Development of Microbody Membrane Proteins during the Transformation of Glyoxysomes to Leaf Peroxisomes in Pumpkin Cotyledons

The microbody transition observed in the cotyledons of somefatty seedlings involves the conversion of glyoxysomes to leafperoxisomes. To clarify the molecular mechanisms underlyingthe microbody transition, we established a method for the preparationof highly purified microbodies. SDS-PAGE and immunoblot analysisof isolated microbodies from pumpkin cotyledons at various stagesshowed that glyoxysomal enzymes are replaced by leaf-peroxisomalenzymes during the microbody transition. Two proteins in glyoxysomalmembranes, with molecular masses of 31 kDa and 28 kDa, werenot solubilized from the membranes with 0.2 M KCl, an indicationthat these proteins are bound tightly with glyoxysomal membranes.Their polyclonal antibodies were raised against the respectivepurified protein. Immunoblot analysis of subcellular fractionsand immunogold analysis confirmed that these proteins were specificallylocalized on glyoxysomal membranes. Analysis of these membraneproteins during development revealed that the amounts of thesemembrane proteins decreased during the microbody transitionand that the large one was retained in leaf peroxisomes, whereasthe small one could not be found in leaf peroxisomes after completionof the microbody transition. The results clearly showed thatmembrane proteins in glyoxysomes change dramatically duringthe microbody transition, as do the enzymes in the matrix. ¹Present address: School of Agriculture, Nagoya University Chikusa,Nagoya, 464-01 Japan.     

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.     

Biosynthesis of a microbody matrix enzyme in greening cotyledons

Earlier work on microbody biosynthesis has shown that glyoxysomal and liver peroxisomal proteins synthesized in the cytosol are made as precursors which are then transferred into the organelles and processed. Here, it is demonstrated that the unprecessed precursor detected in the cytosol after protein synthesis in vivo for an enzyme at the transition stage between glyoxysomes and leaf peroxisomes is indistinguishable from the product of translation in vitro. It is assumed that the transfer of extraorganellarly made precursor across the glyoxysomal membranes is followed by processing of the precursor and oligomerization to the tetrameric or 16-meric form of the enzyme. Oligomerization was, however, also observed in a portion of the cytosolic form.     

Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons

As a step to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in pumpkin (Cucurbita sp. Amakuri Nankin) cotyledons, catalase was purified from glyoxysomes. The molecular weight of the purified catalase was determined to be 230,000 to 250,000 daltons. The enzyme was judged to consist of four identical pieces of the monomeric subunit with molecular weight of 55,000 daltons. Absorption spectrum of the catalase molecule gave two major peaks at 280 and 405 nanometers, showing that the pumpkin enzyme contains heme. The ratio of absorption at 405 and 280 nanometers was 1.0, the value being lower than that obtained for catalase from other plant sources. These results indicate that the pumpkin glyoxysomal catalase contains the higher content of heme in comparison with other plant catalase.

The immunochemical resemblance between glyoxysomal and leaf peroxisomal catalase was examined by using the antiserum specific against the purified enzyme preparation from pumpkin glyoxysomes. Ouchterlony double diffusion and immunoelectrophoretic analysis demonstrated that catalase from both types of microbodies cross-reacted completely whereas the immunotitration analysis showed that the specific activity of the glyoxysomal catalase was 2.5-fold higher than that of leaf peroxisomal catalase. Single radial immunodiffusion analysis showed that the specific activity of catalase decreased during the greening of pumpkin cotyledons.

     

Evidence that a Proliferation of the Endoplasmic Reticulum Precedes the Formation of Glyoxysomes and Mitochondria in Germinating Castor Bean Endosperm

Excised castor bean endosperm halves incubated with CDP-[Me-¹⁴C]cholineactively incorporated this compound into membrane phosphatidylcholine.The capacity of the tissue to synthesize phosphatidyl-[¹⁴C]cholineincreased during the first 3 d of germination and subsequentlydeclined. At the onset of germination phosphatidyl-[^l4C]cholinewas exclusively recovered in the ER membrane fraction. The rateof incorporation into the ER membranes increased strikinglyduring the first 24 h of germination while that into mitochondriaand glyoxysomes remained low. At later developmental stagesan increasing proportion of the newly synthesized phosphatidyl-[¹⁴C]cholinewas present in mitochondria and glyoxysomes; the rate of incorporationinto the membranes of these organelles increased while thatinto the ER membrane began to level off. The kinetics of CDP-[¹⁴C]cholineincorporation into membrane phosphatidylcholine of the majororganelle fractions of 3-d-old endosperm tissue showed thatthe ER was immediately labelled, whereas a lag period precededthe labelling of mitochondria and glyoxysomes. Assuming that the incorporation of CDP-[¹⁴C]choline into phosphatidylcholineserves as a reliable indicator of membrane synthesis, the resultsobtained suggest that a proliferation of ER membranes precedesthe formation of glyoxysomes and mitochondria in germinatingcastor bean endosperm. A comparison of developmental changesin (a) total ER and glyoxysomal phospholipid content and (b)ER and mitochondrial NADH cytochrome c reductase activity providedadditional evidence supporting this conclusion.     

Intraorganellar distribution of superoxide dismutase in plant peroxisomes (glyoxysomes and leaf peroxisomes)

The intraorganellar distribution of superoxide dismutase (SOD) (EC 1.15.1.1) in two types of plant peroxisomes (glyoxysomes and leaf peroxisomes) was studied by determinations of SOD latency in intact organelles and by solubilization assays with 0.2 molar KCl. Glyoxysomes were purified from watermelon (Citrullus vulgaris Schrad.) cotyledons, and their integrity, calculated on the basis of glyoxysomal marker enzymes, was about 60%. Under the same conditions, the latency of SOD activity determined in glyoxysomes was 40%. The difference between glyoxysomal intactness and SOD latency was very close to the percentage of isozyme Mn-SOD previously determined in glyoxysomes (LM Sandalio, LA Del Río 1987 J Plant Physiol 127: 395-409). In matrix and membrane fractions of glyoxysomes, SOD exhibited a solubilization pattern very similar to catalase, a typical soluble enzyme of glyoxysomes. The analysis of the distribution of individual SOD isozymes in glyoxysomal fractions treated with KCl showed that Cu,Zn-SOD II, the major SOD isozyme in glyoxysomes, was present in the soluble fraction of these organelles, whereas Mn-SOD was bound to the glyoxysomal membrane. These data in conjunction with those of latency of SOD activity in intact glyoxysomes suggest that Mn-SOD is bound to the external side of the membrane of glyoxysomes. On the other hand, in intact leaf peroxisomes where only a Mn-containing SOD is present (LM Sandalio, JM Palma, LA Del Río 1987 Plant Sci 51: 1-8), this isozyme was found in the peroxisomal matrix. The physiological meaning of SOD localization in matrix and membrane fractions of glyoxysomes and the possibility of new roles for plant peroxisomes in cellular metabolism related to activated oxygen species is discussed.     

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.     

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.     

Microbody defective mutants of arabidopsis

In germinating fatty seedlings, microbodies are differentiated to leaf peroxisomes from glyoxysomes during greening, and then transformed to glyoxysomes from leaf peroxisomes during senescence. These transformations of microbodies are regulated at various level, such as gene expression, splicing of the mRNA and degradation of microbody proteins. In order to clarify the regulatory mechanisms underlying these transformations of microbodies, we tried to obtain glyoxysome-deficient mutants of Arabidopsis. We screened 2,4-dichlorophenoxybutyric acid (2,4-DB) mutants of Arabidopsis which have defects in glyoxysomal fatty acid β-oxidation. Four mutants can be classified as carrying alleles at three independent loci, which we designatedped1, ped2, andped3, respectively (whereped stands for peroxisome defective). The characteristics of theseped mutants are described. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Evidence for the Presence of a Porin in the Membrane of Glyoxysomes of Castor Bean

Glyoxysomes of endosperm tissue of castor bean (Ricinus communis L.) seedlings were solubilized in a detergent and added to a lipid bilayer. Conductivity measurements revealed that the glyoxysomal preparation contained a porin-like channel. Using an electrophysiological method, which we established for semiquantitative determination of porin activity, we were able to demonstrate that glyoxysomal membranes purified by sucrose density gradient centrifugation contain an integral membrane protein with porin activity. The porin of glyoxysomes was shown to have a relatively small single-channel conductance of about 330 picosiemens in 1 M KCl and to be strongly anion selective. Thus, the glyoxysomal porin differs from the other previously characterized porins in the outer membrane of mitochondria or plastids, but is similar to the porin of spinach (Spinacia oleracea L.) leaf peroxisomes. Our results suggest that, in analogy to the porin of leaf peroxisomes, the glyoxysomal porin facilitates the passage of small metabolites, such as succinate, citrate, malate, and aspartate, through the membrane.     

A proteomic approach towards the identification of the matrix protein content of the two types of microbodies in Neurospora crassa

Microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. Here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and Woronin bodies, from Neurospora crassa and analyzed their protein content by HPLC/ESI‐MS/MS. In the purified Woronin bodies, we unambiguously identified only hexagonal 1 (HEX1), suggesting that the matrix is probably exclusively filled with the HEX1 hexagonal crystal. The proteomic analysis of highly purified glyoxysomes allowed the identification of 191 proteins. Among them were 16 proteins with a peroxisomal targeting signal type 1 (PTS1) and three with a PTS2. The collection also contained the previously described N. crassa glyoxysomal matrix proteins FOX2 and ICL1 that lack a typical PTS. Three PTS1 proteins were identified that likely represent the long sought glyoxysomal acyl‐CoA dehydrogenases of filamentous fungi. Two of them were demonstrated by subcellular localization studies to be indeed glyoxysomal. Furthermore, two PTS proteins were identified that are suggested to be involved in the detoxification of nitroalkanes. Since the glyoxysomal localization was experimentally demonstrated for one of these enzymes, a new biochemical reaction is expected to be associated with microbody function.     

Pumpkin peroxisomal ascorbate peroxidase is localized on peroxisomal membranes and unknown membranous structures

To investigate the roles of peroxisomal membrane proteins in the reversible conversion of glyoxysomes to leaf peroxisomes, we characterized several membrane proteins of glyoxysomes. One of them was identified as an ascorbate peroxidase (pAPX) that is localized on glyoxysomal membranes. Its cDNA was isolated by immunoscreening. The deduced amino acid sequence encoded by the cDNA insert does not have a peroxisomal targeting signal (PTS), suggesting that pAPX is imported by one or more PTS-independent pathways. Subcellular fractionation of 3- and 5-d-old cotyledons of pumpkin revealed that pAPX was localized not only in the glyoxysomal fraction, but also in the ER fraction. A magnesium shift experiment showed that the density of pAPX in the ER fraction did not increase in the presence of Mg(2+), indicating that pAPX is not localized in the rough ER. Immunocytochemical analysis using a transgenic Arabidopsis which expressed pumpkin pAPX showed that pAPX was localized on peroxisomal membranes, and also on a unknown membranous structure in green cotyledons. The overall results suggested that pAPX is transported to glyoxysomal membranes via this unknown membranous structure.     

The origin and turnover of organelle membranes in castor bean endosperm

The origin and turnover of organelle membranes in castor bean (Ricinus communis L. var. Hale) endosperm was examined using choline-¹⁴C as a phospholipid precursor. On sucrose gradients three major particulate fractions were separated; a light membranous fraction (density 1.11-1.13 gram per cm³), the mitochondria (1.18 gram per cm³), and the glyoxysomes (1.24 gram per cm³). Choline-¹⁴C was readily incorporated into lecithin in all three particulate fractions, but the light membranous fraction became labeled first. Incorporation continued into all three fractions for 6 hours, at which time the available choline-¹⁴C had been completely used. Subsequently, ¹⁴C was lost from the three components at distinctly different rates. When an excess of unlabeled choline was added after 1 hour (pulse-chase experiment), incorporation of choline-¹⁴C into glyoxysomes and mitochondria continued for three hours, but at a diminishing rate. This was followed by a period in which the ¹⁴C content of the mitochondria declined at a rate expected, if the half life of lecithin in the membrane were about 50 hours and that of the glyoxysomes 10 hours. These values are close to those calculated from the experiments in which no chase was used. The labeling in the light membrane fraction behaved differently from that of the mitochondria and glyoxysomes following the chase of unlabeled choline. Incorporation continued for only 1 additional hour, and then the ¹⁴C content declined sharply in the subsequent 4 hours. The early kinetics and subsequent interrelationships are those expected if the lecithin in the membranes of mitochondria and glyoxysomes originates in components of the light membrane fraction.     

Serological and developmental relationships between endoplasmic reticulum and glyoxysomal proteins of castor bean endosperm

Glyoxysomes isolated from the endosperm of castor bean (Ricinus communis L.) by sucrose density gradient centrifugation were fractionated into their matrix protein and membrane components. Antisera were raised in rabbits against both the matrix proteins and sodium dodecyl sulphate (SDS)-solubilized membrane proteins. SDS-polyacrylamide gel electrophoresis (PAGE) analysis established that such antisera precipitate all major polypeptide components present in their respective glyoxysomal mixedantigen preparations. Furthermore, when soluble constituents recovered from the microsomal vesicles or solubilized microsomal membranes were challenged with the appropriate glyoxysomal antiserum, serological determinants were again found to be present. Intact endosperm tissue was incubated with [³⁵S]methionine and the kinetics of ³⁵S-incorporation into protein recovered in immunoprecipitates when the glyoxysomal matrix fraction or the soluble fraction released from the microsomes were incubated with anti-glyoxysomal matrix serum were followed. [³⁵S]antigens rapidly appeared in the microsomal fraction whereas a lag period preceded their appearance in glyoxysomes. Interupting such kinetic experiments by the addition of an excess of unlabelled methionine resulted in a rapid decrease in the microsomal content of [³⁵S]antigens and a concomitant increase in glyoxysomal content.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ER endoplasmic reticulum     

The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm.

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.     

Superoxide free radicals are produced in glyoxysomes

The production of superoxide free radicals in pellet and supernatant fractions of glyoxysomes, specialized plant peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons, was investigated. Upon inhibition of the endogenous superoxide dismutase, xanthine, and hypoxanthine induced in glyoxysomal supernatants the generation of O₂⁻ radicals and this was inhibited by allopurinol. In glyoxysomal pellets, NADH stimulated the generation of superoxide radicals. Superoxide production by purines was due to xanthine oxidase, which was found predominantly in the matrix of glyoxysomes. The generation of O₂⁻ radicals in glyoxysomes by endogenous metabolites suggests new active oxygen-related roles for glyoxysomes, and for peroxisomes in general, in cellular metabolism.     

Untersuchungen zur Funktionsänderung der Microbodies in den Keimblättern von Helianthus annuus L.

Summary The enzyme patterns in sunflower cotyledons indicate that the glyoxysomal function of microbodies is replaced by the peroxisomal function of these organelles during the transition from fat degradation to photosynthesis. The separation of the microbody population into glyoxysomes and peroxisomes during this transition period is reported. The mean difference in density between the activity peaks of glyoxysomal and peroxisomal marker enzymes on a sucrose gradient was calculated to be 0.007±0.004 g/cm³ and turned out to be significant (t=7.8>4.04=t _5;0.01). The activity peak of catalase coincides with that of isocitrate lyase in early stages of development, but shifts to the activity peak of peroxisomal marker enzymes during the transition period. No isozymes of the catalase could be detected by gel electrophoresis in the microbodies with the two different functions.During the rise of the peroxisomal marker enzymes no synthesis of the common microbody marker, catalase, could be demonstrated using the inhibitor allylisopropylacetamide. Using D₂) for density labeling of newly-formed catalase, no difference is observed between the density of catalase from cotyledons grown on 99.8% D₂O during the transition period and the density of enzyme from cotyledons grown on H₂O. The activity of particulate glycolate oxidase is reduced 30–50% by allylisopropylacetamide, but is not affected by D₂O. The chlorophyll formation in the cotyledons is strongly inhibited by both substances.     

Kinetin action on the development of microbody enzymes in sunflower cotyledons in the dark

Summary Removal of the roots from etiolated sunflower seedlings (Helianthus annuus L.) at various stages of development resulted in a premature or enhanced decline of the activities of catalase (E.C. 1.11.1.6) and isocitrate lyase (E.C. 4.1.3.1) (glyoxysomes), and hydroxypyruvate reductase (E.C. 1.1.1.26) and glycolate oxidase (E.C. 1.1.3.1) (leaf peroxisomes) in the cotyledons. Treatment of the cuttings with kinetin in the dark inhibited the loss of glyoxysomal enzyme activities and, at the same time, induced a three to fivefold increase in leaf-peroxisomal enzyme activities. The decline of glyoxysomal enzyme activities was also suppressed after application of both kinetin and cycloheximide (50 g/ml). The kinetin-mediated rise of leaf-peroxisomal enzyme activities was strongly curtailed in the presence of cycloheximide. The dose effect curves of kinetin action on the development of glyoxysomal and of leaf-peroxisomal enzyme activities were different, supporting the hypothesis that the mechanisms of kinetin action on the two microbody enzyme systems are different. Nitrogen nutrition of intact seedling effected the development of microbody enzyme activities in a pattern closely resembling that of kinetin action. Presumably endogenous cytokinins produced by the roots are involved in the regulation of microbody enzyme activities in cotyledons of dark-grown sunflower seedlings.