PHOSPHORYLATION OF OPAQUE2 CHANGES DIURNALLY AND IMPACTS ITS DNA-BINDING ACTIVITY

In the maize endosperm, the Opaque2 (O2) basic leucine zipper transcri ptional activator regulates the expression of a subset of the zein see d storage protein gene family, Immunodetection of wild-type or mutant O2 polypeptides fractionated by SDS-PAGE resolved a closely spaced dou blet migrating in the 68- to 72-kD range, whereas by using isoelectric focusing, seven to nine isoforms were detected for each allele. Phosp hatase treatment simplified the protein patterns to a single band corr esponding to the nonphosphorylated component, In vivo and in vitro lab eling confirmed that O2 can be phosphorylated. In protein gel blots pr obed with DNA, only the nonphosphorylated and hypophosphorylated O2 po lypeptides were able to bind an oligonucleotide containing the O2 bind ing sequence. Upon in situ dephosphorylation of the focused isoforms b y phosphatase treatment of the isoelectric focusing filter, the hyperp hosphorylated forms acquired DNA binding activity. The ratio among the Various isoforms remained constant throughout the developmental stage s of endosperm growth but changed from daytime to nighttime, with a si gnificant increase of the hyperphosphorylated forms during the night p eriod, These results indicate that O2 exists in vivo as a pool of diff erently phosphorylated polypeptides and demonstrate that O2 DNA bindin g activity is modulated by a phosphorylation/dephosphorylation mechani sm that appears to be influenced by environmental conditions.