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Item The synthesis of triglyceride and other non-polar acyl lipids by protoplasts and chloroplasts from barley leaf : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science with Honours in Biochemistry at Massey University(Massey University, 1985) McIntosh, Frances JoanProtoplasts, isolated from barley leaf, were incubated with H¹⁴CO₃ and (1-¹⁴C) acetate. The radioactivity was found incorporated into the lipids sulpholipid, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, and monogalactosyl diglyceride as identified by thin layer chromatography. Large quantities were found in an unidentified region referred to in this thesis as USF (Unknowns near the solvent front) . ThisUSF was much less polar than the phospholipids and galactolipids and ran in the same region as plant pigments. USF, by using TLC with several solvent systems, was identified as consisting of acyl lipids; the major proportion was triglyceride (approximately 55%) with diglyceride, free fatty acid and monoglyceride also present. The radioactivity in the triglyceride was found, by GLC of methylated esters, to be contained in the fatty acids; mainly in linoleate (18:2)and palmitate (16:0) and in lesser concentrations in stearate (18:0) and oleate (18:1). Endogenous levels of triglyceride were found to be very low - only 14.8 nmoles /g of fresh tissue. The results of experiments with sections of tissue of different maturity suggested that the high level of incorporation into acyl lipids was related to tissue immaturity. Chloroplasts were isolated by a traditional blending method and by bursting prepared protoplasts. The latter technique produced chloroplasts of a higher quality. Examination of the incubation products when chloroplasts were incubated with (1-¹⁴C) acetate showed once more the appearance of acyl lipids. Triglyceride made up 35% of the USF from chloroplasts incubations. The label accumulated mainly in palmitate (16:0) on the triglyceride. These results suggest that at least part of the triglyceride synthesis in the plant cell is occurring within the chloroplast, contrary to the findings represented in the literature.Item Lipid biosynthesis in isolated barley protoplasts : a thesis ... for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1983) Bell, Louise CynthiaIn most studies of fatty acid and lipid synthesis in plants there has been poor incorporation of radioactive label from acetate into linoleic (18:2) and linolenic (18:3) acids. Consequently the amounts of these fatty acids found in the galactolipids in such studies are much less than their observed endogenous levels. In the present study incorporation of H 14 CO 3 - and (1 14 C) acetate into lipids of barley protoplasts was examined. CO 2 -dependent O 2 evolution rates of the protoplasts were around 180 µmol O 2 /h/mg Chl and intactness was also ascertained by phase contrast microscopy. Incubating proto- plasts with 1mM H 14 CO 3 - or 50 µM (1- 14 C) acetate resulted in 146.2 and 17 nmol/mg Chl being incorporated into lipids respectively after 1 hour. A concentration of 10 mM was optimal for HCO 3 - incorporation and up to 580 nmol/mg Chl was incorporated into lipids at the end of 1 hour. Mg ++i ions used at 2 mM had little effect on HCO 3 - incorporation while PP i appeared to be slightly inhibitory. Acetate assimilation and its incorporation into lipids was markedly affected by pH and pH 5.8 was chosen for the assay medium. In 20 hour incubations 162 nmol acetate/mg Chl was incorporated. About 33% of label from acetate was found in each of palmitic (16:0) and oleic (18:1) acids with less than 9% in each of stearic (18:0), linoleic and linolenic acids. There was little or no incorporation of acetate into DGDG and less than 10% into each of PG, MGDG, PE and U (unknown lipid). Incorporation into PC after 2 1/2 hours was 36.8% then decreased to 8.9%. Acetate incorporation was most significant into U SF (another unknown lipid), being 73.4%. Although acetate was incorporated into a range of glycerolipids, incorporation into constituent 18:2 and 18:3 of these lipids was not significant.