Comprehensive analysis of microtubule‐inhibiting herbicides reveals shared subcellular phenotypes and distinct resistance profiles

BACKGROUND: Herbicides that disrupt microtubules have historically been classified by the Herbicide Resistance Action Committee (HRAC) into microtubule assembly inhibitors (Group 3) and microtubule organization inhibitors (Group 23); however, this classification may not fully reflect their underlying modes of action. RESULTS: Using a unified approach combining subcellular phenotyping in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) cells and resistance profiling in Eleusine indica and Chlamydomonas reinhardtii, we show that all tested herbicides produce indistinguishable, dose-dependent subcellular effects, including microtubule depolymerization, multipolar spindle formation, mitotic arrest, and multinucleation. At the resistance level, the α-tubulin T239I mutation confers strong resistance to all microtubule-inhibiting herbicides except the carbamates and propyzamide, whereas several β-tubulin mutations confer resistance to propyzamide. CONCLUSION: These findings reveal distinct sites of action across chemical classes and demonstrate that resistance profiles - rather than subcellular phenotypes - provide a more robust basis for classification. Our results support redefining Group 3 as α-tubulin binders and relocating propyzamide to Group 23, now described as microtubule-interference herbicides with an unresolved site of action, strengthening the mechanistic foundation for improved classification and resistance-management strategies.