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).Abbreviations ABA abscisic acid - bp base pairs - DPA days post anthesis - HPI hours post imbibition - kb kilobase (pairs) - M _r relative molecular weight - S Svedberg unit (10^-13s)     

Expression and localization of malate synthase during maturation and dessication of castor bean seeds

Summary Enzymatic levels and subcellular localization of malate synthase in maturing seeds of castor bean (Ricinus communis cv. Hale) are reported. Extracts of maturing seeds exhibited moderately high specific activity (9.68 nmoles/min/mg protein) at 15–20 DAP and lower specific activity (0.49) in mature, dry seeds. Subcellular localization of the enzyme during seed maturation was primarily cytosolic (85%). The remainder of the activity in sucrose gradients was located at high density (1.21 g/cm³). Dry seeds did not contain organelle-bound malate synthase activity. In extracts of 4-day germinated seeds the enzyme was present at high specific activity (12.8 nmoles/min/mg protein) with better than 85% of the total activity in glyoxysomes (1.24 g/cm³).Two polypeptides, 62kDa and 66kDa, reactive with anti-malate synthase were detected at high density in sucrose gradients of homogenates of late-maturing seeds (60 DAP); dry seeds; and seeds imbibed for 6 h. One polypeptide, 62 kDa, in 4-day germinated seeds, reacted with anti-malate synthase. Immunoreactive polypeptides in late-maturing and dry seeds were present at approximately 1/760 of the level found in 4-day germinated seeds. We conclude that malate synthase activity is prominent during early seed maturation but is very low and minimally compartmentalized during late maturation. The rapidly sedimenting immunoreactive polypeptides from dry seeds are enzymatically inactive and are presumed to be of no physiological significance.Abbreviations DAP days after pollination - MS malate synthase - EDTA ethylenediamine tetraacetic acid - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis - BSA bovine serum albumin - IgG gamma globulin     

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.     

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.     

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.     

Ontogeny of glyoxysomes in maturing and germinated cotton seeds—a morphometric analysis

Morphometric procedures were used with light and electron microscopy to examine glyoxysome number, volume, shape and distribution as well as mesophyll cell volume, in cotyledons of mature (50 d postanthesis), imbibed (5h) and germinated (24 and 37 h) cotton (Gossypium hirsutum L.) seeds. Additionally, activities of five glyoxysomal marker enzymes in cotyledon extracts were assayed at each of the above ages. Cell volume was determined from photomicrographs of Epon-embedded sections by the point-counting procedure. Analysis of variance showed that cell volume was not different among the tissue segments studied. Glyoxysomes were cytochemically stained for catalase (EC 1.11.1.6) activity with the 3,3-diaminobenzidine-tetrahydrochloride procedure. Analyses involving both phase and electron microscopy, and two separate sterologic calculations for determining the number of glyoxysomes per cell, indicate that glyoxysomes are numerous in mature seeds, persist through desiccation and imbibition, then increase dramatically in volume (seven fold) but not number (a maximum of 1.5-fold), when enzyme activities increase two to six times (depending on the enzyme). During the entire period of increase in glyoxysomal enzyme activities, no ultrastructural evidence was found for glyoxysome formation or destruction. Our data, in contrast to some proposals in the literature, indicate that cottonseed glyoxysomes form during seed maturation, then develop following seed imbibition into pleomorphic organelles by posttranslational accumulation of proteins from the cytosol and transfer of membrane components probably from the endoplasmic reticulum.Abbreviations DAB 3,3-diaminobenzidine tetrahydrochloride - DPA days postanthesis - ER endoplasmic reticulum     

Purification of glyoxysomal polypeptides

Summary A strategy for the rapid purification of proteins from glyoxysomes of castor bean (Ricinus communis cv. Hale) is described. The first step was to separate the proteins in the mixture on the basis of hydrophobicity by reversed phase high performance liquid chromatography using a gradient of increasing acetonitrile concentration. Individual protein peaks were collected and fractionated according to molecular mass by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified polypeptides were used to produce monospecific, polyclonal antibodies. One of these, an anti-catalase antibody, has been employed to assess the subcellular distribution of catalase in endosperm of maturing seeds, dry seeds and seedlings. During seed maturation 45% of the catalase activity was associated with structures sedimenting at high isopycnic densities (1.21 g/cm³). However, in dry seeds, only 6% or less of the catalase activity was associated with these dense particles. In 4-day seedlings 80% of catalase activity was associated with glyoxysomes (1.24 g/cm³). A novel catalase 59 kDa subunit was found in the cytosol of 4-day seedlings and in isolated organelles from maturing and dry seed.Abbreviations AN acetonitrile - CBBR Coomassie brilliant blue R-250 - HPLC high performance liquid chromatography - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Aggregated Forms of Malate and Citrate Synthase are Localized in Endoplasmic Reticulum of Endosperm of Germinating Castor Bean

The endosperm of 3-day germinated seedlings of Ricinus communis was homogenized in the presence or absence of Mg²⁺. When the Mg²⁺ -containing homogenate was fractionated on linear, 20 to 40% sucrose gradients, the endoplasmic reticulum (ER) reached equilibrium at a density of 1.146 grams per cubic centimeter. Absence of Mg²⁺ in the grinding medium resulted in displacement of the ER in the gradient from a density of 1.146 to 1.138 grams per cubic centimeter. At either density, the activities of both malate and citrate synthase were found to overlap the activity of NADH-cytochrome c reductase (an ER marker) in the gradient. Furthermore, this overlap of activities was observed whether the gradients were centrifuged for 3 or 19 hours. An analysis of sedimentation characteristics of the solubilized enzymes revealed that they exist, predominantly, as a 5.2S (s_20,w × 10⁻¹³) form (malate synthase) and a 6.8S form (citrate synthase) in the glyoxysomes and cytosol. When the two enzymes were released from the ER, they appeared as aggregate forms of 70S and 55S, respectively. These results support the conclusion that the synthases are associated with the ER.     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

Canatoxin-, concanavalin A- and canavalin-cross-reactive materials during maturation of Canavalia brasiliensis (Mart.) seeds

The distribution of three cross-reactive materials (CRMs), a toxic protein analogous to canatoxin, CNTX-CRM, a lectin analogous to concanavalin A, Con A-CRM, and a major storage protein, canavalin-CRM, was investigated during successive stages of maturation of Canavalia brasiliensis Mart. seeds. The data obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunological analyses indicated that these proteins share extensive homology with the analogous proteins found in Canavalia ensiformis seeds. The changes in CNTX-CRM and Con A-CRM levels throughout the maturation process were assayed by rocket immunoelectrophoresis. Synthesis of Con A-CRM was detectable at 30 days post-anthesis (DPA) while its hemagglutinating activity appeared only at 35 DPA. The CNTX-CRM was detected as a biologically active protein from 30 DPA onwards. The behavior of CNTX-CRM during maturation of C. brasiliensis seeds was quite distinct from that of Con A-CRM, pointing to different biological roles of these proteins in the seed.     

Comparative studies of glyoxysomes from various Fatty seedlings

The separation of various organelles from cotton cotyledon (Gossypium hirsutum L.), cucumber cotyledon (Cucumis sativus L.), peanut cotyledon (Archis hypogaea L.), pine megagametophyte (Pinus ponderosa Laws), and watermelon cotyledon (Citrullus vulgaris Schrad.) by sucrose density gradient centrifugation was found to be similar to that described for castor bean endosperm (Ricinus communis L.). Equilibrium densities were 1.12 to 1.13 g cm³ for endoplasmic reticulum, 1.17 to 1.19 g/cm³ for mitochondria, and 1.25 g/cm³ for glyoxysomes. Isolated glyoxysomes from different fatty seedlings have striking similar specific activities of individual enzymes. The only exception is alkaline lipase activity which, when assayed with an artificial substrate, varies some 10-fold in glyoxysomes from different fatty seedlings. The properties of individual enzymes in glyoxysomes from different fatty seedlings are qualitatively similar as regard to sub-organelle localization and behavior in the presence of KCl and Triton X-100. In pine megagametophyte, the glyoxysomes and not the mitochondria are the intracellular site for the breakdown of stored lipid.     

Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

A novel defensin from the lentil Lens culinaris seeds

A novel 47-residue plant defensin was purified from germinated seeds of the lentil Lens culinaris by ammonium sulfate precipitation, gel filtration, chromatography, and RP-HPLC. The molecular mass (5440.41 Da) and complete amino acid sequence (KTCENLSDSFKGPCIPDGNCNKHCKEKEHLLSGRCRDDFRCWCTRNC)¹ of defensin, termed Lc-def, were determined. Lc-def has eight cysteines forming four disulfide bonds. The total RNA was isolated from lentil germinated seeds, RT-PCR and subsequent cloning were performed, and cDNA was sequenced. A 74-residue predefensin contains a putative signal peptide (27 amino acid) and a mature protein. Lc-def shows high sequence homology with legumes defensins, exhibits an activity against Aspergillus niger, but does not inhibit proteolytic enzymes.     

Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development

Oat (Avena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [³⁵S]sulfate into protein. Incorporation of [³⁵S]sulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the α and β globulin subunits were present 2 to 4 DPA, but the full complement of globulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis.     

Inhibition of linolenic Acid synthesis and modification of chilling resistance in cotton seedlings

The temperature at which cotton seeds (Gossypium hirsutum L.) germinated influenced the fatty acid composition of the polar lipids of developing root tips. Seeds were germinated at 15, 20, 25, and 30 C. As the temperature decreased the linolemic acid content of the polar lipid fraction increased. Sandoz 9785[4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone] reduced the low temperature-induced increase in linolenic acid content of the polar lipids and reduced seedling ability to withstand 8 C chilling. The results are consistent with the conclusion that chilling resistance in cotton seedlings is related to the level of linolenic acid in the polar lipids in the developing root tips.     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Microsomal phosphatidate phosphatase in maturing safflower seeds

An assay system comprising sodium phosphatidate, phosphatidylcholine, and bovine serum albumin has been developed for the reproducible determination of phosphatidate phosphatase activity in maturing seeds of safflower (Carthamus tinctorius L.). The activity was detected in both membrane and soluble fractions, and the microsomal phosphatidate phosphatase was characterized. The optimum pH for Pi release was 6.7, and the activity depended on the concentration of Mg²⁺. Phosphatidylcholine and bovine serum albumin stimulated the phosphatase reaction. This phosphatase was highly specific for phosphatidate; lysophosphatidate, and water-soluble phosphate esters did not serve as substrate. The specific activity was approximately 20 nanomoles per minute per milligram of protein, which was close to that of glycerol-phosphate acyltransferase and higher than that of diacylglycerol acyltransferase. Furthermore, the activity per seed was enough to account for the rate of triacylglycerol accumulation in vivo. The step of diacylglycerol formation by phosphatidate phosphatase does not appear to be rate-limiting for triacylglycerol synthesis during seed maturation.     

Lipases in the storage tissues of peanut and other oil seeds during germination

The castor-bean endosperm-the best-studied material of reserve lipid hydrolysis in seed germination-was previously shown to have an acid lipase and an alkaline lipase having reciprocal patterns of development during germination. We studied oil seeds from 7 species, namely castor bean (Ricinus communis L.), peanut (Arachis hypogaea L.), sunflower (Helianthus annus L.), cucumber (Cucumis sativus L.), cotton (Gossypisum hirsutum L.), corn (Zea mays. L.) and tomato (Lycopersicon esculentum Mill.). The storage tissues of all these oil seeds except castor bean contained only alkaline lipase activity which increased drastically during germination. The pattern of acid and alkaline lipases in castor bean does not seem to be common in other oil seeds. The alkaline lipase of peanut cotyledons was chosen for further study. On sucrose gradient centrifugation of cotyledon homogenate from 3-d-old seedlings, about 60% of the activity of the enzyme was found to be associated with the glyoxysomes, 15% with the mitochondria, and 25% with a membrane fraction at a density of 1.12 g cm^-3. The glyoxysomal lipase was associated with the organelle membrane, and hydrolyzed only monoglyceride whereas the mitochondrial and membrane-fraction enzymes degraded mono-, di- and triglycerides equally well. Thus, although the lipase in the glyoxysomes had the highest activity, it had to cooperate with lipases in other cellular compartments for the complete hydrolysis of reserve triglycerides.     

Glyoxysomes in megagamethophyte of germinating ponderosa pine seeds

Decoated ponderosa pine (Pinus ponderosa Laws) seeds contained 40% lipids, which were mainly stored in megagametophytic tissue and were utilized or converted to sugars via the glyoxylate cycle during germination. Mitochondria and glyoxysomes were isolated from the tissue by sucrose density gradient centrifugation at different stages of germination. It was found that isocitrate lyase, malate synthase, and catalase were mainly bound in glyoxysomes. Aconitase and fumarase were chiefly localized in mitochondria, whereas citrate synthase was common for both. Both organelles increased in quantity and specific activity of their respective marker enzymes with the advancement of germination. When the megagametophyte was exhausted at the end of germination, the quantity of these organelles and the activity of their marker enzymes decreased abruptly. At the stage of highest lipolysis, the isolated mitochondria and glyoxysomes were able to synthesize protein from labeled amino acids. Both organellar fractions contained RNA and DNA. Some degree of autonomy in glyoxysomes is indicated.