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R N Trelease

Publications and source records attributed to R N Trelease.

At least 37 records · Page 2Linked to original sources
PubMed1991

Acquisition of membrane lipids by differentiating glyoxysomes: role of lipid bodies.

Glyoxysomes in cotyledons of cotton (Gossypium hirsutum, L.) seedlings enlarge dramatically within 48 h after seed imbibition (Kunce, C.M., R.N. Trelease, and D.C. Doman. 1984. Planta (Berl.). 161:156-164) to effect mobilization of stored cotton-seed oil. We discovered that the membranes of enlarging glyoxysomes at all stages examined contained a large percentage (36-62% by weight) of nonpolar lipid, nearly all of which were triacylglycerols (TAGs) and TAG metabolites. Free fatty acids comprised the largest percentage of these nonpolar lipids. Six uncommon (and as yet unidentified) fatty acids constituted the majority (51%) of both the free fatty acids and the fatty acids in TAGs of glyoxysome membranes; the same six uncommon fatty acids were less than 7% of the acyl constituents in TAGs extracted from cotton-seed storage lipid bodies. TAGs of lipid bodies primarily were composed of palmitic, oleic, and linoleic acids (together 70%). Together, these three major storage fatty acids were less than 10% of both the free fatty acids and fatty acids in TAGs of glyoxysome membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) constituted a major portion of glyoxysome membrane phospholipids (together 61% by weight). Pulse-chase radiolabeling experiments in vivo clearly demonstrated that 14C-PC and 14C-PE were synthesized from 14C-choline and 14C-ethanolamine, respectively, in ER of cotyledons, and then transported to mitochondria; however, these lipids were not transported to enlarging glyoxysomes. The lack of ER involvement in glyoxysome membrane phospholipid synthesis, and the similarities in lipid compositions between lipid bodies and membranes of glyoxysomes, led us to formulate and test a new hypothesis whereby lipid bodies serve as the dynamic source of nonpolar lipids and phospholipids for membrane expansion of enlarging glyoxysomes. In a cell-free system, 3H-triolein (TO) and 3H-PC were indeed transferred from lipid bodies to glyoxysomes. 3H-PC, but not 3H-TO, also was transferred to mitochondria in vitro. The amount of lipid transferred increased linearly with respect to time and amount of acceptor organelle protein, and transfer occurred only when lipid body membrane proteins were associated with the donor lipid bodies. 3H-TO was transferred to and incorporated into glyoxysome membranes, and then hydrolyzed to free fatty acids. 3H-PC was transferred to and incorporated into glyoxysome and mitochondria membranes without subsequent hydrolysis. Our data are inconsistent with the hypothesis that ER contributes membrane lipids to glyoxysomes during postgerminative seedling growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Transport↗
PubMed1991

Intracellular localization of phosphatidylcholine and phosphatidylethanolamine synthesis in cotyledons of cotton seedlings.

Subfractionation of clarified cotyledon homogenates of cotton (Gossypium hirsutum L.) seedlings on sucrose gradients revealed a single coincident peak of cholinephosphotransferase (EC 2.7.8.2) (CPT) and ethanolaminephosphotransferase (EC 2.7.8.1) (EPT) activities, which equilibrated with the main peak of Antimycin A-insensitive NADH:cytochrome c reductase (CCR) activity. The small percentage of CPT and EPT activities (less than 5% of the total) in glyoxysome-enriched pellets equilibrated with cytochrome c oxidase activity, not with catalase activity. Preincubation of microsomes (containing 83% of total CPT and EPT activities) in 0.2 millimolar MgCl(2) followed by subfractionation on sucrose gradients resulted in peak CPT and EPT activities equilibrating with peak CCR activity at 24% (w/w) sucrose. Preincubation of microsomes with (14)C-CDPcholine (or (14)C-CDPethanolamine) resulted in synthesis and incorporation of (14)C-phosphatidylcholine (PC) (or (14)C-phosphatidylethanolamine, PE) into membranes at the same density. Increasing the Mg(2+) concentration to 2.0 millimolar facilitated binding of ribosomes and caused a concomitant shift in density (to 34% w/w sucrose) of peak CPT, EPT, and CCR activities. Under these conditions, newly synthesized and incorporated (14)C-PC (or PE) was recovered in these membranes. Transmission electron microscopy of this fraction confirmed binding of ribosomes to membranes. Radiolabeling in vivo of cotyledons with [methyl-(14)C] choline chloride or [1,2 ethanolamine-(14)C] ethanolamine hydrochloride resulted in a linear incorporation of radiolabel into PC or PE in a time dependent manner. Subfractionation of homogenates of radiolabeled cotyledons on sucrose gradients showed that membranes sedimenting at 24% (w/w) sucrose (ER) contained the majority of radiolabeled PC and PE with a minor peak at 40% (w/w) sucrose (mitochondria), but no radioactive PC or PE was recovered in glyoxysomes. These results indicate that ER in cotyledons of germinated cotton seedlings is the primary subcellular site of PC and PE synthesis. This is similar to the situation in endosperm tissue but distinctly different from root and hypocotyl tissue where Golgi are a major subcellular site of PC and PE synthesis.

Journal Article↗
PubMed1990

Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism.

Blotting, Western↗
PubMed1990

Characterization of a cDNA encoding cottonseed catalase.

A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

Amino Acid Sequence↗
PubMed1990

Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase.

A cDNA clone encoding the glyoxysomal enzyme isocitrate lyase (ICL) (EC 4.1.3.1) was isolated from a library prepared from cotton (Gossypium hirsutum L.) cotyledon poly(A)+ RNA. The clone is 1893 basepairs (bp) in length and contains a 1728 bp open reading frame encoding a polypeptide of 576 residues (Mr = 64,741). The deduced amino acid sequence of cotton ICL is 85.2%, 90.3% and 41.1% identical to ICL from rapeseed, castor bean and E. coli, respectively. Cotton ICL has a C-terminal tripeptide of A-R-M which is a putative trafficking signal for peroxisome (glyoxysome) proteins.

Amino Acid Sequence↗
PubMed1990

Development and regulation of three glyoxysomal enzymes during cotton seed maturation and growth.

The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton (Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s).

Blotting, Western↗
PubMed1990

Biogenesis of catalase in glyoxysomes and leaf-type peroxisomes of sunflower cotyledons.

Eight charge isoforms of catalase (EC 1.11.1.6.) appeared in the peroxisomes of sunflower cotyledons during growth after germination (2.5 days of dark, continuous light thereafter). In the light, when glyoxysomes were transformed to leaf-type peroxisomes, the five more-basic forms (CAT 1 through CAT 5) became more prominent, while amounts of the three more-acidic forms (CAT 6 through CAT 8) decreased considerably. The isoforms CAT 1 through CAT 5 were hybrids of 55- and 59-kDa subunits, whereas CAT 6 through CAT 8 contained 55-kDa subunits exclusively. The catalase translation products changed during the transition of glyoxysomes to leaf-type peroxisomes. Polyadenylated RNA from 2-day-old cotyledons directed synthesis of 56-kDa subunits, whereas 59-kDa subunits predominated after in vitro translation of RNA from 4-day-old cotyledons. Both translation products were processed to lower molecular weight forms in vivo. The 56-kDa translation products were precursors for 55-kDa subunits in glyoxysomes. It could not be decided however, whether the 59-kDa precursors were processed to 56-kDa or 55-kDa subunits, because both subunits of lower molecular weight were present in leaf-type peroxisomes. Some of the 59-kDa precursors escaped proteolytic processing and formed hybrid isoforms (CAT 1 through CAT 5) with mature 55-kDa subunits. This type of isoform formation, i.e., condensation of mature and unprocessed subunits, has not yet been described for other plant catalases. In summary, the results showed that the postgerminative changes in the number and abundance of catalase isoforms resulted from changes in translation (transcription) of catalase precursors and assembly of proteolytically processed and unprocessed subunits into tetramers within peroxisomes acquiring leaf peroxisomal function.

Catalase↗
PubMed1990

Inhibition of Cottonseed Choline- and Ethanolaminephosphotransferases by Calcium during Postgerminative Growth.

Activities of choline- and ethanolaminephosphotransferase (CPT and EPT) were reproducibly high in microsomes from imbibed seeds of cotton (Gossypium hirsutum, L.). Initial studies showed that both activities dramatically declined during postgerminative growth when demand for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesis was high. Addition of CaCl(2) (0.1 millimolar) or aliquots of supernatant fractions (150,000g, 60 minutes) from cotyledons of 48-hour-old seedlings to imbibed-seed microsomes reduced the CPT and EPT activities to levels approximating those found in 48-hour microsomes. Inhibition by supernatants was completely reversed by adding EGTA (1.0 millimolar), but not by boiling the supernatants. EGTA (1.0 or 5.0 millimolar) relieved inhibition in cellular fractions whether it was added to the homogenization media or the assay reaction mixtures. A time course of CPT and EPT activities in cellular fractions prepared with 1.0 millimolar EGTA showed that activities were well developed in imbibed seeds, doubled coincidentally to a peak at 36 hours, then declined during the next 12 hours to levels approximating those in imbibed seeds. Greater than 90% of the CPT and EPT activities were pelletable (150,000g, 60 minutes) at all ages examined. Calcium apparently was artificially released upon homogenization, to a progressively greater extent in older cotyledons, and severely inhibited CPT and EPT activities. This is the only time course of CPT and EPT activities reported for cotyledons of any oilseed; it is substantially different from that in oil-storing endosperm.

Journal Article↗
PubMed1989

Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum.

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.

Journal Article↗
PubMed1988

Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase.

As part of our research on peroxisome biogenesis, catalase was purified from cotyledons of dark-grown cotton (Gossypium hirsutum L.) seedlings and monospecific antibodies were raised in rabbits. Purified catalase appeared as three distinct electrophoretic forms in non-denaturing gels and as a single protein band (with a subunit Mr of 57,000) on silver-stained SDS/polyacrylamide gels. Western blots of crude extracts and isolated peroxisomes from cotton revealed one immunoreactive polypeptide with the same Mr (57,000) as the purified enzyme, indicating that catalase did not undergo any detectable change in Mr during purification. Synthesis in vitro, directed by polyadenylated RNA isolated from either maturing seeds or cotyledons of dark-grown cotton seedlings, revealed a predominant immunoreactive translation product with a subunit Mr of 57,000 and an additional minor immunoreactive product with a subunit Mr of 64000. Labelling studies in vivo revealed newly synthesized monomers of both the 64000- and 57,000-Mr proteins present in the cytosol and incorporation of both proteins into the peroxisome without proteolytic processing. Within the peroxisome, the 57,000-Mr catalase was found as an 11S tetramer; whereas the 64,000-Mr protein was found as a relatively long-lived 20S aggregate (native Mr approx. 600,000-800,000). The results strongly indicate that the 64,000-Mr protein (catalase?) is not a precursor to the 57,000-Mr catalase and that cotton catalase is translated on cytosolic ribosomes without a cleavable transit or signal sequence.

Catalase↗
PubMed1988

Regulation of isocitrate lyase gene expression in sunflower.

A cDNA sequence that encodes a portion of sunflower (Helianthus annuus L.) seedling isocitrate lyase was selected from a lambda gt11 cDNA library derived from sunflower seedling cotyledon poly(A)(+) messenger RNA. The library was screened for bacteriophage recombinants that expressed antigens which reacted with antisera directed against cotton seed isocitrate lyase. The isolated cDNA hybridized with a 2 kilobase RNA species that was first detectable in maturing sunflower embryos 19 days after flowering and remained at a constant low level through seed desiccation. The prevalence of this transcript in sunflower cotyledons increased by about 10-fold within 2 days after inhibition in darkness, and transcript levels began to decrease by 5 days after imbibtion. During the first 2 days of germination and growth of sunflower seedlings in light, the rate of isocitrate lyase mRNA accumulation was greater than the rate observed during this period in dark-grown seedlings, giving peak levels about 2-fold higher than corresponding levels in dark-grown seedlings. Illumination of seedlings also promoted an earlier, and more rapid decline of isocitrate lyase transcripts. Peak levels of isocitrate lyase mRNA preceded a corresponding peak in immunologically detectable isocitrate lyase polypeptides by about 24 hours. Isocitrate lyase expression in sunflower cotyledons is developmentally regulated and is modulated, in seedlings, by exposure to light. Mechanisms that control these processes appear to function primarily at the level of mRNA accumulation and are likely to involve changes in transcription rates and/or mRNA stability.

Journal Article↗
PubMed1987

Cottonseed malate synthase : purification and immunochemical characterization.

Malate synthase (EC 4.1.3.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose). Large-scale preparation of glyoxysomes, a main step in all other published procedures, was not involved. The purified enzyme and that extracted from glyoxysomes appears to be a dodecamer with a native molecular weight of 750,000 (sedimentation coefficient of >20 Svedberg units [S] on sucrose gradients) composed of identical subunits (molecular weight approximately 63,000). The monomer (5S) occurs in the cytosol. Polyclonal antibodies raised in rabbits were judged to be monospecific for malate synthase by immunotitration, double immunodiffusion, and western blotting. Double immunodiffusion experiments revealed only partial immunological identity between the 5S (cytosolic) and 20S (glyoxysomal forms, although complete identity was observed between the 5S form in immature and germinated seeds, and the 20S form in immature and germinated seeds. Cross-reactivity of the cotton antimalate synthase serum was observed with extracts from five other oilseeds. Western blot analyses showed that malate synthase protein was not present in immature seeds prior to appearance of enzyme activity, but when present, subunit molecular weight was indistinguishable in immature, desiccated, and germinated seeds.

Journal Article↗
PubMed1987

Cottonseed malate synthase : biogenesis in maturing and germinated seeds.

The activity of malate synthase (MS) (EC 4.1.3.2) appears and increases during cotton (Gossypium hirsutum L.) seed maturation, persists through desiccation and imbibition, then increases again following germination. The research reported herein is a comparative study of the synthesis and acquisition of MS into glyoxysomes as they occur in maturing and germinated seeds. Rate-zonal centrifugation of cotyledon extracts revealed that the 5 Svedberg unit (S) cytosolic form of MS was the only form present at 42 days postanthesis (DPA) when activity was first detectable. At later stages (48 DPA, 0 day, 26 hours, and 48 hours), both the 5S and glyoxysomal 20S forms were present, with the 20S form becoming much more prevalent. Western blot analyses revealed that no other form(s) of MS were present in the phosphate-buffered gradients, and that 5S and 20S forms had the same subunit molecular weight in maturing and germinated seeds. Comparisons of radiospecific activity of MS immunoprecipitates following in vivo labeling with [(35)S]methionine for varying time intervals provided strong evidence for a 5S-precursor to 20S-product relationship during both seed maturation and seedling growth. Comparisons of MS labeled in vivo and in vitro in wheat germ and rabbit reticulocyte lysates programmed with poly(A)(+)RNA (from maturing and germinated seeds) revealed no detectable differences in subunit molecular weights. These results reinforced our other data indicating that MS was synthesized in the cytosol and acquired by glyoxysomes in both maturing and germinated cotton seeds without involvement of an intervening aggregate pool in the endoplasmic reticulum, or via processing of a cleavable precursor molecule. MS was translated from poly(A)(+)RNA extracted from 28 DPA cotton seeds. This was nearly 2 weeks before MS activity or protein was detected in vivo. This finding invites further study on the regulation of RNA transcripts during maturation.

Journal Article↗
PubMed1986

Heterogeneity of catalase in maturing and germinated cotton seeds.

To investigate possible charge and size heterogeneity of catalase (EC 1.11.1.6) in cotton (Gossypium hirsutum L. cv Deltapine 62), extracts of cotyledons from different developmental ages were subjected to nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Special precautions (e.g. fresh homogenates, reducing media) were necessary to prevent artefacts due to enzyme modification during extraction and storage. When the gels were stained for enzyme activity, two distinct electrophoretic forms of catalase were resolved in extracts of maturing and mature cotton seeds. In germinated seeds, three additional cathodic forms were detected revealing a total of five electrophoretic variants. In green cotyledons, the two anodic forms characteristic of ungerminated seeds were less active; whereas, the most cathodic form was predominant. All forms of catalase were found in isolated glyoxysomes. Corresponding electrophoretic patterns were found on Western blots probed with anticatalase serum; no immunoreactive, catalytically inactive forms were detected. Western blots of sodium dodecyl sulfate-polyacrylamide gels revealed only one immunoreactive (55 kilodaltons) polypeptide in cotton extracts of all developmental ages. Results from isoelectric focusing and Ferguson plots indicate that the electrophoretic variants of catalase are charge isomers with a molecular weight of approximately 230,000.

Journal Article↗
PubMed1981

Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination : IV. beta-OXIDATION.

Microbodies were isolated by zonal-rotor sucrose density gradient centrifugation from cotton (cv. DP 61) seeds at two distinct stages of embryogenesis (38 and 50 days after anthesis) and after 48 hours postgerminative growth. In all cases, beta-oxidation activity (palmitoyl-coenzyme A (CoA)-dependent reduction of acetylpyridine adenine dinucleotide or production of acetyl-CoA) and activities of the enzymes palmitate:CoA ligase, acyl-CoA oxidase, enoyl hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, plus catalase, were localized exclusively in the microbody fractions, i.e. none of the activities were associated with mitochondria. Acyl-CoA dehydrogenase activity could not be detected in any of the gradient fractions or in homogenates.Glyoxysomes isolated from cotyledons of 48-hour-germinated seeds were capable of beta-oxidation of acyl-CoAs of various chain lengths and degrees of unsaturation and were the sole site of 3-cis-2-trans enoyl-CoA isomerase activity. Direct measurement of the isomerase is the first demonstration of an enzyme required for unsaturated fatty acid catabolism in a higher plant. Palmitoyl-carnitine was not oxidized by any of the organelle fractions.Subfractionation of glyoxysomes by osmotic shock revealed that none of the beta-oxidation enzymes were tightly membrane-associated.

Journal Article↗
PubMed1981

Role of malate synthase in citric Acid synthesis by maturing cotton embryos: a proposal.

Cotton embryos from 34 to 54 days after anthesis were analyzed for organic acids, and enzymes associated with organic acid metabolism. During this developmental period, embryos accumulated citrate. Malate synthase activity appeared at 46 days after anthesis and increased rapidly to 54 days. Of other enzymes examined, only citrate synthase activity increased during this period. As isocitrate lyase activity was absent from cotton embryos during maturation, an alternative source of glyoxylate would be required for in vivo malate synthase activity. Of several metabolic sources tested, glycine was converted to glyoxylate via a transamination reaction.Halves of 50-day (mature) cotton embryos incorporated radioactivity from [1-(14)C]acetate, [1-(14)C]glyoxylate, and [1-(14)C]glycine into organic acids. Embryo halves incubated with [(14)C]glyoxylate plus [(3)H]acetate synthesized double-labeled malate and citrate. Radioactive citrate isolated from 50-day cotton embryos incubated with [1-(14)C]acetate was degraded; label was distributed as follows: 55% in C(1), 33% in C(5), and 12% in C(6). Taken together, these data strongly suggest participation of malate synthase in citrate production in vivo.Separation of organelles by sucrose density gradient sedimentation revealed that malate synthase, malate dehydrogenase, and citrate synthase were compartmentalized together only in the peroxisome fraction (1.24 grams per milliliter). Peroxisomes isolated from 50-day embryos, when incubated with glyoxylate and [(3)H]acetyl-CoA, synthesized labeled malate and citrate, but only radioactive citrate accumulated. Incubations with glycine plus alpha-ketoglutarate, in place of glyoxylate, also resulted in synthesis of radioactive citrate.A metabolic scheme illustrating the participation of cotton embryo peroxisomes in citrate synthesis is proposed. This scheme suggests a function for plant peroxisomes not previously elucidated. The ontogenetic and metabolic relationship between these organelles and glyoxysomes active in gluconeogenesis during postgerminative growth remains to be examined.

Journal Article↗
PubMed1981

Enzyme development and glyoxysome characterization in cotyledons of cotton seeds.

Unimbibed, mature cotton seeds (cv. Deltapine 61) were found to possess activity for all gluconeogenesis-related enzymes examined, except for isocitrate lyase activity. This indicates that transcription and translation of most enzymes needed for postgerminative growth takes place during seed maturation. This is in contrast with the generalization that "germination" enzymes are synthesized de novo from previously untranslated mRNAs conserved in dry seeds. All enzyme activities increased 3-fold or greater following imbibition, and most remained constant after reaching their peak. Notable exceptions were activities for three beta oxidation enzymes and fructose bisphosphatase, which decreased precipitously after peaking with other enzyme activities.Standard sucrose gradient procedures with swing-out rotors were not useful for isolating cotton glyoxysomes. Satisfactory and reproducible results ultimately were obtained with sucrose gradients constructed in a Beckman JCF-Z zonal rotor. Specific activities of glyoxysomal enzymes were 2- to 3-fold lower than those reported for other oil seeds, except malate dehydrogenase which was 10-fold lower. Electron microscopy revealed that protein body fragments were the primary contaminant of glyoxysome fractions. Glyoxysomes were subfractionated by osmotic shock treatments to evaluate sub-organelle localization of constituent enzymes, several of which have not been examined in other oil seeds.

Journal Article↗