Functional validation of EXLB1 in leaf and stomatal development in tobacco

Leaf morphological traits are the key determinants of tobacco yield and stress responses, and cell wall synthesis and metabolism play an important role in leaf development. However, the underlying regulatory mechanism of cell wall on leaf morphology remains largely unknown in tobacco. Here, we compared two pairs of tobacco cultivars (DMC vs. DX3 and DMC vs. WM3) with distinct leaf traits, and identified a cell wall-related expansin gene, NtEXLB1, based on transcriptome data analysis. The homolog NbEXLB1 from N. benthamiana is highly expressed in the mature leaf, and the NbEXLB1 protein is localized to the cell wall. NbEXLB1 gene overexpression promoted leaf expansion with larger abaxial pavement cells, decreased stomatal density and aperture in N. benthamiana. NbEXLB1 CRISPR mutant plants exhibited smaller pavement cells and more stomata, while the leaf area did not show any significant difference. Furthermore, overexpression of NbEXLB1 remarkably enhanced drought tolerance and resistance to Rhizoctonia solani infection. These findings provide mechanistic insights into how cell wall regulation coordinates leaf development, and highlight NbEXLB1 as a promising genetic resource for improving leaf productivity and stress resilience in tobacco breeding.