Studies on the Entry of Fructose - 2 , 6 - Bisphosphate into Chloroplasts 1

The regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6P2) has an important function in controlling the intermediary carbon metabolism of leaves. Fru-2,6-P2 controls two cytosolic enzymes involved in the interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate (fructose-1,6-bisphosphatase and pyrophosphate,fructose-6-phosphate 1-phosphotransferase) and thereby controls the partitioning of photosynthate between sucrose and starch. It has been demonstrated that Fru-2,6-P2 is present mainly in the cytosol. Here we present evidence that Fru2,6-P2 can be taken up by isolated intact chloroplasts but at a very slow rate (about 0.01 micromoles per milligram of chlorophyll per hour). This uptake is time and concentration dependent and is inhibited by PPi. When provided a physiological concentration of Fru-2,6-P2 (10 micromolar), chloroplasts accumulated up to 0.6 micromolar Fru-2,6-P2 in the stroma. Elevated plastid Fru-2,6-P2 levels had no effect on overall photosynthetic rates of isolated chloroplasts. The results indicate that, while Fru-2,6-P2 enters isolated chloroplasts at a sluggish rate, caution should be exercised in ascribing physiological importance to effects of Fru-2,6P2 on chloroplast enzymes. Fru-2,6-P23 plays a key regulatory role in the intermediary carbon metabolism of leaves (4, 10, 16). It does so by controlling two cytosolic enzymes involved in the interconversion ofFru1,6-P2 and Fru-6-P: cytosolic FBPase, which is strongly inhibited by Fru-2,6-P2 (5, 9), and PFP, which is activated by Fru-2,6-P2 (10, 16). PFP activation is mainly in the glycolytic or forward direction (production of Fru-1,6-P2). High levels of Fru-2,6-P2 thus seem to favor glycolytic carbon flow, whereas low levels of Fru-2,6-P2 result in the production of hexose-monophosphates and hence sucrose synthesis (9, 17, 18). The relative activities that synthesize and degrade Fru-2,6P2 (Fru-6-P,2K and Fru-2,6-P2-ase) determine the concentra' Supported by a grant from the National Science Foundation. 2Recipient of an EMBO Long Term Fellowship. Present address: Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. 3Abbreviations: Fru-2,6-P2, fructose-2,6-bisphosphate; Fru1,6-P2, fructose-1,6-bisphosphate; Fru-6-P, fructose-6-phosphate; FBPase, fructose-1,6-bisphosphatase (EC 3.1.3.1 1); PFP, pyrophosphate,fructose-6-phosphate l-phosphotransferase (EC 2.7.1.90); Fru6-P,2K, fructose-6-phosphate, 2-kinase (EC 2.7.1); Fru-2,6-P2-ase, fructose-2,6-bisphosphatase (EC 3.1.3); Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). tion of Fru-2,6-P2 and thus the flow of carbon in the cell. In plants the major forms of these activities are found on different cytosolic proteins (13, 14). Typical concentrations of cytosolic Fru-2,6-P2 are in the micromolar range (5, 17), although for CAM plants higher concentrations (about 100 liM) have been reported (6). Although Fru-2,6-P2 is found mainly in the cytosolic fraction of leaves, the question arises as to whether Fru-2,6-P2 can enter chloroplasts and, if so, whether it affects photosynthesis. We have, therefore, studied Fru-2,6-P2 uptake by isolated intact chloroplasts and now report that, while Fru-2,6P2 entry is observed, uptake is limited and quite slow. Furthermore, as elevated levels of Fru-2,6-P2 were found to have no effect on photosynthesis, the physiological significance of Fru-2,6-P2 uptake and function in chloroplasts is questionable. MATERIALS AND METHODS