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Citrus species are prone to suffer from copper (Cu) toxicity because of improper application of Cu-based agrochemicals. Copper immobilization mediated by pectin methylesterase (PME) in the root cell wall (CW) is effective for Cu detoxification. However, the underlying mechanisms of the structural modification and stress responses of citrus root CW pectin to Cu toxicity have been less discussed. In the present study, seedlings of 'Shatian pummelo' (Citrus grandis L. Osbeck) and 'Xuegan' (Citrus sinensis L. Osbeck), which differ in Cu tolerance, were irrigated with nutrient solution containing 0.5 (as control), 100, 300 or 500 μM Cu for 18 weeks in sandy culture or 24 h in hydroponics. At the end of treatments in the 18-week sandy culture, Cu toxicity on CW pectin content, Cu distribution, degree of pectin methylesterification (DPM) and the PME enzyme activity were discussed. At the genome-wide level, PME gene family was identified from the two citrus species, and qRT-PCR array of citrus PMEs under control and 300 μM Cu stress for 18 weeks were performed to screen the Cu-responsive PME genes. Moreover, the candidate genes that responded to Cu toxicity were further examined within 24 h. The results showed that Cu toxicity increased the root CW pectin content. The root CW pectin under Cu toxicity was remodeled by upregulation of the expression of the Cu-responsive PME genes followed by increasing PME activity, which mainly promoted low methylesterased pectin level and the Cu content on root CW pectin. Compared with C. sinensis, C. grandis root CW had a lower DPM and higher Cu content on the Cu-stressed root CW pectin, contributing to its higher Cu tolerance. Our present study provided theoretical evidence for root CW pectin remodeling in response to Cu toxicity of citrus species., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)