Comparative transcriptomics and network analysis define gene coexpression modules that control maize aleurone development and auxin signaling

Abstract

Abstract The naked endosperm1 (nkd1), naked endosperm2 (nkd2), and thick aleurone1 (thk1) genes are important regulators of maize (Zea mays L.) endosperm development. Double mutants of nkd1 and nkd2 (nkd1,2) show multiple aleurone (AL) cell layers with disrupted AL cell differentiation, whereas mutants of thk1 cause multiple cell layers of fully differentiated AL cells. Here, we conducted a comparative analysis of nkd1,2 and thk1 mutant endosperm transcriptomes to study how these factors regulate gene networks to control AL layer specification and cell differentiation. Weighted gene coexpression network analysis was incorporated with published laser capture microdissected transcriptome datasets to identify a coexpression module associated with AL development. In this module, both Nkd1,2+ and Thk1+ appear to regulate cell cycle and division, whereas Nkd1,2+, but not Thk1+, regulate auxin signaling. Further investigation of nkd1,2 differentially expressed genes combined with published putative targets of auxin response factors (ARFs) identified 61 AL‐preferential genes that may be directly activated by NKD‐modulated ARFs. All 61 genes were upregulated in nkd1,2 mutant and the enriched Gene Ontology terms suggested that they are associated with hormone crosstalk, lipid metabolism, and developmental growth. Expression of a transgenic DR5–red fluorescent protein auxin reporter was significantly higher in nkd1,2 mutant endosperm than in wild type, supporting the prediction that Nkd1,2+ negatively regulate auxin signaling in developing AL. Overall, these results suggest that Nkd1,2+ and Thk1+ may normally restrict AL development to a single cell layer by limiting cell division, and that Nkd1,2+ restrict auxin signaling in the AL to maintain normal cell patterning and differentiation processes.