Opaque-2 regulates a complex gene network associated with cell differentiation and storage function of maize endosperm

Development of the cereal endosperm involves cell differentiation processes that enable nutrient uptake from the maternal plant, accumulation of storage products and their utilization during germination. However, little is known about the regulatory mechanisms that link cell differentiation processes with those controlling storage product synthesis and deposition, including the activation of zein genes by the maize (Zea mays) bZIP transcription factor Opaque-2 (O2). In this study, we mapped in vivo binding sites of O2 in B73 endosperm, and compared the results with genes differentially expressed in B73 and B73o2. We identified 186 putative direct O2 targets and 1,677 indirect targets, encoding a broad set of gene functionalities. Examination of the temporal expression patterns of O2 targets using real-time RT-PCR experiments revealed at least two distinct modes of O2-mediated gene activation. We also showed that two O2-activated genes, bZIP17 and NAKED ENDOSPERM2 (NKD2) encoded transcription factors, which can in turn co-activate other O2-network genes with O2. NKD2 (with its paralog NKD1) was previously shown to be involved in regulation of aleurone development. Collectively, our results provide novel insights into the complexity of the O2-regulated network and its role in regulation of endosperm cell differentiation and function. RNAs were isolated from endosperm of a maize o2 mutant (in B73 background) and a wild-type B73 at 15 days after pollination (DAP). Chromatin immunoprecipitation (ChIP) was performed using an anti-O2 antibody on 15-DAP endosperm of B73 in comparison to B73o2. All the RNAs and immunoprecipitated DNA were sequenced using an Illumina HiSeq 2500 platform.