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Keywords:

  • acetaldehyde;
  • C-6 compounds;
  • high temperature;
  • isoprene;
  • methanol;
  • photoinhibition.

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Among the volatile organic compounds (VOCs) emitted by plants, some are characteristic of stress conditions, but their biosynthesis and the metabolic and environmental control over the emission are still unclear. We performed experiments to clarify whether (1) the emission following wounding can occur at distance from the wounding site, from VOC pools subjected to metabolic signals; and (2) the emission of biogenic VOCs generated by membrane damage (e.g. consequent to wounding or ozone exposure) can also be induced by exposure to high light and high temperature, recurrent in nature. In Phragmites australis, leaf cutting caused large and rapid bursts of acetaldehyde both at the cutting site and on parts of the cut leaf distant from the cutting site. This emission was preceded by a transient stomatal opening and did not occur in conditions preventing stomatal opening. This suggests the presence of a large pool of leaf acetaldehyde whose release is under stomatal control. VOCs other than isoprene, particularly acetaldehyde and (E)-2-hexenal, one of the C-6 compounds formed by the denaturation of membrane lipids, were released by leaves exposed to high temperature and high light. The high-temperature treatment (45 °C) also caused a rapid stimulation and then a decay of isoprene emission in Phragmites leaves. Isoprene recovered to the original emission level after suspending the high-temperature treatment, suggesting a temporary deficit of photosynthetically formed substrate under high temperature. Emission of C-6 compounds was slowly induced by high temperature, and remained high, indicating that membrane denaturation occurs also after suspending the high-temperature treatment. Conversely, the emission of C-6 compounds was limited to the high-light episode in Phragmites. This suggests that a membrane denaturation may also occur in conditions that do not damage other important plant processes such as the photochemistry of photosynthesis of photoinhibition-insensitive plants. In the photoinhibition-sensitive Arabidopsis thaliana mutant NPQ1, a large but transient emission of (E)-2-hexenal was also observed a few minutes after the high-light treatment, indicating extensive damage to the membranes. However, (E)-2-hexenal emission was not observed in Arabidopsis plants fumigated with isoprene during the high-light treatment. This confirms that isoprene can effectively protect cellular membranes from denaturation. Our study indicates that large, though often transient, VOC emissions by plants occur in nature. In particular, we demonstrate that VOCs can be released by much larger tissues than those wounded and that even fluctuations of light and temperature regularly observed in nature can induce their emissions. This knowledge adds information that is useful for the parameterization of the emissions and for the estimate of biogenic VOC load in the atmosphere.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Plants release in the atmosphere large quantities of carbon emitted as volatile organic compounds (VOCs). Biogenic VOCs are important factors in the chemistry of the troposphere as they can be involved in mechanisms of ozone, aerosol and particulate formation (Andreae & Crutzen 1997). In unperturbed leaves, isoprenoids (isoprene and monoterpenes) are the most abundant VOCs (Guenther et al. 1995). Methanol, acetaldehyde and C-6 compounds are also emitted in large quantities (Fall 2003; Heiden et al. 2003). These three classes of VOCs are however, generally associated to mechanical wounding or to the occurrence of stresses.

Methanol formation is likely occurring from the demethylation of pectins in cell walls (Galbally & Kirstine 2002). Large but transient release of methanol in the atmosphere is therefore associated to cell wall damage occurring because of wounding (Karl et al. 2001). Methanol can be also released by rapidly expanding leaves in which cell walls loosen continuously (Nemecek-Marshall et al. 1995) and in senescing leaves, perhaps again because of irreversible decomposition of cell walls (Fall 2003).

Acetaldehyde is predominantly formed by the enzymatic oxidation of ethanol, which is formed in roots under anoxic conditions and is then translocated to leaves through the transpiratory stream (Kreuzwieser, Scheerer & Rennenberg 1999). Consistently, large fluxes of acetaldehyde have been observed when roots are flooded and are exposed to anoxia (Kreuzwieser et al. 2000). However, transient fluxes of acetaldehyde are emitted by leaves upon darkening and cannot be explained by ethanol transformation in leaves. Acetaldehyde might also be formed in leaves by cytosolic pyruvate when this is transiently abundant, such as immediately after darkening (Karl et al. 2002). However, experiments using inhibitors of acetaldehyde oxidation have recently brought evidences against this mechanism (Graus et al. 2004). It has been proposed that acetaldehyde may be formed upon darkening by acetyl-CoA, although no conclusive evidence for this mechanism has been collected yet (Graus et al. 2004). Acetaldehyde is also emitted following wounding (Fall et al. 1999) and ozone stress episodes (Cojocariu et al. 2005). The release of acetaldehyde is associated to the amount of ozone to which the leaves have been exposed, but the biochemical origin of this pool of acetaldehyde is still elusive (Cojocariu et al. 2005).

C-6 compounds are formed from the breakdown of membrane lipids, predominantly from unsaturated fatty acids, under the action of lipoxygenase and hydroperoxide lyase enzymes (Hatanaka 1993). The first C-6 compound formed by these reactions should be an aldehyde (Z)-3-hexenal, which may be therefore isomerized into (E)-3-hexenal and (E)-2-hexenal. Each aldehyde can be transformed in its corresponding alcohol [(Z)-3-hexenol (E)-3-hexenol and (E)-2-hexenol, respectively] by alcohol dehydrogenase, and leaf alcohols can then produce hexenolacetates (Croft, Jüttner & Slusar 1993). C-6 compounds are emitted from wounded leaves, producing the typical green odour (Hatanaka 1993). The sequence of events leading to the formation and emission of primary and secondary C-6 compounds upon wounding has been clearly observed by rapid detection of volatiles by proton-transfer reaction mass-spectrometry (Fall et al. 1999). More recently, emission of C-6 compounds has been reported as a consequence of other stresses such as insect feeding and ozone exposure (Heiden et al. 2003). However, the emission of C-6 compounds can be largely delayed (up to several hours) with respect to the occurrence of these stresses, indicating a much longer induction period. In addition, there is no clear sequence in the emission of primary and secondary C-6 compounds under these conditions (Heiden et al. 2003). However, good relationships between ozone uptake and C-6 compound emissions, and between ozone uptake and the induction time course were observed (Beauchamp et al. 2005), indicating that the emission was probably related to the damage caused by ozone to membranes.

There are several unanswered questions regarding the stress-induced emission of VOCs, which can now be more easily addressed with the use of novel technologies. Loreto & Sharkey (1993) showed that isoprene emission can vary distant from wounding or rough handling sites. They suggested that the emission was stimulated upon generation of an electric signal. Here we investigated whether wounding could induce an emission of other volatiles in a leaf part distant from the wounding site, that is whether, as in the case of isoprene, a much larger surface that that directly wounded can release these compounds in the atmosphere. Emission measurements distant from wounding sites also improve understanding plant metabolism of volatile compounds. In particular, those compounds that are present in intercellular pools and are influenced by metabolic signalling (e.g. hydraulic, hormonal, electrical) can be revealed. Here we bring an indication that this is the case of acetaldehyde.

As discussed earlier, de novo emission of many biogenic VOCs has been demonstrated in response to very stressful conditions, resulting in permanent leaf damage, such as after wounding (Fall 2003) or after exposure to elevated ozone level (Beauchamp et al. 2005). As a second goal of this study, we wanted to determine whether the induction of the same volatiles also occurs as a response to abiotic stresses to which plants are commonly exposed, and which often only cause transitory damage, namely episodes of high light and elevated temperatures. We demonstrate that this is again the case, and suggest that emission of (E)-2-hexenal is a sensitive indicator of membrane damage under these recurrent stress conditions.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Plant materials and growth conditions

Phragmites australis was selected as the case-study species for these experiments. Phragmites is an aquatic plant that often copes with anoxic conditions and emits isoprene (Loreto & Velikova 2001). Plants of Phragmites were grown for 2 months, between May and July, in 50 L pots under optimal water and nutrient conditions. Phragmites plants were grown in a greenhouse under natural light. The light intensity during the day did not exceed 1000 µmol photons m−2 s−1 at the canopy level during sunny days. A total photon load of 30.2 and 12.5 mol m−2 over the 15 h photoperiod of July was recorded at the canopy level during sunny and overcast days, respectively. The air temperature ranged between 25 and 32 °C during the day, and between 15 and 25 °C at night, with mean temperatures of 28.7/19.6 °C (day/night).

The high-light experiment was repeated in Arabidopsis thaliana mutants (NPQ1) in which the xanthophylls cycle was hampered, consequently preventing the de-epoxidation of violaxanthin (Niyogi et al. 1998) and making these plants sensitive to photoinhibition. Arabidopsis plants were grown in a climatized phytotron (Sanyo Gallenkampf, Loughborough, UK) in small pots with a 2 : 1 : 1 (v/v/v) mixture of peat : sand : perlite, regularly watered and fertilized with a half-strength Hoagland solution to avoid nutrient and water stress. Plants were grown at low light (180 ± 30 µmol m−2 s−1) during a 10 h photoperiod, and under a 22/15 °C (day/night) air temperature.

Experimental conditions

Wounding

A part of a Phragmites leaf (5.9 cm2) was clamped in a gas-exchange cuvette and exposed to a flux of synthetic air reflecting the ambient air composition (80% N2, 20% O2 and 370 µmol m−1 CO2) but deprived of contaminants and VOCs. The leaf was exposed to white light (1000 µmol photons m−2 s−1) provided by an Osram-Power Star 1000 HQT source (Osram, Munich, Germany) or darkened. The leaf was maintained at a temperature of 30 °C and at a relative humidity of 40%. Temperature and humidity were controlled as shown by Loreto et al. (1996). The cuvette air inlet and outlet were connected to the gas-exchange system. This included a proton transfer reaction mass-spectrometer (PTR-MS; Ionicon, Innsbruck, Austria) for the online determination of VOCs by their characteristic protonated parent ions or fragments without the need for preconcentration or chromatography (Lindinger, Hansel & Jordan 1998), and a LI-6262 infrared gas analyser (Li-Cor, Lincoln, NE, USA) for measurements of H2O and CO2 exchanges and calculation of transpiration, stomatal conductance and photosynthesis. The system (including equipment for environmental control and manipulation) is virtually identical to that used by Loreto & Velikova (2001) but the portable gas-chromatograph was replaced by the PTR-MS in the new configuration. The PTR-MS was set in a single ion mode to record traces of protonated methanol (m 33), acetaldehyde (m 45), isoprene (m 69) (Z)-3-hexenal [m 99 and 81 (= m 99 – a H2O molecule)] (Z)-3-hexenol and (E)-2-hexenol [m 101 and m 83 (= m 101 – a H2O molecule)] and (E)-2-hexenal (m 57). For each compound, the instrument was daily calibrated with gaseous standard.

When photosynthesis, stomatal conductance and VOC emissions were steady, the leaf was wounded with a sharp pair of scissors. The leaf area inside the cuvette was wounded by rapidly opening the cuvette and cutting half of the blade vertical to the main vein (a 2 cm cut) before reinserting the wounded leaf in the cuvette. When wounding the leaf outside the cuvette, the blade was cut partially or totally and at different distances from the cuvette, as specified later. The cut length was again 2 cm, as for the wounding inside the cuvette. The part that was completely cut was either immediately placed in water or left in air to dehydrate rapidly. In some cases, the cutting was incomplete and part of the blade was left attached to the stem (as for the cutting inside the cuvette). In another set of experiments, the completely cut part was rapidly placed in a vial where 12CO2 in the air stream was replaced with 13CO2 to see whether VOCs emitted by the leaf part inside the cuvette became labelled by 13C. Alternatively, the leaf part enclosed in the cuvette (not the cut surface) was exposed to 13CO2. Labelling was carried out for 15 min. Further details of the labelling protocol and measurements can be found in Loreto et al. (1996). In this experiment however, the 13C labelling was followed by an online measuring spectra of the isotopes of compounds by PTR-MS.

High temperature

The same setting used for the wounding experiment was also used for the high-temperature experiment. When all physiological parameters were steady, the temperature of the illuminated Phragmites leaf was increased rapidly to 45 °C and maintained at this very high temperature for 40 min while maintaining the relative humidity at 40–45% as previously indicated (Loreto et al. 1996). The temperature was then again decreased to 30 °C and measurement was collected for 30 more minutes during this recovery time.

High light

A  Phragmites leaf was exposed to 500 µmol photons m−2 s−1 until steady gas exchange was recorded. This light intensity was chosen because photosynthesis was close to light saturation but no photoinhibition was observed after a 2 h exposure (data not shown). The light intensity at the leaf level was controlled, interposing neutral screens between the light source and the leaf. The leaf was then suddenly exposed to 2000 µmol photons m−2 s−1 for 30 min and the light was then decreased to 500 µmol m−2 s−1 to monitor the recovery from the high-light exposure. This treatment was occasionally repeated on several cycles. The gas-exchange system used for the high-light experiment with Phragmites was that illustrated in the wounding experiment.

Arabidopsis whole plants maintained at 25 °C were exposed to a light intensity of 380 µmol m−2 s−1 until steady gas exchange was recorded. As for Phragmites, this light intensity was chosen because photosynthesis was close to light saturation but no photoinhibition was observed after the 2 h exposure. The plants were then exposed for 90 min to a light intensity of 1600 µmol m−2 s−1 at 16 °C before restoring the original conditions. A batch of Arabidopsis plants was maintained in an atmosphere enriched with isoprene (3 µmol m−1) before and during the high-light treatment, as explained in details by Loreto et al. (2001). This experiment was carried out in a special glass cuvette that allowed to enclose a whole plant of Arabidopsis and to minimize the release of non-biogenic hydrocarbons by the cuvette and by soil (Thöll et al. 2006). In this experiment, measurements were carried out before and after the photoinhibitory treatment because the different temperature during the treatment could have independently affected the emission rates.

Photoinhibition of Phragmites and Arabidopsis was also assessed by chlorophyll fluorescence. The optic fibre of a Walz Mini-PAM modulated fluorometer (Walz, Effelrich, Germany) was attached to the leaves darkened for 15 min before and after the treatment, and the photoinhibition index Fv/Fm (the ratio between variable and maximal fluorescence) was recorded.

All experiments (wounding, high temperature and high light) were repeated on at least four different fully expanded leaves (the fifth to sixth leaf in the leaf profile starting from the apex) of different Phragmites plants (one leaf per plant). The high-light experiment was repeated in at least four different Arabidopsis plants. In Phragmites, the central part of the leaf was clamped in the cuvette. Data from a single measurement per experiment are shown, but they are representative of the whole data set in terms of characterization of the emission profile and with respect to the time course of the emission. To corroborate this statistically, the difference between the mean values (n = 4) recorded before treatments, those recorded after treatments, and at the peak emission during the treatments, was assessed by t-test (NS = non-significant, ***P < 0.01).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Wounding

Wounding of the leaves inside the cuvette generated a rapid and high burst of methanol followed by the typical emission of C-6 compounds and acetaldehyde (Fig. 1). We were not able to detect differences in the time course of the induction of the different C-6 compounds in Phragmites leaves. Interestingly, a burst of isoprene was also observed, slightly delayed with respect to the emission of C-6 compounds and acetaldehyde. The emission of all VOCs, with the exception of acetaldehyde, was O2-dependent as it was not detected in a N2 atmosphere (data not shown).

image

Figure 1. Time course of the emission of volatile organic compounds (VOCs) following leaf cutting. The emission was detected at the wounding site. A piece of Phragmites leaf inside the cuvette was cut at the time identified by the arrow. Isoprene (m = 69), methanol (m = 33) and acetaldehyde (m = 45) are identified by symbols (solid circles, hollow triangles and solid triangles, respectively). C-6 compounds are identified by the two dashed lines that represent (Z)-3-hexenal (m 81 + m 99) and the sum of (Z)-3-hexenol and (E)-2-hexenol (m 83 + m 101). The mean values (n = 4) recorded before wounding and at the peak emission after wounding are statistically different (t-test, P < 0.01) for all compounds shown.

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When the leaves were wounded outside the cuvette, completely detaching the piece of leaf in the cuvette from the stem, a large burst of acetaldehyde was observed within a few seconds (Fig. 2). The burst was preceded by a sudden and transient increase of transpiration, stomatal conductance and photosynthesis. When the leaves were cut at a longer distance (8 cm instead of 5 cm) from the part enclosed in the cuvette, the burst of acetaldehyde was attenuated and somehow delayed but still visible (Fig. 2). The stimulation of transpiration and stomatal conductance was also about 20% of that observed when cutting the leaf at a shorter distance. A small emission of acetaldehyde was seen again when recutting at a shorter distance the same leaf that was previously cut 8 cm away from the cuvette. This emission was about 30% of the emission observed with the first cut (data not shown). No other VOC was emitted when the wounding was remote. Acetaldehyde emission was not observed when the leaf part inside the cuvette was darkened immediately (seconds to minutes) before wounding. No emission of acetaldehyde was also observed when the cut was incomplete and part (1 cm) of the blade remained attached to the stem, or when the cut leaves were immediately placed in water instead of leaving them in air (data not shown). The emission of acetaldehyde was not labelled by 13C in either one of the labelling experiments (labelling the air flowing over the cut part, or the air flowing over the leaf part in the cuvette, data not shown). Finally, no ethanol emission was associated to the burst of acetaldehyde emission after remote wounding.

image

Figure 2. Time course of the emission of acetaldehyde following leaf cutting. The emission was detected in a Phragmites leaf disc clamped in a gas-exchange cuvette, at two distances from the wounding site. The leaf was cut outside the cuvette at the time identified by the arrow, as detailed in the text. Photosynthesis and stomatal conductance of the leaf disc inside the cuvette are also shown (solid and hollow circles, respectively) for the leaf cut at a 5 cm distance from the cuvette. Stomatal conductance is also shown (hollow squares) for the leaf cut at an 8 cm distance from the cuvette.

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High temperature

Exposure to 45 °C induced a very rapid increase of isoprene emission in P. australis leaves. Isoprene emission however, was not sustained but, after reaching a maximum in about 30 min, it decayed to a level lower than that measured before exposure to high temperature (Fig. 3). The emission of (E)-2-hexenal, a product of linolenic acid breakdown, notably increased with a delay of a few minutes with respect to isoprene emission, but then a constantly high emission level was maintained during the high-temperature treatment (Fig. 3). Methanol, acetaldehyde and other C-6 compounds were emitted in low amount, but followed the same emission kinetics as (E)-2-hexenal. The emissions of these compounds started immediately after the initiation of the stress treatment, reaching their maximum approximately after 45 min.

image

Figure 3. Time course of the emission of volatile organic compounds (VOCs) following exposure to high temperature of Phragmites leaves. The sequence of temperature exposure (30–45 °C and 45–30 °C) is shown in the top part of the figure. Isoprene (m = 69), methanol (m = 33) and (E)-2-hexenal (m = 57) are identified by symbols (solid circles, hollow triangles and hollow circles, respectively). Other C-6 compounds are identified by the lines that represent (Z)-3-hexenal (m 81 + m 99) and the sum of (Z)-3-hexenol and (E)-2-hexenol (m 83 + m 101). The mean values of isoprene, methanol and (E)-2-hexenal emissions (n = 4) recorded before the high-temperature treatment, at peak emission during the high-temperature treatment, and at the end of the recovery period, were statistically separated with a t-test (***< 0.01, NS = mean values non-significantly different from those recorded before treatment).

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When the leaves were exposed again to 30 °C after the high-temperature treatment, isoprene emission increased back to its original level (Fig. 3). The emission of methanol and acetaldehyde decayed during this recovery period, while the C-6 compounds, particularly (E)-2-hexenal, were still emitted at very high levels.

The high-temperature treatment was also performed under anaerobic conditions. Replacing O2 with N2 determined a steady but low increase of the emissions of methanol, acetaldehyde and (E)-2-hexenal while decreased to very low levels the emissions of all other VOCs (Fig. 4). When the temperature was raised to 45 °C, a massive increase in the emission of acetaldehyde was immediately observed while the emissions of methanol and (E)-2-hexenal did not seem to be affected by high temperature. The emission of all other C-6 compounds and isoprene was possibly quenched further under high temperature, although the very low emission rates made difficult this evaluation.

image

Figure 4. Time course of the emission of volatile organic compounds (VOCs) following exposure to high temperature of Phragmites leaves in an O2-deprived atmosphere. The sequence of treatments (O2 removal and exposure to 45 °C) is shown by the lines in the top part of the figure. Acetaldehyde (m = 45), methanol (m = 33) and (E)-2-hexenal (m = 57) are identified by symbols (solid triangles, hollow triangles and hollow circles, respectively). Other VOCs, unaffected by the treatment (isoprene and other C-6 compounds), are identified by the lines. The mean values of acetaldehyde, methanol and (E)-2-hexenal emissions (n = 4) recorded before and at the end of the high-temperature treatment were statistically separated with a t-test (***< 0.01, NS = mean values non-significantly different).

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High light

Exposure to high light also caused an increase of the emission of many VOCs. We recorded no change in the photoinhibition index Fv/Fm following the high-light treatment (Fv/Fm was always > 0.73). The stimulation of isoprene emission was very rapid, while the stimulation of the emission of acetaldehyde and C-6 compounds occurred with a certain delay (Fig. 5). The stimulation of VOC emission was fully reversed once the leaves were exposed again to moderate light.

image

Figure 5. Time course of the emission of volatile organic compounds (VOCs) following exposure to high light of Phragmites leaves. The high-light treatment (2000 µmol photons m−2 s−1) was initiated at the time shown by the first arrow and stopped at the time shown by the second arrow. Isoprene (m = 69), acetaldehyde (m = 45) and (E)-2-hexenal (m = 57) are identified by symbols (solid circles, solid triangles and hollow circles, respectively). Other VOCs (C-6 compounds), are identified by the lines. The mean values of isoprene, acetaldehyde and (E)-2-hexenal emissions (n = 4) recorded before the high-light treatment, at peak emission during the high-light treatment, and at the end of the recovery period, were statistically separated with a t-test (***< 0.01, NS = mean value non-significantly different from those recorded before treatment).

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Arabidopsis NPQ1 mutants, unable to carry out the xanthophylls cycle, were susceptible to photoinhibition and started to increase the emission of (E)-2-hexenal, the most abundant of C-6 compounds, within minutes from the end of the high-light treatment (Fig. 6). High emissions of C-6 compounds, and in particular of (E)-2-hexenal, were observed in these plants for several minutes after the treatment, but then declined again to rates similar to those observed before the high-light treatment. However, when plants were fumigated with isoprene before, during and after the high-light treatment, the increase of C-6 emissions was not observed, and the photochemistry was less inhibited by the treatment, as shown by the photoinhibition index Fv/Fm (Fig. 6).

image

Figure 6. Time course of the emission of (E)-2-hexenal (m = 57) following exposure to high light of Arabidopsis NPQ1 plants. The whole plants were exposed to high-light treatment (1600 µmol photons m−2 s−1 at 16 °C for 90 min) but some plants were fumigated with exogenous isoprene (3 µmol mol−1 in the air entering the cuvette) before and during the treatment.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Wounding

Wounding caused the well-known burst of VOCs observed by many authors (e.g. Fall et al. 1999). The rapid and transient emission of methanol suggests that we have observed the depletion of a small aqueous pool of methanol, possibly evaporating after wounding (Fall 2003). C-6 compounds should be formed in sequence from the breakdown of membrane lipids under the action of lipoxygenase (Hatanaka 1993). The sequence of formation of C-6 compounds, with the parent compound (Z)-3-hexenal, emitted first and then decaying when its isomerization into (E)-3-hexenal and (E)-2-hexenal (Fall et al. 1999), was not observed in this experiment. Perhaps membrane denaturation occurs very fast in Phragmites, or C-6 compounds are very rapidly released with the very high transpiration rates of this plant (Loreto & Velikova 2001).

We hypothesized that some stress-induced VOCs that build large intercellular pools and/or are influenced by hydraulic or electric signals (Loreto & Sharkey 1993) could also be emitted distantly from the wounding sites. A very large and transient emission of acetaldehyde was observed from the piece of leaves distant from wounding, once the wounding was complete and the leaf piece was detached from the stem. No other VOC was emitted under these conditions. Acetaldehyde can be formed in leaves by at least two different pathways, the enzymatic oxidation of ethanol translocated from roots (Kreuzwieser et al. 1999), or the pyruvate overflow mechanism, quenching excess of pyruvate especially formed under a light–dark transition (Karl et al. 2002). In our case, the emission of acetaldehyde occurred also when leaves were previously detached from the stem, and recut at shorter distance from the cuvette where acetaldehyde emission was measured. Moreover, no ethanol emission was associated to acetaldehyde burst. Thus, acetaldehyde emitted as a consequence of remote wounding may not be formed by ethanol transported from roots. Acetaldehyde was only emitted when the wounding completely separated the leaf from the stem, and was preceded by an increase of stomatal conductance. Stomatal opening, and the consequent, transient, increase of photosynthesis and transpiration [known as Ivanov effect (Milburn 1979)], therefore drives acetaldehyde out of leaves. Consistent with this observation, no acetaldehyde burst was observed when stomatal opening after cutting was prevented by darkening the cuvette, and when cutting was incomplete and the Ivanov effect was absent. Acetaldehyde has a partition coefficient between the gaseous and liquid phase which is intermediate between isoprene and methanol (Niinemets et al. 2004), and acetaldehyde emission should consequently be relatively insensitive to stomatal movement (Kreuzwieser et al. 2001). This is not consistent with our results.

The burst of acetaldehyde may be explained if a large pool of acetaldehyde is present in Phragmites leaves. Whether this pool is always present, or is formed after the wounding, is not known. Because Phragmites is an aquatic plant species subjected to anoxic conditions, it may have constitutively high levels of acetaldehyde in the leaves (Kreuzwieser et al. 2000). However, bursts of acetaldehyde emission remotely from the wounding site were observed also from poplar and velvet bean leaves (data not shown). Alternatively, acetaldehyde may be formed in large amount from pyruvate upon wounding by a yet unknown mechanism. In this case, acetaldehyde should be formed at the wounding site and then translocated away from this site through the transpiration stream. However, acetaldehyde formed by pyruvate should not enter the transpiration stream. In addition, a certain amount of 13C labelling can be found in pyruvate-derived acetaldehyde after a short exposure to air containing 13CO2, indicating that pyruvate can be partially of chloroplastic origin, or that two sources of cytosolic pyruvate contribute to acetaldehyde formation (Karl et al. 2002). In our experiment, however, no 13C was incorporated in the emitted acetaldehyde when exposing to 13CO2 atmosphere either the wounded part, or the leaf disc into the cuvette where acetaldehyde emission was detected. Clearly, more experiments are needed to determine the biochemical origin of the acetaldehyde that is emitted remotely upon wounding. The finding that acetaldehyde can be emitted by much larger surfaces than those directly wounded may have important consequences when estimating global biogenic emissions of this VOC in the atmosphere.

High temperature

While VOC emission from wounding, mostly attributable to biotic stresses, has been clearly measured in past, previously-referenced studies, little work has been done to investigate VOC emissions as a consequence of environmental stresses or rapid fluctuations of environmental factors, often experienced by plants in nature (Graus et al. 2004; Beauchamp et al. 2005). The second objective of our work was to expand this knowledge, possibly identifying VOC emissions which could reveal metabolic damage in response to recurrent stressful factors such as high temperature and high light. A large emission of many VOCs after a high-temperature treatment was detected in our study. The rapid stimulation of isoprene emission was expected, because it is known that the optimal temperature for isoprene (and isoprene synthase activity) is around 42 °C (Monson et al. 1992). The drop of isoprene while maintaining leaf at high temperature was also previously described and attributed to a regulation of isoprene synthesis rather than to enzyme denaturation (Singsaas & Sharkey 2000). In our experiment, the temperature to which leaves were exposed was higher than in the experiment of Singsaas & Sharkey (2000), and this may explain why isoprene emission dropped even further. We also hypothesize that isoprene drop was made stronger by the inefficient supply of photosynthetic carbon into the pathway of isoprene formation. This speculation is supported by the rapid reduction of photosynthesis during the high-temperature treatment, to about 30% of the rates measured at 30 °C (data not shown). Because, contrary to the studies of Singsaas & Sharkey (2000), isoprene emission was totally inhibited by the high-temperature treatment, isoprene synthase denaturation might also have played a role. However, as in Singsaas & Sharkey (2000), a rapid reconstitution of the original rates of isoprene emission (and photosynthesis) during the recovery at 30 °C was observed. This time course is not consistent with the time likely required to reconstitute proteins denatured by heat.

Methanol, acetaldehyde and C-6 compounds also increased during the high-temperature treatment but more slowly than isoprene and far more slowly than during the wounding stress. (E)-2-hexenal was the C-6 compound predominantly emitted during the high-temperature treatment. This compound was not observed in the profile of C-6 compounds emitted after wounding (Fig. 1) and this suggests that the high-temperature stress caused a different level or a different pattern of membrane denaturation, not involving oxidation of emitted compounds into alcohols and actetates (Croft et al. 1993). The emission of (E)-2-hexenal and all other VOCs, with the exception of isoprene, peaked about 100 min after beginning the high-temperature treatment. Heiden et al. (2003) also noted the different time course of induction of C-6 compounds when exposing plants to abiotic stresses (ozone, in their case). They reasoned that this may reveal emission of constitutive (wounding) versus induced (ozone) C-6 compounds, with induced compounds needing more time to activate their biosynthesis. This may apply also to the environmental stresses studied in this work, although C-6 compound emission was stimulated by high temperature more rapidly than by ozone. However, it is likely that the time course of the induction of C-6 compound emissions reflects the time course of membrane degradation caused by high temperature.

Interestingly, the large emission of (E)-2-hexenal was sustained over time, and remained high along the entire recovery period at moderate temperature. This may indicate that membrane damage continues for longer time than the actual high-temperature stress episode. It also shows that plants exposed to high temperature may release in the atmosphere a very relevant amount of VOCs for a very long period, another circumstance that should be taken into consideration for modelling purposes.

We repeated the same high-temperature treatment maintaining the piece of leaf in the cuvette under anaerobic conditions to determine which of the heat-induced VOCs was O2-dependent. Acetaldehyde emission was stimulated by this treatment. While it is known that acetaldehyde is formed by ethanol translocated from roots when these are anoxic (Kreuzwieser et al. 1999), it was surprising to observe that acetaldehyde stimulation could occur following the establishment of anaerobic conditions in a piece of leaf only. The likely source of acetaldehyde is in this case sugar breakdown (Kimmerer & MacDonald 1987). As for the wounding experiment, this also indicates that acetaldehyde can be formed locally, perhaps from a pool of ethanol that is large even when roots are not under anoxic conditions. The emission of C-6 compounds generally decreases when leaves are subjected to anaerobic conditions (Fall et al. 1999). However, in heat-stressed leaves under anaerobic conditions, the emission of (E)-2-hexenal slightly increased. This may suggest either the presence of an alternative pool of these compounds, or an O2-independent pathway of synthesis, different from the O2-dependent lipoxygenase-catalysed cleavage of α-linolenic acid (Hatanaka 1993).

High light

Even the exposure to short periods of high light can induce the emission of VOCs. We used two treatments, one of which did not cause photoinhibition and slowly reversible photochemical damage to Phragmites leaves, while the other did inhibit the photochemistry of sensitive Arabidopsis mutants. As for the high-temperature treatment, the induction of isoprene in Phragmites leaves under high-light intensity was expected (Loreto & Sharkey 1990). More interestingly, our experiment shows that acetaldehyde and C-6 compound emission can also be highly stimulated under high-light intensity. The acetaldehyde emission may be stimulated by either an accelerated rate of synthesis or by stomatal opening caused by high light. Under the high-light treatment, Phragmites stomata opened by about 30% (data not shown). As discussed before, stomatal opening was not believed to influence acetaldehyde emission (Kreuzwieser et al. 2001), but our results suggest that there may be a strong stomatal control on acetaldehyde emissions because of wounding or high-light exposure.

The emission of C-6 compounds, formed predominantly from the denaturation of membrane lipids (Hatanaka 1993), suggests that a certain damage to the membrane may occur even in absence of other photoinhibition symptoms in Phragmites under high light. It is remarkable that, contrary to what was observed with the high-temperature treatment, the emission of C-6 compounds dropped once the light intensity was dimmed. This is also indicative of a very transient stress, or may indicate that even these compounds form pools inside leaves and that their emission is dependent on stomatal opening. If the emission of C-6 compounds reveals an early stress conditions, their measurement may become very important as stress indicator in physiopathological practices.

The experiment with the photoinhibition-sensitive Arabidopsis NPQ1 confirmed that, when leaves are heavily photoinhibited, as indicated by the fluorescence record, a large increase of emission of a C-6 compounds, especially (E)-2-hexenal, may occur. Remarkably, in this case the increase in the emission of (E)-2-hexenal was recorded after the high-light treatment and not during it. We did not follow the emission during the treatment because of the concurrent decrease of temperature, also affecting the volatility of VOCs. Also noticeable is that the emission of (E)-2-hexenal after the high-light treatment was not sustained over time, but slowly decayed to the level observed before the high-light treatment. We speculate that (E)-2-hexenal emission after high-light exposure in sensitive Arabidopsis leaves is related to the occurrence of a second episode of membrane degradation and that (E)-2-hexenal emission decays when no further unsaturated fatty acid is formed from the breakdown of membrane lipids. Interestingly, no (E)-2-hexenal emission was observed when Arabidopsis leaves were previously fumigated with isoprene. This is an important confirmation that isoprene may have a very strong protective action against the denaturation of membrane lipids, as first hypothesized by Sharkey & Singsaas (1995). Again, as for the high-temperature treatment, it is important to underline that the detection by sensitive PTR-MS measurements of emissions of C-6 compounds may be used as a very sensitive stress indicator.

CONCLUSIONS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

We have further expanded our knowledge that VOC emission by plants is influenced by environmental factors. It has been shown that wounding can induce emission of acetaldehyde distant from the wounding site and that large emissions of VOCs, especially C-6 compounds and acetaldehyde, can occur in response to exposure to transiently high-temperature and light levels. This should be taken into account when parameterizing emissions of these compounds for modelling purposes. In general, our study indicates that emissions of acetaldehyde and C-6 compounds may be larger than previously estimated, and that these compounds may be released in large quantities also in conditions that are very common in nature, being very sensitive indicators of stress in plants.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES

This work was supported by the European Commission (contract MC-RTN-CT-2003-504720, ‘ISONET’), and by the European Science Foundation scientific programme VOCBAS.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  • Andreae M.O. & Crutzen P. (1997) Atmospheric aerosols: biogeochemical sources and role in atmospheric chemistry. Science 276, 10521058.
  • Beauchamp J., Wisthaler A., Hansel A., Kleist E., Miebach M., Niinemets U., Schurr U. & Wildt J. (2005) Ozone induced emissions of biogenic VOC from tobacco: relationships between ozone uptake and emission of LOX products. Plant, Cell & Environment 28, 13341343.
  • Cojocariu C., Escher P., Haeberle K.-H., Matyssek R., Rennenberg H. & Kreuzwieser J. (2005) The effect of ozone on the emission of carbonyls from leaves of adult Fagus sylvatica. Plant, Cell & Environment 28, 603611.
  • Croft K.P.C., Jüttner F. & Slusarenko A.J. (1993) Volatile products of the lipoxygenase pathway evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae pv phaseolicola. Plant Physiology 101, 1324.
  • Fall R. (2003) Abundant oxygenates in the atmosphere: a biochemical perspective. Chemical Reviews 103, 49414951.
  • Fall R., Karl T., Hansel A., Jordan A. & Lindinger W. (1999) Volatile organic compounds emitted after leaf wounding: on-line analysis by proton transfer-reaction mass spectrometry. Journal of Geophysical Research 104, 1596315974.
  • Galbally I.E. & Kirstine W. (2002) The production of methanol by flowering plants and the global cycle of methanol. Journal of Atmospheric Chemistry 43, 195229.
  • Graus M., Schnitzler J.P., Hansel A., Cojocariu C., Rennenberg H., Wisthaler A. & &. Kreuzwieser J. (2004) Transient release of oxygenated volatile organic compounds during light-dark transitions in grey poplar leaves. Plant Physiology 135, 19671975.
  • Guenther A.B., Hewitt C.N., Erickson D., et al. (1995) A global model of natural volatile organic compound emissions. Journal of Geophysical Research 100, 88738892.
  • Hatanaka A. (1993) The biogeneration of green odour by green leaves. Phytochemistry 34, 12011218.
  • Heiden A.C., Kobel K., Langebartels C., Schuh-Thomas G. & Wildt J. (2003) Emissions of oxygenated volatile organic compounds from plants, Part I: emissions from lipoxygenase activity. Journal of Atmospheric Chemistry 45, 143172.
  • Karl T., Fall R., Jordan A. & Lindinger W. (2001) On-line analysis of reactive VOCs from urban lawn mowing. Environmental Science and Technology 35, 29262931.
  • Karl T., Curtis A.J., Rosenstiel T.N., Monson R.K. & Fall R. (2002) Transient releases of acetaldehyde from tree leaves – products of a pyruvate overflow mechanism? Plant Cell & Environment 25, 11211131.
  • Kimmerer T.W. & MacDonald R.C. (1987) Acetaldehyde and ethanol biosynthesis in plants. Plant Physiology 84, 12041209.
  • Kreuzwieser J., Scheerer U. & Rennenberg H. (1999) Metabolic origin of acetaldehyde emitted by poplar (Populus tremula × P. alba) trees. Journal of Experimental Botany 50, 757765.
  • Kreuzwieser J., Kuehnemann F., Martis A., Rennenberg H. & Urban W. (2000) Diurnal pattern of acetaldehyde emission by flooded poplar trees. Physiologia Plantarum 108, 7986.
  • Kreuzwieser J., Harren F.J.M., Laarhoven L.J.J., Boamfa I., Lintel-Hekkert S., Scheerer U., Hüglin C. & Rennenberg H. (2001) Acetaldehyde emission by leaves of trees – correlation with physiological and environmental parameters. Physiologia Plantarum 113, 4149.
  • Lindinger W., Hansel A. & Jordan A. (1998) Proton-transfer-reaction mass spectrometry (PTR-MS): on-line monitoring of volatile organic compounds at pptv levels. Chemical Society Reviews 27, 347354.
  • Loreto F. & Sharkey T.D. (1990) A gas-exchange study of photosynthesis and isoprene emission in Quercus rubra L. Planta 182, 523531.
  • Loreto F. & Sharkey T.D. (1993) Isoprene emission by plants is affected by transmissible wound signals. Plant, Cell & Environment 16, 563570.
  • Loreto F. & Velikova V. (2001) Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products and reduces lipid peroxidation of cellular membranes. Plant Physiology 127, 17811787.
  • Loreto F., Ciccioli P., Cecinato A., Brancaleoni E., Frattoni M. & Tricoli D. (1996) Evidence of the photosynthetic origin of monoterpenes emitted by Quercus ilex leaves by 13C labelling. Plant Physiology 110, 13171322.
  • Loreto F., Mannozzi M., Maris C., Nascetti P., Ferranti F. & Pasqualini S. (2001) Ozone quenching properties of isoprene and its antioxidant role in leaves. Plant Physiology 126, 9931000.
  • Milburn J.A. (1979) Water Flow in Plants. Longman, London, UK.
  • Monson R.K., Jaeger C.H., Adams W.W., Driggers E.M., Silver G.M. & Fall R. (1992) Relationship among isoprene emission rate, photosynthesis, and isoprene synthase activity as influenced by temperature. Plant Physiology 98, 11751180.
  • Nemecek-Marshall M., MacDonald R.C., Franzen J.J., Wojciechowski C. & Fall R. (1995) Methanol emission from leaves: enzymatic detection of gas phase methanol and relation of methanol fluxes to stomatal conductance and leaf development. Plant Physiology 108, 13591368.
  • Niinemets U., Loreto F. & Reichstein M. (2004) Physiological and physicochemical controls on foliar rolatile organic compound emissions. Trends in Plant Science 9, 180186.
  • Niyogi K.K., Grossman A.R. & Björkman O. (1998) Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. The Plant Cell 10, 11211134.
  • Sharkey T.D. & Singsaas E.L. (1995) Why plants emit isoprene. Nature 374, 769.
  • Singsaas E.L. & Sharkey T.D. (2000) The effects of high temperature on isoprene synthesis in oak leaves. Plant, Cell & Environment 23, 751757.
  • Thöll D., Boland W., Hansel A., Loreto F., Roese U.S.R. & Schnitzler J.-P. (2006) Practical approaches to plant volatile analysis. Plant Journal 45, 540560.