The Development of Glyoxysomes in Peanut Cotyledons and Maize Scutella

The development of glyoxysomes during germination has been studied in isolated peanut (Arachis hypogaea L.) cotyledons and in maize (Zea mays L.) scutella. In peanut cotyledons isocitratase, malate synthetase, and protein associated with the glyoxysomal fraction increase simultaneously from the 3rd to the 8th day of incubation. In scutella of germinating maize seeds the specific activities of isocitratase, malate synthetase, and catalase associated with the glyoxysomes rise until the 4th day of germination and then decline while the total amount of protein present in the fraction stays constant during the first 5 days. If the peanut cotyledons are cultured in 2% glucose, the development of isocitratase and malate synthetase is severely inhibited, but the level of the glyoxysomal protein is not measurably affected.     

DEVELOPMENTAL STUDIES ON GLYOXYSOMES IN RICINUS ENDOSPERM

The development of glyoxysomes and their associated enzymes, isocitrate lyase and malate synthetase, was studied in the endosperm of castor bean seeds during germination and early growth in darkness. The protein content of the glyoxysome fraction, separated by sucrose density centrifugation, increased linearly from day 2 to day 4 and declined subsequently, while maximum enzyme activities were reached at day 5. The specific activities of the enzymes in the glyoxysomes increased until day 5 and remained constant thereafter. At all stages of germination the only organelle with isocitrate lyase activity was the glyoxysome, but at the earlier stages a greater portion of the total activity was recovered in the soluble form. Malate synthetase was found primarily in the glyoxysomes after day 4, but at earlier stages part of the activity appeared at regions of lower density on the sucrose gradient. It was shown that this particulate malate synthetase activity was due to glyoxysomes broken during preparation, and that, as a result of this breakage, isocitrate lyase was solubilized. We conclude that both enzymes are housed in the glyoxysome in vivo throughout the germination period, and that the rise and fall in enzyme activities in phase with fat breakdown correspond to the net production and destruction of this organelle.     

Development of Endoplasmic Reticulum and Glyoxysomal Membrane Redox Activities during Castor Bean Germination

Redox activities, NADH:ferricyanide reductase, NAD(P)H:cytochrome reductases, and NADH:ascorbate free-radical reductase, are present in endoplasmic reticulum (ER) and glyoxysomal membranes from the endosperm of germinating castor bean (Ricinus comminus L. var Hale). The development of these functions was followed in glyoxysomes and ER isolated on sucrose gradients from castor bean endosperm daily from 0 through 6 days of germination. On a per seed basis, glyoxysomal and ER protein, glyoxysomal and ER membrane redox enzyme activities, and glyoxylate cycle activities peaked at day 4 as did the ER membrane content of cytochrome P-450. NADH:ferricyanide reductase was present in glyoxysomes and ER isolated from dry seed. This activity increased only about twofold in glyoxysomes and threefold in ER during germination relative to the amount of protein in the respective fractions. The other reductases, NADH:cytochrome reductase and NADH:ascorbate free-radical reductase, increased about 10-fold in the ER relative to protein up to 4 to 5 days, then declined. NADPH:cytochrome reductase reached maximum activity relative to protein at day 2 in both organelles. The increases in redox activities during germination indicate that the membranes of the ER and glyoxysome are being enriched with redox proteins during their development. The development of redox functions in glyoxysomes was found to be coordinated with development of the glyoxylate cycle.     

Subcellular Localization and Developmental Changes of Aspartate-alpha-Ketoglutarate Transaminase Isozymes in the Cotyledons of Cucumber Seedlings

The total activity of aspartate-α-ketoglutarate transaminase in the cotyledons of cucumber (Cucumis sativus L.) seeds increased 17-fold during the first 2 days of germination in darkness and then declined gradually to 20% of the peak activity after 10 days. Exposure of the seedlings to light at day 3 accelerated the decline. The enzyme in the cotyledon extracts from seedlings at various ages was resolved into six distinct isozymes by starch gel electrophoresis. Isozymes 1 and 2 were glyoxysomal isozymes with different developmental patterns. Isozyme 1 followed the developmental pattern of the total enzyme activity in darkness, and was rapidly eliminated upon illumination. Isozyme 2 increased in activity to a peak at day 2 and declined rapidly thereafter, and disappeared completely at day 6; this developmental pattern was independent of light. No major difference in the optimal pH for activity, substrate specificity, and reversibility was observed between isozymes 1 and 2. The combined developmental pattern of isozymes 1 and 2 during germination correlated with that of the glyoxysomes. Isozyme 3 was located in the cytosol and its developmental pattern followed that of the total activity. Isozymes 4,5, and 6 were plastid isozymes and appeared only after 2 days of germination. Unlike many other chloroplast enzymes, the appearance of the chloroplast transaminase isozymes was under temporal control and was independent of illumination. No enzyme activity was detected in isolated mitochondria. The findings illustrate a complicated cellular control system for the appearance of various organelle-specific transaminase isozymes and thus the amino acid metabolism during germination.     

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

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

Lipase Activities in Castor Bean Endosperm during Germination

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal “ghosts” at equilibrium density 1.21 g/cm³ on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.     

Cytochemical localization of catalase in glyoxysomes isolated from maize scutella

The localization of catalase in isolated maize scutellum glyoxysomes was investigated by means of the diaminobenzidine histochemical reaction. Only the membranes of the glyoxysomes become heavily stained after incubation with diaminobenzidine and H₂O₂. If the glyoxysomes are lysed with Tricine buffer at pH 9, 70% of the catalase is solubilized, while the remaining 30% is tightly bound to an insoluble fraction formed mostly by glyoxysomal membranes. This suggests that catalase may be present also in the matrix of the glyoxysomes. The lack of staining of the matrix with diaminobenzidine is probably due to the high concentration of catalase in the membranes of the organelles.     

Histochemical Studies of Scutellum During Germination of Oat Seed by Light Microscopy and Immunofluorescent Localization of Oat Globulin

The protein, phytin, lipid and starch contents of the scutellum of oats showed marked changes during the first three days after seed germination. Protein and phytin disappeared almost completely during the first two days after seed germination. The degradation at lipid was much slower. In the scutellum of the ungerminated seed very few starch granules were seen. At days-1 and -2 after germination the starch contents increased, but at day-3 the starch contents decreased. Immunofluorescent localization of oat globulin indicated that the oat globulin was sequestered mostly, if not exclusively, in the protein bodies. The degradation of the oat globulin inside the protein bodies was very rapid. At day-3 after seed germination it disappeared almost completely. When excited by the ultraviolet light the walls of both the epithelial and parenchyma cells of the scutellum autofluoresced intensely. As germination progressed further, the autofluorescence in the walls of the epithelial cells gradually faded away, whereas the autofluorescence in the walls of the parenchyma cells did not.     

Non-Coordinate Genetic Alterations in the Expression of Catalase and other Glyoxysomal Enzymes during Maize Development

In the scutellum of maize during post-germinative development,the primary form of catalase expressed is the product of theCat2 structural gene, CAT-2. The developmental time-course ofCAT-2 protein follows a rapid increase with a peak at approximately4–5 d alter germination and a subsequent decline. An inbredstrain of maize, A337, has been found to exhibit a similar generalizedprofile with the significant exception that the level of CAT-2protein present in the scutellum is far above that in the ‘typical’maize lines exemplified by W64A. Our data suggest that the higherlevels of CAT-2 exhibited in A337 are due to increased synthesisand accumulation of more CAT-2 protein, and not merely to enzymeactivation. A comparison of A337 and W64A showed that the twolines are similar with respect to number of glyoxysomes andwith the exception of catalase, other microbody associated enzymesexhibit similar activity levels and developmental profiles.Thus, the results presented suggest that the catalasc developmentalprogramme characteristic of line A337 is not due to a concurrentincrease and subsequent decline in the number of glyoxysomesformed in the scutellum during this developmental period butis instead due to a greater level of CAT-2 protein. The datafurther support our earlier findings that the genes coding forglyoxysomal enzymes in maize are non-coordinately regulated. Key words: Gene regulation, glyoxysomes, catalase, glyoxysomal enzymes     

Site of production of gibberellin-like substances in germinating barley embryos

Summary It has been shown that gibberellin-like substances are produced in the scutellum of barley embryos during the first two days of germination (at 25°). This production is not stimulated by mevalonic acid or sugar, but it is inhibited by CCC. On the third day the activity of the scutellum ceases and the axis probably commences to produce gibberellins. Both gibberellic acid and gibberellin A₁ appear to be present, the latter predominating.     

Regulation of Glyoxysomal Enzyme Expression in Maize

The activity levels of three glyoxysomal enzymes (catalase, isocitric lyase, and malate synthase) were measured in the scutellum following germination of the inbred lines W64A, R6-67, and A16. In W64A, as in most maize lines examined, germination was accompanied by a rapid and synchronous increase in the activities of all three enzymes, and reached a peak at about day 4 and declined thereafter. In R6-67, catalase activity continues to increase past day 4 and reaches its highest activity level on later days. In A16, catalase activity is very low due to the lack of expression of the Cat2 gene. Despite these significant differences in catalase expression, the levels of the other two glyoxysomal enzymes did not differ in these inbred lines. Artificial inhibition of catalase in W64A by exogenous application of 10^–4 M aminotriazole did not inhibit germination, nor did it alter the levels of the other two glyoxysomal enzymes. Similarly, application of 10^–4 M itaconate to W64A seeds inhibited the appearance of isocitric lyase, but did not inhibit germination or alter the levels of malate synthase or catalase. Comparative cell fractionation and immunological studies were conducted with W64A and A16 and their microbodies were observed under the electron microscope. Cell fractionation studies were also conducted with W64A seeds germinated in the presence of aminotriazole or itaconate. Thus, our results suggest that the expression of these three glyoxysomal enzymes is not regulated coordinately in the maize scutellum.     

Role of the endoplasmic reticulum in glyoxysome formation in castor bean endosperm

Homogenates of the endosperm of castor bean (Ricinus communis var. Hale) were prepared at intervals during germination and fractionated on sucrose gradients. Early in germination when glyoxysomes were being produced, a substantial proportion (50%) of the activities of malate synthetase and citrate synthetase was recovered in the membranes of the endoplasmic reticulum (mean density 1.12 grams per cubic centimeter). This proportion declined to less than 10% at 4 days when the glyoxysomes were fully developed.     

Carboxypeptidases of germinating triticale grains

Presence of five carboxypeptidases was found in endosperm of germinating triticale grains, while two of them in scutellum. Changes of their activities during four days of germination suggest that carboxypeptidase II plays an important role at initial stage of germination, while carboxypeptidases I and III - at subsequent stages of the process. High activity of carboxypeptidase II both in scutellum and endosperm of dry grains accompanied by its decrease during germination, and on the other hand, the appearance of carboxypeptidases I and III activities at the 2^nd and 3^rd day of the process seems to confirm such functions of these enzymes. Experiments with GA₃ indicated that carboxypeptidase I was synthesized in scutellum, and carboxypeptidase III — in aleurone layer. Carboxypeptidases I and II cleave N-CBZ-Phe-Ala, and carboxypeptidase III — N-CBZ-Ala-Met and N-CBZ-Ala-Phe as substrates with the highest rate.     

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds

Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.     

STUDIES ON THE SOLUBLE CARBOHYDRATES AND CARBOHYDRATE PRECURSORS IN GERMINATING SOYBEAN SEED

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light-grown soybean cotyledons increased approximately 5-fold during the first 9 days of germination, whereas that of dark-grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.     

Development of Microbodies in Sunflower Cotyledons and Castor Bean Endosperm during Germination

In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2- to 3-fold, and thereafter it remained about constant in light or darkness.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

Starch degradation in the cotyledons of germinating lentils

Starch, total amylolytic and phosphorylase activities were determined in lentil cotyledons during the first days of germination. Several independent criteria show that the amylolytic activity is due mainly to an amylase of the α type. Starch is degraded slowly in the first days; during this time, α- and β-amylase activity are very low, while phosphorylase increases and reach a peak on the 3rd day. On the 4th day, there is a more rapid depletion of starch which coincides with an increase in α-amylase activity. By polyacrylamide gel electrophoresis of the crude starch-degrading enzyme, five bands were obtained: one phosphorylase, three α-amylases, and one β-amylase. Based on their heat lability or heat stability, two sets of α-amylase seem to exist in lentil cotyledons.     

Quantitative changes of fatty acids in soybean cotyledons during senescence and regreening

The quantity of total fatty acids in soybean cotyledons during aging, senescence and regreening has been studied. The greatest change in the fatty acid profile during the initial greening of the cotyledons (4–7 days after germination) was a 130% increase in the content of linolenate. Linoleate, as in the case of the other fatty acids, declined in the first 4 days and then increased by 7 days. Following the 10th day after germination, the quantity of palmitate, linoleate, and linolenate decreased continuously through senescence to 20–28% of the maximum quantity of each. When the cotyledons were regreened by removal of the epicotyl 15 or 16 days after germination, linolenate was present in quantities substantially higher than in the senescing cotyledon. On the 22nd day after germination, the quantity of linolenate in regreened tissue was 140% greater than that in senescing tissue of the same age. By contrast, the quantity of linoleate was only 30–40% greater in regreening tissue and the quantity of most of the other fatty acids was similar in both tissues. Similar changes in the quantity of chloroplast fatty acids were observed during this period. Removal of the epicotyl resulted in a higher level of chloroplast linolenate. During aging, the total chlorophyll and the number of chloroplasts reached a maximum on the 10th day and decreased rapidly during senescence. The amount of chlorophyll per chloroplast remained relatively constant during this period whereas the quantity of linolenate per chloroplast decreased during senescence. It is suggested that major structural changes observed in chloroplast membranes may be related to changes in fatty acid composition, but are not dependent on changes in chlorophyll concentration.     

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

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