Species-Specific Differences in Acetyl Coenzyme A Synthesis of Chloroplasts Hans-Jürgen Treede, Burgi Riens, and Klaus-Peter Heise Institut für Biochemie der Pflanze der Universität Göttingen. Untere Karspüle 2. D-3400 Göttingen, Bundesrepublik Deutschland Z. Naturforsch. 41c, 733-740 (1986); received March 21/April 17, 1986 Acetyl-CoA and Fatty Acid Synthesis, Chloroplasts The present investigation indicates that photosynthetically active chloroplasts can synthesize acetyl-CoA either from acetate via acetyl-CoA synthetase (ACS) or from pyruvate via the pyru­ vate dehydrogenase complex (PDC). Both enzyme systems have been assayed in rapidly prepared extracts of chloroplasts isolated from spinach, peas and maize mesophyll. Their kinetic properties showed few species-specific differences. The differing pyruvate and acetate concentrations within the corresponding leaf tissues have been interpreted, therefore, as constituting a major factor determining the relative involvement of both acetyl-CoA synthesizing systems within the different types of chloroplasts. The idea that acetate originates from mitochondria and pyruvate from the cytosol has been supported by nonaqueous fractionation studies. Diffusion-mediated faster up­ take of acetate may indicate a predominant role of the ACS in spinach chloroplasts. Higher cellular pyruvate/acetate-ratios (2—5) in pea and maize leaves may enhance pyruvate uptake into chloroplasts and thus PDC-driven acetyl-CoA synthesis in pea and maize mesophyll chloroplasts. Maize mesophyll chloroplasts even show a light-driven pyruvate uptake accompanied by a stimu­ lated acetyl-CoA and fatty acid formation. Assuming light-dependent increasing parameters in the stroma space, like Mg2+-concentrations, pH and ATP, as further control criteria in chloroplast acetyl-CoA formation, the ACS appears better adapted to the circumstances in illuminated chloroplasts because of the fact that 1. the ACS requires these cofactors altogether; 2. the PDC is stimulated by increasing pH (up to 8) and Mg-levels (up to 5 m M ) alone. Introduction spinach chloroplasts from 3-PGA by plastidic iso­ enzymes [9]. Thus, in spinach leaves, acetate may be Recent investigations indicate the coexistence of provided by acetyl-CoA hydrolysis in mitochondria two pathways for acetyl-CoA synthesis in photosyn­ [10] while pyruvate may be predominantly synthe­ thetically active chloroplasts of different species sized in the cytosol [11, 12] and to a lesser degree [1—5]. One operates through the action of acetyl- within the chloroplasts [9]. Because of deficient CoA synthetase (ACS), the other through the pyru­ acetyl-CoA hydrolyzing activities in pea mitochon­ vate dehydrogenase complex (PDC). It has been dria [13], a generalization of these species-specific proposed that both enzymes are primarily controlled findings appears questionable. However, it has been by the stromal levels of their substrates acetate and recently shown that especially in spinach chloroplasts pyruvate [5]. Both metabolites seem to originate 1. the calculated stromal metabolite levels appear to mainly from outside the chloroplast and to enter the favor acetate as substrate for acetyl-CoA- and sub­ plastid by either diffusion [6—8] or carrier mediated sequent fatty acid formation [5]; 2. the acetyl-CoA transport (postulated for pyruvate concentrations synthesized from acetate(via ACS) seems to inhibit lower than 1 mM only) [8]. There is also evidence its formation from pyruvate [5] at the expense of supporting an additional formation of pyruvate in branched-chain amino acids by feedback control [14]- Abbreviations: ACS, acetyl-CoA synthetase; ACP, acyl- The pathway of acetyl-CoA formation has been carrier protein; BSA, bovine serum albumin; CoA. co­ studied here for those chloroplasts which are not lim­ enzyme A; CS, citrate synthetase; DTE, dithioerythritol; ited in pyruvate content. Stromal accumulation of GAP-DH, glyceraldehyde 3-phosphate dehydrogenase; PDC, pyruvate dehydrogenase complex; PEP-CX. phos- pyruvate in this case in question is either due to en­ phoenolpyruvate carboxylase; 3-PGA, 3-phosphoglycerate. hanced cellular concentrations of this metabolite or Reprint requests to Dr. K.-P. Heise. by light-dependent active transport as recently Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen shown for maize mesophyll chloroplasts [15], These 0341 - 0382/86/0700 - 0733 $01.30/0 chloroplasts are suitable for in vitro studies of the 734 H.-J. Treede et al. ■ Species-Specific Differences in Acetyl Coenzyme A Synthesis of Chloroplasts two coexisting acetyl-CoA synthesizing enzymes CoA trapping with dithioerythritol [3], Incubation within the chloroplast. conditions for acetyl-CoA and fatty acid synthesis in maize mesophyll chloroplasts are given in the legends to tables and figures and extraction proce­ Materials and Methods dures and measurement of labelled fatty acids were The cultivation of spinach [16], peas [5], and maizeas described in [23]. [15] has been described earlier. The overall meta­ bolite levels have been determined in protein free Results extracts of the different leaf tissues. A known quanti­ ty (on a chlorophyll or protein base) of leaf sections 1. Overall levels of acetate and pyruvate (without middle rib) was homogenized in liquid N2. Before studying in detail the species-dependent The resulting fine powder was moderately acidified capacity of photosynthetically active chloroplasts for with 5% HC104, gradually thawed under continued the synthesis of acetyl-CoA from either pyruvate homogenization, kept 15 min at 4 °C, centrifuged or acetate, we measured concentrations of both and neutralized with 5m KOH-1 m Tricine modified metabolites in protein-free extracts of leaf tissues. according to Stitt et al. [17], Aliquots of the eluates Losses during the elution of the homogenates were were analyzed for either acetate [5] or pyruvate with minimized by working at low temperatures and by enzymatic kits from Boehringer Mannheim (FRG) using moderate acidification and neutralization ac­ with a double beam spectrophotometer (Sigma cording to Stitt et al. [17], Aliquots of the eluates ZFP 22). Chi. was assayed according to Arnon [18] were stored in liquid N2 prior to assay. and protein after the method by Lowry [19]. Thein As shown in Table I, the observed significant differ­ vivo-distribution of metabolites in leaf cells has been ences in the endogenous acetate and pyruvate levels investigated by a nonaqueous fractionation techni­ of the leaf tissues suggest species-specific variations in que of dry-powders from spinach leaves prepared by the availability of both precursors for acetyl-CoA for­ thorough homogenization and subsequent lyophiliza- mation. Thus, in extracts from spinach the acetate tion of quickly frozen leaf material according to concentration (59 ± 20 nmol-mg-1 Chi) exceeded Gerhardt et al. [20]. that of pyruvate (20 ± 10 nmol • mg-1 Chi) by a factor Isolation, purification and critical examination of of 3. In corresponding measurements with peas and intact spinach and pea chloroplasts was carried out as maize a 2—5-fold higher pyruvate than acetate level in [5, 16] and that of maize mesophyll chloroplasts as was determined. Pea and maize tissues contained in [21]. The preparation methods of crude chloro­ about 3—9-fold more pyruvate but only half of the plast extracts including the determinations of ACS acetate level found with spinach. Pronounced changes and PDC activities have been described elsewhere in the intracellular acetate and pyruvate concentra­ [5, 16]. Acetyl-CoA formation by intact chloroplast tions of the same species appear to take place de­ suspensions has been measured by two radiochemi­ pending on the physiological state of the leaf mate­ cal methods based either on the adsorption of the rial. Thus, a decrease of pyruvate in leaf cells in the 14C-labelled acetyl-CoA to charcoal [22] or on acetyl- light and its increase after darkening has been inter- Table I. Determination of acetate and pyruvate concentrations in protein-free extracts of dif­ ferent leaf tissues. Results are mean values of 5 — 10 experiments. Metabolite concentrations Leaf tissue [nmol-mg 1protein] [nmol • mg Chi] Overall concentrations* [M’M] pyruvate acetate pyruvate acetate pyruvate acetate Spinach 1 ± 0.5 3 ± 1 20 ± 10 59 ± 20 33 ± 16 98 ± 33 Peas 3.5 ± 1 2 ± 0.5 66 ± 19 38 ± 10 111 ± 32 63 ± 16 Maize 11 ± 2.5 2.5 ± 1 185 ± 42 39 ± 23 308 ± 70 65 ± 38 * For estimations of overall concentrations it has been assumed that 1 mg Chi is approximately equally distributed in 600 ^1 of cell sap. H.-J. Treede et al. • Species-Specific Differences in Acetyl Coenzyme A Synthesis of Chloroplasts 735 preted in terms of an inhibition of glycolysis in the phyll cell with 200 (xl-mg-1 Chi in space being sub­ light and its reversal in the dark [24], For estimation divided into chloroplast (25 jil-mg-1 Chi), mito­ of the endogenous overall concentrations of both chondrial, cytosolic (20 |xl-mg_1 Chi) and vacuolar substrates it has been assumed that 1 mg of chloro­ compartment (150 ^il-mg“1 Chi) [20]. From this phyll is approximately equally distributed in 600 (u.1 of point of view, the mesophyll as predominant cell cell sap. This value has been determined by dehydra­ type appears to make up one third of the total cell tion of either leaf sections or of their homogenates sap in spinach leaf tissue (600 (il-mg-1 Chi) only. and showed little variation within the different leaf tissues investigated. Based on this assumption en­ 2. Intracellular distribution of acetate and pyruvate dogenous acetate concentrations were generally be­ tween 30 and 130 and between 65 and 130 jxm in At present there is little information concerning spinach leaves (Table I). These values agree better the cellular distribution and the stromal level of both with those recently reported for young spinach leaves metabolites. Thus, preliminary rough estimates of (70 |xm) [2] than with the significantly higher con­ the stromal pyruvate amounts were in the order centrations (> 1 m M ) found in earlier determina­ 0.1 mM in both spinach [26] and pea chloroplasts [8].