| Sucrose Phosphate Synthase (SPS, UDP glucose: D-fructose-6-phosphate-2-glucosyltransferase; EC 2.4.1.14) is a key enzyme in the sucrose biosynthesis pathway. ATP-dependent, Ca+2-independent activation of SPS (pH optimum of 7.2 to 7.5) was demonstrated in vascular bundles of the sweet orange (Citrus sinensis L. Osbeck cv. Washington navel). To further characterize the activation, SPS at a low state of activation was purified from dark-treated citrus leaves and used as substrate for the purification of the putative kinase. SPS purification followed by SDS-PAGE resolved two bands of 110 and 50 kD, respectively. A kinase purified from the vascular bundles of citrus fruit was confirmed to activate purified leaf SPS and incorporate [gamma-32P] ATP into the 110 kD subunit of the enzyme. The sucrose phosphate synthase kinase was also detected in vascular bundles of leaves, but not in the leaf lamina, nor the photosynthetic exocarp of citrus fruit (flavedo). Purified sucrose phosphate synthase kinase from the vascular bundles of citrus fruit resolved in two bands by SDS-PAGE of 90 and 45 kD, respectively. A phosphatase, localized in the fruit vascular bundles and acting antagonistically to the kinase, was also demonstrated. Localization of sucrose phosphate synthase kinase in the vascular bundles suggested possible involvement of the enzyme in the regulation of fruit sink strength. By comparing the activities of the enzymes involved in phloem unloading and sucrose metabolism (acid invertase, sucrose synthase, SPS) and factors regulating the enzymes in whole fruit and vascular bundles of fruit of leafy and leafless inflorescences during the 9 weeks after petal fall, the role of the enzymes in controlling sink strength was elucidated. Taken together, the results provide evidence that citrus fruit that are stronger sinks are characterized by high sucrose synthase activity, low SPS activity, and low sucrose phosphate synthase kinase activity in their vascular bundles and a low ratio of sucrose to hexoses. Exogenous GA3 shifted the activities of SPS and sucrose phosphate synthase kinase in favor of a strong sink, suggesting a role for endogenous GA in maintaining sink strength. |
