Control of enzyme activities in cotton cotyledons during maturation and germination

Immature Glycine max (L.) Merrill (20-10-20, per cent as N-P205-K20 equivalent) in water at weekly intervals beginning at 4 weeks after seeding.Seed Culture. Seeds were selected and cultured as previously described (16). Single excised seeds without pods were transferred aseptically into presterilized 1 25-ml Erlenmeyer flasks containing 20 ml of liquid medium. Standard medium was prepared as previously described ( 15), except that Na2MoO4 was omitted and the complete medium was sterilized by ultrafiltration. Alterations to the standard medium included 5-ml additions of 62.5 mm glutamine and/or 146 mm sucrose solution at 4, 8, and 12 d during culture; adjusting initial medium to 73, 146 (standard), and 296 mM sucrose and 31.25, 62.5 (standard), and 125 mm glutamine (in all combinations); addition of 100 to 300 mm mannitol and adjustment to 73 mM sucrose in otherwise standard medium; and addition of 0.01 to 100 ,gM ABA and adjustment to 73 mm sucrose in otherwise standard medium. ABA was dissolved and diluted in 95% ethanol, and 5 ul ofethanol solution was added to 20 ml of medium to obtain the final concentrations indicated. Osmolality was measured by a freezing point osmometer. Harvested seeds were air dried to 12% water content before dry weight determinations. Starting dry weight was estimated from starting fresh weight by a regression equation (16). Seeds which germinated precociously were excluded from seed weight and seed growth rate calculations (16). Air dried seeds were germinated on rolled paper towels at 25°C for 3 or 5 d unless otherwise indicated. Experimental results from two to four replicate experiments are expressed as frequency, percentage, or numerical mean ± SE.Postharvest Germination. To evaluate the effect of growth medium on postharvest germination, seeds were harvested after 14 d in culture to optimize germination potential (16). At this time axis tissues were yellow and the edges of cotyledons were starting to yellow. Delayed harvest resulted in declining postharvest germination potential (16). After excluding seeds which precociously germinated or failed to grow, cultured seeds were harvested aseptically and air dried to 12% water content during 2 weeks at 25C and 60 to 70% RH. Dry seeds (embryos) were humidified overnight to about 17% water content before germination on paper towels saturated with glass distilled H20 at 25°C for 3 or 5 d unless otherwise indicated.Control seeds (matured on plants) were

Section: Methodssupporting

confidence: 86%

Precocious Germination during In Vitro Growth of Soybean Seeds

Obendorf

Wettlaufer²

1984

“…Analysis of viviparous mutants of corn (3,24) and Arabidopsis (17,19) have shown that precocious germination of these embryos is ( 16) and a-amylase in barley (14), but stimulates synthesis of embryospecific proteins in rapeseed (6), rice (25), and wheat (28). Furthermore, ABA appears to play a role in the maturation of cotton embryos since young embryos cultured on ABA acquire a subset of proteins including a battery of enzymes normally appearing late in embryogeny (5,9).…”

Section: Discussionmentioning

confidence: 99%

Role of ABA in Maturation of Rapeseed Embryos

Finkelstein¹,

Tenbarge²,

Shumway³

et al. 1985

279

168

Development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without abscisic acid (ABA) was compared with normal development in situ to investipte the role of ABA in embryo maturation. Endogenous ABA levels were measured by radioimmunoassay, and sensitivity to ABA was assayed in terms of its ability to suppress precocious germination and stimulate accumulation of storage protein and storage protein mRNA. During development in situ, the levels of endogenous ABA and 12S storage protein mRNA both reach their peaks just before the embryos begin to desiccate. The ABA levels during this phase of development also correlate with the time required in culture before germination is evident. Following these peaks, increasing concentrations of exogenous ABA are required to both suppress germination and continue storage protein accumulation in vitro. Thus, both endogenous ABA and ABA sensitivity decline during maturation. The concentrations of exogenous ABA required to suppress germination at these later stages result in abnormally high levels of endogenous ABA and appear to be toxic. These results are consistent with the hypothesis that in maturing rapeseeds, low water content rather than ABA prevents germination during the later stages of development.Embryogenic development ceases during maturation, the final phase of seed development in most angiosperms. During this phase, the seeds desiccate and enter a period of developmental arrest which prevents them from germinating before environmental conditions favor seedling growth. Precocious DMSO (50% v/v), was added to the basal medium to give final concentrations of 1 and10 uM. In all cases, the media ingredients were mixed, pH was adjusted to 5.5 with 1.0 N KOH, and powdered agar (Difco-Bacto; Difco Laboratories) was added to 0.7% (w/v). The medium was autoclaved and dispensed 10 ml/ dish. The dishes were sealed with Parafilm (American Can Co.) and cultured at 28°C in continuous light from cool white fluorescent bulbs (General Electric).Extraction of ABA. Crude extracts for use in radioimmunoassay were prepared as described below, based on the method of Weiler (30). A conical sintered-glass homogenizer (Duall, Kontes of Illinois, Evanston, IL) attached to a Tri-R stirrer (Tri-R Instruments, Rockville Centre, NY) was used to grind 50 to 150 mg of tissue in 2.5 ml of 90% (v/v) methanol containing 10 mg/ L 2,6 di-t-butyl-4-methylphenol (BHT). Each sample was divided into two tubes, and 150 ng of ABA (mixed isomers, grade IV, Sigma) were added to one tube as an internal standard of recovery efficiency. Samples were stored in the dark at 4°C for 48 h, with intermittent shaking. Extracts were cleared by centrifugation for 3 min at 12,500 g, diluted 5-fold with H20, and immunoassayed within 2 d.Radioimmunoassay. Endogenous ABA was measured by radioimmunoassay according to the procedure of Weiler (30) using rabbit anti-ABA-human serum albumin serum (Miles-Yeda Ltd., Naperville, IL). This serum does not distinguish between the (+) and (-) ...

“…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).…”

mentioning

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...