Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

MOLECULAR AND FUNCTIONAL ANALYSIS OF TWO MUTANT ALLELES AT THE OPAQUE-2 LOCUS IN ZEA MAYS

LAZZARI B.*, CICERI P.**, GIANAZZA E.***, CARZANIGA R.****, FAORO F.****, GENGA A.*, VIOTTI A.*

* Istituto Biosintesi Vegetali, CNR, Via Bassini 15, I-20133 Milano, Italy

lazzari@ibv.mi.cnr.it

** Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0116

*** Istituto di Scienze Farmacologiche, Università degli Studi di Milano, Via Balzaretti 9, I-20133 Milano, Italy

**** Centro Patogeni Generalizzati Colture Agrarie, CNR, Via Colombo 60, I-20133 Milano, Italy

opaque-2, phosphorylation, subcellular localization

The Opaque2 bZip transcriptional activator is involved in the transcriptional regulation of a subset of zein genes in the subaleurone layers of the maize endosperm cells. Three wild-type (O2W1, O2W2 and O2W3) and four mutant alleles (o2T, o2It, o2-52 and o2-676) were considered in this study. The wild-type alleles produce polypeptides with highly conserved amino acid sequence having only a short deletion/insertion of a PE motif within the amino terminal part of the protein. The o2T, o2It and o2-52 mutants have been cloned by RT-PCR and sequenced. The o2T mRNA is characterized after 645 bases from the ATG of the longest ORF by a 25 nucleotide deletion that introduces a stop codon and produces a truncated polypeptide (o2T) missing both the basic and the L-Zipper motifs. Similarly, a deletion is present also in the o2-52 allele, producing a truncated polypeptide that is longer than the former and contains the basic domain and a part of the L-zipper motif. Conversely, the O2It and O2-676 polypeptides contain both these motives but show some amino acid modification within the basic region leading to non functional proteins. SDS-PAGE analysis performed on both protein extracts and in vitro translated wild-type and mutant proteins confirms the presence of the sequence-deduced polypeptides. Western analyses were conduced both with N-term and COOH-term specific antibodies, confirming the absence of the COOH-terminal region in the truncated polypeptides. IPG-IEF (Immobilized pH Gradient-IsoElectric Focusing) analysis of the same alleles was performed both on native and phosphatase-treated protein extracts, showing that there is a high heterogeneity of charge that is lost after phosphatase treatment. This indicates that O2s are multiply phosphorylated. In the wild-types and in the o2It and o2-676 proteins at least nine isoforms were detected with the non- and hypo-phosphorylated forms (mono-and di-phosphorylated) more abundant in respect to the hyper-phosphorylated ones. The o2T and the o2-52 polypeptides were, however, fractionated into four isoforms, suggesting the occurrence of three phosphorylation events. In order to investigate the functional meaning of this multi-phosphorylation process we performed south-western experiments on both wild-type and mutant O2s before and after phosphatase treatment. The functional analysis reveals that only the hypo-phosphorylated forms of the wild-types are able to bind the O2 target sequence, while the hyper-phosphorylated forms acquire DNA binding activity only after phosphatase treatment. In addition, immunogold detection experiments were carried out in order to localize these polypeptides at the subcellular level. These studies reveal that more than 75% of the wild-type O2 is present in the nucleus. The o2It and o2-676 mutants are similarly localized into the nucleus while the o2T remains confined into the cytoplasm. In conclusion the different extent of phosphorylation modulates O2 DNA-binding activity and follows well-defined subcellular events.