Phosphorylation of a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase, is regulated physiologically and developmentally in rosette leaves of Arabidopsis thaliana

The phosphorylation status of 6-phosphofructo-2-kinase/ fructose-2,6-bispho sphate 2-phosphatase (EC 2.7.1.105/ EC 3.1.3.46) in rosette leaves of Arabi dopsis was examined. Immunoblotting with specific antisera detected 96-kDa and! 92-kDa bands in the crude protein extracts from rosette leaves of Arab idopsis. Incubation of protein samples with alkaline phosphatase before SDS -PAGE reduced the 96-kDa band with concomitant increase of the 92-kDa band, suggesting that the former is a phosphorylated form of the latter. In acco rdance with this result, 96-kDa and 92-kDa bands were immuno-precipitated f rom the crude protein extracts from [P-32]orthophosphate-labeled rosettes o f Arabidopsis; and, the former was heavily labeled, the latter faintly labe led. Analysis of phospho-amino acid residues derived from the [P-32]-labele d 96-kDa band revealed that the phosphorylation occurred on serine and thre onine residues, excluding the possibility that the phosphorylated band repr esent a phospho-histidine intermediate that is known to form in the phospha tase reaction. The relative level of the 96-kDa band over the 92-kDa band i n whole rosette extracts changed diurnally and was highest at the beginning of nighttime. Furthermore, the 96-kDa band was highly enriched in the extr acts of very young rosette leaves, suggesting that the phosphorylation stat us of 6-phosphofructo-2-kinase/fructose2,6-bisphosphate 2-phosphatase is re gulated physiologically and developmentally in Arabidopsis.