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ABSTRAC1This paper describes a technique for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the vacuole of spinach (Spinacia okracea U.S.A. hybrid 424) leaves. Spinach leaves were freeze stopped and the frozen tissue was ground and lyophilized. The dry material was homogenized by sonication in a mixture of carbon tetrachloride and heptane, and fractionated by density gradient centrifuption.Measurements of the activity of marker enzymes in various subcellular compartments show the chloroplastic material mainly appearing in the lightest fractions and the cytosolic material in the middle of the gradient, whereas most of the vacuolar material is found in the heaviest fraction. Using the measured distributions of metabolites and of marker enzymes in each fraction of the gradient, the subcellular distribution of the metabolite can be calculated.As a frst application, the new fractionation technique was used to investigate the subcellular contents of malate and sucrose in spinach leaves. The results show striking diurnal changes of sucrose and malate, with both substances primarily located in the vacuolar compartment. About three times more malate is present at the end of the day than at the end of the night. The sucrose content in the vacuole fails from a maximum of 45 millimolars at the end of the day to an almost undetectable value of approximately I millimolar at the end of the night.An understanding of the metabolism of a plant cell requires information about the metabolite levels in the various metabolic compartments, such as the chloroplast, the mitochondria, the vacuole, and the cytosol. This demands a procedure for tissue fractionation during which the total content and the subcellular distribution of metabolites in these compartments is not altered. Recently, methods have been developed for the assay of subcellular metabolite levels in plant protoplasts in which the protoplasts were ruptured by passage through a nylon net or a capillary tube followed by immediate filtration of the particles either through a layer of silicone oil (8, 15, 18) or a combination of membrane filters (12). The measurement of subcellular metabolite levels in whole leaves is more difficult; one possible approach is to use a nonaqueous fractionation procedure similar to that developed in 1932 by Behrens (2) for the fractionation of nuclei. In this method the tissue is frozen and lyophilized, the dried material homogenized in nonaqueous solvents, and the resulting homogenate is fractionated by centrifugation. The absence of water prevents the enzymic interconversion of metabolites, and the polar enzymes and metabolites are not extracted during the treatment of the cell material with the nonpolar ' Supported by the Deutsche Forschungsgemeinschaft.solvents. This method has also been successfully applied to the separation of chloroplasts (9, 17). The general principle was to purify a chloroplast fraction by several centrifugation steps. Such procedure involves the loss of a large portion ofchloroplasts...

The alterations of subcellular metabolite levels during the day in spinach leaves have been investigated using nonaqueous density gradient centrifugation to separate chloroplasts, cytosol, and vacuole. The results provide direct evidence for the role of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase in regulating sucrose synthesis in leaves and also show that the phosphate translocator is kinetically limiting in vivo.

The subceliular distribution of fructose 2,6-bisphosphate in spinach (Spinacia oleracea) leaves was studied using nonaqueous fractionation, showing that all, or almost all, is located in the cytosol. The amount of fructose 2,6-bisphosphate present in leaves during the diurnal cycle was measured and compared to the accumulation of starch and sucrose, and the amounts of selected phosphorylated intermediates in the leaf. Upon illumination, the level offructose 2,6-bisphosphate decreases, but prolonged illumination leads to an increase in the level to above that found in the dark, which accompanies the onset of rapid accumulation of starch in the leaf.F2,6P22 plays an important role in the regulation of glycolysis and gluconeogenesis in animal tissues (5). Recently, it has been shown that leaves contain substantial quantities of F2,6P2 (1, 10), that both the stromal FBPase (10) and the cytosolic FBPase (1, 10) are inhibited by F2,6P2, and that the cytosol contains a PFP which is stimulated by F2,6P2 (1, 10). However, the role of F2,6P2 in photosynthetic metabolism was not revealed by these investigations. We have now studied the distribution of F2,6P2 between the chloroplast and cytosol in whole leaves, and investigated the extent to which the level of F2,6P2 varies during the light and dark.

The occurrence of O2-insensitive photosynthesis at high quantum flux and moderate temperature in Spinacia oleracea was characterized by analytical gas exchange measurements on intact leaves. In addition photosynthetic metabolite pools were measured in leaves which had been rapidly frozen under defined gas conditions. Upon switching to low 0°in 02-insensitive conditions the ATP/ADP ratio fell dramatically within one minute. The P-glycerate pool increased over the same time. Ribulose bisphosphate initially declined, then increased and exceeded the pool size measured in air. The pools of hexose monophosphates and UDPglucose were higher at a partial pressure of 02 of 21 millibars than at 210 millibars. These results are consistent with the hypothesis that the rate of sucrose synthesis limited the overall rate of assimilation under O2-insensitive conditions.In this article we ask whether manipulating the ambient 02 concentration provides a way of recognizing a situation in which sucrose or starch synthesis limit the rate of photosynthesis. During photosynthesis, CO2 and Pi are converted to triose-P in the chloroplast. The triose-P is converted to sucrose in the cytosol, or starch in the chloroplast, liberating Pi for use in subsequent CO2 fixation. The production of triose-P in the chloroplast requires electron transport, regeneration of RuBP3 and activity of Rubisco. As all these processes are also needed for photorespiration, there is normally a stimulation of photosynthesis when the ambient 02 concentration is lowered to suppress photorespiration. However, photorespiration does not compete for the reactions by which triose-P is converted into end products such as sucrose and starch. This means that the rate of photosynthesis would not be reduced by photorespiration when photosynthesis is limited by the utilization of photosynthesis products provided there is adequate electron transport and carbon reduction cycle activity to cope with the additional fluxes required when photorespiration occurs. The rate of photosynthesis would then be independent of the ambient 02 level. It has been observed that decreasing the 02 concentration from 210 to 21 mbar does not always lead to an increase of photosynthesis, especially when the light intensity is high and the temperature is below 20°C (3,12,14,24). It has been proposed that this 02 insensitivity of photosynthesis is due to a limitation of photosynthate utilization, in particular triose-P utilization for sucrose and starch synthesis (17, 18). When 02 sensitivity is lost photosynthesis also fails to respond to CO2 (18) indicating that the phenomenon is not simply an effect of 02 on pseudo-cyclic photophosphorylation.A limitation ofthe utilization of phosphorylated intermediates would lead to a fall of the stromal Pi. Many studies have shown that the rate of CO2 fixation in isolated chloroplasts depends on the stromal Pi level. Upon lowerng the stromal Pi concentration from 7 to 2 mM, CO2 fixation was decreased by 40% (11) and this inhibition of CO2 fixation was acco...