Comprehensive analysis of Cytokinin response factors revealed PagCRF8 regulates leaf development in Populus alba × P. glandulosa

Abstract

Cytokinin response factors (CRFs) are a specialized group of APETALA2/Ethylene Responsive Factor (AP2/ERF) family transcription factors found exclusively in plants, where they play essential roles in responding to cytokinins and regulating plant development and stress responses. This study focused on the analysis of CRF family members in Populus trichocarpa and ‘84 K’ poplar (Populus alba × P. glandulosa), elucidating their evolutionary patterns and expression characteristics. Notably, compared to the 12 CRFs in Arabidopsis thaliana, only 11 members were identified in poplar, with five paralogous pairs generated from the whole-genome duplication events. And differential expression patterns were observed in pairs-2, 4, and 5, particularly highlighting the high expression of PagCRF8 in shoot apical and young leaves, as confirmed by promoter::GUS staining. Furthermore, overexpression of PagCRF8 in poplar led to distinct phenotypic changes, including leaf shrinkage, reduced leaf area, and an increased leaf length/width ratio. Microscopic analysis demonstrated irregular and enlarged leaf cells, elongated cells in leaf veins, and a reduction in trichomes on the leaf surface in response to PagCRF8 overexpression. In summary, this study provides a comprehensive understanding of the evolutionary and functional characteristics of the poplar CRF gene family, while also highlighting the involvement of PagCRF8 in controlling leaf development in poplar.