Identification of Stomatal Opening Enhancers in Vetch and Alfalfa and a Preliminary Investigation into Their Potential for Yield Improvement

Screening for stomatal opening enhancers and their application via foliar spraying represents a feasible strategy to increase CO₂ assimilation flux by augmenting stomatal aperture, thereby enhancing photosynthesis and promoting plant growth. However, the lack of relevant research on forage crops has significantly limited the implementation of this strategy in forage production. In this study, using vetch (Vicia sativa) and alfalfa (Medicago sativa) as experimental materials, we first established a stable observation system tailored for evaluating stomatal opening regulation in forages: incubating abaxial epidermal peels in a solution containing 0.5% KCl (pH 6.0) under light conditions for 4 h resulted in stably opened stomata. Utilizing this system, we systematically screened the regulatory effects of 14 stomatal opening modulators, including signaling molecules, phytohormones, and amino acids. The results indicated that stomatal opening in both vetch and alfalfa exhibited pronounced concentration-dependent and species-specific responses to the modulators. Supplementation with appropriate concentrations of EGTA, GA₃, MT, His, and Pro significantly promoted stomatal opening in vetch, with increases ranging from 21% to 35%. In contrast, appropriate concentrations of Ca²⁺, H₂O₂, MJ, His, Glu, Met, Arg, and Ala effectively enhanced stomatal opening in alfalfa, with increases of 8% to 34%. To further validate the reliability of the screening system, we selected Met, which showed no regulatory effect on vetch stomata but enhanced opening in alfalfa, for foliar application validation. The results demonstrated that Met treatment had no significant effect on stomatal aperture in vetch but significantly increased it in alfalfa, consistent with the initial screening results. This consistency further confirmed the reliability of our established screening system for identifying stomatal opening enhancers in forages. Correspondingly, foliar Met application did not affect vetch growth but significantly promoted alfalfa growth, increasing biomass by 18%. In conclusion, this study established a stable screening system for stomatal opening enhancers specifically for vetch and alfalfa and successfully identified several species-specific enhancers using this system. Foliar application of these species-specific enhancers effectively increased stomatal aperture and promoted growth in target forage species, demonstrating promising potential for enhancing forage yield.