Spatiotemporal Metabolome and Single-Nucleus Transcriptome Integration Illuminates an Auxin Gradient Orchestrated by NtTAC1 Underlying Leaf Angle Regulation in Tobacco.

Plant architecture is key to crop yield, with leaf angle being critical for high-density cultivation. Although TAC1 represents a promising regulator of leaf angle for breeding, its molecular mechanism remains poorly understood, particularly at single-nucleus resolution. Here, we performed single-nucleus RNA sequencing on NtTAC1 knockdown lines exhibiting reduced leaf angle. This analysis generated a transcriptional atlas comprising 20 distinct clusters corresponding to 14 cell types and identified the endodermis as a central regulatory hub. Weighted gene co-expression network analysis and trajectory inference revealed that the auxin transporter NtPIN3 acts as a key downstream effector of NtTAC1. The two genes are co-expressed in endodermal cells and promote their differentiation from meristematic cells. Spatial metabolomics further demonstrated that NtTAC1 suppression elevates auxin levels and alters its spatial distribution, resulting in asymmetric auxin accumulation preferentially in the abaxial region and consequent reduction in leaf angle. Silencing NtPIN3 recapitulated the NtTAC1 disruption phenotype, confirming that the NtTAC1-NtPIN3 axis regulates both auxin asymmetry and cell wall remodelling. Consistently, both knockdown lines exhibited enhanced lignin deposition, linking disrupted auxin flow to secondary wall thickening. Moreover, CRISPR/Cas9-mediated editing of SlTAC1 in tomato suppressed SlPIN3 expression, indicating evolutionary conservation of this module. Collectively, our findings uncover a cell-type-resolved mechanism underlying leaf angle regulation and provide a mechanistic framework for precision engineering of crop architecture adapted to high-density cultivation.