Abstract

Introduction L-theanine, a nitrogen compound uniquely synthesized in tea plant roots, is a core determinant of tea flavor and a key carrier for nitrogen. Its metabolic dynamics are tightly linked to root development, yet whether auxin signaling participates in regulating theanine biosynthesis and the underlying molecular mechanisms remain unclear. Objectives This study aims to elucidate the molecular pathways and key regulatory factors governing auxin signaling in the regulation of root-specific theanine biosynthesis in tea seedlings. Methods Changes in free amino acids (FAA), ethylamine, and endogenous hormone contents in roots, stems, and leaves of tea seedlings across five developmental stages (S1-S5) were detected by HPLC, GC–MS, and LC-MS, respectively. A theanine biosynthesis regulatory network was constructed by integrating time-series transcriptome and weighted gene co-expression network analysis (WGCNA). Additionally, exogenous indole-3-acetic acid (IAA) treatment, yeast one-hybrid assays, in vivo function validation, and DAP-seq were employed to screen and characterize key regulatory factors. Results During tea seedling radicle development, theanine content rapidly increase from 10% to over 80% of the total FAA. Multi-Omics correlation analysis revealed a feedback inhibition relationship between rapid theanine biosynthesis and auxin signaling levels. Exogenous IAA treatment and in vivo/in vitro assays confirmed that the CsZAT6/CsZAT12-CsAlaDC modules, which respond to auxin signaling, exert bidirectional regulation on theanine metabolism: CsZAT6 positively regulates theanine biosynthesis by activating the expression of CsAlaDC (the rate-limiting enzyme for ethylamine production), while CsZAT12 negatively regulates theanine biosynthesis by repressing CsAlaDC expression. Conclusion This study uncovered that CsZAT6 and CsZAT12, as core response factors to auxin signaling, differentially regulate CsAlaDC expression to modulate theanine biosynthesis rate and n