Online detection of plant stress volatile compounds

The identification of the natural defence response in plants relies on the application of highly sensitive analytical methods. This section reviews the development and application of new laser-based techniques to enable detection of natural defence (volatile) molecules with unprecedented sensitivity, versatility and reliability.

Many of the components of the natural defence response in plants are volatile organic compounds that are emitted as a response to pathogen attack. The detection of these compounds presents several problems especially because of their great variety, low concentration (generally in the ppb (10-9) or ppt (10-12) range) and the rapidity of the processes involved, which can occur in a matter of a few minutes, as it has been demonstrated in the case of the plant response to stress.19

The techniques developed for on-line detection of volatile compounds in other fields have to fulfil a number of specific requirements for application in either plant physiology or plant pathology investigations, namely:

• high sensitivity to detect ppb and ppt concentrations

• high selectivity allowing clear differentiation between several compounds and the ability to analyse different gases simultaneously using a single instrument

• excellent time resolution for real-time measurements

• automatic operation allowing day and night analysis.

Typically, the methods used for trace analysis of volatile compounds can be separated into spectroscopic and non-spectroscopic techniques. Of the non-spectroscopic techniques, the most used are chemiluminiscence, mass spectrom-etry (MS) and gas chromatography (GC). While the former two techniques have been used mainly as laboratory tools, GC has achieved outstanding features for a wide variety of gases at detection limits as low as a part per trillion (pptv) with a high degree of reliability, especially with the implementation of commercial GC-MS instrumentation. In plant science it has been used, for example, for the detection of the ethylene emission as a stress response in more complex plants.4,20 The main drawback of GC is that previous sample preparation or preconcen-tration is usually needed which, together with the slow time response of the technique, limits the temporal resolution of the analysis. Moreover, the system is generally not automatic.

Spectroscopic techniques are generally based on absorption measurements, especially in the infrared (IR) wavelength region. IR gas analysers with broadband thermal sources of radiation have been used in investigations of plant defence molecules, but these are generally industrial analysers and designed to detect one single particular gas. Thus, the simultaneous measurement of different gases, which is necessary in the study of many plant processes, is not allowed. The availability of tunable laser light sources has favoured the development of many spectroscopic techniques, among them are differential optical absorption spectroscopy (DOAS),21-23 light detection and ranging (LIDAR),23-26 Fourier transform IR spectroscopy27-29 and tunable diode laser absorption spectroscopy (TDLAS).30,31 These techniques have been applied in the detection and analysis of volatile organic compounds, especially in environmental applications, but any one of them presents several drawbacks for the detection of natural defence molecules in plants, particularly their lack of sensitivity and/or selectivity.

One of the most interesting developments in the detection of volatile compounds released by the plants during the past few years has been so-called laser photoacoustic spectroscopy (LPAS) which has allowed the identification of many key molecules and the unravelling of signalling plant defence mechanisms, as described below. The technique is based on the photoacoustic effect, that is, the

Temporal & spectral laser control

IR laser

Sampling cuvette

Amplifier

n F

Grating iSl

PA cell

scrubber

Grating

E control

Coojing trap

Fig. 12.2 Schematic representation of the LPAS set-up for the detection of plant volatile emissions.

generation of acoustic waves as a consequence of light absorption as was first reported by A. Graham Bell in 1880.32 A comprehensive description of the physical principles behind LPAS is out of the scope of this chapter and can be found elsewhere.33-35 Thus only a basic explanation is given.

The effect is originated by the absorption of photons of a suitable wavelength and energy by the gas molecules, which then become excited to a higher rovibrational state. Neglecting spontaneous radiative decay, the absorbed energy is subsequently transferred by intermolecular collisions to translational energy, and thereby to heat. When a gas sample is collected in a closed cell, the heating of the gas molecules will produce an increase in the cell pressure. By modulating the light intensity (e.g. turning the light source on and off) pressure variations are produced which create a sound wave susceptible to detection by a sensitive microphone. Figure 12.2 shows a schematic view of the LPAS experimental system.

The microphone signal depends on (1) the number of absorbing molecules present in the gas, (2) the absorption strength of the molecules at a specific light frequency, and (3) the intensity of the light. Thus, for practical trace gas detection, the light source must satisfy two conditions: it should be narrow banded and tunable in order to reach the specific wavelength of the molecule and it should have high intensity because the absorption signal is proportional to it.

As the absorption processes of interest are related to rovibrational transitions, it is necessary to work in the IR region, where each molecular gas has its own

'fingerprint' absorption spectrum whose strength can vary strongly over a short wavelength interval. Specifically, the range preferred for spectroscopic applications varies between 3 and 20 |mm. Although in some cases a high intensity continuous lamp is used as IR source,36,37 an infrared laser provides both high intensity and narrow band tunable light and is therefore ideal for photoacoustic (PA) detection techniques. CO2 and CO lasers are commonly used as a light source for PA detection of gases38,39 because they provide relatively high continuous wave (cw) powers, typically 100W and 20W, respectively, over this wavelength region. Pulsed laser sources have been also used for LPAS investigations, but there is much less work published on pulsed photoacoustic.40

The main disadvantage of CO2 and CO laser sources is that their tunability is only moderate. They are only line tunable, which may cause interference problems, with a rather large spacing between the laser lines and cover a relatively short range of wavelengths. Several alternatives have been proposed to overcome these limitations: the use of other CO2 isotopes or high pressure CO2 lasers for CO2-LPAS and a CO overtone laser for CO-LPAS are the more relevant suggestions. Moreover other laser sources have been used in order to implement a broadly tunable source with a narrow bandwidth into PA systems, especially with the rapid development of solid state lasers; among them, tunable III-V diode lasers, diode-pumped solid state lasers or distributed feedback diode lasers, allow the development of compact tunable IR laser radiation with a variety of applications in LPAS.41,42 Finally, several applications of LPAS using an optical parametric oscillator system has been reported by different groups43-45 and is certainly the most promising technique for the enhancement of the tunability of PA systems.

Despite the disadvantages mentioned, CO and CO2 lasers are still the most commonly used IR light sources in photoacoustic spectrometers. In order to show the versatility and main features of this equipment, Table 12.1 shows the limits of detection (LoD) for several compounds reached by the LPAS technique in the Department of Molecular and Laser Physics at the University of Nijmegen.46

Table 12.1 gives a clear idea of the multiple applications of LPAS with respect to the detection and reliable analysis of volatile organic compounds in various fields, like environmental chemistry,47-51 although one of the main applications of LPAS remains in the field of the plant sciences52-55 owing to the specific requirements mentioned above. In particular, LPAS is widely applied in monitoring the volatile defence compounds released by the plants.56-63

As indicated in the introduction to this chapter, ethylene plays an important role in a number of plant physiological processes. LPAS has proved to provide a reliable method of detecting this plant hormone at ppt levels35,64 in an instantaneous and continuous manner; as a consequence there are many LPAS investigations of ethylene emission from fruits and plants under different environmental conditions.61,65-70 Figure 12.3 shows the evolution of ethylene emission of a cherry tomato under different conditions.68 The experiment starts under anaerobic conditions and at t = 5.6h the normoxic conditions are restored, yielding a sudden and huge increment in ethylene emission during a period of about 45min. The ability of the technique to follow the process in real-time (data are registered

Table 12.1 Limits of detection for laser photoacoustic spectroscopy

Compound

LoD (ppbv)

Compound

LoD

CO

Carbon disulphide CS2

0.01

Methane CH4

1

Acetaldehyde CH3CHO

0.1

Dimethylsulphide S(CH3)2

1

Water (vapour) H2O

0.1

Ammonia NH3

1

Nitrogen dioxide NO2

0.1

Trimethylamine N(CH3)3

1

Sulphur dioxide SO2

0.1

Ethanol CH3CH2OH

3

Nitrous oxide N2O

1

Pentane CH3(CH2)3CH3

3

Nitric oxide NO

1

Methanethiol CH3SH

10

Acetylene C2H2

1

Hydrogen sulphide H2S

1000

Ethane C2H6

1

Carbon dioxide CO2

1000

Ethylene C2H4

1

CO2

Ammonia NH3

0.005

Ethylene C2H4

0.01

Ozone O3

0.02

Hydrogen sulphide H2S

0.04

Time (hour)

Fig. 12.3 Ethylene emission of a cherry tomato under different anaerobic conditions as measured by the LPAS technique. The rapidity of the plant response and the ability of the technique to follow it are noticeable (adapted from de Vries et al.68).

Time (hour)

Fig. 12.3 Ethylene emission of a cherry tomato under different anaerobic conditions as measured by the LPAS technique. The rapidity of the plant response and the ability of the technique to follow it are noticeable (adapted from de Vries et al.68).

every 2min) together with its high sensitivity (variations of few picolitres per minute can be detected) are remarkable.

LPAS has been also extensively used in the detection of ethanol and acetalde-hyde to investigate the rate of alcoholic fermentation in plant tissue during anoxic

Fig. 12.4 Ethanol and acetaldehyde emission rates from stored pears as measured by the LPAS technique (adapted from Ref. 74).

and hypoxic treatments in several harvested fruits.19,71-73 Monitoring the trace gases released by the stored fruits and vegetablesgivesinformationonthemeta-bolic processes occurring in the crops (rateoffermentation,ripeningstage,etc.) and is important in order to optimise the storage conditions. As an example, Fig. 12.4 shows the ethanol productionratefromstoredpears asmeasuredby LPAS.74 These studies have opened up newwaysofunderstandingandimprov-ing the natural defence mechanisms ofstoredfruit,asitwill beseeninmore detail in section 12.6.

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