl₃, 100 mM NaN₃ and 5 mM of an-hydroxyacid in 0.1M Pipes or 0.1M Tris-maleate buffer both adjusted to pH 7.8. Ten aliphatic -hydroxyacids with chain lengths between 2 and 8 carbon atoms and two aromatic substrates were tested. The -hydroxyacid oxidase in the kidney exhibited a markedly different substrate specificity than the corresponding enzyme in the liver. Thus glycolate gave a negative reaction while two aromatic substrates, mandelic acid and phenyllactic acid, stained prommently. With aliphatic substrates a stronger reaction was obtained in Pipes than in theTris-maleate buffered incubation media. The best reaction in the kidney was obtained with hydroxybutyric acid. These cytochemical findings were confirmed by the luminometric determination of the oxidase activity in isolated purified peroxisome fractions. By electron microscopy the electron dense reaction product of cerium perhydroxide was found in the matrix of peroxisomes in the proximal tubules. The intensity of reaction varied markedly in neighbouring epithelial cells but also in different peroxisomes within the same cell. Thus heavily stained particles were seen next to lightly reacted ones. These observations establish the substrate specificity of -hydroxyacid oxidase in the rat kidney and demonstrate the marked heterogeneity in the staining of renal peroxisomes for this enzyme.     

Microbodies in fungi: a review

Summary Microbodies are ubiquitous organelles in fungal cells, occurring in both vegetative hyphae and spores. They are bounded by a single membrane and may contain a crystalloid inclusion with subunits spaced at regular intervals. Typically, they contain catalase which reacts with the cytochemical stain 3,3-diaminobenzidine to yield an electron-opaque product, urate oxidase,l--hydroxy acid oxidase andd-amino acid oxidase. Their fragility and the necessity to disrupt the tough fungal cell wall before isolating them make them difficult to isolate. Analysis of enzymes in purified or partially purified microbodies from fungi indicates that they participate in fatty acid degradation, the glyoxylate cycle, purine metabolism, methanol oxidation, assimilation of nitrogenous compounds, amine metabolism and oxalate synthesis. In organisms where microbodies are known to contain enzymes of the glyoxylate cycle, they are known as glyoxysomes; where they are known to contain peroxidatic activity, they are known as peroxisomes. In some cases microbodies contain enzymes for only a portion of a pathway or cycle. Thus, they must be involved in metabolic cooperation with other organelles, particularly mitochondria. The number, size and shape of microbodies in cells, their buoyant density and their enzyme contents may vary with the composition of the medium; their proliferation in cells is regulated by the growth environment. The isolation from the same organism of microbodies with different buoyant densities and different enzymes suggests strongly that more than one type of microbody can be formed by fungi.     

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.     

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b₅ reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm⁻³, a membrane fraction at 1.17 g × cm⁻³ containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient.     

Peroxisomes in the Alga Vaucheria are Neither of the Leaf Peroxisomal Nor of the Glyoxysomal Type*,1

Microbodies were isolated from the freshwater alga Vaucheria sessilis as well as from a marine Vaucheria. The organelles equilibrated on sucrose gradients at densities 1.23 g . cm^?3 and 1.24g . cm^?3, respectively. On electron micrographs they showed an ovoid or spheroid shape with a diameter of 0.5 to 0.8 μm. Besides catalase, the peroxisomes of both algae possess glycolate oxidase and glutamate-glyoxylate aminotransferase, but no other leaf-peroxisomal enzymes. Instead, the enzymes malate synthase and isocitrate lyase, which are markers of glyoxysomes in higher plants, are constituents of the peroxisomes in the marine as well as in the freshwater alga. Citrate synthase, aconitase, malate dehydrogenase and enzymes of the fatty acid β-oxidation pathway are located exclusively in the mitochondria. Therefore, the peroxisomes from Vaucheria do not belong to either the type of leaf peroxisomes or to the type of glyoxysomes.