lntact plastids from cauliflower (Brassica oleracea var Prince de Bretagne) buds were isolated according to the method described by Journet and Douce (E.P. Journet and R. Douce [1985] Plant Physiol 79: [458][459][460][461][462][463][464][465][466][467]. lncubation of these plastids with various 14C-labeled compounds revealed that glucose-6-phosphate can act as a precursor for starch synthesis. However, significant rates (incorporation of 120 nmol glucose mg-' protein h-') could only be observed when both 3-phosphoglyceric acid and ATP were present as well. Starch synthesis i n isolated plastids was strongly dependent upon the intactness of the organelle. The presence of a high-affinity ATP/ADP translocator with a K,,, for ATP of 12 ~L M was demonstrated by uptake experiments with [14C]ATP. ADP inhibited both ATP uptake and effector-stimulated starch synthesis. Effector-stimulated glucose-6-phosphate-dependent starch synthesis was not significantly influenced by fructose-6-phosphate or 2-deoxyglucose-6-phosphate but was strongly inhibited by triose phosphate and inorganic phosphate. Starch synthesis was also inhibited by 4,4'-diisothio-cyanostilbene-2,2'-disulfonate, which is known to be a potent inhibitor of the chloroplast phosphate translocator. The data presented here support the view that starch biosynthesis in heterotrophic tissues is powered by increasing levels of cytosolic 3-phosphoglyceric acid and ATP when glucose-6-phosphate is available.

We recently developed a method of purifying amyloplasts from developing maize (Zea mays L.) endosperm tissue [Neuhaus, Thom, Batz and Scheibe (1993) Biochem. J. 296, [395][396][397][398][399][400][401]. In the present paper we analyse how glucose 6-phosphate (Glc6P) and other phosphorylated compounds enter the plastid compartment. Using a proteoliposome system in which the plastid envelope membrane proteins are functionally reconstituted, we demonstrate that this type of plastid is able to transport ["%C]Glc6P or [$#P]P i in counter exchange with P i , Glc6P, dihydroxyacetone phosphate and phosphoenolpyruvate. Glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate do not act as substrates for counter exchange. Besides hexose phosphates, ADP-glucose (ADPGlc) also acts as a substrate for starch synthesis in isolated maize endosperm amyloplasts. This process exhibits saturation kinetics with increasing concentrations of exogenously supplied ["%C]ADPGlc, reaching a maximum at

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Abstract. Isolated amyloplasts from cauliflower buds are capable of mobilizing starch. This mobilization is strongly dependent upon the intactness of the plastids and is linear with time for up to 30 min. The degradation of starch occurs via a hydrolytic breakdown and is stimulated by ATP-dependent phosphorylation of products of this hydrolysis. The rate of phosphorolytic stimulation of starch degradation is negligible. Carbohydrates derived from starch degradation do not appear to enter the oxidative pentose-phosphate pathway. Phosphorylation of hydrolytically solubilized intermediates leads to the synthesis of dihydroxyacetone phosphate and hexose phosphates. 3-Phosphoglyceric acid acts as an inhibitor of starch mobilization. The export of labelled phosphorylated intermediates from amyloplasts containing 14C-labelled starch implies the presence of an amyloplastic phosphate translocator in these plastids. The physiological role of varying concentrations of 3-phosphoglyceric acid is discussed with respect to the regulation of a metabolic cycle of simultaneous starch synthesis and degradation.