Dissection of Developmental Programs and Regulatory Modules Directing Endosperm Transfer Cell and Aleurone Identity in the Syncytial Endosperm of Barley

Endosperm development in barley starts with the formation of a multinucleate syncytium, followed by cellularization in the ventral part of the syncytium generating endosperm transfer cells (ETCs) as first differentiating subdomain, whereas aleurone (AL) cells will originate from the periphery of the enclosing syncytium. Positional signaling in the syncytial stage determines cell identity in the cereal endosperm. Here, we performed a morphological analysis and employed laser capture microdissection (LCM)-based RNA-seq of the ETC region and the peripheral syncytium at the onset of cellularization to dissect developmental and regulatory programs directing cell specification in the early endosperm. Transcriptome data revealed domain-specific characteristics and identified two-component signaling (TCS) and hormone activities (auxin, ABA, ethylene) with associated transcription factors (TFs) as the main regulatory links for ETC specification. On the contrary, differential hormone signaling (canonical auxin, gibberellins, cytokinin) and interacting TFs control the duration of the syncytial phase and timing of cellularization of AL initials. Domain-specific expression of candidate genes was validated by in situ hybridization and putative protein-protein interactions were confirmed by split-YFP assays. This is the first transcriptome analysis dissecting syncytial subdomains of cereal seeds and provides an essential framework for initial endosperm differentiation in barley, which is likely also valuable for comparative studies with other cereal crops.

Automated summary

Endosperm development in barley involves the formation of a multinucleate syncytium and subsequent cellularization, leading to the differentiation of endosperm transfer cells (ETCs) and aleurone cells (AL). The positional signaling in the syncytial stage determines the cell identity in the cereal endosperm. This study used morphological analysis and RNA-seq to investigate the developmental and regulatory programs directing cell specification in early endosperm. The results revealed specific characteristics and identified signaling pathways and transcription factors involved in ETC and AL specification.