Red light improved photosynthesis and defence-related metabolism in tomato in a leaf level-dependent manner

• Nocturnal red light treatment improved photosynthetic efficiency in various leaf levels. • Older leaves benefited from red light exposure based on the enhanced CO 2 assimilation. • Red light treatment resulted in significant changes to the accumulation of starch and sugars. • Metabolites such as 4-guanidinobutyric acid, fucose and quinic acid increased by red light. • Some metabolites, such as myo-inositol and N-acetylaspartate decreased upon red light. • Red light influenced plant height and leaf area, enhancing the defence responses of plants. This study explores the long-term effects of nocturnal red light treatment on tomato plants, focusing on photosynthesis, metabolism, and defence mechanisms. The impact of red light treatment applied at night to both young and old leaves at various times of day was investigated. The results showed that, after three weeks, nocturnal red light treatment improved photosynthetic efficiency in both upper and lower leaves, particularly in the morning and at noon. This enhancement in photosynthesis was associated with higher CO 2 assimilation. Interestingly, older leaves, which usually demonstrate reduced photosynthetic activity, also benefited from red light exposure. Additionally, red light treatment resulted in significant changes to the accumulation of starch and sugars. Higher levels of starch were found in the leaves in the afternoon, alongside increased levels of sucrose in both leaves. Metabolomics analysis revealed that the level of several defence-related metabolites, including 4-guanidinobutyric acid, fucose and quinic acid, increased following exposure to red light. This suggests that red light applied at night could have a priming effect, enhancing defence against pathogens. Conversely, the levels of some metabolites, such as myo-inositol and N-acetylaspartate, decreased, indicating adjustments in plant stress responses. Red light influenced plant height and leaf area and enhanced callose content in older leaves. Moreover, red light can hard plants against fungal pathogens, such as Botrytis cinerea . These results suggest that exposure to red light at night is an effective, environmentally friendly approach to enhance the resilience of tomato plants and optimise greenhouse farming practices, particularly through supplemental or interlighting systems.