Clarifying the Opaque: Identification of Direct Targets of Maize Opaque2

What do we need from a seed? This is not the subject of a long-lost Dr. Seuss book, but rather a serious agronomic question. Indeed, artificial selection by humans has driven dramatic changes in crops, so that seeds remain on the stem rather than falling to the ground (shatterproof) and germinate at sowing time—not sooner and not later. Given the major role that seeds play in our diet, a predominant role since the domestication of grains and the establishment of agriculture, recent efforts have also turned to improving the nutritional value of grain crops. For example, maize (Zea mays) seeds contain mainly starch and prolamins, a grass-specific type of storage protein known in maize as zeins and including α-, β-, γ-, and δ-type zeins, which have very little lysine.

Recent work (reviewed in Azevedo and Arruda, 2010) has aimed to improve the lysine content of maize. The opaque2 (o2) mutant, first described in 1964 (Mertz et al., 1964), changes the composition of the maize endosperm, and o2 seeds have ∼70% more lysine than control seeds. Although o2 mutant seeds have high lysine content, the mutant phenotype also includes other undesirable pleiotropic effects that affect agronomic performance; these phenotypes have proven difficult to disentangle. O2 encodes a bZIP transcription factor, and Li et al. (2015) combine RNA sequencing (RNA-Seq) based transcriptome analysis with chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of protein binding to identify the direct and indirect targets of O2. RNA-Seq identified ∼1000 genes that show different transcript levels between wild-type and o2 endosperm; similarly, ChIP-Seq identified ∼1000 genes with O2 binding sites. Comparison of these two sets found 35 direct regulatory targets of O2 in multiple pathways (see figure), including zein-encoding genes, as expected, and other transcription factors, as well as genes involved in stress resistance and nitrogen metabolism. Regulation of the known O2 target, cyPPDK, which encodes a cytoplasmic pyruvate orthophosphate dikinase, indicates that O2 also affects partitioning of carbon. Comparison of sequence motifs also identified known and novel cis-acting O2 target sites. The authors also showed that the effects of O2 on lysine catabolism, and O2 transcription, likely occur via indirect mechanisms, rather than direct binding.

O2 transcriptional regulation in maize endosperm. O2 targets expression of the genes encoding a subset of the zein storage proteins but also regulates genes and long, noncoding RNAs (lncRNAs) that participate in multiple processes. (Reprinted from Li et al. [2015], Figure 9.)

Despite the initial promise offered by the o2 mutant, efforts to increase lysine contents in maize have lagged due to the complexities of the aspartate pathway (including feedback inhibition and catabolic activities) and complex transcriptional regulation by O2. Now, some 50 years later, deployment of genetic modifiers of o2 in quality protein maize, targeted modifications of the aspartate and lysine catabolic pathways, and RNA interference strategies to limit synthesis of zein in favor of higher lysine storage proteins such as α-globulin, have begun to bear fruit in high-lysine varieties of maize. The identification of O2 targets and binding sites and the resulting model of O2 regulation provide additional valuable tools for the efforts to improve the nutritional quality of this important food and feed crop. These findings may also find applications in efforts to improve maize as an industrial or biofuels feedstock, which might require grain with low protein contents.