Date of Award

12-2011

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Biological Sciences

Advisor

Sparace, Sam A

Committee Member

Bielenberg , Doug G

Committee Member

Paul , Kim

Abstract

A method for the isolation of intact physiologically active plastids from rapidly developing soybean (Glycine max L.) somatic embryos has been developed for the in vitro study of lipid metabolism. Using de novo fatty acid biosynthesis from 14C-acetate as a marker for physiological functionality, the greatest rates of fatty acid biosynthesis were recovered in 3000 x g fractions that were isolated in the presence of 0.5 M sorbitol, with essentially no activity occurring in the 3000 x g supernatant. Plastids purified on 10% Percoll were approximately 70 and 97 % free from mitochondrial and ER contamination, respectively, as judged by marker enzymes analysis. Isolated plastids have an absolute requirement for exogenously supplied ATP, coenzyme A and bicarbonate for in vitro fatty acid biosynthesis. The greatest rates of fatty acid biosynthesis were observed in the presence of up to 7 mM, 0.35 mM and 60 mM of each of these cofactors, respectively. Although not required for activity, fatty acid biosynthesis was improved by about 100 % by the addition of both MgCl2 and glycerol-3-phosphate. The addition or omission of NADH and NADPH had little or no effect. Fatty acid biosynthesis was optimal at pH 8 in 50 mM Tricine buffer. Under optimum conditions, maximum rates of fatty acid biosynthesis ranged from 400 to 800 nmoles acetate/hr-mg chlorophyll. In comparison to acetate, there was a strong preference for pyruvate as a precursor for fatty acid biosynthesis which was followed by glucose-6-phosphate and glucose, while malate was relatively ineffective as a precursor. Radioactivity from 14C-acetate was recovered almost exclusively in palmitic and oleic acid. Similarly, radioactivity from 14C-acetate or glycerol-3-phosphate was recovered mainly in phosphatidylcholine, phosphatidic acid and neutral lipids, with smaller amounts of phosphatidylglycerol and the plant sulfolipid. Although plastids from soybean somatic embryos are pigmented green with chlorophyll, light has essentially no effect on plastid lipid metabolism. Our observations indicate that soybean embryo plastids more closely physiologically resemble heterotrophic plastids than photosynthetic plastids.

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Biochemistry Commons

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