Metabolism of 14 C-Maltose in Avena fatua Seeds During Germination

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Measurement of Oxygen Uptake. The uptake of 02 was measured by means of a Gilson differential respirometer. Sterile precautions were taken as described previously (4). Each flask contained 10 seeds. Two flasks were included in each treatment.Incorporation of 1'C-Leucine into Protein. Lots of 10 seeds were husked, pricked at the dorsal side of the endosperm (a measure which facilitates entry of the applied compounds without affecting germination), sterilized in 1 % NaOCI for 2 min, washed, placed in a 25-mi Erlenmeyer flask with 2 ml of water, shaken at 21 -:-1 C for a period of time, then transferred to 1 ml of 1 mM leucine with 2.5 ,uc of uniformly labeled '4C-leucine (specific radioactivity 290 mc/mmole, New England Nuclear Corp.), and further shaken for 2 hr. The seeds were then washed in ice-cold, nonradioactive leucine solution, homogenized, extracted in 4 ml of 0.2 M NaCl plus 0.1 mm leucine, and centrifuged at 12,000g for 15 min. The pellet was suspended in 4 ml of the salt solution and recentrifuged. One twentieth of the pooled 12,000g supernatant fraction was precipitated with an equal volume of 15% trichloroacetic acid, and the precipitate (soluble protein) was collected on a Millipore filter. The filter was dried and the radioactivity was determined in a Nuclear Chicago gas flow detector with a counting efficiency of 40% for carbon-14. The pellet was suspended in water and precipitated in the same way as above. Approximately 80% of the radioactivity associated with the acid-insoluble material could be released by pronase, a potent proteolytic enzyme of bacterial source. Thus, this material is considered as insoluble protein.Microautoradiography of Seeds Exposed to 3H-Leucine. To locate the sites of protein synthesis, the seeds were exposed to L-leucine-4, 5-3H (specific radioactivity 6.0 c/mmole, Schwarz BioResearch), then washed in nonradioactive leucine, fixed, dehydrated and infiltrated with paraffin according to the technique of Feder and O'Brien (7). Sections 5 to 8 ,u thick were placed on a slide, dewaxed, coated with Kodak nuclear track emulsion type NTB 3, exposed, stained with toluidine blue-acid fuchsin, mounted, and examined under the microscope (10). RESULTSRespiratory Intensity. The rate of respiration (02 uptake) in dormant seeds was low as compared to that of nondormant seeds; the difference could be detected from the early hours of soaking (Fig. 1). In both kinds of seeds, the rate increased with time. However

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confidence: 99%

Respiration and Protein Synthesis in Dormant and After-ripened Seeds of Avena fatua

Chen

Varner

1970

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“…The neutral fraction was further chromatographed on Whatman No. 1 filter paper, using ethylacetate-pyridine-water (8 :2: 1) as the solvent, and the label associated with sugars was determined (4). In some experiments, the alcohol-insoluble residue was hydrolyzed with 6 N HCl at 105 C overnight, and the compounds released were analyzed for radioactivity.…”

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confidence: 99%

Patterns of Food Utilization by the Germinating Lettuce Seeds

WonMok¹,

Chen²

1974

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The embryo excised from seed of Grand Rapids lettuce (Lactuca sativa L.) can be cultured in distilled water. Complete digestion of the endosperm and transfer of nutrients from the endosperm to the embryo occur in the germinating seed with fat as the source of food. The fat is utilized for respiration, synthesis of amino acids, and to a degree, converted to sucrose. '4C-Glucose administered to the seed is quickly converted to sucrose in the endosperm and translocated to the embryo. Radioactivity associated with the glucose remains predominantly in the carbohydrate fraction, and much of it is incorporated into what is believed to be cell wall polysaccharides. Relatively little isotope is distributed in the amino or organic acids.The seed of Grand Rapids lettuce has long been a favorite subject for germination studies, especially in connection with the hormonal and phytochrome actions (2, 6-8, 11, 12, 16, 17) and with the dormancy mechanism (9,10,18

“…Breaking dormancy generally increases percentage germination and rate of seedling growth. Measurable biochemical differences between dormant and nondormant seeds exist in inhibitor content (16) and in metabolism of carbohydrates (2,7,18), organic acids (5,12), and nucleic acids (15,19). However, it is not clear whether the observed biochemical differences result from breaking dormancy or from other causes.…”

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confidence: 92%

Metabolic Changes in Partially Dormant Wheat Seeds during Storage

Anderson

1970

Seed deterioration generally is characterized by the loss of seedling vigor followed by the complete loss of germination (9). Some physiological and biochemical changes associated with deterioration have been reported recently (2,3 Germination Tests. Seeds were surface-disinfected for 5 min with 1% sodium hypochlorite, rinsed three times with sterile distilled water, and germinated on sterilized absorbent toweling in distilled water or 10-3 M GA3 at 25 C in the dark. Seeds were also germinated in rolled towels at 20 C (4). Only those seeds which produced normal seedlings at 4 days were counted as germinated.Pathological Tests. The percentage of seeds from each storage condition infected with storage fungi was determined by placing surface-disinfected seeds on 10% NaCl malt agar (8) and storing the plates in a high humidity box in the laboratory for 10 to 20 days. The fungi growing from the seeds were classified as either storage or field fungi.Glucose Utilization. Seeds were imbibed in sterile water containing 50 Ag/ml each of penicillin G and streptomycin sulfate, then incubated in glucose-U-14C as described by Abdul-Baki (1). The CO2 was collected in either hyamine hydroxide or saturated Ba(OH)2. After incubation, the seeds were washed three times with ice-cooled water and were frozen until they were fractionated by a modification of the procedure described by Abdul-Baki (1). Seeds were homogenized in ice-cooled water, boiled for 45 min, cooled, made to 80% (v/v) ethanol, and kept at 4 C overnight. The preparation was centrifuged, and the supernatant was decanted and saved. The pellet was washed three times in 80% ethanol, and the washes were combined with the original supernatant. The supernatant contained the unutilized sugars, organic and amino acids, and other ethanol-soluble components (1). The precipitate contained the radioactivity which had been converted into polysaccharides and proteins.Aliquots of the ethanol-soluble and ethanol-insoluble (resuspended in water) fractions were taken for measurement of radioactivity by liquid scintillation using 10 ml of scintillation solution (6). Radioactivity in the BaCO3 was determined in this scintillator whereas the '4CO2 in hyamine was detected in a scintillator containing 4 g of 2, 5-diphenyloxazole plus 50 mg of 1 ,4-di-2-(5-phenyloxazolyl)benzene per liter of toluene.