Transcriptional and Epigenetic Control of Storage-Protein Gene Expression in Maize Endosperm

Endosperm is a terminal organ for storage of proteins and carbohydrates. In Zea mays, the major storage proteins are alcohol-soluble prolamins called zeins, exclusively expressed in endosperm starting about 10 days after pollination. Transcription of the zein-gene superfamily is characteristic for three outstanding features: tissue specificity, developmental timing, and its strength. Their promoters contain a conserved cis-element, called prolamin-box (P-box), recognized by the trans-activator P-box Binding Factor (PBF). Because of the lack of null mutants in all grass species, its physiological role in storage-protein gene expression has been elusive. In contrast, a null mutant of another endosperm-specific trans-activator Opaque2 (O2) has been shown to be required for the transcriptional activation of subsets of this superfamily by binding to the O2 box. Therefore, we used RNAi to knock down Pbf expression and found that only 27-kDa g- and 22-kDa a-zein gene expression was affected, whereas the level of other zeins remained unchanged. Still, transgenic seeds had an opaque seed phenotype. Combination of PbfRNAi and o2 resulted in further reduction of a-zein expression and a more severe opaque phenotype. We also tested the interaction of promoters and constitutively expressed PBF and O2. Whereas transgenic promoters could be activated, endogenous promoters appeared to be not accessible to transcriptional activation, presumably due to differential chromatin states. Although analysis of the methylation of binding sites of PBF and O2 correlated with the expression of endogenous 22-kDa a-zein promoters, a different mechanism seems to apply to the 27-kDa g-promoter, which does not undergo methylation changes.