Histological and Single-nucleus Transcriptome Analyses Unveil the Specialized Functions of Ligular Sclerenchyma Cells and Key Regulators of Leaf Angle in Maize

Abstract

Leaf angle (LA) is a crucial factor affecting planting density and yield in maize. However, the regulatory mechanisms underlying LA formation remain largely unknown. In this study, we conducted a comparative histological analysis of the ligular region across various maize inbred lines, revealing that LA size is significantly influenced by a two-step regulatory process involving initial cell elongation followed by subsequent lignification in the ligular adaxial sclerenchyma cells (SC). We performed both bulk and single-nucleus RNA sequencing, generated a comprehensive transcriptomic atlas of the ligular region, and identified numerous genes enriched in the hypodermal cells that may influence their specialization into SC. Furthermore, we functionally characterized two genes encoding atypical basic helix-loop-helix (bHLH) transcription factors, bHLH30 and its homolog bHLH155, respectively, which are highly expressed in the elongated adaxial cells. Genetic analyses revealed that bHLH30 and bHLH155 positively regulate LA expansion, and molecular experiments demonstrated their ability to activate the transcription of genes involved in cell elongation and lignification of SC. These findings highlight the specialized functions of ligular adaxial SC in LA regulation by restricting the further extension of ligular cells and enhancing mechanical strength. The transcriptomic atlas of ligular region at single -nucleus resolution not only deepens our understanding of LA regulation but also identifies numerous potential targets for optimizing plant architecture in modern maize breeding.