Pyruvate Dehydrogenase Complex from Germinating Castor Bean

Subceliular organelles from castor bean (Ricuns commnwuis) endosperm were isolated on discontinuous sucrose gradients from germinating seeds which were 1 to 7 days postbit o. Marker enzyme activities of the organeUes were measured (fumarase, catalase, and triose phosphate isomerase) and the homogeneity of the organelle fractions was examined by electron microscopy. Pyrumate dehydrogenase complex activity was measured only in the mitochondrial fraction and attempts to activate or release the enzyme from the proplastid were not successful. A pathway is proposed for the most efficient use of endosperm carbon for de novo fatty acid biosynthesis that does not require the presence of the pyruvate dehydrogenase complex in the proplastid to provide acetyt-coenzymeA. Recent studies with developing castor beans have shown that the carbon source for fatty acid synthesis is sucrose, which is transported from the leaves to the endosperm (12, 20, 24-26). Yamada et al. (24, 25), using radioactive fatty acid precursors, have shown that isolated proplastids are able to convert sucrose to fatty acids. However, Simcox et al. (20) have examined the subcellular distribution of the intermediate enzymic steps responsible for conversion of sucrose to pyruvate and their findings do not entirely support those of Yamada and co-workers. Dennis' group (20) found that fatty acids could not be synthesized directly from sucrose in proplastids. Rather, hexose-P synthesis from sucrose occurs in the cytosol along with the first oxidative step in the pentose-P pathway (glucose-6-P dehydrogenase). The glucose-6-P produced in the cytosol is transported into the proplastids where conversion to pyruvate dccurs. The PDC3 catalyzes the oxidative decarboxylation of pyruvate to yield the acetyl-CoA needed for fatty acid synthesis. The complex is generally associated with the mitochondria in eucaryotes (17). Reid et al. (18) have demonstrated the presence of the complex in proplastids in developing castor bean endosperm and Elias and Givan (7) have reported that pea chloroplasts also contain PDC. Both ofthese nonmitochondrial locations have been confirmed in our laboratory (16; manuscript in preparation). In the case of developing endosperm, the location of the complex within the proplastid allows the generation of needed acetyl-CoA inside the organelle and eliminates the need for transport of acetyl-CoA from the mitochondria. In germinating castor bean endosperms the situation is different, ' This work was supported by National Science Foundation Grant PCM 77-11390 and Missouri Agricultural Experiment Station, Series No. 8374. 2To whom requests for reprints may be addressed. 3Abbreviations: PDC: pyruvate dehydrogenase complex; MOPS: 4(Nmorpholino-propane sulfonic acid; TPI: triose phosphate isomerase. according to recent work by Fritsch and Beevers (8). They have demonstrated the presence of ATP citrate lyase in proplastids of germinating endosperm. This extramitochondrial source of acetylCoA presumably would supply the precursor for fatty acid biosynthesis. Another possible source of acetyl-CoA in proplastids is PDC and Fritsch and Beevers (8) have also reported measuring proplastid PDC. However, the proplastid PDC activity was low (30-fold lower) compared to that measured in the mitochondria. This is in contrast to the developing endosperm in which the mitochondrial and proplastid PDC activities appear to be essentially equal (18). The carbon source of fatty acid precursors in germinating endosperm has not been identified. Vick and Beevers (22) have shown that castor bean fatty acids are synthesized primarily in the proplastids and that maximum synthesis occurs in the first 3 days of germination, prior to maximum development of glyoxysomes and mitochondria. It is in the newly synthesized glyoxysomes where fatty acid ,-oxidation occurs (5, 9). These findings support the hypothesis that the proplastids of the germinating endosperm synthesize the fatty acids de novo for construction of the membranes of the organelles which then can facilitate further degradation of the stored lipid (6, 13, 22) for gluconeogenesis (1). This report describes studies on the subcellular localization of the pyruvate dehydrogenase complex in the germinating castor bean as well as in the developing endosperm tissue. A proposal is made for more efficient use of the stored carbon: that acetyl-CoA for proplastid fatty acid synthesis is generated via a proplastid ATP citrate lyase instead of a proplastid PDC. MATERIALS AND METHODS Chemicals. Coenzyme A and NAD+ were purchased from P/L Biochemicals. Potassium pyruvate, TES, Tricine, MOPS, thiamine pyrophosphate, cysteine-HCl, malate, DL-glyceraldehyde-3-phosphoric acid, and a-glycerophosphate dehydrogenase were obtained from Sigma Chemical Co. Triton X-100 was purchased from Packard and NaH"4CO3 was from ICN. Cellulysin and Macerase were obtained from Calbiochem. Plant Material. Castor beans (Ricinus communis var. N5, McNair Seed Company, Plainview, Tex.) were soaked for 18 h in running tap water and then germinated in Vermiculite for I to 7 days in the dark at 30 C. Seeds were also planted in the horticultural gardens and developing castor beans were harvested 30 to 40 days after pollination. Organelle Isolation. Three or more days after imbibition the developing cotyledons were separated from the endosperm tissue of seeds. To isolate the subcellular organelles the endosperm tissue was homogenized by gently crushing with a mortar and pestle for 2 min. Fifteen ml of grinding medium was used per 10 g of tissue. The grinding medium contained 0.2 M Tricine, 1 mm EDTA, 1 mM MgCl2, 0.1% (w/v) BSA, and 0.4 M sucrose, pH 7.5 (3). The homogenate was filtered through two layers of cheesecloth and one layer of Miracloth (Chicopee Mills, Milltown, N. J.) and 314 www.plantphysiol.org on November 11, 2017 Published by Downloaded from Copyright © 1980 American Society of Plant Biologists. All rights reserved. PDC FROM CASTOR BEAN ENDOSPERM centrifuged at 12,000g for 10 min; the pellet was gently resuspended in about 2 ml of ginding medium for 10 g of original tissue. All isolation steps were performed at 4 C. Discontinuous sucrose density gradients were used to resolve subcellular organelles in the resuspended 480-12,000g fraction. The gradients consisted of a 4-ml cushion of 60% sucrose overlaid with 8 ml 51%, 8 ml 43%, 8 ml 35%, and 7 ml 25% sucrose. Sucrose solutions were w/w in 10 mm Tes (pH 7.5). The 51% sucrose contained 1 mM MgCI2 and the 43, 35, and 25% sucrose solutions contained 0.5 mm MgCl2. From 1 to 2 ml of the resuspended organelle fraction was layered onto the gradients which were centrifuged at 4 C in a Beckman SW 27 rotor. The gradients were centrifuged at 96,300g for 1 h using a Beckman L2-65B ultracentrifuge. An ISCO density gradient fractionator was used to fractionate the gradients from the top into 1.5-ml samples and to record the Ao0. When organelles were isolated from developing castor bean endosperm, the ratio grinding media to tissue was 1:1 (v/w). Otherwise, the isolation procedure was identical to that described for germinating endosperm tissue. Subcellular organelles were also isolated from protoplasts which were obtained by enzymic digestion of germinating endosperm tissue according to the methods described by Nishimura and Beevers (14). The ruptured protoplast preparation was layered on the sucrose gradient described above as well as the gradient reported by Nishimura and Beevers (14). Enzyme Assay. Each fraction from gradient was assayed for fumarase (21), TPI (21), catalase (21), and PDC. PDC was assayed at room temperature using a recording spectrophotometer to monitor NADH formation at 340 nm. The standard assay mixture contained 85 ,umol MOPS (pH 7.5), 0.21 yimol thiamine pyrophosphate, 1 jmol MgCl2, 2.4 ,imol NAD+, 0.12 ,umol Li CoA, 2.6 !Lmol cysteine-HCl, 1 ,umol potassium pyruvate, and enzyme complex in a total volume of 1 ml. The final pH of the assay mixture was 7.35. Triton X-100 was added to all assays to a fmal concentration of 0.1% to ensure organelle rupture. Ribulose bisP carboxylase activity was measured radiochemically using NaH"4CO3 at 30 C according to the method of Benedict et al. (2). Electron Microscopy. Organelles were prepared from 3-day germinating castor bean endosperm as described above. Mitochondrial and proplastid fractions were collected from two gradients, pooled, and then gently mixed with the appropriate amount of 50%o glutaraldehyde to a final concentration of 3%. The organelles were fixed for 3 h at 4 C, then again carefully diluted (over a 10-min period) with 10 mM Tes (pH 7.5) to a final sucrose concentration of 15% (w/w), and then pelleted at 18,000g in an SW 27 rotor for 10 min. Both pellets were washed twice with the Tes buffer and then resuspended in molten 4% agar. When hardened the agar was chopped into 1-mm2 pieces which were postfixed in 1% OS04 (in Tes) for 3 h at 4 C. The samples were washed twice with Tes, dehydrated with an acetone series, and then embedded in Epon. The tissue was thin sectioned and photographed at the Electron Microscopy Facility, University of Missouri, Columbia. A 0.5-ml portion of each organelle fraction was not fixed so that organelle marker enzymes (fumarase, TPI, and PDC) could be assayed and thus provide an estimate of the homogeneity of the fractions being prepared for electron microscopy.