P i e t r o Ciceri,a.l E l i s a b e t t a Gianazza,b B a r b a r a Lazzari,a G u i d o Lippoli,a A n n a m a r i a Genga,a Gisela Hoschek,c R o b e r t J. Schmidt,c a n d A n g e l o Viottiag2 a lstituto Biosintesi Vegetali, Consiglio Nazionale delle Ricerche, Via Bassini 15, 1-201 33 Milan, ltaly lstituto di Scienze Farmacologiche, Universita degli Studi di Milano, Via Balzaretti 9, 1-201 33 Milan, ltalyDepartment of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-01 16In the maize endosperm, the Opaque2 (02) basic leucine zipper transcriptional activator regulates the expression of a subset of the zein seed storage protein gene family. lmmunodetection of wild-type or mutant 0 2 polypeptides fractionated by SDS-PAGE resolved a closely spaced doublet migrating in the 68-t o 72-kD range, whereas by using isoelectric focusing, seven t o nine isoforms were detected for each allele. Phosphatase treatment simplified the protein patterns t o a single band corresponding t o the nonphosphorylated component. In vivo and in vitro labeling confirmed that 0 2 can be phosphorylated. In protein gel blots probed with DNA, only the nonphosphorylated and hypophosphorylated 0 2 polypeptides were able t o bind an oligonucleotide containing the 0 2 binding sequence. Upon in situ dephosphorylation of the focused isoforms by phosphatase treatment of the isoelectric focusing filter, the hyperphosphorylated forms acquired DNA binding activity. The ratio among the various isoforms remained constant throughout the developmental stages of endosperm growth but changed from daytime t o nighttime, with a significant increase of the hyperphosphorylated forms during the night period. These results indicate that 0 2 exists in vivo as a pool of differently phosphorylated polypeptides and demonstrate that 0 2 DNA binding activity is modulated by a phosphorylation/dephosphorylation mechanism that appears t o be influenced by environmental conditions.
INTRODUCTIONProtein phosphorylation and dephosphorylation, catalyzed by protein kinases and phosphatases, respectively, are important control mechanisms for severa1 biological processes in yeast, plant, and animal cells. These processes include cell division, perception and transduction of externa1 stimuli, hormone sensitivity, and control of metabolic pathways (Ranjeva and Boudet, 1987;Ma, 1993). The control of gene expression at the transcriptional leve1 is determined by cisacting elements and trans-acting factors that modulate the expressivity of a gene in response to the above-mentioned biological processes. In particular, transcription factors themselves are often regulated or modulated in their capacity to transactivate a gene by post-translational modifications involving phosphorylation (reviewed in Hunter and Karin, 1992;Karin and Hunter, 1995).During cereal seed maturation, starch, proteins, and oil, which are the main storage products, accumulate during de-' Current address: Department of Biology and Center for Molecular Genetics,...