Purification and Light-Dependent Molecular Modulation of the Cytosolic Fructose-1,6-Bisphosphatase in Sugarbeet Leaves

Abstract

Cytosolic fructose-1,6-bisphosphatase (FBPase) was purified 472-fold from sugarbeet (Beta vulgaris L.) leaves by ammonium sulfate fractionation, anion-exchange chromatography (DEAE Sepharose), cation-exchange chromatography (S-Sepharose), gel filtration (Sephacryl S-300), and hydrophobic interaction chromatography (Phenyl Sepharose). The dissociated polypeptide (molecular mass of 37 kD) was used to generate polyclonal antibodies. Western blot analysis revealed a single band that was identified as the cytosolic FBPase. Enzyme activity and protein and transcript levels were measured under various light and dark conditions in growth chamber-grown plants. FBPase protein level remained unchanged during a diurnal cycle, but enzyme activity and transcript levels were highest and lowest at the end of the light and dark periods, respectively. Light-dependent increase in the enzyme activity and transcript level was gradual, occurring several hours after the onset of light. At the end of an extended dark period (48 h), FBPase activity was negligible, protein level was unchanged, and transcript level had declined (but considerable amounts of transcript remained). Neither activity nor protein and transcript were detected in etiolated leaves. Nearly 24 h of continuous exposure to light was required before the FBPase protein and activity reached maximal levels. Unlike the chloroplastic FBPase, which is light activated (direct regulation), changes in the cytosolic FBPase activity and transcription appear to be light dependent in an indirect manner. The data provide first evidence on the coarse control of this enzyme via a light-dependent modulation of transcription and posttranslational modification.