Oxidation of Paraffins by Plant Tissues

Paraffins are found in almost every organism, plants (2), animals (13), and microorganisms (14). The most common biological source of paraffins appears to be the surface lipids of plants (2), the chain length of the most abundant paraffin being most often C,9 or C31. These natural paraffins can be readily catabolized by soil organisms (5) and to a limited extent by anlimals (6). The mechanism of paraffin oxidation was elucidated with bacteria (12) and with enzymes isolated from them (4,15,16). Young leaves of many plants readily synthesize paraffins from fatty acids (8). Experiments on the time course of incorporation of labeled substrates into paraffins of Brassica oleracea failed to show any turnover, possibly because the paraffins are ejected onto the surface of the leaves as soon as they are synthesized (7). Attempts to show catabolism by providing Co, paraffin to excised leaves, leaf tissue slices, or leaf homogenates failed. The extremely low solubility of these very long paraffins, and the resulting lack of uptake made it almost impossible to find out whether plants possess the capacity to oxidize their natural paraffins. In order to circumvent these problems a much smaller paraffin, hexadecane-l-14C, was used as substrate. This communication describes experimental results which show that plants do possess the ability to oxidize the terminal methyl carbon of this paraffin to a carboxyl group. Hexadecane-l-'4C (47.2 mc/mmole, Nuclear-Chicago) contained polar impurities and was therefore purified by column chromatography on activated SilicAR cc-4 100 to 200 mesh (Mallinckrodt Chemical Works. St. Louis, Missouri) with hexane as the solvent (9). The purified hexadecane-l-14C contained no radiochemical impurity as determined by thin-layer chromatography. A sample of this labeled paraffin (50 juc) was dissolved in 4 to 5 ml ethyl ether and 3 (rops of Tween-20 were added. The ether was evaporated with a stream of nitrogen, and the residue shaken with 10 ml of water to give a stable emulsion of the paraffin. Young leaves of broccoli, pea and tobacco, second from the apex and not fully expanded, were sliced (2 X 10 mm) with a razor blade. These slices when incubated with the purified hexadecane, produced radioactive lipids more polar than paraffin as shown in table I. Although Table I. Oxidation of Hexadecane-1-l4C by Leaf Slices of Broccoli, Pea, and Tobacco In experiment 1, 1 g of leaf slices was incubated with 2 X 106 cpm hexadecane-l-14C for 4 hr at 30° in 3 ml water. In experiment 2, 3 g of pea hypocotyl slices were incubated with 4 X 106 cpm hexadecane-l-14C for the periods indicated at 30° in 3 ml water. At the end of the incubation period, tissue slices were ground in a 2:1 mixture of chloroform and methanol, and the total lipids extracted as described before (9). Aliquots of the total lipids were chromatographed on silica gel G plates with hexane as the developing solvent in an unlined tank. Only paraffins migrated with the solvent front. The oxidation products such as fatty alcohols, acids and phospholipids remained at the origin. The radioactivity in this fraction is shown in the table.