THE TRANSCRIPTIONAL ACTIVATOR OPAQUE-2 CONTROLS THE EXPRESSION OF A CYTOSOLIC FORM OF PYRUVATE ORTHOPHOSPHATE DIKINASE-1 IN MAIZE ENDOSPERMS

The maize Opaque-2 (OZ) protein is a transcription factor of the basic /leucine-zipper class, involved in the regulation of endosperm protein s including the 22 kDa alpha-zein storage proteins and b32 protein. In this study we have focussed our attention on the relationship between O2 and the cyPPDK1 gene, which encodes a cytoplasmic pyruvate orthoph osphate dikinase (PPDK) isoform. The results of this study showed that PPDK activity is detectable in wild-type maize endosperms, while in o 2 mutant endosperms, the levels of PPDK protein, mRNA, and enzymatic a ctivity are reduced, indicating that O2 is involved in the regulation of cyPPDK1 in this tissue. By employing transient expression experimen ts in tobacco mesophyll protoplasts, we have demonstrated that the O2 protein can activate expression of a chloramphenicol acetyl transferas e reporter gene placed under the control of the cyPPDK1 promoter. An i n vitro binding assay and DNaseI footprint analysis demonstrated that a specific sequence in the cyPPDK1 promoter can be recognized and prot ected by maize O2 protein. The regulation by the O2 locus of cyPPDK1 r eported here, and control of alpha-zein synthesis by O2 suggest that t he O2 protein may play a more general role in maize endosperm developm ent than previously thought.