Arsenic (As) affects several key physiological and metabolic processes, resulting in reduced seed germination and plant growth. The aim of this work was to study the impact of arsenite (AsIII) treatment on the metabolism of glycerophospholipids (MGPLs), evaluating the turnover activity (TA) of major GPLs, the expression of genes involved in this metabolism and also related to stress response. Total antioxidant activity (TAA) under AsIII-induced stress was evaluated as well as the implication of the circadian clock (CC) on the parameters studied. For this purpose, tobacco (Nicotiana tabacum) hairy roots (HRs) synchronized with a 12/12 h light/dark photoperiod were used. The TA of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) showed a circadian behavior, especially under untreated conditions. However, the As treatment significantly modified the TA profiles of all analyzed GPLs, by inducing an opposite oscillation (PC and LysoPC) or increasing their TA (LysoPE and cardiolipin) during both the light and dark phase. Curiously, under As-treatment, the relative expression of key genes involved in the MGPLs (NtLPAT2 and NtCEK4) was increased and showed a circadian oscillation, while under control conditions, no significant changes were detected. NtPHT4
1 gene, involved in As-induced stress, showed a circadian expression profile, which was conserved under AsIII treatment but with an antiphase performance. Moreover, TAA was significantly affected in AsIII-treated HRs, mainly during the dark phase. Our results show that AsIII treatment affected the parameters studied and constitute valuable evidence to unravel the plant responses and the CC implication under AsIII-induced stress.