Regulation of Sucrose Synthesis by Cytoplasmic Fructosebisphosphatase and Sucrose Phosphate Synthase during Photosynthesis in Varying Light and Carbon

The aim of this work was to investigate whether sucrose synthesis in the cytosol of leaf cells is regulated in response to the supply of energy and organic carbon from the chloroplast. Fluxes into sucrose and metabolite levels in wheat (Triticum aestivum var Timmo) leaf protoplasts were compared in a range of light intensities and CO2 concentrations, showing that sucrose-phosphate synthase and the cytosolic fructose-l,6-bisphosphatase are inhibited in situ when the supply of trioseP from the chloroplasts decreases. Such a regulation might aid CO2 fixation in limiting conditions by permitting stromal metabolites to be maintained at higher levels than would otherwise be possible. pools are depleted, or whether the enzymes of sucrose synthesis are actually regulated by smaller fluctuations of selected metabolites. This would promote CO2 fixation in limiting conditions by permitting higher levels of metabolites to be maintained in the stroma. There is already evidence that protoplasts and leaves can contain substantial levels of metabolites in the dark (25). We have now studied in more detail the fluxes and metabolite levels in wheat leaf protoplasts during photosynthesis in varying light intensities and CO2 concentrations. The results show that both the cytosolic FBPase and sucrose P synthase are involved in regulating the rate of sucrose synthesis in response to the capacity of the chloroplast to provide trioseP. The trioseP produced in the stroma during photosynthesis represents an important branch point in metabolism. Some is metabolized further in the stroma to regenerate RuBP2 which is needed to support more CO2 incorporation, while some is exported via the Pi translocator in exchange for Pi. The two processes must be balanced to achieve rapid CO2 fixation. If export of trioseP should exceed the rate at which new trioseP is being synthesized from CO2 and Pi, the levels of metabolites in the stroma would decrease and the operation of the Calvin cycle would be impaired or eventually stopped (see 26). With isolated chloroplasts, this balance is artificially achieved by optimizing the Pi concentration used in the medium. When CO2 fixation is restricted by low pH, C02, or light, the maximal rates of CO2 fixation in the new conditions are attained only when the Pi in the medium is lowered (6), decreasing the rate at which carbon is withdrawn from the chloroplasts, so that it matches the lower rate ofCO2 fixation. This minimizes the depletion of the stromal metabolite levels, which would otherwise lead to an additional inhibition ofphotosynthesis. In situ, most of the exported trioseP is converted to sucrose in the cytosol, releasing Pi which reenters the chloroplast. In analogy to isolated chloroplasts, there will be a marked depletion of the stromal metabolite pools during photosynthesis in limiting light or CO2, unless the rate of sucrose synthesis is readjusted to match the rate of CO2 fixation. The question arises whether photosynthetic cells undergo large fluctuations in the levels of metabolites because sucrose synthesis is restricted only when the metabolite 'Supported by the Deutsche Forschungsgemeinschaft. 2 Abbreviations: RuBP, ribulose 1,5-bisphosphate; FBP, fructose 1,6bisphosphate; FBPase, fructose-1,6-bisphosphatase; PGA, 3-phosphoglyceric acid; SBP, sedoheptulose 1,7-bisphophosphate. MATERIALS AND METHODS Protoplast Preparation. Protoplasts were prepared from wheat (Triticum aestivum var Timmo) leaves as in Lilley et al. (18) and were stored at 4°C in the dark. Medium. Protoplasts were incubated in 0.42 M sorbitol (for silicone oil centrifugation) or 0.32 M sorbitol plus 0.1 M raffinose (for other experiments). Raffmose was included to provide a isopycnic medium to prevent sedimentation of protoplasts (see 18). The medium always contained 10 mm KC1, 0.5 mM MgC12, 0.2% (w/w) PVP, and 0.05% BSA. Buffer and NaHCO3 concentration varied with the experiment (see below and figures). Light Intensity. In experiments where light intensity was varied, 50 mm Hepes (pH 7) and 1.2 mm NaHCO3 were used. Protoplasts were illuminated in saturating light for 8 min, before altering the light intensity. Light intensity was measured with a Kipp and Zonen flight meter and was corrected for light absorbed by the vessels containing the protoplasts. The protoplasts were diluted to 20 to 25 ,g Chl/ml and held in containers less than 9 mm in diameter to minimize self-shielding. As light sources, two 220-w tungsten-halogen lamps in projectors were used, and the intensity was controlled by varying the voltage applied. CO2 Concentration. To vary the CO2 concentration rapidly, the pH in the medium was adjusted (pK C02, HCO3 = 6.85). Protoplasts were initially illuminated in 10 mm Mes at pH 5.5 with 0.25 to 0.5 mM NaHCO3 (equivalent to 0.22-0.43 mM C02) for 6 to 9 min. During this time, the C02 removed by fixation (0.15-0.2 mM) could be measured by 02 evolution or ' C incorporation. Then, by adding 10 mm Tris (HCO3-free) to raise the pH to 8, the CO2 concentration was decreased to 1 to 5 AtM. Alternatively, bicarbonate was included in the Tris to maintain the CO2 at higher concentrations. The experiments were carried out in an 02 electrode to monitor 02 evolution and 02 concentration. In experiments where ['4CJbicarbonate was added, the specific activity (55 Ci/mol) was taken into account in calculating the total CO2 plus