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

Summary

This protocol provides a standardized within-chamber cutting approach to resolve the onset, peak timing, and kinetics of wound-induced volatile emissions, together with simultaneous gas-exchange measurements.

Abstract

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.

Introduction

Plants respond to injury by releasing a rapid chemical signal, but capturing that signal accurately remains technically challenging. Within seconds after herbivory, wind damage, or mechanical disturbance, plants release a transient burst of volatile organic compounds (VOCs) that activate internal defenses and prime neighboring plants^1,2,3,4. These wound-induced VOCs (wVOCs) include green leaf volatiles (GLVs), methanol, short-chain alcohols and aldehydes, terpenoids, and oxylipin derivatives. They arise either from de novo synthesis in mesophyll cells or from the rupture of storage structures such as resin ducts and trichomes. As immediate stress signals, wVOCs coordinate physiological defenses, mediate plant–plant communication, and interact with atmospheric chemistry^1,5,6,7.

Because natural herbivory combines mechanical damage with salivary and microbial elicitors, experimental studies use simulated wounding to isolate responses to physical injury alone⁸. However, the transient nature of wVOCs makes them inherently difficult to capture. Conventional methods, such as static headspace sampling coupled with gas chromatography-mass spectrometry (GC-MS), often miss the initial emission burst and distort its kinetic profile^9,10. Equilibration delays, elevated incubation temperatures, and extended sampling lines increase residence time and distort the recorded signal, particularly for reactive compounds such as sesquiterpenes^9,11,12. Together, these artifacts reduce signal magnitude and obscure the temporal dynamics of wound-induced volatile emissions, thereby complicating the interpretation of plant stress responses.

Many studies also interrupt the measurement to wound leaves outside the chamber. This interruption destabilizes gas flow, alters wound timing, and reduces control over incision precision^13,14,15. As a result, the first minutes after injury are often lost or distorted¹³, precisely when emission rates peak and characteristic kinetic patterns emerge^3,6,16,17. Some approaches moved leaf excision inside large-volume chambers using Teflon blades or mechanical punches^12,18,19. However, these setups often led to sealing instability, inconsistent cut lengths, and uneven incisions due to cutter shifting or differences in leaf toughness. The large chamber volume further delayed air turnover and broadened the volatile signal¹⁶. Together, these experimental limitations prevent accurate characterization of the temporal dynamics of wound-induced emissions under stable physiological conditions.

To overcome these challenges, this study presents a real-time within-chamber wounding protocol that integrates a surgical-grade cutter into a gas exchange system coupled to proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS). The protocol enables synchronous leaf wounding and volatile detection under controlled environmental conditions while simultaneously measuring volatile fluxes and gas exchange at high temporal resolution. By performing leaf excision within the measurement chamber, the protocol minimizes sampling delays associated with conventional approaches, preserves the temporal dynamics of rapidly emitted wound-induced volatiles, and illustrates a standardized cutting procedure across leaves with different mechanical properties. This temporal resolution enables direct characterization of the onset, peak timing, and species-specific kinetics of wound-induced volatile emissions.

The protocol was evaluated using three species with contrasting anatomical and physiological characteristics (Quercus rubra, Acer platanoides, and Gossypium hirsutum) to demonstrate its applicability across diverse leaf types. Applying identical measurement conditions across all species demonstrates that the protocol can resolve wound-induced VOC dynamics over a broad range of emission patterns while minimizing experimental variability. The approach is broadly applicable to studies requiring high-temporal-resolution measurements of wound-induced VOCs under controlled environmental conditions. Together, these features provide a standardized framework for investigating rapid plant stress responses and facilitate the application of the protocol across species and experimental systems.

Protocol

All plants were grown under controlled-environment conditions in growth chambers. The reagents and the equipment used are listed in the Table of Materials.

1. Construction and installation of the within-chamber leaf cutter

1. Fabricate a rotating leaf cutter designed to fit the internal dimensions (4 cm × 2 cm) of the standard measuring head.
2. Attach a razor blade to a stainless-steel rod (1 mm diameter) using silver solder to ensure mechanical stability.
   CAUTION: Razor blades are extremely sharp. Wear cut-resistant gloves during handling. After use, dispose of razor blades according to institutional laboratory safety procedures for sharp objects. Perform silver soldering under appropriate ventilation and wear protective eyewear.
3. Insert the blade-holding rod into a stainless-steel syringe needle (outer diameter 1.6 mm) to allow free axial rotation.
4. Seal both ends of the needle using a low-emission polytetrafluoroethylene (PTFE) stopper to maintain airtight conditions while preserving rotation.
5. Mount a manual turning knob at the proximal end of the rod to enable controlled rotation of the internal cutter.
6. Attach two flexible supports made from spring brass sheet (0.2 mm thickness) to the outer surface of the needle to secure the cutter to the lower chamber half.
7. Create a triangular incision in the lower chamber gasket to allow blade insertion without contact with the leaf surface.
8. Seal the insertion point using a low-emission sealing putty to prevent gas leakage.
   NOTE: Before proceeding, verify that the cutter installation has not compromised chamber sealing. Confirm that the chamber closes completely, the thermocouple remains in contact with the leaf surface, and that the gas-exchange system indicates stable airflow with no evidence of leakage. Proceed only after stable chamber conditions have been confirmed. Select blade length to produce a cut of approximately 10 mm. The integrated cutting mechanism and its position within the leaf chamber are shown in Figure 1B.

Figure 1: Modified gas-exchange system with an integrated within-chamber leaf cutter and volatile sampling lines.​  (A) Overview of the modified gas-exchange system, showing the PTR-TOF-MS sampling port and the GC–MS sampling line positioned near the chamber outlet for volatile collection. (B) Close-up view of the opened chamber showing the integrated cutting mechanism and the region in which the leaf is positioned during wounding. The cutter is hermetically integrated into the chamber, allowing leaf wounding without opening the enclosure during measurement. This configuration enables real-time monitoring of volatile emissions and gas exchange under controlled environmental conditions. Please click here to view a larger version of this figure.

2. Configuration of volatile sampling pathways

1. Install a Teflon T-piece (1/16″ or 1/8″ ID) adjacent to the chamber outlet (Figure 1A) to minimize tubing length between the chamber and the volatile analyzer.
2. Connect the outlet to the VOC collection system using inert tubing (e.g., FEP or PTFE; 1.6 mm inner diameter). Install the T-piece immediately adjacent to the chamber outlet to keep the tubing between the chamber and the volatile analyzer as short as practicable, thereby minimizing internal volume, residence time, and adsorption losses.
3. Maintain chamber airflow at 750 µmol s^-1.
   NOTE: Short inert tubing reduces adsorption and residence time for reactive compounds.

3. Preparation of plant material

1. Grow Quercus rubra, Acer platanoides, and Gossypium hirsutum under a controlled-environment chamber at 25 °C day / 20 °C night, 60% relative humidity, 1000 µmol m^-2 s^-1 light intensity, and 16 h/ 8 h photoperiod for 4 months.
2. Maintain plants under ambient atmospheric CO₂ concentration (~430 ppm) throughout the experiment.
3. Water all plants every two days until the soil reaches field capacity.
4. Select fully expanded, non-senescent leaves for experiments of comparable developmental stage.

4. Leaf wounding and VOC collection experiments

1. Enclose the leaf and set environmental conditions
   1. Enclose a fully expanded attached leaf in the cuvette equipped with the internal cutter.
   2. Set block temperature to 25 °C, relative air humidity to 60%, actinic light intensity to 1000 µmol m^-2 s^-1 (90% red, 10% blue), and airflow to 750 µmol s^-1.
   3. Allow the leaf to acclimate under the selected environmental conditions until steady-state values of net CO₂ assimilation, stomatal conductance, and baseline VOC signals are reached, typically within 20–30 min after leaf enclosure.
2. Perform gas exchange, fluorescence, and VOC monitoring
   1. Begin simultaneous measurements of gas exchange and VOCs using 1 Hz resolution.
   2. After completing gas exchange measurements, record the steady-state fluorescence yield (F_s) and then apply a saturating pulse (8000 µmol m^-2 s^-1, 0.6 s) via the chamber fluorimeter (3055-FL) to determine maximum fluorescence yield (F_m′) in light-adapted leaves.
   3. Turn off the actinic light and dark-adapt the leaf for 20 min.
   4. Record leaf dark respiration.
   5. Measure the initial fluorescence (F_₀) using weak modulated measuring light (0.03 µmol m^-2 s^-1), followed by a saturating pulse to obtain maximum fluorescence in dark-adapted leaves (F_m).
   6. Turn the actinic light back on.
   7. Continue VOC and gas-exchange measurements under the same environmental conditions until the pre-wounding baseline is stable.
      ​NOTE: Before wounding, confirm that the selected environmental conditions remain stable and that net CO₂ assimilation, transpiration, and baseline VOC signals have reached steady-state values. Proceed only after these conditions have been maintained under the selected measurement settings.
3. Leaf wounding and VOC collection
   1. Insert the first VOC cartridge into the outlet line and connect it to a portable constant-flow air sampling pump. Draw chamber air through the cartridge at a constant flow rate of 200 mL min^-1. Use a multi-bed stainless-steel adsorbent cartridge (10.5 cm length, 3 mm inner diameter) suitable for C3–C17 volatile capture, packed with Carbotrap C 20/40 mesh (0.2 g), Carbopack B 40/60 mesh (0.1 g), and Carbotrap X 20/40 mesh (0.1 g), arranged from weakest to strongest adsorption strength in the sampling direction.
      ​NOTE: Before sampling, condition cartridges by passing high-purity helium through the cartridge at 200 mL min^-1 for 3 h at 250 °C. Cartridge sampling enables subsequent offline GC-MS analysis for compound identification when required by the experimental objectives. Although cartridge collection forms part of the workflow described here, the representative results presented in this study are derived from continuous PTR-TOF-MS measurements. Match adsorbent choice to the volatility range and polarity of the target VOCs, as well as to the sampling conditions.
   2. Collect pre-wounding VOCs for 15 min at 200 mL min^-1.
   3. Replace cartridge and collect VOCs for 2 min.
   4. Rotate the internal cutter 360° to excise the leaf without opening the chamber and return the blade to its initial position.
      NOTE: Position the leaf within the chamber so that the incision is made in the enclosed lamina while maintaining complete chamber sealing. Whenever permitted by leaf size and morphology, avoid cutting through the primary vein and keep the incision position as consistent as possible among samples. Because major vein damage can substantially increase wound-induced volatile emissions, consistent wound placement improves experimental reproducibility⁵.
      CAUTION: Ensure the chamber is fully sealed before cutting.
   5. Continue VOC collection with the same cartridge for 13 min.
   6. Insert a third cartridge and collect VOCs for 15 min.
   7. Operate PTR-TOF-MS continuously during all periods for real-time detection.
      NOTE: Before and after each plant measurement, collect an empty-chamber background sample under the same environmental conditions. Use these background measurements for baseline correction of VOC emission data during data analysis.

5. Volatile detection and instrument operation

1. Set PTR-TOF-MS inlet and drift tube temperatures to 80 °C, inlet flow of 100 mL min^-1, water vapor flow 4.0 mL min^-1, ion current 4.0 mA, drift chamber pressure to 2.1 mbar, drift tube field density ratio 115 Td, and TOF-MS module pressure of 1.2 × 10^-7 mbar.
2. Record volatile signals at a temporal resolution of 1 Hz with a 400 ps sample interval.
3. Calibrate the PTR-TOF-MS before each experiment using a certified gas standard mixture containing representative VOCs from the major plant volatile classes. Process calibration data and calculate VOC concentrations using PTR-MS Viewer software following the procedures described^13,16.

6. Offline GC-MS analysis of cartridge samples

NOTE: The following procedure describes the offline analysis of cartridge samples collected during the workflow above. GC-MS analysis provides complementary compound identification, whereas the representative results presented in this study are based on continuous PTR-TOF-MS measurements.

1. Thermally desorb cartridges using an automated thermal desorption unit coupled to a GC-MS system following the procedure described by Kännaste et al.²⁰.
2. Identify compounds by comparison with authentic standards and the NIST mass spectral library. Perform mass spectrometric detection using electron impact ionization (70 eV) and confirm compound identities by matching both retention times and mass spectra with authentic standards when available.
   NOTE: Cartridge sampling follows the modified protocol from Kännaste et al.²⁰ for C3–C17 volatile capture.

7. Data analysis

1. Subtract the empty-chamber background from all emission traces before analysis. Define the pre-wounding baseline for each monitored mass as the mean signal during a fixed interval immediately before cutting.
2. Use the cutting time as the common reference point for all monitored masses. Determine emission onset as the first detectable increase in the baseline-corrected VOC signal following leaf cutting.
   NOTE: Emission onset was defined as the first consecutive increase above the pre-wounding baseline that continued into the rising phase of the emission burst, thereby excluding isolated fluctuations attributable to instrumental noise.
3. Calculate the time to peak as the interval between the time of cutting and the maximum emission signal. Calculate the maximum slope of the emission burst from the first derivative of the baseline-corrected signal using the mean of the forward and backward slopes between adjacent data points¹³. Define peak magnitude as the maximum observed emission value during the burst.

Results

To evaluate whether volatile sampling position influences signal detection, we compared two configurations using controlled gas injections into the leaf chamber. In one setup, volatiles were sampled directly at the chamber outlet of the modified gas-exchange chamber (Figure 1). In contrast, in the other, air passed through the gas-exchange analyzer tubing before reaching the detector. Recording both signals simultaneously enabled direct comparison of their temporal dynamics.

Discussion

The protocol presented here enables high-temporal-resolution analysis of wound-induced volatile emissions under controlled experimental conditions. By integrating the cutting event directly into the leaf chamber and continuously monitoring volatile signals (Figure 1), the system captures the emission response without the distortions introduced by delayed sampling, chamber opening, or leaf manipulation outside the measurement setup. These disturbances can shift both the apparent timing and th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GC-MS system with TD-20 thermal desorber	Shimadzu	GCMS-QP2010 Plus; TD-20	GC-MS system with TD-20 thermal desorber. www.shimadzu.com
GFS-3000 Gas Exchange System	Walz GmbH	GFS-3000	Leaf gas-exchange measurements. www.walz.com
High-purity helium (99.999%)	Local supplier	N/A	Used for adsorbent cartridge conditioning and GC-MS thermal desorption. www.airliquide.com (or equivalent supplier).
Isoprene calibration standard	Sigma-Aldrich Chemie GmbH	Custom	Custom isoprene calibration standard (3.43 ppm). www.sigmaaldrich.com
Low-emission sealing putty	Local supplier	N/A	Used to seal the cutter insertion point in the chamber.
Multi-bed stainless-steel adsorbent cartridge	Supelco	Custom	10.5 cm length × 3 mm i.d.; packed with Carbotrap C (0.2 g, 20–40 mesh), Carbopack B (0.1 g, 40–60 mesh), and Carbotrap X (0.1 g, 20–40 mesh). Used for optional offline GC-MS identification of VOCs. www.sigmaaldrich.com
Portable constant-flow air sampling pump	SKC Inc.	210-1003MTX	Portable constant-flow air sampling pump.
PTR-TOF 8000 mass spectrometer	Ionicon Analytik GmbH	PTR-TOF 8000	VOC detection. www.ionicon.com
Silver solder wire	Local supplier	N/A	Used to attach a razor blade to a stainless steel rod.
Spring brass sheet (0.2 mm thickness)	Local supplier	N/A	Flexible supports used to secure the cutter.
Stainless steel rod (1 mm diameter)	Local supplier	N/A	Cutter support rod.
Stainless steel syringe needle (1.6 mm outer diameter)	Local supplier	N/A	Housing for a rotating cutter blade.
Teflon stoppers	Local supplier	N/A	Used to seal the cutter housing while allowing rotation.
Teflon T-piece connector	Local supplier	N/A	Installed near the chamber outlet to reduce tubing length.
Teflon tubing (FEP or PTFE)	Local supplier	N/A	VOC sampling line between the chamber and the detectors.
VOC calibration gas mixture	Ionimed GmbH	Custom	1 ppm of each compound in N2; used for PTR-TOF-MS calibration. www.ionimed.com