ABSTRACIIn vivo pool sizes of a range of metabolites have been determined in subcellular fractions of darkened and illuminated mesophyll protoplasts of Avena sativa L. These estimations were made by combining a method of rapid protoplast fractionation with enzymic cycling techniques. Results are given for reduced and oxidized pyridine nucleotides, triose phosphates, 3-phosphoglycerate, inorganic phosphate, aspartate, malate, oxaloacetate, glutamate, 2-oxoglutarate, and citrate, from chloroplasts, mitochondria, and a fraction representing the remainder of the protoplast. The results indicate distinct differences of compartmented levels of certain metabolites between darkened and illuminated protoplasts.Cells of green tissue have two major systems for energy production, viz. that in chloroplasts and that in mitochondria. In the dark, ATP formed by oxidative phosphorylation is exported from mitochondria to energy consuming sites in the cell, while a back flow of ADP maintains phosphorylation (15). Under illumination there is additional production of ATP by chloroplasts: the latter exceeds considerably that by mitochondria (26).From metabolite determinations on nonaqueously fractionated leaf tissue, Heber and Santarius (12) suggested that mitochondrial respiration of green tissue possibly is inhibited in the light. This hypothesis was deduced from an observed increase in the extrachloroplast ATP/ADP ratio after the onset of illumination, and invoked to the operation of the TP2/PGA shuttle across the inner chloroplast membrane (10,12). Such an increase of the ATP/ADP ratio also has been observed by other authors (8,22). In addition, Goller et al. (6) showed that the incubation of oat mesophyll protoplasts with permeable and specific inhibitors of the light-dependent electron transport is almost without effect on the cytosolic adenylate ratio in the light. These results were taken as evidence that light has no direct effect on oxidative phosphorylation and that, potentially, mitochondria are equally active under darkness and light.Recent experiments with isolated animal and plant mitochondria (5,27) ratio is not the primary factor in the control of respiration in isolated mitochondria. Therefore, Dry and Wiskich (5) concluded that, if leaf mitochondrial respiration is inhibited in the light, the ATP/ADP ratio would be of minor importance in regulation, i.e. respiration may be controlled in more than one way (27). Information on in vivo pool sizes of metabolites in various compartments is limited and mainly has been derived from nonaqueous fractionations of leaf tissue into chloroplast and extrachloroplast material (e.g. Heber and coworkers [11][12][13]29]). A better insight into mechanisms regulating mitochondrial respiration is only possible, however, when pool sizes of a wider range of metabolites within different cellular compartments are known. Importantly, knowledge of how environmental perturbations affect the pool sizes is necessary.In the present report we have combined our method of rapid fractionation of m...