Characterization of Glucose-6-Phosphate Incorporation into Starch by Isolated Intact Cauliflower-Bud Plastids

Intact 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: 458-467). Incubation 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-1 protein h-1) could only be observed when both 3-phosphoglyceric acid and ATP were present as well. Starch synthesis in isolated plastids was strongly dependent upon the intactness of the organelle. The presence of a high-affinity ATP/ADP translocator with a Km for ATP of 12 [mu]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[prime]-diisothio-cyanostilbene-2,2[prime]-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.