Import of in-vitro-synthesized glyoxysomal malate dehydrogenase into isolated watermelon glyoxysomes

Glyoxysomal malate dehydrogenase (gMDH; EC 1.1.1.37) is synthesized by a reticulocyte system in the presence of watermelon mRNA (Citrullus vulgaris Schrad., var. Kleckey's Sweet No 6) as a cytosolic, higher-molecular-weight precursor (41 kdalton). We now show that this precursor is posttranslationally sequestered by a crude glyoxysomal fraction or by glyoxysomes purified on a Percoll^R gradient to a proteolytically protected form (60 min proteinase-K treatment at 4° C) with the size of the gMDH subunit (33 kdalton). In the presence of buffer instead of organelles a complete degradation of the precursor is obtained. The in-vitro organelle import, however, depends upon the presence of proteases such as proteinase K or trypsin. After short proteolytic treatments (e.g. 10 min proteinase K at 4° C), the correct processing of the MDH precursor is obtained even in the absence of organelles. This product, however, is not sequestered in vitro to a protease-resistant form by glyoxysomes. The possibility is discussed that under in-vivo conditions pre-gMDH is processed on the outside of the glyoxysomal membrane and transferred immediately after processing into the organelle presumably as a gMDH monomer followed by refolding and dimerization.Abbreviations gMDH glyoxysomal malate dehydrogenase - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - TPCK-trypsin trypsin treated with l-1-tosylamide-2-phenylethyl chloromethyl ketone Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

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.     

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     

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     

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k _i=2.6 mmol · l^-1 for gCS, k _i=0.33 mmol · l^-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)⁺RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)⁺RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)⁺RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - gCS glyoxysomal citrate synthase - gMDH glyoxysomal malate dehydrogenase - k _i inhibitor constant - mCS mitochondrial citrate synthase - OAA oxaloacetate - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Fate of glyoxysomal malate dehydrogenase from watermelon after heterologous in-vivo translation in xenopus oocytes

Total poly A⁺-mRNA from watermelon cotyledons was translated in Xenopus laevis oocytes. Watermelon glyoxysomal malate dehydrogenase was found as its higher molecular weight precursor (pre-gMDH) and accumulates over at least 48 hours of translation. Organelle separation and immunocytochemistry located the watermelon pre-gMDH in the cytosol of the oocyte. The heterologous translation product from oocytes can be imported into isolated glyoxysomes from endosperm of castor bean. This import was correct in terms of protection against proteolysis and cleavage of the presequence within the limits of accuracy. We conclude that watermelon pre-gMDH accumulates in mRNA-injected oocytes as an import competent cytosolic precursor.     

The uptake of acylated anthocyanin into isolated vacuoles from a cell suspension culture of Daucus carota

Anthocyanin-containing vacuoles were isolated from protoplasts of a cell suspension culture of Daucus carota. The vacuoles were stable for at least 2 h as demonstrated by the fact that they showed no efflux of anthocyanin. The uptake of radioactively labelled anthocyanin was time-dependent with a pH optimum at 7.5, and could be inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. Furthermore, the transport was specific, since vacuoles from other plant species showed no uptake of labelled anthocyanin, and strongly depended on acylation with sinapic acid, as deacylated glycosides were not taken up by isolated vacuoles. Hence, it is suggested that the acylation of anthocyanin, which is also required for the stabilization of colour in vacuoles, is important for transport, and that acylated anthocyanin is transported by a selective carrier and might be trapped by a pH-dependent conformational change of the molecule inside the acid vacuolar sap.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - PVP polyvinylpyrrolidone - TLC thin-layer chromatography     

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)     

Expression of a single gene encoding microbody NAD-malate dehydrogenase during glyoxysome and peroxisome development in cucumber

A full-length cDNA clone encoding microbody NAD⁺-dependent malate dehydrogenase (MDH) of cucumber has been isolated. The deduced amino acid sequence is 97% identical to glyoxysomal MDH (gMDH) of watermelon, including the amino terminal putative transit peptide. The cucumber genome contains only a single copy of this gene. Expression of this mdh gene increases dramatically in cotyledons during the few days immediately following seed imbibition, in parallel with genes encoding isocitrate lyase (ICL) and malate synthase (MS), two glyoxylate cycle enzymes. The level of MDH, ICL and MS mRNAs then declines, but then MDH mRNA increases again together with that of peroxisomal NAD⁺-dependent hydroxypyruvate reductase (HPR). The mdh gene is also expressed during cotyledon senescence, together with hpr, icl and ms genes. These results indicate that a single gene encodes MDH which functions in both glyoxysomes and peroxisomes. In contrast to icl and ms genes, expression of the mdh gene is not activated by incubating detached green cotyledons in the dark, nor is it affected by exogenous sucrose in the incubation medium. The function of this microbody MDH and the regulation of its synthesis are discussed.     

Purification and Characterization of Glyoxysomal Enzymes from Germinating Pumpkin Cotyledons

Four glyoxysomal enzymes, malate synthase, malate dehydrogenase,3-hydroxyacyl-CoA dehydrogenase and citrate synthase, were purifiedfrom glyoxysomes of germinating pumpkin cotyledons. Molecularweights of their subunits were as follows: malate synthase,60,000; malate dehydrogenase, 33,000; 3-hydroxyacyl-CoA dehydrogenase,72,000 and citrate synthase, 45,000. Malate synthase and 3-hydroxyacyl-CoAdehydrogenase activities were exclusively localized in glyoxysomes,whereas malate dehydrogenase and citrate synthase activitieswere found in both glyoxysomes and mitochondria. Monospecificantibodies against malate dehydrogenase and citrate synthaseinhibited their activities present in glyoxysomes but in mitochondria.Immunocytochemical analysis using the protein A-gold techniquecombined with Lowicryl K4M embedding showed that the antigenicsites for these enzymes were found exclusively in glyoxysomes.These data indicates that malate dehydrogenase and citrate synthasepresent in glyoxysomes are immunologically different from thosein mitochondria, respectively. ¹ This is paper No. 9 in the series "Analytical Studies on MicrobodyTransition". ³ Present address: Meiji Institute of Health Science, Naruta,Odawara, Kanagawa 250, Japan. ⁵ Present address: Department of Biology, Faculty of Science,Kobe University, Rokkoudai, Nada, Kobe 657, Japan. (Received December 23, 1987; Accepted January 27, 1988)     

Electron transport in purified glyoxysomal membranes from castor-bean endosperm

Glyoxysomes isolated from castor-bean (Ricinus communis L.) endosperm were treated with water, 0.2 M KCl, 1 M KCl, or 0.1 M Na₂CO₃. Glyoxysomal sacs, i.e. membranes which retained some visible matrix, resulted from the treatments with water and KCl. Glyoxysomal ghosts, i.e. intact membranes free of matrix, were only obtained following treatment with carbonate. The ghosts were free of activities of matrix enzymes, particularly palmitoyl-CoA oxidation, isocitrate dehydrogenase (EC 1.1.1.42) and isocitrate lyase (EC 4.1.3.1), and contained only negligible amounts of malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37), -hydroxyacyl-CoA dehydrogenase (EC 1.1.1.98) and catalase (EC 1.11.1.6). Distribution and appearance of membrane-associated particles in the protoplasmic and ectoplasmic faces of freeze-fracture replicas of the glyoxysomal membrane were the same in intact tissue, isolated glyoxysomes, and ghosts. Membranes purified by treatment with 0.2 M KCl or 0.1 M carbonate catalyzed the reduction of cytochrome-c when NADH or NADPH was provided as the electron donor. -Oxidation, localized in the matrix, could be linked to reduction of cytochrome-c or ferricyanide when purified membranes were combined with the matrix supernatant. Cytochrome-c could also be reduced by coupling enzyme activities in the matrix, NADP-isocitrate dehydrogenase or malate dehydrogenase, with those of the membrane. These results indicate that electrons from -oxidation, malate oxidation or isocitrate oxidation can be transferred directly to the redox components of the glyoxysomal membrane. We, therefore, conclude that any NADH and NADPH formed by enzymes in the matrix can be recycled continuously within the organelle.Abbreviations EF ectoplasmic face - ER endoplasmic reticulum - PF protoplasmic face     

Fluorescence immunohistochemical localization of malate dehydrogenase isoenzymes in watermelon cotyledons : a developmental study of glyoxysomes and mitochondria

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination.     

cDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons

A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a gt11 cDNA library constructed from the poly(A)⁺ RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal signals that target proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.     

Purification and comparative properties of microsomal and glyoxysomal malate synthase from castor bean endosperm

Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent K_m for acetyl-CoA of 10 μm and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (K_i 110 μm) and glycolate (K_i 150 μm) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating ³⁵S-labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [³⁵S]methionine-labeled endosperm tissue.     

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.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

The activity of powdery-mildew haustoria after feeding the host cells with different sugars,as measured with a potentiometric cyanine dye

The biotrophic parasite Erysiphe graminis f. sp. hordei produces haustoria within the cells of its host Hordeum vulgare. To determine the physiological activity of these haustoria, the electric potential across the membranes in the mitochondria of the haustorium was studied. The membrane potential was estimated with the fluorescent potentiometric cyanine dye 3,3-dibutyloxacarbocyanine iodide. The addition of depolarizing agents (carbonylcyanide m-chlorophenylhydrazone, 2,4-dinitrophenol or KCN) to infected cells resulted in an increase of fluorescence after the addition of low concentrations or a decrease of fluorescence after the addition of higher concentrations. When the infected host cell was fed with increasing concentrations of d-glucose (25, 50, 75 mM), corresponding decreases of fluorescence were measured immediately in the mitochondria of the fungal haustoria. Sucrose induced a similar reduction of fluorescence about 20 min late. d-Galactose and d-fructose induced a somewhat smaller reduction of fluorescence, l-glucose and d-glucitol had no effect. The results indicate that haustoria take up glucose from the host cells immediately. Sucrose, d-galactose and d-fructose seem to require time to be metabolized before their products reach the fungal haustorium or mitochondria.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DiOC₄(3) 3,3-dibutyloxacarbocyanine iodide - DNP 2,4-dinitrophenol     

Uptake of [14C]sucrose in isolated minor-vein networks of Commelina benghalensis L

Maceration with pectinase (4.5h) of Commelina benghalensis L. leaves stripped at either side yielded isolated vein networks consisting of four to five secondary veins and tertiary cross veins (=minor veins). Examination with Evans Blue and injection of Fluorescein F showed that 80% of the veins were viable. Proof of normal functioning of isolated minor veins was that [¹⁴C]sucrose fed to an apical vein network attached to the remaining intact part of the leaf was absorbed and finally arrived in the petiole. Sucrose uptake by veins obeyed Michaelis-Menten kinetics (K _m 5·10^-4 mol l^-1; V _max (light) 3.2 mol h^-1 g^-1 fresh weight, V _max (dark) 1.5 mol h^-1 g^-1 fresh weight). A linear component, not inhibited by carbonylcyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid, was present. Maximal uptake took place at 5 mmol l^-1 K⁺; concentrations of K⁺ higher than 10 mmol l^-1 decreased the rate of uptake. The uptake rates by isolated veins and veins in situ (in disks) were in the same order of magnitude. Altogether, isolated veins promise to be a useful system for the study of loading.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediamine tetraacetic acid - PCMBS p-chloromercuribenzenesulfonic acid     

Influence of cell turgor on sucrose partitioning in potato tuber storage tissues

Sucrose uptake and partitioning in potato (Solanum tuberosum L.) tuber discs were examined under a range of mannitol and ethylene-glycol concentrations. Mannitol caused the same changes in turgor over a wide range of incubation periods (90 min-6 h), indicating that it did not penetrate the tissue. In comparison, ethylene glycol reduced turgor losses but did not eliminate them, even after 6 h. Between 100 mM and 300 mM mannitol, turgor fell by 350 kPa, compared with 35 kPa in ethylene glycol. Uptake experiments in mannitol alone showed that total sucrose uptake was strongly correlated with both osmotic potential and with turgor potential. In subsequent experiments sucrose uptake and partitioning were examined after 3 h equilibration in 100 mM and 300 mM concentrations of mannitol and ethylene glycol. Total sucrose uptake and the conversion of sucrose to starch were enhanced greatly only at 300 mM mannitol, indicating an effect of turgor, rather than osmotic potential on sucrose partitioning. The inhibitors p-chloromercuribenzenesulfonic acid and carbonylcyanide m-chlorophenylhydrazone (CCCP) both reduced sucrose uptake, but in quite different ways. p-Chloromercuribenzenesulfonic acid reduced total sucrose uptake but did not affect the partitioning of sucrose to starch. By contrast, CCCP inhibited total uptake and virtually eliminated the conversion of sucrose to starch. Despite this, sucrose uptake in the presence of CCCP continued to increase as the mannitol concentration increased, indicating an increase in passive transport at higher mannitol concentrations. Increased sucrose uptake above 400 mM mannitol was shown to be the result of uptake into the free space. The data show that starch synthesis is optimised at low but positive turgors and the relation between sucrose partitioning and the changing diurnal water relations of the tuber are discussed.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid     

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863–871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.). Received: 20 December 1997 / Accepted: 18 March 1998