Wild-type opaque2 and defective opaque2 polypeptides form complexes in maize endosperm cells and bind the opaque2-zein target site

The Opaque2 (O2) basic leucine (Leu)-zipper transcriptional activator controls the expression of several genes in maize (Zea mays). We investigated the phosphorylation extent of wild-type O2 and mutant-defective or mutant-truncated o2 polypeptides in endosperm cells, their subcellular localization, participation in complex formation, and involvement in functional activity. Besides wild type, four mutant alleles (o2T, o2-52, o2It, and o2-676) producing o2 polypeptides and a null transcript allele (o2R) were considered. Observing the effects of these mutations, multiphosphorylation events in O2 or o2 proteins were confirmed and further investigated, and the involvement of both the nuclear localization signal (NLS)-B and Leu-zipper domains in proper targeting to the nucleus was ascertained. The absence of these domains in the o2T and o2It-S mutant-truncated forms holds them within the cytoplasm, where they are partially phosphorylated, whereas the presence of NLS-B and a partial Leuzipper domain in o2-52 distributes this mutant-truncated form in both cytoplasm and nucleus. Although mutated in the NLS-B domain, the o2It-L and o2-676 mutant-defective forms are, respectively, partially or completely distributed into the nucleus. Only wild-type O2 and mutant-defective o2 polypeptides bearing the Leu-zipper are able to form complexes whose components were proven to bind the O2-zein target site by in vitro analyses. The transcription of a subset of H-zein genes as well as H-zein polypeptide accumulation in several o2-mutant-defective genotypes indicate the in vivo involvement of o2-mutant-defective proteins in O2-zein target site recognition. The gathered information broadens our knowledge on O2 functional activity and our view on possible quality protein maize trait manipulation or plant transformation via the utilization of cisgenic elements.