Structural and cytochemical characterization of three specialized peroxisome types in soybean

Structural and cytochemical comparisons were made between three peroxisome types in soybean [ Glycine max (L.) Merr. cv. Centennial]. Leaf peroxisomes were densely granular organelles with an amorphous nucleoid and were generally located in close proximity to the chloroplasts. Catalase (EC 1.11.1.6) and glycolate oxidase (EC 1.1.3.1) were localized in these peroxisomes although glycolate oxidase was absent from the nucleoid region. Glyoxysomes, present in the etiolated cotyledons, were coarsely granular organelles that were generally in close proximity to lipid bodies. Malate synthase (EC 4.1.3.2), catalase, and glycolate oxidase were present throughout the matrix. Although peroxisomes were found in both infected and uninfected nodule tissue, uninfected interstitial cell peroxisomes were the most developed. These organelles contained a core surrounded by a less electron-opaque periphery that frequently was in close association with (but distinct from) a network of smooth endoplasmic reticulum. Of the enzymes studied, only catalase and urate oxidase (EC 1.7.3.3) were detected in the nodule peroxisomes. Neither enzyme was detected in the peripheral area of the peroxisome. These data indicate that peroxisomes in the three tissue types have organelle associations, internal structures, enzyme constitutions and packaging that reflect their metabolic differences.     

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.     

Peroxisomes of soybean (Glycine max) root nodule vascular parenchyma cells contain a "nodule—specific" urate oxidase

The cortex of soybean ( Glycine max L. cv. Centennial) nodules contain an organellerich layer of vascular parenchyma tissue, which encircles the elaborate vascular tissue of the nodule. Peroxisomes with small, electron-opaque nucleoids are found in the vascular parenchyma cells. Positive cytochemical staining for catalase (EC 1.11.1.6) confirms their morphological identification as peroxisomes. Activities of both glycolate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were detected cytochemically in these peroxisomes. Nodule-specific urate oxidase was localized principally in the nucleoid region of these vascular parenchyma peroxisomes, as indicated by immunogold labelling using antibodies against nodulin-35, the nodule-specific urate oxidase. The density of urate oxidase immunogold labelling in the vascular parenchyma peroxisome nucleoid is similar to that of the more well-characterized interstitial cell peroxisomes of the infected zone. These results show that the induction of nodule-specific urate oxidase may be induced in tissue outside of the infected zone.     

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.     

Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

cDNA cloning and differential gene expression of three catalases in pumpkin

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.     

Control by phytochrome of urate oxidase and allantoinase activities during peroxisome development in the cotyledons of mustard (Sinapis alba L.) seedlings

The peroxisomal enzyme, urate oxidase (EC 1.7.3.3), and the next enzyme of the urate pathway, allantoinase (EC 3.5.2.5), demonstrate a lightmediated rise of activity in the cotyledons of mustard (Sinapis alba L.). The capacity of the peroxisomes for urate breakdown, marked by the time course of urate oxidase, develops distinctly later than the two other peroxisome functions (fatty acid breakdown, glyoxysomal function; glycolate breakdown, leaf peroxisomal function). The light effect on urate oxidase and allantoinase is mediated through the phytochrome system in all three seedling organs (cotyledons, hypocotyl, radicle), as revealed by induction/reversion experiments with red/far-red light pulses and continuous irradiation with far-red light (high irradiance reaction of phytochrome). Both enzyme activities can be induced by phytochrome in the seedling cotyledons only during a sensitive period of about 48 h prior to the actual light-mediated rise of activity, making it necessary to assume the existence of a long-lived intermediate (transmitter) in the signal response chain connecting enzyme formation to the phytochrome system. Detailed kinetic investigation, designed to test whether urate oxidase and allantoinase are controlled by phytochrome via the same signal response chain (coordinate induction), revealed large differences between the two enzymes: (i) a different onset of the loss of reversibility of a red light induction by a far-red light pulse (=onset of transmitter formation=coupling point; 48 h/24 h after sowing for urate oxidase/allantoinase); (ii) a different onset of the response (=onset of competence for transmitter= starting point; 72 h/48 h); (iii) full loss of reversibility (=completion of transmitter formation) is reached at different times (independence point, 90 h/52 h). These differences show that phytochrome controls urate oxidase and allantoinase via separate signal response chains. While urate oxidase can be localized in the peroxisomal fraction isolated from crude organelle extracts of the cotyledons by density gradient centrifugation, most of the allantoinase activity found in the peroxisomal fraction did not appear to be an integral part of the peroxisome but originated presumably from adhering membrane fragments.Abbreviations AL allantoinase, EC 3.5.2.5 - CAT catalase, EC 1.11.1.6 - GO glycolate oxidase, EC 1.1.3.1 - ICL isocitrate lyase, EC 4.1.3.1 - UO urate oxidase, EC 1.7.3.3. P_r - P_fr red and far-red absorbing forms of phytochrome On the occasion of his 80th birthday we dedicate this paper to Prof. Dr. phil., Dr. mult. h.c. Kurt Mothes, pioneer in research on metabolism of urates     

Absence of CeCl3-detectable peroxisomal glycolate-oxidase activity in developing raphide crystal idioblasts in leaves of Psychotria punctata Vatke and roots of Yucca torreyi L.

Three peroxisomal enzymes, glycolate oxidase, urate oxidase and catalase were localized cytochemically in Psychotria punctata (Rubiaceae) leaves and Yucca torreyi (Agavaceae) seedling root tips, both of which contain developing and mature calcium-oxalate raphide crystal idioblasts. Glycolate-oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6) activities were present within leaftype peroxisomes in nonidioblastic mesophyll cells in Psychotria leaves, while urate-oxidase (EC 1.7.3.3) activity could not be conclusively demonstrated in these organelles. Unspecialized peroxisomes in cortical parenchyma of Yucca roots exhibited activities of all three enzymes. Reactionproduct deposits attributable to glycolate-oxidase activity were never observed in peroxisomes of any developing or mature crystal idioblasts of Psychotria or Yucca. Catalase localization indicates that idioblast microbodies are functional peroxisomes. The apparent absence of glycolate oxidase in crystal idioblasts of Psychotria and Yucca casts serious doubt that pathways involving this enzyme are operational in the synthesis of the oxalic acid precipitated as calcium-oxalate crystals in these cells.Abbreviations AMPD 2-amino-2-methyl-1,3-propandiol - CTEM conventional transmission electron microscopy - DAB 3,3-diaminobenzidine tetrahydrochloride - HVEM high-voltage electron microscopy     

A Survey of Plants for Leaf Peroxisomes

Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose.In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 mumoles x min(-1) x g(-1) wet weight or a specific activity of 0.02 to 0.05 mumole x min(-1) x mg(-1) protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 mumoles x min(-1) x g(-1) wet weight or 90 to 300 mumoles x min(-1) x mg(-1) protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial.Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 mumole x min(-1) x mg(-1) protein; for catalase. 8000 mumoles x min(-1) x mg(-1) protein, and for malate dehydrogenase, 40 mumoles x min(-1) x mg(-1) protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 mumole of glycolate oxidase x min(-1) x g(-1) tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane.Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration.Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation.     

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.     

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.     

An activated-oxygen-mediated role for peroxisomes in the mechanism of senescence of Pisum sativum L. leaves

The possible involvement of peroxisomes and their activated-oxygen metabolism in the mechanism of leaf senescence was investigated in detached pea (Pisum sativum L.) leaves which were induced to senesce by incubation in complete darkness for up to 11 d. At days 0, 3, 8, and 11 of senescence, peroxisomes were purified from leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. Xanthine-oxidoreductase activity increased with senescence, especially the O ₂ ^{. -} -producing xanthine oxidase (EC 1.1.3.22). The activities of H₂O₂-generating Mn-superoxide dismutase (EC 1.15.1.1) and urate oxidase (EC 1.7.3.3) were also enhanced by senescence, whereas catalase (EC 1.11.1.6) activity was severely depressed. Hydrogen peroxide concentrations increased significantly in senescent leaf peroxisomes. During the progress of senescence, glycollate oxidase (EC 1.1.3.1) and hydroxypyruvate reductase (EC 1.1.1.81), two marker enzymes of photorespiratory metabolism, gradually decreased in activity and disappeared. At the same time, the activities of malate synthase (EC 4.1.3.2) and isocitrate lyase (EC 4.1.3.1), key enzymes of the glyoxylate cycle, which were undetectable in presenescent leaves, increased dramatically upon induction of senescence. Ultrastructural studies of intact leaves showed that the population of peroxisomes and mitochondria increased with senescence. Results indicate that peroxisomes could play a role, mediated by activated oxygen species, in the oxidative mechanism of leaf senescence, and further support the idea, proposed by other authors, that foliar senescence is associated with the transition of leaf peroxisomes into glyoxysomes.Abbreviation Mn-SOD (manganese-containing) superoxide dismutase The authors thank Dr. A.J. Sánchez-Raya (Unidad de Fisiología Vegetal, Estación Experimental del Zaidín, Granada, Spain) for his valuable help in measuring ethylene production, and Dr. G. Barja de Quiroga (Departamento de Biología Animal II, Universidad Complutense, Madrid, Spain) for carrying out the malondialdehyde determinations by HPLC. This work was supported by grant PB87-0404-01 from the DGICYT and the Junta de Andaluc'ia (Research Group # 3315), Spain.     

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.     

Development of Enzymes of the Glyoxylate Cycle during Senescence of Pumpkin Cotyledons

The presence and activities of isocitrate lyase (EC 4.1.3.1 [EC] )and malate synthase (EC 4.1.3.2 [EC] ) were studied during senescenceof pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). Afterincubation of detached cotyledons in permanent darkness, theactivities appeared and increased up to the eighth day and thendeclined, while the activities of catalase (EC 1.11.1.6 [EC] ), glycolateox-idase (EC 1.1.3.1 [EC] ), and hydroxypyruvate reductase (EC 1.1.1.81 [EC] )decreased dramatically. After fractionation of cell organellesby sucrose density gradient, we detected isocitrate lyase andmalate synthase activities in peroxisomal fractions. The activityof the two key enzymes of the glyoxylate cycle also increasedduring senescence in vivo and we confirmed the presence of thetwo enzymes in the peroxisomal fractions after sucrose gradientcentrifugation. At every point examined, the level of malatesynthase was demonstrated by immunoblotting. It is concludedthat the development of isocitrate lyase and malate synthaseactivities represents the transition from leaf peroxisomes toglyoxysomes and that such a phenomenon is associated with senescence. (Received January 25, 1991; Accepted March 22, 1991)     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

The stability of the lactose and citrate plasmids in Lactococcus lactis subsp. lactis biovar. diacetylactis

We have studied the intraperoxisomal location of catalase in peroxisomes of methanol-grown Hansenula polymorpha by (immuno)cytochemical means. In completely crystalline peroxisomes, in which the crystalline matrix is composed of octameric alcohol oxidase (AO) molecules, most of the catalase protein is located in a narrow zone between the crystalloid and the peroxisomal membrane. In non-crystalline organelles the enzyme was present throughout the peroxisomal matrix. Other peroxisomal matrix enzymes studied for comparison, namely dihydroxyacetone synthase, amine oxidase and malate synthase, all were present throughout the AO crystalloid. The advantage of location of catalase at the edges of the AO crystalloids for growth of the organism on methanol is discussed.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demonstrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

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.     

Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana

Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.     

Cell infrastructure and some enzyme activities in rice coleoptiles grown in air and in anoxia

The rice ( Oryza saliva L. cv. S-6) cells in anaerobic coleoptiles maintained their ultra-structure. Most of the organelles did not show significant changes as compared to those from aerobic tissues. However, the number of mitochondria was reduced by 34% and they showed enlarged cristae. Most affected were unspecialized micro-bodies: Their number was reduced by 80% under anaerobiosis and both matrix and membrane structure appeared altered. The activities of the unspecialized microbody enzymes, glycolate oxidase (EC 1.1.3.1), urate oxidase (EC 1.7.3.3) and catalase (EC 1.11.1.6) were alt reduced by anoxia. Catalase decreased to the same extent as the number of microbodies.