Wound Response in Plants: An Orchestrated Flavor Symphony

Chapter 5

Wound Response in Plants: An Orchestrated Flavor Symphony 1

P. Dunphy, F. Boukobza , S . Chengappa, A. Lanot, and J. Wilkins

Downloaded by UNIV LAVAL on July 12, 2016 | http://pubs.acs.org Publication Date: September 7, 2000 | doi: 10.1021/bk-2000-0763.ch005

Plant Science Unit, Unilever Research Laboratory, Bedfordshire, MK44 1LQ, United Kingdom

Wound response in plants is a complex ordered collection of localised and systemic effects with a temporal span extending from seconds to hours and days. Important components of the armoury of the plant are volatile molecules emitted as part of the first line of defence. Many of the aroma signature compounds of leaves, fruits and vegetables are formed and/or released in response to wound damage. This rapid temporal subtlety can be captured by application of real time aroma analysis using atmospheric pressure chemical ionisation mass spectrometry (APCI-MS). This technique has been utilised to demonstrate the duality of tomato leaf response to mechanical damage by releasing volatiles derived principally from the terpenoid and oxylipin pathways. The wound response of the edible Genus Allium on the other hand employs a remarkably rapid and complex series of transformations involving unstable volatile organosulfur compounds.

The ability of plants to maintain their 'milieu intérieur' in response to changing environment has evolved to a greatly developed state; this to a large extent dictated by their sessile existence. This capability has been highly refined in the area of plant wound response. Plants manage repair of tissue damage by invoking constitutive defense measures and induced responses effectively sealing off damaged tissue before opportunistic microbes invade the wounded site (1,2). This involves activation of genes for structural wound repair and may involve strategies to signal distal tissue including systemin and proteinase inhibitor gene activation (3,4). These responses also include defence initiatives that serve to minimise further ingress and even elicit in many cases the mutualistic support of natural enemies of the aggressor by release of wound induced volatiles from the plant. These compounds may have Current address: Division of Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, United Kingdom.

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© 2000 American Chemical Society

Roberts and Taylor; Flavor Release ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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potent biological properties acting as phytoalexins, recruiting cues for predators of the invading organism or airborne warning signals for distal parts of the plant or neighboring plants (5-9). From a flavor point of view many of these components contribute significantly to the aroma signature of the particular fruits, vegetables or herbs and to their perceived quality. Classically these wound response aroma molecules fall into two functional groups, those generally stable molecules endogenously present in the plant and the de novo produced, generally unstable compounds derived enzymatically from non-volatile precursors (10). The plant therefore possesses a number of distinct but overlapping armories of responses falling into the above two categories that not only nullify or minimize the assault but also counter-attack the aggressor - a good model perhaps for military strategists. A key component in the response strategy is the ability to rapidly deploy defence initiatives over a timescale of fractions of a second through minutes to hours; aroma volatile release responses to wounding encompasses this whole timespan (11,12). The short timescale of responses are exemplified by the generic oxylipin cascade (7) and the group specific alk(en)yl cysteine sulfoxide pathway of the Genus Allium (13). The ability to measure the very rapid formation and transformation of trace volatiles in complex structures such as viable plant tissues has, until now, been a significant challenge to scientists. Chemical ionisation mass spectrometry (CI-MS) is a powerful technique for the identification and quantitation of mixtures of volatile organic molecules in the vapor phase depending on ion-molecule reactions rather than electron impact processes. The CI method generally results in less fragmentation of the charged molecular species and simpler spectra. A variety of proton transfer reaction systems operating at medium pressure [e.g. proton transfer reaction-MS (PTR-MS)] and atmospheric pressure chemical ionisation-MS (APCI-MS) have been developed using protonated water (H O ) as the primary reactant ion (14-16). Protonated water in this mode has several advantages including non-interaction with the natural components of air and reactivity, in the proton transfer mode, with most organic molecules as well as tolerance of water in the system; a very useful advantage for a wide range of applications. Reaction occurs as follows:H 0 +R->RH +H 0 where R = reactant gas; R H = product ion These CI-MS systems are capable of real time monitoring of complex mixtures in the gas phase over the concentration range of parts per million and in some cases as low as a few parts per trillion by volume. A number of applications of this type based on A P C I - M S and PTR-MS have been documented (15,17,18). This paper describes the release of aroma compounds in response to mechanical damage in plants exemplified by:- constitutively derived stable volatile molecules endogenous to the plant - induced unstable compounds derived by enzymatic transformation of stable nonvolatile precursors. The strategy for intercellular segregation of responses by cellular specialisation within the same plant tissue will be exemplified by consideration of the +

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photosynthetic tomato leaf surface whilst the intracellular segregation of precursors and enzymes will be demonstrated in both tomato leaf epidermal cells and cells of the garlic clove. The volatile responses of these mechanically damaged tissues will be probed in real time using A P C I - M S in conjunction with solid phase micro extraction-GC-MS (SPME-GC-EI-MS) and solvent extraction for molecular characterisation.


EXPERIMENTAL Materials. Mature garlic bulbs were obtained locally and single cloves carefully removed for experimental purposes. Leaves from greenhouse grown tomatoes of the processing variety BOS 3155 were picked from the second apical branch; flavor measurements were carried out from the adaxial surface. Authentic standard reference chemicals of the highest purity were purchased from Aldrich or the Fluka Chemical Company. Real Time Headspace Analysis. Experiments were performed using A P C I - M S with gaseous sample introduction. This represents a non-standard mode of operation since APCI is generally used for solute analysis in the liquid phase, principally from H P L C eluates. The production and detection of intact protonated molecular species was encouraged by the use of a low cone (skimmer) voltage potential. APCI-MS. Volatiles release measurements were carried using the Finnigan Navigator Mass Spectrometer fitted with a new probe design incorporating a Silcosteel® (Restek Corporation) flexible silica coated tube (id 2 mm) heated to 100°C. The instrument end of the steel tube was adjacent to the corona needle whilst the other end was connected via PTFE tubing to the reaction chamber. This provided an inert and heated transfer line directly from the sample chamber to the ionisation zone of the mass spectrometer to prevent vapour condensation and minimise unwanted chemical reactions. Nitrogen was used as the bath gas for the API region. The source temperature was 100°C; corona discharge 3kV and a cone voltage of 20V. Acquisition was in the positive ion mode and continuous from m/z 20-300. The reaction chamber was a screw threaded clear glass vial (od 29mm χ 81mm length with a capacity of 40mL) with a PTFE-silicone septum kept in place via an open topped phenolic enclosure. The air inlet (length 40mm) to the reaction chamber and outlet (length 60 mm) to the APCI probe ferrule connector was via flexible PTFE tubing (id 2mm and od 4mm). The flow rate from the reaction vessel to the A P C I was 1.25mL/sec under the venturi conditions employed and the tubing lengths and diameters indicated. The reaction vessel was filled to approx. 50% of its volume by adding inert #4 Ballotine beads (870-1275μπι diameter). For the studies with garlic a single garlic clove (~3.5g) was placed on top of the glass beads. Damage to the clove was achieved via a syringe needle (65-85μ diameter) passing through the septum and penetrating the garlic clove to a depth of ~7.5mm via two rapid repetitions without opening the vessel or interrupting gas flow. For studies with tomato leaf, the PTFE


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tube connected to the probe inlet was placed ~ l c m above the leaf surface which was still attached to the intact plant to be treated, and in the open atmosphere. Volatiles were drawn directly into the A P C I for analysis. A P C I - M S - M S The air above the sample was drawn into the megaflow APCI interface of a Micromass Quattro I Mass Spectrometer. Nitrogen was used as bath gas for the A P I region. The source temperature was 100°C. The corona discharge pin was at 2.5 k V . Positive ion continuum M S data were acquired, generally from m/z 10 to 300. The cone potential was 20 V . M S / M S experiments were performed using collision energy of 30 V , with argon as the collision gas.


Extraction and Analysis of Volatiles.

Garlic and Onion. a) SPME. A 100 μιη polydimethylsiloxane sampling fibre of a Supelco S P M E syringe was introduced into a headspace vial (4 m L capacity) containing approx. l g of a freshly chopped garlic clove (cut into about 7-10 pieces) and retained in this position, at room temperature, for 2 min. The fibre was retracted and introduced into an A T A S Optic-2 P T V G C Injector fitted with a 2 mm injection liner, splitless injection, with a purge off of 0.5 min, injector temperature 150°C, for a 1 min desorption period. The helium carrier gas flow was 1.0 mL/min. (For GC-EI-MS see section c below). b) Direct Solvent Extraction. A single garlic clove (about 3g) was submerged in 20mL H P L C grade hexane in a 50ml beaker. The clove was sectioned five times under the solvent and swirled for 20sec. A l m L aliquot of the hexane extract was removed and 1 μΐ transferred to the G C injector held at 240°C. Analysis was by G C EI-MS (see section c below). c) GC-EI-MS Analysis. A n HP5890 G C was employed using a DB624 column (30m χ 0.25mm χ 1.5μ film thickness); G C temperature programme, 40°C(0 min) to 250°C at 10°C/min followed by holding at the same temperature for 4 min. The G C column was coupled directly by a transfer line heated at 250°C into a Finnigan Mass Lab Trio-1000 mass spectrometer, source temperature 200°C, operating at 70eV electron ionisation (EI+) mode, acquiring full scan data from m/z 30-200 over a G C run time of 30 min. Tomato Leaf and Stem Analysis by S P M E . Direct volatile analysis of leaf or stem trichomes was achieved by gently rubbing the plant surface with the exposed S P M E fibre (ΙΟΟμπι polydimethyl solixane [PDMS] (ex Supelco). The fibre was withdrawn back into its protective sheath until ready for injection, which was carried out by inserting the fibre holder into the G C M S injection port where the fibre was exposed for 2 min at 200°C to allow needle absorbed components to desorb onto the G C column. GC-EI-MS employed a HP 5890 G C with an HP5-MS column (30 m χ 0.25 mm χ 0.25μ film thickness). The G C temperature programme was set at 50°C (1 min) to 160°C at 10°C/ min, then to 240°C at 20°C /min. The G C column was coupled directly, via a heated transfer line heated at 250°C, into an HP 5972A M S D ; source temperature 250°C,


48 EI (70eV source); acquisition m/z 30-300. Helium was used as the carrier gas at a constant flow of 1 .OmL/min. For peak identification mass spectra were compared with those of known standards from the NIST M S Library (1992 edition). The identification of the terpene hydrocarbons was confirmed, where possible, using authentic standards. Scanning Electron Microscopy (SEM). Samples of upper leaf surface from the second apical spur of greenhouse-grown tomatoes were prepared by mounting leaf tissue (20 χ 10 mm) onto an S E M stub followed by quenching in liquid nitrogen and transferring to an Oxford CP2000 S E M preparation chamber. Surface frost was removed by holding at -98°C at 5 χ 10" tonfor 5min. The sample was then cooled to -110°C and the surface coated with Gold/Palladium, 6mA at 5 χ 10" millibar Argon for 20 sec followed by recovery of the vacuum to 5 χ 10" ton*. The coated sample was finally transferred under vacuum to a Jeol J S M 6301 F E S E M and images recorded at 5kV and at -165°C on a Cressington cold stage at a working distance of 48mm. 7


1

7

RESULTS AND DISCUSSION Brushing and Crushing of Tomato Leaves. Gentle handling of tomato plants produces a persistent and characteristic aroma. Visual inspection of the adaxial leaf surface and stem revealed in both cases a down like surface appearance. Close examination of the leaf surface by S E M revealed projecting from the epidermis two types of structural outgrowth or trichomes namely hair-like and capitate. The capitate glandular hairs, which are characteristic of the Solanaecae family, are reported to contain a four-celled head surrounding an intercellular space containing essential oil drops (19,20). Gentle brushing of the leaf and stem trichomes was carried out by gently stroking the exposed needle of the S P M E fibre across the trichome layer, parallel to the epidermal surface for a period of about 3 sec. The fibre was then heat desorbed and analysed by GC-EI-MS. Figure 1 shows the SPME-GC-TIC chromatograms for both stem and leaf trichomes. The leaf gland volatiles were dominated by monoterpene hydrocarbons (65% of the total ion current) of which β-phellandrene was the major component. Sesquiterpene hydrocarbons accounted for ca. 30% with (E)-caryophyllene the major contributor. These findings were in keeping with previous observations on tomato leaf volatile components with the exception that the C aldehydes (Z)-3- and (E)-2-hexenal and hexanal were present at trace levels reflecting slight tissue bruising during sampling (27). Comparison of the volatiles from stem trichomes in Figure 1 showed a much greater predominance of monoterpene hydrocarbons (about 91% of the total ion current) and a much lower level (about 1%) of sesquiterpene hydrocarbons. The significance of these compositional differences on the same plant is not clear. It should be noted however that the wild and cultivated potato species Solanum berthaultii Hawkes and S. tuberosum L. respectively contain two types of foliar glandular trichomes (types A and B). The type A glands contained sesquiterpenes as the major volatile constituents whilst, in type Β glands, the C hydrocarbons were 6

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Wound Response in Plants: An Orchestrated Flavor Symphony

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