NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2 interact to regulate gene networks in maize endosperm development.

NAKED ENDOSPERM1 (NKD1), NKD2 and OPAQUE2 are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these three factors in co-regulating gene networks, we developed a set of nkd1, nkd2 and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the eight genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 days after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2 and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early, then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2 and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.