Abstract:

Objective: Under laboratory conditions simulating roadside NO₂ exposure, this study investigated the leaf morphological, physiological, biochemical, and metabolomic responses of the urban roadside greening plant Bougainvillea × buttiana ‘Miss Manila’ to long-term NO₂ exposure, aiming to clarify the mechanisms underlying plant responses to NO₂ and to provide a theoretical basis for the precise selection and management of urban greening plants. Method: The common ornamental cultivar B. × buttiana ‘Miss Manila’ was used as the experimental material. In a fully automated closed fumigation system, plants were exposed to three treatments: clean air as the control (CK), class I NO₂ concentration (T1, 0.02 μL·L^?1), and class II NO₂ concentration (T2, 0.04 μL·L^?1). During the treatment period, plant phenotype, photosynthetic parameters, and metabolic regulation related indicators were dynamically monitored. After exposure, leaf nitrogen metabolism, microstructure, and metabolomic profiles were comparatively analyzed. Result: 1) With prolongation of fumigation time, NO₂ significantly inhibited the growth and photosynthetic rate of B. × buttiana ‘Miss Manila’ and altered the contents of physiological regulatory substances. Under the class II concentration, the leaf integrity index and the activities of superoxide dismutase and catalase decreased by 50.14%, 31.58%, and 46.81%, respectively, whereas the contents of malondialdehyde, proline, and soluble protein increased by 43.50%, 149.02%, and 22.72%, respectively. 2) Compared with the control, T2 treatment significantly reduced leaf chlorophyll content, net photosynthetic rate, and photochemical efficiency, damaged the leaf cuticular structure, disrupted epidermal cells and chloroplast structure, significantly enhanced the activities of nitrogen metabolism-related enzymes, and increased oxidized nitrogen content by 158.43%. 3) After conducting correlation analysis and cluster analysis on the 22 selected indicators, two major groups were divided on their basic response patterns: the first group comprised chlorophyll fluorescence parameters and antioxidant enzyme indicators, and the second group comprised nitrogen forms and nitrogen metabolism-related enzyme indicators. Compared with the control, the overall response trend of B. × buttiana ‘Miss Manila’ under T1 and T2 treatments was similar, but their temporal trajectories were non-linear. 4) NO₂ stress induced systemic metabolic responses, with significant changes in the contents of multiple metabolites, including L-leucine and α-linolenic acid. These changes mainly involved metabolic pathways of amino acids, α-linolenic acid, and betalain. KEGG enrichment analysis showed that metabolic pathways such as betalain biosynthesis and glutathione metabolism had relatively high enrichment factors, and the substrate content in the corresponding pathways significantly decreased, showing dose-dependent responses. Conclusion: Inhibition of key metabolite synthesis and disruption of cellular structure may be important causes of the reduced resistance in B. × buttiana ‘Miss Manila’ under NO₂ stress, and NO₂ exposure markedly alters its metabolic profile. Overall, high-concentration and long-term NO₂ exposure have stronger negative effects, whereas short-term exposure to low-concentration NO₂ shows a certain low-dose stimulatory effect. Therefore, in roadside environments with long-term high NO₂ concentrations, B. × buttiana ‘Miss Manila’ should be planted in combination with other plant species rather than as a single dominant species.

Key words: nitrogen dioxide, road vegetation, Bougainvillea × buttiana ‘Miss Manila’, physiological characteristics, microstructure, metabolomics