RNA Sequencing of Cleanly Isolated Early Endosperms Reveals Coenocyte-to-cellularization Transition Features in Maize

Early endosperm development in maize (Zea mays) is essential for creating a functional endosperm for filling, but its rapid and dynamic process remains largely unknown. The coenocytic stage is a particular stage with rapid nuclear division without cytokinesis. From 48-144 h after pollination (HAP), endosperm mainly undergoes four cellular processes: coenocyte, cellularization, cell proliferation, and differentiation. Although the high temporal-resolution transcriptome data within 144 HAP of maize kernel development have been investigated, due to technical limitations, the samples contained the maternal nucellus and the embryo sac; as a consequence, many endosperm-specifically-expressed genes might be over-looked. In this study, we isolated early endosperms by free hand and laser-capture microdissection (LCM) and generated high-resolution transcriptome data from 48 to 144 HAP with an interval of 24 h. Through weighted gene co-expression network analysis (WGCNA), we identified nine distinct modules of co-expressed gene sets, of which Module 7 was composed of 5,555 genes that showed the highest expression levels at the coenocytic stage. In Module 7, there were 391 genes not expressed in nucellus, and thus were named as the Coenocyte-Expressed (CE) Gene Set. These genes were involved in transcriptional regulation and auxin-activated signaling pathway. Consistent with the stage transition of early endosperm development, the co-expressed gene sets and enriched gene function modules were changed accordingly. We verified the reliability of the transcriptome data by in situ hybridization. Our work provides a valuable gene resource for early endosperm development studies in the future.