Mechanisms of coumarin against copper toxicity in citrus leaves based on physiological, transcriptomic, and metabolomic analysis

Citrus trees are mainly cultivated in acidic soil with high available Cu. The objective of this study was to reveal the mechanisms of coumarin (COU) against Cu toxicity in citrus leaves. ‘Xuegan’ (Citrus sinensis (L.) Osbeck) seedlings were submitted to 100 (COU100) or 0 (COU0) μM COU and 400 (Cu400) or 0.5 (Cu0.5) μM CuCl2 for 24 weeks. Thereafter, the study used widely targeted metabolome, RNA-Seq, Fourier transform infrared (FTIR) spectroscopic analysis, and physiological methods to examine metabolite abundances, gene expression levels, and some related physiological parameters (electrolyte leakage, nonstructural carbohydrates (NCs), total free amino acids, total soluble proteins, total flavonoids, and total phenolics) in leaves; FTIR spectra in leaf cell wall (CW) materials (CWMs); and Ca and Cu concentrations in leaves and CWMs. The results suggested that COU100 alleviated Cu toxicity in leaves by a combination of factors: (a) maintaining the homeostasis of Ca and Cu, as well as the structure and function of CWs and plasma membranes; (b) alleviating Cu400-induced leaf senescence and growth inhibition due to less damages to CWs, cell division, and cell growth; (c) enhancing the capability to keep a balance between reactive oxygen species formation and scavenging; and (d) reducing Cu400-induced alterations in primary and secondary metabolisms. Leaves of COU0-treated seedlings (LCOU0) exhibited some adaptive responses to Cu400. For example, Cu400 increased the fractions of Ca, Cu, and pectin in CWs and the accumulation of NCs and secondary metabolites, and upregulated some antioxidants and genes related to antioxidant defense in LCOU0.