De Novo Synthesis of Isocitritase in Peanut ( Arachis hypogaea L.) Cotyledons

Developmental patterns of glyoxylate cycle and photosynthetic acivities have been correlated with electropboretic profiles of cotyledonary RNA and protein in both light-and dark-grown cucumber seedlngs (Cucumis sadvus L.) Cytoplasmic rRNA increases 10-fold between days 0 and 5, and the steepest increase coincides with the most rapid rise in activities of the glyoxysomal enzymes, isocitrate lyase and malate synthase. Chloroplast rRNA and ribulose bisphosphate (RuBP) carboxylase begin rising at day 3, followed about a day later by increases in glyoxylate reductase activity and chbrophyl content. Of these phototrophic indicators, only chlorophyll requires light for its initial appearance. Sodium dodecyl sulfate gel electrophoresis of total and soluble cotyledonary protein showed several developmental patterns, including: (a) Seed germination in fat-storing species requires a functional glyoxylate cycle to effect net gluconeogenesis from the acetyl-CoA derived by , oxidation of storage triglycerides (3). Although much is already known about the developmental physiology of the glyoxylate cycle enzymes and the glyoxysomal compartment in which they are localized (3,8,14,15,29,35), little is understood about the regulatory mechanisms which underlie the developmentally orchestrated expression of this specific metabolic capability. We are interested both in the level(s) at which the activities of glyoxysomal enzymes are regulated during germination of cucumber (Cucumis sativus) and also in the control mechanisms involved in the subsequent transition from heterotrophy to autotrophy that takes place in the cotyledon upon emergence and greening.This function, and enzyme activities which occur in cotyledons of lightand dark-grown cucumber seedlings and attempts to relate these changes to developmental changes in cotyledonary protein and RNA, thus providing a characterization of the system necessary for further, more specific studies on the regulation of the glyoxysomal and peroxisomal enzymes. A preliminary report of this work has already been presented (2). MATERIALS AND METHODSCulture Conditions. Seeds of C. sativus L. var. Long Green Ridge or Improved Long Green were cold-imbibed (16 hr at 4 C) in distilled H20, then planted at a depth of about 1 cm in trays of Vermiculite underlaid with soil. Trays were either kept in continuous darkness (dark-grown) or illuminated for 12 hr/day with a mixture of fluorescent and incandescent lamps at an approximate intensity of 6,500 lux (light-grown). The temperature was in both cases maintained at 26 to 28 C for 12 hr, followed by a night depression to 22 C for 12 hr. Germination time was measured in days from planting of the cold-imbibed seeds.Harvesting of Cotyledons. Cotyledons were harvested at daily intervals from day 0 (cold-imbibed only) to day 7, with harvesting initiated about 1 hr after the onset of illumination for the lightgrown seedlings.

Section: Resultssupporting

confidence: 91%

Regulation of Glyoxysomal Enzymes during Germination of Cucumber

Becker¹,

Leaver²,

Weir³

et al. 1978

“…This increase is a common feature of all fatty seeds (2,4,7,12,14,18,20). The increase in activity of glyoxysomal enzymes is due to de novo enzyme synthesis and not merely to activation (13,16,19,29). This de novo synthesis of new glyoxysomal proteins is believed by many to be accompanied by the formation of an increasing number of new microbodies (glyoxysomes) (6,12,20,24,25,30).…”

Section: Introductionmentioning

confidence: 99%

Comparison of the Glyoxysomes and the Glyoxysomal Enzymes in Maize Lines with High or Low Oil Content

Tsaftaris¹,

Scandalios²

1983

Seed germination in fat-storing species requires a functional glyoxylate cycle to effect net gluconeogenesis from the acetyl-CoA derived by fl-oxidation of storage triglycerides (4,5,15). The number of fully functional glyoxysomes and the activity of the glyoxysomal enzymes, catalase (EC 1.1 1.1.6), malate synthase (EC 4.1.3.2), and isocitrate lyase (EC 4.1.3.1), undergo a well-characterized increase and subsequent decline in activity, with peak activity corresponding to the period ofmaximum lipid metabolism (2,5,12,20). This increase is a common feature of all fatty seeds (2,4,7,12,14,18,20). The increase in activity of glyoxysomal enzymes is due to de novo enzyme synthesis and not merely to activation (13,16,19,29). This de novo synthesis of new glyoxysomal proteins is believed by many to be accompanied by the formation of an increasing number of new microbodies (glyoxysomes) (6,12,20,24,25,30). Despite this common coordinated expression ofthe three enzymes in most inbred maize lines, mutant lines have been identified having the typical developmental profile for malate synthase and isocitrate lyase but an altered profile for catalase (21,22,26). This suggests the existence of genetic regulatory circuits which affect one of the glyoxysomal enzymes independently of the other enzymes and of the formation of the organelle per se. MATERIALS AND METHODS Growth of Seedlings. The lines with high and low oil used in this study were kindly provided by Dr. W. J. Dudley, University of Illinois. Seeds were surface sterilized for 10 min in 1% NaOCl solution (20% commercial Clorox) and soaked in sterile distilled H20 for 24 h. The seeds were then placed in plastic containers on several layers ofgermination paper (Kimpak) moistened with H20 and grown at 230C in darkness.Preparation of Crude Extracts. Five scutella from each day postimbibition were isolated and ground with sand in a prechilled mortar with 25 mm glycylglycine buffer (pH 7.4; 1 ml/scutellum), and the extracts were centrifuged at 15,300 g for 30 min at 4°C. The supernatants were used in in vitro assays and/or subjected to electrophoresis as described below. For immunoelectrophoresis, the extracts were prepared using 0.2 ml buffer/0.1 g scutellar tissue.Enzyme Assays. Catalase was assayed spectrophotometrically according to Beers and Sizer (3) Cell Fractionation Studies. Glyoxysome isolation was accomplished essentially as described by Briedenbach and Beevers (6). In each experiment, approximately 50 scutella (2.5 g) were isolated from seeds at 4 d postimbibition and minced with a razor blade in 5 ml of cold grinding medium. The homogenate was passed through four layers of cheesecloth and centrifuged for 5 min at 490g. Following lipid removal, the supernatant was then layered onto a sucrose gradient (5 ml of 600%o, 8 ml of 48%, 7 ml of 40o, 5 ml of 35%, and 2 ml of 25% sucrose; w/v). The gradient was always prepared 24 h before use, then centrifuged at 113,000g for 3.5 h in a SW-27 rotor at 4°C using a Beckman L5-65 ultracentrifuge. After centrifugation, t...

“…These observations seem to have ruled out the latter two possibilities. The experiments with cycloheximide, a potent inhibitor of protein synthesis (2,7,16), under different conditions (Tables I and II), suggest that the increase of phytase activity in the cotyledon with an increased period of soaking is due to the synthesis of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Metabolism of Inositol Phosphates

Mandal

Biswas

1970

The degradation of phytin in germinating mung bean seeds has been found to be associated with the increased activity of phytase in the cotyledon. In the differentiated embryo the increase of this activity is very low all throughout the growth periods studied. Phytase appears in the cotyledon during germination. No activity has been detected in the cotyledons of unsoaked seeds. Cycloheximide (10-6 M) inhibits the appearance of phytase by 61% during 24 and 48 hours after the start of germination. This phytase increase is dependent on the synthesis of new RNA in the cotyledon. Synthesisof DNA is not detected inthe cotyledon during germination.Phytase or myoin-ositol hexaphosphate phosphohydrolase (EC 3.1.3.8) which carL hydrolyze myoinositol hexaphosphate to myoinositol and inorganic phosphate has long been known (23). Pears (18) showed that in ungerminated wheat the aleurone portion contained 39%, endosperm 34%, and scutellum 15% of the total phytase activity of the whole seeds. It has also been shown by different groups of workers that phytase activity of different grains increases during germination (1,12,15). Many reports are now available on the characteristics of the enzyme phytase. Nagai and Funahashi (17) reported a phytase, purified 1500-fold from wheat bran, which shows broad substrate specificity at pH 5.0. Gibbins and Norris (6) purified phytase and an acid phosphatase from the cotyledons of germinated Phaseolus vulgaris, the pH optimum of which has been recorded at 5.0. In the present study the activity of phytase in the cotyledons of mung bean seeds (Phaseolus aureus) has been found to be increased after soaking for 24 hr. The pH optimum for this system has been recorded at 7.5 instead of 5.0, the level reported with various preparations from different seeds. Very little phytase activity has been detected in the cotyledons of mung bean seeds after soaking for 6 hr. Regarding the appearance of phytase activity there may be two possibilities: (a) de novo synthesis or (b) activation of enzyme protein already present in the seeds. From the present study it appears that phytase is synthesized de novo in the cotyledon of mung beans after soaking. Research Institute, Pussa, Bihar. "4C-Amino acid mixture, 2-14C-uridine, and 2-'4C-thymidine were obtained from Radiochemical Centre, Amersham, Buckinghamshire, England. Cycloheximide was procured from Nutritional Biochemical Corporation, Cleveland, Ohio. Chromomycin A3 was obtained as a gift from Takeda Chemical Industries, Ltd., Osaka, Japan. MATERIALS AND METHODSIsolation of Phytate. Phytate was isolated from mung bean by extraction with 1 M HCl04 (4) and separated by gradient elution chromatography on a Dowex 1-Cl column (19).Germination Procedure. The dry seeds of mung beans (Phaseolus aureus) were washed with water for 5 min and then covered with water (3 ml/g of seed) in a large Petri dish with seeds dispersed uniformly over the dish. The Petri dish was then covered, allowing for a little air passage, and kept at 35°in an incubator. After the seeds ...