Metabolomics combined with physiology reveal how white clover (Trifolium repens L.) respond to 6PPD stress

As a ubiquitous tire antioxidant, N-(1,3-Dimethyl-butyl)-N'-phenyl-p-phenylene- diamine (6PPD) exists widely in various environmental media and has been detected at high levels in the environment. However, the effects of 6PPD on plants are still poorly understood. In this study, a hydroponic experiment was carried out to investigate the response of white clover (Trifolium repens L.) stressed by 6PPD on physiology and metabolomics. The results indicated that the length of stem and root, as well as biomass were significantly reduced after 500 μg L^-1 6PPD treatment. Photosynthetic performances including photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO₂ concentration (Ci), transpiration rate (Tr) and chlorophyll content of leaves decreased in all treatments except 500 μg L^-1 of 6PPD. The malondialdehyde (MDA) content in the shoot of white clover increased by 66.33 % when exposed to 500 μg L^-1 of 6PPD compared to control group (CK). Hydrogen peroxide and superoxide anion presented a U-shape trend and began to increase at 500 μg L^-1. Besides, peroxidase and catalase significantly decreased compared to CK after exposure to 500 μg L^-1. Metabolic analysis of clover showed that 6PPD treatment induced changes in 10 metabolic pathways of white clover. Metabolites were significantly down-regulated after exposure to 500 μg L^-1 in shoot, while significantly down-regulated in all treatment groups except 500 μg L^-1 in root. These findings may provide a novel perspective for phytotoxicity assessment and phytoremediation of 6PPD.