Key message <p>MAP65-1 phosphorylation mediates Cr(VI)-induced microtubule disruption in plants.</p> Abstract <p>Chromium (Cr) exists in two prevalent forms in the environment: trivalent Cr(III) and hexavalent Cr(VI). While Cr(III) can benefit plants as a micronutrient, Cr(VI) is highly toxic and disrupts essential physiological, biochemical, and cellular processes in plants. Plant cell microtubules (MTs) are particularly susceptible to Cr(VI) toxicity, with MAP65-1, a key microtubule-associated protein, playing a crucial role in stabilizing MTs by forming cross-bridges between them. The stability function of MAP65-1 is influenced by its phosphorylation status: phosphorylation causes MAP65-1 to detach from MTs during different cell cycle phases. This study investigated how Cr(VI) affects MAP65-1 presence on cortical MTs in <i>Arabidopsis thaliana</i> roots and hypocotyls. Seedlings expressing GFP:AtMAP65-1 and its non-phosphorylatable variant, AtMAP65-1^9A (GFP:AtMAP65-1^9A), were exposed to 100&#xa0;μM potassium dichromate (K₂Cr₂O₇, Cr(VI)) on ½ MS solid medium for 24 or 48&#xa0;h. Confocal laser scanning microscopy (CLSM) revealed that Cr(VI) began impacting cortical MTs within 24&#xa0;h. The signal intensity of GFP indicated a significant reduction in MAP65-1 presence, whereas the GFP:AtMAP65-1^9A signal remained stable and MTs were unaffected. These results suggest that Cr(VI) toxicity to MTs is linked to MAP65-1’s phosphorylation status.</p>