High-Throughput Monitoring of Plant VOC Kinetics with a Novel Autosampler and the Vocus PTR-TOF

The combination of the fast, ultra-sensitive Vocus PTR-TOF and this novel autosampler, allows quasi-simultaneous, real-time monitoring of over 100 samples with a single mass spectrometer – providing a unique solution for VOC research in plant science and other fields.

Marc Pfander, Lingfei Hu, Meng Ye,  and Matthias Erb, Institute of Plant Sciences, University of Bern, Switzerland
Manuel Hutterli and Felipe Lopez, TOFWERK, Thun, Switzerland

The interactions of plants with their environment determines their survival in nature and yields in agriculture. Plants can release specific VOCs (volatile organic compounds) to attract beneficial organisms and can detect VOCs as environmental cues.  Time-resolved monitoring of VOC exposure and emissions for large numbers of individual plants is critical for understanding the dynamic roles and mechanisms of VOC-dependent plant-environment interactions. 

Traditionally, plant scientists collect trace level VOCs on traps and then measurement them offline with gas chromatography (GC). This process is notoriously slow, with a single sample typically taking more than 30 min to analyze, thus limiting the total number of samples which may be included in a study.  The Vocus PTR-TOF mass spectrometer provides fast, real-time VOC measurements at ultralow concentrations while preserving molecular identity – therefore no prior concentration, separation, or sample preparation is needed. The Vocus PTR-TOF is thus well suited for real-time measurement of the fragrances which plants release and perceive.  In addition to enabling highly time-resolved measurements of VOC emissions from single sources (or single plants), the speed and sensitivity of the Vocus PTR-TOF creates the possibility to sample many plants in a very short time as a form of high throughput phenotyping.  

In the context of an ongoing research collaboration between the University of Bern Institute of Plant Sciences and TOFWERK, with support from Abon Life Sciences and Bibus AG,  a Vocus PTR-TOF was integrated into a newly developed autosampler (patent pending) to study VOC kinetics of 102 plants in a near-simultaneous fashion (see feature image above).  During operation, each volatile source containing a plant is purged by a clean air flow that carries the emitted VOCs directly to the Vocus PTR-TOF for analysis. The autosampler then cycles between individual containers, measuring VOC emissions and plant responses to different stimulants, treatments, or environmental conditions. In this experiment each plant is measured for 15 seconds with a <5 second transition between samples. Complete mass spectra (0-500 Th) are saved and plotted at 5 Hz  (for example, Figure 1). 

Figure 1 . (Top) An example of an autosampler cycle measuring a series of 17 plants. Concentrations of select VOCs are plotted at 5 Hz (5 unique mass spectra per second). In this case, 17 individual plants were sampled with a measurement time of only 10 seconds per plant . Between individual plants, a fast zero air measurement is performed for 5 seconds. The auto sampler devised here is capable of resolving individual plant kinetics for 102 plants with time resolution better than 30 minutes. It is evident from the 5 Hz time-series that the signal-to-noise of the Vocus is excellent even at concentrations approaching single digit pptv concentrations. The response time of the instrument, even for sticky compound such as terpenes, is on the order of 100 ms or better. (Bottom) An example mass spectrum from a single plant averaged for the 15 second measurement cycle shows that hundreds of compounds are detected and monitored in seconds.

Figure 2 presents the results for 1 of 17 individual plants during a 10-hour experiment measuring the plants kinetic response to a simulated attack from Spodoptera littoralis larvae which eat maize leaves. The plant responds by emitting a variety of VOCs with different kinetics, some of which can be perceived as warning cues by other plants. 

Figure 2. The emission kinetics of selected VOCs over time after a simulated insect attack of one of the plants. Initially, green leaf volatiles such as hexenal are rapidly emitted after the leaves are scratched and exposed to insect regurgitate. After 30 minutes, indole starts being released as a priming cue that can induce defenses in neighboring plants. Terpenes, which can attract predators of herbivores, are released at later time points. The quasi-simultaneous sampling of over 100 plants using one mass spectrometer provides a unique solution to for systematic studies of a large number of samples, thus allowing for genetic mapping and large-scale comparative approaches in plant science and other fields.

This work was funded through the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-2016-STG 714239 to M. Erb).  

Want to see the auto sampler in action? The following video shows the application of our autosampler for the measurement of VOC emitted from plants.