Integration of single‐nuclei transcriptome and bulk RNA‐seq to unravel the role of AhWRKY70 in regulating stem cell development in Arachis hypogaea L.

Peanut stem is a vital organ to provide mechanical support and energy for aerial tissue development. However, the transcriptional regulatory mechanisms underlying stem development at a single-cell resolution remain unclear. Herein, single-nuclei isolation coupled with fluorescent-activated cell sorting was employed to construct a cell atlas of peanut seedling stems using microdroplets-based single-nuclei RNA-sequencing. This approach yielded 29 308 cells with 53 349 expressed genes underlying the identification of five cell types characterized by known marker genes. Additionally, 2053 differentially expressed genes (DEGs) were identified across different cell types. Furthermore, 3306 core-DEGs involved in cell development trajectories were used to construct a transcription factor (TF) interaction network, providing insights into specific biological pathways and transcriptional regulation dynamics underlying cell-type differentiation. Additionally, 1446 DEGs associated with different cell-cycle profile were identified, revealing that peanut stem elongation and cell expansion are closely linked to auxin-responsive pathway. This was supported by the examination of endogenous phytohormones and the identification of 10 hormone-responsive DEGs. Moreover, AhWRKY70 was localized in the nucleus and is highly enriched in stem cortex and xylem cells and exhibits a tissue-specific expression pattern that regulates stem growth. Overexpression of AhWRKY70 in Arabidopsis led to accelerated stem growth by modulating the phytohormone signalling pathway, influencing the expression of sixteen auxin and ethylene-responsive genes as demonstrated by transcriptome sequencing. In conclusion, the single-cell atlas provides a foundational dataset for understanding gene expression heterogeneity in peanut seedling stems. The elucidation of AhWRKY70 function expands our understanding of the roles of WRKY family members in peanut.