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Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoil gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl-CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction.or leucoplast glycolytic enzymes are more than adequate to account for the triacylglycerol synthesis of castor endosperm (17). However, glycolytic intermediates have not been well characterized as carbon sources for fatty acid synthesis (18). In this study, we present data regarding the kinetics and exogenous cofactor requirements for fatty acid synthesis in isolated leucoplast preparations using pyruvate, malate, and acetate as carbon sources. We show that malate and pyruvate support much higher rates of fatty acid synthesis than does acetate and the metabolism of both these substrates alleviates any requirement for externally added reductant. Based on the measurement of leucoplast-associated NADP+-malic enzyme activity, we suggest that cytosolic malate may be a source of carbon for fatty acid synthesis in vivo. MATERIALS AND METHODS Plant MaterialCastor plants (Ricinus communis L. cv Baker 296) were greenhouse grown under natural light supplemented with 16 h fluorescent light.Storage lipids make up 45 to 50% of the dry weight of castor bean (Ricinus communis) endosperm (9, 15). During development, the conversion of sucrose to triacylglycerols is the major metabolic activity of this tissue (9). Dennis (11) presented a model indicating the possible pathways of triacylglycerol synthesis from sucrose. The model is primarily based on enzyme compartmentation studies (12,17,22,23,27) and suggests that glycolytic intermediates may cross the leucoplast membrane and serve as carbon skeletons for fatty acid synthesis. Acetate is another possible precursor.Acetate incorporation into fatty acids has been used to study the cofactor requirements for fatty acid biosynthesis (6,16,18,25,26). It is unlikely that acetate is the in vivo carbon precursor of fatty acid synthesis because of its relatively low cellular concentration and the low efficiency of acetate utilization for de novo fat...

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The rate of respiratory O , consumption by Chlamydomonas reinhardtii cell suspensions was greater after a period of photosynthesis than i n the preceding dark period. This "light-enhanced dark respiration" (LEDR) was a function of both the duration of illumination and the photon fluence rate. Mass spectrometric measurements of gas exchange indicated that the rate of gross respiratory O , consumption increased during photosynthesis, whereas gross respiratory CO, production decreased in a photon fluence ratedependent manner. The rate of postillumination O , consumption provided a good measure of the O, consumption rate in the light.LEDR was substantially decreased by the presence of 3-(3,4-dichloropheny1)-1,l-dimethylurea or glycolaldehyde, suggesting that LEDR was photosynthesis-dependent. The onset of photosynthesis resulted in an increase in the cellular levels of phosphoglycerate, malate, and phosphoenolpyruvate, and a decrease in whole-cell ATP and citrate levels; all of these changes were rapidly reversed upon darkening. These results are consistent with a decrease i n the rate of respiratory carbon flow during photosynthesis, whereas the increase in respiratory O, consumption during photosynthesis may be mediated by the export of photogenerated reductant from the chloroplast. We suggest that photosynthesis interacts with respiration at more than one level, simultaneously decreasing the rate of respiratory carbon flow while increasing the rate of respiratory O , consumption.

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