EXO70A1 governs both the timing and patterning of secondary cell wall deposition: evidence from an in vitro tracheary element system for individual-cell imaging

Introduction Mechanisms governing secondary cell wall (SCW) deposition during tracheary element (TE) differentiation are poorly understood, largely due to the inaccessibility of deeply embedded TEs within plant tissues. Although the Arabidopsis thaliana exocyst subunit EXO70A1 has been characterized as essential for SCW patterning through in vivo studies, its stage-specific roles throughout TE development, particularly regarding the initiation of SCW formation, remain unresolved. Methods Here, we establish an in vitro TE induction system in Arabidopsis that aligns with the distinct stages of TE development defined in vivo . Using this system, we visualized EXO70A1 dynamics and characterized the detailed phenotypes of loss-of-function mutants in individual developing TEs. Results We discovered that plasma membrane-localized EXO70A1 puncta undergo a distinct transition from random to linear localization dynamics during TE development. Disruption of EXO70A1 led to disorganized SCW patterns and irregular cell morphologies. Crucially, we observed that exo70a1 cells initiate lignified SCW deposition prematurely compared to wild-type controls. Discussion These findings redefine EXO70A1 as a multifaceted regulator essential for TE development, revealing a previously unrecognized function for EXO70A1 in the temporal regulation of SCW initiation. Consequently, this platform not only offers a robust tool for dissecting targeted secretion but also establishes a critical foundation for future studies with broader downstream applications, such as single-cell omics analysis of TE development.