Regulation of sucrose phosphate synthase in vascular bundles of Washington navel orange fruit (Citrus Sinensis L. Osbeck) by a protein kinase and a protein phosphatase

Abstract

Sucrose phosphate synthase (SPS) is activated by a novel ATP-dependent, Ca-independent kinase and inactivated by a protein phosphatase in vascular bundles isolated from citrus fruit (Citrus sinensis L. Osbeck, cv. Washington navel orange). The SPS purified from dark-treated citrus leaves was used as the substrate for purification of the protein kinase and phosphatase. Sucrose phosphate synthase kinase (SPSK) purified from citrus fruit vascular bundles was resolved in a single band of 45 kDa by SDS-PAGE. The SPSK activated SPS and incorporated 32P from [γ-32P]ATP into the 110 kDa subunit of SPS. The sucrose phosphate synthase phosphatase (SPSP) was purified to a single band of 31 kDa determined by SDS-PAGE. The SPSP was able to dephosphorylate SPS previously phosphorylated by SPSK. The SPSP was shown to be a novel protein phosphatase 2C (PP2C) based on its insensitivity to okadaic acid, activation by Fe3+ and inhibition by Mg2+, Fe2+ and Mn2+. Activation of SPS by a Ca2+-independent kinase and inactivation by a PP2C in citrus constitutes a novel mechanism regulating SPS activity in plants.