Malate- and Pyruvate-Dependent Fatty Acid Synthesis in Leucoplasts from Developing Castor Endosperm

Abstract

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...

Distribution of Pyruvate Dehydrogenase Complex Activities between Chloroplasts and Mitochondria from Leaves of Different Species

“…The substrate pyruvate for cpPDC could be provided by a stromal glycolytic pathway starting with 3-phosphoglycerate (Murphy and Leech, 1978;Hoppe et al, 1993). Altematively, malate could be imported from the cytosol and converted into pyruvate by a stromal NADPmalic enzyme (Smith et al, 1992), or pyruvate could be imported directly to the stroma (Smith et al, 1992). Furthermore, acetyl-COA could be produced in the stroma from acetate by an acetyl-COA synthetase (Kuhn et al, 1981).…”

Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers

“…Leucoplasts were isolated according to the method described by Kang and Rawsthorne (1994), and were purified using a discontinuous Percoll gradient (Smith et al, 1992). The gradient consisted of 2 ml 70% Percoll, 4 ml of each of 50, 35, 22 and 10% PBF-Percoll, and leucoplasts were collected from the 22-35% interface after 8 min of centrifugation at 9200 g using a Sorvall HB-6 swing-out rotor.…”

Bacterial-type Phosphoenolpyruvate Carboxylase (PEPC) Functions as a Catalytic and Regulatory Subunit of the Novel Class-2 PEPC Complex of Vascular Plants

“…Although no definitive physiological role has been established for Class-2 PEPC, it was hypothesized to support carbon flux to malate required for storage of lipid synthesis in developing COS (10). Our interest in COS PEPC was sparked by discoveries that: (i) exogenous L-malate supported maximal rates of fatty acid synthesis by purified leucoplasts from developing COS (21), and (ii) L-malate import from the cytosol into the leucoplast stroma is catalyzed by a malate/P i translocator within the COS leucoplast envelope (22).…”