A Within-Chamber Cutting Method for Real-Time Quantification of Leaf Wounding Volatiles and Gas Exchange

Volatile organic compounds (VOCs) released by plant leaves play key roles in stress signaling, plant-atmosphere interactions, and plant defense. Among these, wound-induced VOCs (wVOCs) are emitted within seconds of mechanical damage, herbivory, or environmental disturbance. Their emission dynamics depend strongly on the timing, severity, and method of tissue disruption. Yet, accurate quantification remains challenging due to mechanical artifacts, variable exposure conditions, and delays between injury and measurement. This study presents a standardized within-chamber leaf excision protocol for real-time monitoring of wVOCs and gas exchange. A surgical-grade cutter was integrated into a portable gas-exchange chamber to enable clean, controlled cuts within a sealed chamber under stable light, humidity, CO2, and temperature conditions. A proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS) continuously measured volatile emissions at the chamber outlet, minimizing delay and signal distortion. This setup resolves emission onset, peak timing, maximum rise rate, and total release with high temporal fidelity. Application of the method to Quercus rubra, Acer platanoides, and Gossypium hirsutum demonstrated its ability to resolve distinct wound-induced emission patterns across contrasting leaf types. By eliminating delays associated with conventional sampling, this method resolves the full kinetic trajectory of wound-induced emissions and overcomes major limitations of previous approaches. It provides a robust framework for studying rapid stress responses in plant physiology, ecological biochemistry, and plant-atmosphere interactions.