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.     

Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor to glyoxysomal citrate synthase.

Glyoxysomal citrate synthase in pumpkin is synthesized as a precursor that has a cleavable presequence at its N-terminal end. To investigate the role of the presequence in the transport of the protein to the microbodies, we generated transgenic Arabidopsis plants that expressed beta-glucuronidase with the N-terminal presequence of the precursor to the glyoxysomal citrate synthase of pumpkin. Immunogold labeling and cell fractionation studies showed that the chimeric protein was transported into microbodies and subsequently was processed. The chimeric protein was transported to functionally different microbodies, such as glyoxysomes, leaf peroxisomes, and unspecialized microbodies. These observations indicated that the transport of glyoxysomal citrate synthase is mediated by its N-terminal presequence and that the transport system is functional in all plant microbodies. Site-directed mutagenesis of the conserved amino acids in the presequence caused abnormal targeting and inhibition of processing of the chimeric protein, suggesting that the conserved amino acids in the presequence are required for recognition of the target or processing.     

Pumpkin malate synthase. Cloning and sequencing of the cDNA and northern blot analysis.

A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979-nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed.     

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.     

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.     

Glyoxysomal Malate Dehydrogenase in Pumpkin: Cloning of a cDNA and Functional Analysis of Its Presequence

Glyoxysomal malate dehydrogenase (gMDH) is an enzyme of theglyoxylate cycle that participates in degradation of storageoil. We have cloned a cDNA for gMDH from etiolated pumpkin cotyledonsthat encodes a polypep-tide consisting of 356 amino acid residues.The nucleotide and N-terminal amino acid sequences revealedthat gMDH is synthesized as a precursor with an N-terminal extrapeptide.The N-terminal presequence of 36 amino acid residues containstwo regions homologous to those of other micro-body proteins,which are also synthesized as large precursors. To investigatethe functions of the N-terminal presequence of gMDH, we generatedtransgenic Arabidopsis that expressed a chimeric protein consistingof rß-glucuroni-dase and the N-terminal region ofgMDH. Immunologi-cal and immunocytochemical studies revealedthat the chimeric protein was imported into microbodies suchas gly-oxysomes and leaf peroxisomes and was then subsequentlyprocessed. Site-directed mutagenesis studies showed that theconserved amino acids in the N-terminal presequence, Arg-10and His-17, function as recognition sites for the targetingto plant microbodies, and Cys-36 in the presequence is responsiblefor its processing. These results correspond to those from theanalyses of glyoxysomal citrate synthase (gCS), which was alsosynthesized as a large precursor, suggesting that common mechanismsthat can recognize the targeting or the processing of gMDH andgCS function in higher plant cells. (Received July 10, 1997; Accepted November 22, 1997)     

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.     

Thiolase mRNA translated in vitro yields a peptide with a putative N-terminal presequence

Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48 539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.     

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.     

The malate synthase gene of cucumber

The complete sequences of a full-length cDNA clone and a genomic clone encoding the Cucumis sativus glyoxysomal enzyme malate synthase, have been determined. The sequences have enabled us to identify putative control regions at the 5 end of the gene, three introns, and possible alternative polyadenylation sites at the 3 end. The deduced amino acid sequence predicts a polypeptide of 64961 molecular weight, which has 48% identity with that of Escherichia coli. Comparison of the sequence of malate synthase from cucumber with that from E. coli and with other glyoxysomal and peroxisomal enzymes, shows that a conserved C-terminal tripeptide is a common feature of those enzymes imported into microbodies.     

A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein.

A plant cysteine endopeptidase with a molecular mass of 35 kD was purified from microbodies of germinating castor bean (Ricinus communis) endosperm by virtue of its capacity to specifically process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro. Processing of the glyoxysomal malate dehydrogenase precursor occurs sequentially in three steps, the first intermediate resulting from cleavage after arginine-13 within the presequence and the second from cleavage after arginine-33. The endopeptidase is unable to remove the presequences of prethiolases from rape (Brassica napus) glyoxysomes and rat peroxisomes at the expected cleavage site. Protein sequence analysis of N-terminal and internal peptides revealed high identity to the mature papain-type cysteine endopeptidases from cotyledons of germinating mung bean (Vigna mungo) and French bean (Phaseolus vulgaris) seeds. These endopeptidases are synthesized with an extended pre-/prosequence at the N terminus and have been considered to be processed in the endoplasmic reticulum and targeted to protein-storing vacuoles.     

Peroxisomal enzyme activities in attached senescing leaves

Recently it has been demonstrated that detached leaves show glyoxysomal enzyme activities when incubated in darkness for several days. In this report glyoxylate-cycle enzymes have been detected in leaves of rice (Oryza sativa L.) and wheat (Triticum durum L.) from either naturally senescing or dark-treated plants. Isolated peroxisomes of rice and wheat show isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2) and -oxidation activities. Leaf peroxisomes from dark-induced senescing leaves show glyoxylic-acid-cycle enzyme activities two to four times higher than naturally senescing leaves. The glyoxysomal activities detected in leaf peroxisomes during natural foliar senescence may represent a reverse transition of the peroxisomes into glyoxysomes.This work was supported by CNR Italy, special grant RAISA, subproject 2, paper no. 26.     

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.

     

Ontogeny of microbodies (glyoxysomes) in cotyledons of dark-grown watermelon (Citrullus vulgaris Schrad.) seedlings

The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT catalase - DAB 3,3 diaminobenzidine tetrahydrochloride - ER endoplasmic reticulum - GOX glycolate oxidase - HPR hydroxypyruvate reductase - HVEM high-voltage electron microscopy - ICL isocitrate lyase - MS malate synthase - RER rough endoplasmic reticulum In the figures bars represent 0.1 m (if not stated otherwise)     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy

Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function. We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A- gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.     

Sequence homologies between glyoxysomal and mitochondrial malate dehydrogenase

The comparison of mitochondrial and glyoxysomal malate dehydrogenase (EC 1.1.1.37) from cotyledons of germinating watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) by means of serological methods and peptide patterns revealed a high degree of homology. The N-terminal sequence analysis yielded a distinct presequence of eight or nine amino-acid residues, respectively, which is followed by an almost identical stretch of at least 20 amino-acid residues. A very similar domain has been recognized for mitochondrial malate dehydrogenase from porcine heart and yeast, and for Escherichia coli malate dehydrogenase.Abbreviations gMDH glyoxysomal malate dehydrogenase - mMDH mitochondrial malate dehydrogenase - SDS sodium dodecyl sulfate     

Effect of light on the development of glyoxysomal functions in the cotyledons of mustard (Sinapis alba L.) seedlings

The degradation of storage fat in the cotyledons of mustard seedlings is unaffected by phytochrome and photosynthesis (irradiation with continuous red or far-red light from sowing of the seeds) although light imposes a strong constraint on the translocation of organic matter from the cotyledons into the seedling axis. Likewise, the development and disappearance of glyoxysomal enzyme activities (isocitrate lyase, malate synthase, citrate synthase) takes place independently of light. It is concluded that the mobilization of storage fat (fatcarbohydrate transformation) is independent of photomorphogenesis. The surplus of carbohydrate produced from fat in the light seems to be converted to starch grains in the plastids, which function as a secondary storage pool in the cotyledons.Abbreviations CS citrate synthase - ICL isocitrate lyase - MS malate synthase