PLANT PHYSIOLOGY , Vol 101, Issue 1 57-64, Copyright © 1993 by American Society of Plant Biologists
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MOLECULAR BIOLOGY AND GENE REGULATION |
Purification and Light-Dependent Molecular Modulation of the Cytosolic Fructose-1,6-Bisphosphatase in Sugarbeet Leaves
E. Khayat, C. Harn and J. Daie
Plant Science Department, Lipman Hall, Rutgers University, P.O. Box 231, New Brunswick, New Jersey 08903
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.
