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

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 1.07

MOLECULAR, FUNCTIONAL AND CELLULAR ANALYSES OF WILD-TYPE AND MUTANT ALLELES OF THE OPAQUE-2 REGULATORY LOCUS

B. LAZZARI*, C. COSENTINO*, F. GAVAZZI*, S. DOLFINI**, A. VIOTTI*

*) Istituto Biologia e Biotecnologia Agraria, CNR, Milano

**) Dipt. Genetica e Microorganismi, Università, Milano

opaque-2 alleles, o2-polypeptydes, cellular location

The Opaque2 b-ZIP transcriptional activator is involved in the transcriptional regulation of several genes in the subaleurone layers of the maize endosperm cells. Three wild-type and five mutant alleles (o2T, o2It, o2-676 o2-52, and o2R) were considered in this study. The wild-type alleles encode polypeptides with highly conserved amino acid sequence having only a short hypervariable region within the amino terminal part of the protein. The o2R allele is a null o2-transcript allele and thus represents a suitable control in these analyses. The o2T allele is characterised after 645 bases from the ATG by a 25 nucleotide deletion that introduces a stop codon thereby producing a truncated polypeptide (o2T) missing both the basic and the L-Zipper motifs. The o2-52 allele contains an insertion of 4 base pairs 796 bases downstream from the start codon, which causes a frame shift, giving rise to another truncated polypeptide that lacks all the carboxy terminal part downstream from the second leucine of the L-Zipper. Conversely, the o2It allele encodes for two different polypeptides, o2It-S and o2It-L. The o2-676 polypeptide contains both the basic and the L-Zipper motifs but show an amino acid substitution within the basic region leading to a non-functional protein. Western analyses with specific amino terminal and carboxy terminal O2-antisera confirm the presence of differentially shortened o2 polypeptides. Biochemical analyses of native wild-type and mutant polypeptides followed by IPG-IEF fractionation show that the native proteins are highly heterogeneous by charge, and this was associated to a different extent of protein phosphorylation. In the wild-type and in the o2It-L, o2-676 and o2.52 proteins six to nine isoforms were detected with the non- and hypo-phosphorylated forms more abundant in respect to the hyper-phosphorylated ones. The o2T and o2It-S polypeptides were, however, fractionated into three to four isoforms.

In order to investigate the functional meaning of the-phosphorylation process and domain region we performed southwestern experiments on both wild-type and mutant O2s before and after phosphatase treatment and in addition immuno-detection to localise these polypeptides at the subcellular level. 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. Localisation studies reveal that more than 75% of the wild-type O2 is present in the nucleus. The o2It-L, o2-52 and o2-676 mutants are similarly localised into the nucleus while the o2T remains confined into the cytoplasm. In conclusion, wild-type and mutant polypeptides reveal functional and cellular behaviours mediated by their structural and biochemical features.