Optimizing light environments is essential for maximizing crop productivity and resource-use efficiency in plant factories with artificial lighting. Specifically, the frequency of light-dark cycles within a 24-h period interacts with the intrinsic circadian rhythms of plants, thereby influencing carbon metabolism and photosynthetic efficiency. In this study, we examined the effects of different light-dark cycle frequencies on the growth and photosynthetic activity of cucumber and tomato seedlings. The light-dark cycle frequencies were established as follows: Cycle 1 (16/8 h), 2 (8/4 h), 4 (4/2 h), and 8 (2/1 h). Three cultivars of each fruit type with distinct phenotypic traits were used as experimental materials: cucumber (spiny, white, and dark green) and tomato (pink, intermediate, and red). Cucumber demonstrates notable metabolic plasticity, effectively synchronizing their circadian clock, even under segmented light-dark cycles. Despite the frequent changes in light conditions, both FV/FM and PIABS increased. Additionally, increased stomatal conductance directly enhanced net photosynthetic rates, thereby significantly promoting growth. In contrast, tomato growth was notably inhibited as the frequency of the light-dark cycle increased. Continuous dark periods lasting less than 3-4 h disrupted circadian rhythms. Consequently, non-stomatal limitations became apparent, characterized by the accumulation of intercellular CO2 concentrations and a pronounced decline in net photosynthetic rates despite the stomata remaining open. An increase in DI0/RC indicated that the photosynthetic system was considerably damaged. Partial least squares discriminant analysis corroborated these contrasting strategies by identifying chlorophyll content, intercellular CO2 concentration, and PIABS as the primary discriminative indicators. Customized lighting designs that accommodate species-specific characteristics are essential for plant factories that use artificial illumination. Although multiple light-dark cycles can improve production efficiency in adaptable crops, such as cucumber, a single light-dark cycle maintaining continuous darkness is recommended for tomato to prevent physiological disruption.
