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The role of acetylcholine (ACh) on plant root- shoot communication was investigated using the root-split system of Vicia faba L. In the experiments, slight osmotic stress caused the decrease of ACh content in root tips and the xylem sap transported up per time unit from root tip to the shoot when the water potential of the shoot was kept unchanged. It also caused the decrease of ACh content in the abaxial epidermis. The decrease was highly correlative to the changes of transpiration rate, suggesting that the decrease of ACh content probably functions as a signal to regulate stomatal behavior. The effect of osmotic stress might be mainly through the inhibition of the ACh synthesis in root tip; thus further influences the ACh content in root tip, xylem sap and abaxial epidermis and resulting in the changes of stomatal behavior. These results provide new evidence that plants transduce positive and negative signals among roots and shoots to coordinate stomatal behavior and adapt to variable environments.