Lineage-specific response to melatonin in stomatal regulation across vascular plants

Melatonin (N-acetyl-5-methoxytryptamine) is a conserved indoleamine that regulates diverse physiological processes, including stomatal movement in angiosperms. However, whether this regulation is conserved across different plant lineages remains uncertain. Here, we examined the stomatal responses to exogenous melatonin in 17 representative plant species: seven angiosperms, two gymnosperms, seven ferns, and one lycophyte. In seed plants, melatonin induced dose-dependent stomatal closure, accompanied by the accumulation of reactive oxygen species (ROS), nitric oxide (NO), and cytosolic Ca2+; this response was abolished by ROS and NO scavengers or synthesis inhibitors. In contrast, ferns and the lycophyte exhibited melatonin-induced ROS production but showed no NO or Ca2+ accumulation or stomatal closure. Melatonin application attenuated or completely disrupted circadian-driven stomatal closure under constant light in seed plants. In contrast, rhythmic stomatal closure was not observed under constant light in ferns or lycophytes, with the exception of one semi-aquatic species, Marsilea quadrifolia, which exhibited a rhythmic increase in stomatal conductance that was not influenced by exogenous melatonin treatment. These results indicate that the signaling pathway for melatonin-induced stomatal movement is present in seed plants but absent in ferns and lycophytes, suggesting that variation in melatonin sensitivity may be related to lineage-specific differences in nighttime stomatal behavior.