SEARCH

SEARCH BY CITATION

Keywords:

  • Populus;
  • carbon-11;
  • DOXP pathway;
  • isoprene emissions;
  • jasmonic acid

ABSTRACT

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

A new approach for pulse labelling of plants using the short-lived positron emitting radioisotope carbon-11 (half-life: 20.4 min) as 11CO2 is reported together with its application to measuring [11C]isoprene emissions from intact leaves capturing information associated with: (1) rate of emission; (2) the relative contribution of recently fixed carbon to isoprene biosynthesis; and (3) the transit time for tracer movement through the leaf and biochemical pathways associated with isoprene biosynthesis. This approach was applied to study the response of certain Populus species to exogenous treatments of jasmonic acid (JA), a plant hormone implicated in signal transduction linked to defence response against herbivory. Twelve hours after treatment of single intact leaves of aspen (Populus tremuloides) with a 1 m m JA spray, isoprene emissions from those leaves increased 1.5 times the controls from 35.4 ± 2.2 to 53.1 ± 4.8 nmol m−2 s−1. [11C]Isoprene emissions from the same leaves, reflecting the isoprene that was derived from recently fixed carbon, increased much more, to 2.2 times the controls. This increase coincided with a change in emitted [11C]isoprene from 0.31 to 0.68% of 11C fixed in the leaf tissue, while the tracer transit time remained constant at 12.5 min. These results suggest that JA had no effect on enzyme kinetics involved in isoprene biosynthesis, but did impact the source of recent carbon feeding that pathway. Studies with poplar (Populus nigra clone NC 5271) showed similar trends in systemic emissions (from an untreated leaf on the same plant).


INTRODUCTION

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

2-Methyl-1, 3-butadiene (isoprene) is one of several biogenic volatile organic compounds (BVOCs) emitted by many woody plant species (Kesselmeier & Staudt 1999) that exert strong influences over the chemical and radiative properties of the troposphere (Trainer et al. 1987; Chameides et al. 1988; Fuentes et al. 2000). BVOCs represent the single most important source of photochemically reactive compounds to the troposphere and are implicated in the dynamics of both tropospheric ozone (Brasseur & Chatfeld 1991; Fehsenfeld et al. 1992) and methane (Jacob & Wofsy 1988; Wuebbles et al. 1989). Numerous surveys and inventories of vegetation have demonstrated that members of the genera Quercus (oaks) and Populus (poplars) are large emitters of isoprene in North America and Europe (Harley, Monson & Lerdau 1999; Holdren, Westberg & Zimmerman 1979; Lamb et al. 1993; Simpson et al. 1995).

The biochemical and physiological controls over isoprene biosynthesis have received considerable attention in past years because of efforts to develop process-based models of isoprene emission that can be used to predict the response of emissions to environmental and land-use change. Empirical models of isoprene emission (e.g. Guenther et al. 1993) are quite useful for predicting how emissions respond to short-term changes in light and temperature, but several ecological studies have demonstrated that the isoprene emission capacity responds to growth conditions and environmental perturbations, and current models do not account for changes in emission capacity (Guenther et al. 1993, 1995; Fall & Wildermuth 1998). The recent discovery of a novel biosynthetic pathway for isoprene production has opened up new possibilities for understanding the regulation of isoprene emission across multiple time-scales and for developing improved models of emissions (Niinemets et al. 1999; Zimmer et al. 2000).

Isoprene is biosynthesized within the chloroplast from dimethylallyl pyrophosphate by isoprene synthase (Silver & Fall 1995) acting through the 1-deoxy- d-xylulose-5-phosphate (DOXP)/methyl erithritol 4-phosphate pathway requiring glyceraldhyde-3-phosphate (G3P) and pyruvate photosynthetic substrates to fuel the process (Rohmer et al. 1993; Zeidler et al. 1997; Schwender et al. 1996, 1997; Lichtenthaler 1999; Kreuzwieser, Schnitzler & Steinbrecher 1999; Fisher et al. 2000; Rohdich et al. 2000). Isoprene is not stored in leaf tissue, is not metabolized, diffuses rapidly through leaves to the intercellular air space, and is generally present at concentrations far below the saturation vapour pressure; these factors allow one to treat steady-state emission as a direct reflection of isoprene biosynthesis (Fall & Monson 1992; Delwiche & Sharkey 1993 Monson et al. 1995; Lerdau & Keller 1997).

The fundamental understanding of this pathway has allowed more detailed studies of its function within plants, and of the impact that isoprene production has on cellular metabolism. Most efforts to understand isoprene's function have considered its role as a protectant against either high temperatures (Sharkey & Singaas 1995; Singaas et al. 1997, 1999; Sharkey, Chen & Yeh 2001) or oxidative stress (Loreto et al. 2001; Affek & Yakir 2002). More recently it has been suggested that isoprene production plays a role in cell-level carbon and mineral balance and may be linked to the activities of biosynthetic pathways that compete for substrate (Lerdau, Guenther & Monson 1997; Rosenstiel et al. 2003).

It is well recognized that the metabolite pathways involved with the synthesis of certain plant volatiles respond to wounding and damage (Pare & Tumlinson 1999). Early work on isoprene and wounding demonstrated that emissions decreased substantially when lateral or remote leaflets of velvet bean (Mucuna deeringeniana L.) and kudza (Pueraria montana var. lobata Wild. Ohwi) were mechanically damaged (Loreto & Sharkey 1993). Partial defoliation of entire branches of field-grown eastern cottonwood (Populus deltoides) gave similar results (Funk, Jones & Lerdau 1999). A general consensus formed that this decrease in emissions resulted from the wound-induced reallocation of carbon to specialized wound-response pathways (Karban & Baldwin 1997; Strauss & Agrawal 1999) and away from the DOXP pathway. Other studies, however, have reported increases in the emissions of other volatiles in response to wounding (Ping et al. 2001; Rose & Tumlinson 2004).

Clearly, wound responses will elicit a complex sequence of events at both the molecular and biochemical level that culminate in an integrated response by the plant. Damage may elicit signals that increase metabolic activity and the synthesis of isoprene, while the loss of photosynthetic tissue may lead to a decrease in substrate availability and a net decline in isoprene biosynthesis. The possible roles of wound signals as regulators of isoprene biosynthesis have not, however, been investigated. To deepen our understanding of these processes, it is necessary to dissect the integrated response of the plant into its individual component processes. Since the biosynthesis of jasmonic acid (JA) has been implicated as one of several important signal transduction pathways that result in the early induction of defences in response to herbivory (Creelman & Mullet 1997; Wasternack & Parthier 1997) we chose to investigate its impact on the isoprenoid pathway.

Here we present the first of a series of papers addressing key aspects on the effects of exogenous JA treatment on re-allocation of carbon resources relevant to plant metabolism. Our specific goals were to: (1) validate this method of using carbon-11 in isoprene emission studies; (2) examine the effects of JA on isoprene emission rates; and (3) determine whether the carbon sources for isoprene production change in response to JA. Carbon-11 (t1/2 = 20.4 min) has previously been used in numerous investigations of photosynthate transport within intact plants (for a recent review see Minchin & Thorpe 2003), however, this work represents the first known application to phytogenic volatile emissions.

METHODS

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

Production and manipulation of 11CO2 for rapid pulse labelling

11CO2 was produced on the BNL 41 inch (Japan Steel Works, Ltd, Tokyo, Japan) cyclotron in a 212-mL volume gas target pressurized to 200 psi with scientific grade N2 (99.9995% purity; MG Industries, Malvern, PA, USA). Irradiation using 17 MeV H+ generated 11C via the 14N(p,α)11C nuclear transformation as 11CO2 (Ferrieri & Wolf 1983). The radioactive gas was immediately released from the target after bombardment (Fig. 1), and the tracer concentrated through selective adsorption onto a bed of Ni(0) catalyst (Fig. 2). The catalyst bed was comprised of 135 mg of silica support Ni catalyst (Shimadzu, Inc., Kyoto, Japan) mixed with molecular sieve 4 Å (100 mesh; Alltech, Inc., Lancaster, PA, USA). The material was contained within a stainless steel tube using standard high performance liquid chromatography column frits (Alltech, Inc.). Prior to use, the catalyst was heated to 400 °C under H2 flow to ensure complete reduction of the Ni metal. Adsorption of 11CO2 from the target N2 gas stream was quantitative for a target gas flow rate set at 1.5 L min−1 and a bed temperature ranging between 20 and 30 °C. Once the tracer was concentrated on the bed, the module was resealed and rapidly heated to 400 °C using resistance cartridge heaters. In the absence of extraneous H2, 11CO2 rapidly desorbed from the Ni(0) surface without effect on its chemical integrity. The desorbed 11C was passed through an oxidation furnace to ensure all 11C was oxidized to CO2 and swept to the experimental platform in a controlled flow of air mixed with a desired level of unlabelled CO2 and a portion diverted to the leaf chamber. The catalyst within the module was restored to its reduced state by flushing it with H2 for a few minutes prior to reducing temperature back to ambient.

image

Figure 1. Schematic representation of the experimental platform used for pulse administration of 11CO2. The platform included a complete interface with the cyclotron target, a tracer pulse generator, leaf chamber and gas exchange instrumentation. The leaf chamber had its own red/blue LED light source providing a maximum irradiance of 800 µmol m−2 s−1. This chamber resided within a larger environmental chamber that maintained ambient light (average 400 µmol m−2 s−1) and temperature.

Download figure to PowerPoint

image

Figure 2. Schematic representation of the 11CO2 pulse generator module. This module consisted of a Ni(0) catalyst bed that quantitatively removed 11CO2 from the target gas, and allowed it to be introduced as a discrete pulse in the plant gas mixture.

Download figure to PowerPoint

We used a source gas of zero air (0% CO2 in air; Praxair, Inc., Danburg, CT, USA) blending it with 800 p.p.m. of CO2 in air to a level of 420 p.p.m. CO2 using electronic mass flow controllers on the gas feed stream (MKS Industries, Wilmington, MA, USA), with a total flow rate of 400 mL min−1. The gas flow was split on exiting the pulse module (Fig. 1) yielding a flow rate of 150 mL min−1 that first passed through a dew point generator (Li-Cor model LI-610; Li-Cor Inc., Lincoln, NE, USA), then through the leaf chamber, and exiting through the sample cell of an infrared gas analyser (IRGA, model LI-6262; Li-Cor, Inc.). The other split component, 250 mL min−1, was routed directly through the reference cell of the IRGA to provide gas exchange information. Outflow was also temporarily diverted to a gas chromatograph immediately prior to 11CO2 administration to measure total isoprene emissions.

The leaf chamber was fabricated from Plexiglas on a split clamshell hinge that allowed us to seal targeted leaves between neoprene rubber gaskets. The chamber housed a bank of red/blue LED lights providing an irradiance of 800 µmol m−2 s−1. It also had four inlet ports mounted across the top half and four exhaust ports mounted across the bottom to provide adequate gas mixing. The 150 mL min−1 flow rate of air was sufficient to void a 30-s radiotracer pulse from the chamber volume within 3 min. Leaf surface temperatures were constantly monitored using fine wire thermocouples (Omega Engineering, Inc., Stamford, CT, USA) The leaf chamber was housed within a larger environmental chamber that maintained air temperatures between 20 and 30 °C with 1 °C accuracy, and an average light irradiance of 450 µmol m−2 s−1, using a water-filtered 300-W greenhouse lamp.

The leaf chamber was also equipped with a PIN diode gamma radiation detector (Bioscan, Inc., Washington, DC, USA) to provide direct measurement of 11CO2 in the leaf chamber. Typically, time-activity data from this detector (Fig. 3) reveals information about the pulse magnitude, tissue fixation and subsequent loss of tracer from the leaf reflecting a combination of phloem transport of mobile [11C]photosynthate, emission of [11C]BVOC, and photorespiration of 11CO2. The amount of carbon fixation was used to normalize measurements from the same plant when re-labelled. We typically administered 20 mCi pulses of 11CO2. Where it was necessary to vary the amount of carbon-11 in the pulse, the gas target was irradiated for different times. PIN diode detector response was periodically calibrated by replacing the leaf with a silica thin layer chromatography strip (Fisher Scientific, Inc., Fairlawn, NJ, USA) spotted with known amounts of aqueous [11C]carbonate.

image

Figure 3. Decay corrected time-activity data (measured by the PIN diode detector) illustrating the shape of the pulse passing through the leaf chamber. Features of this curve include measurement of pulse size and leaf fixation of 11C for data normalization, and leaf loss of tracer reflecting a combination of sugar export and volatile emissions (BVOCs and CO2).

Download figure to PowerPoint

Measurement of leaf [11C] isoprene emission

Administration of discrete pulses of 11CO2 allowed us to assess three parameters regarding [11C]isoprene emission. First, we measured transit times reflecting speed of tracer movement through the isoprenoid pathway. Second, we measured integrated flux and related it to amount of recently fixed carbon. Third, we measured changes in the source of carbon for isoprene biosynthesis over time providing insight into whether treatments changed the relative proportions of recently fixed carbon and/or old carbon sources.

Air from the leaf chamber was exhausted through a well-shielded soda-lime CO2 trap (Alltech Inc.; 80–100 mesh) to capture 11CO2 from the pulse and from any subsequent leaf 11CO2 emissions, as well as any radiolabelled volatile monoterpenes. We verified that isoprene was not retained by the soda-lime packing, but that monoterpenes were. This was accomplished using a short column (6 in. × 1/4 in o.d) packed with soda lime particles (20–40 mesh) coupled to a thermal conductivity detector of a gas chromatograph. We observed no loss of isoprene when passed through this packed column compared against standard samples passed through the empty column. A similar test for monoterpene retention by soda-lime was performed using β-pinene. Unlike isoprene, this higher molecular weight substance was completely retained.

The exhaust from this first soda-lime trap passed to an auxiliary platform that housed an auto-sampling capillary gas chromatograph with a photometric detector to quantify total isoprene emissions. Retention time and detector response were calibrated using isoprene standards. A by-pass on the GC sampling loop allowed gas to pass to a quartz furnace filled with 20 g of copper oxide (Aldrich, Milwaukee, WI, USA; 30–60 mesh) and maintained at 800 °C to oxidize [11C]isoprene to 11CO2. The 11CO2 from this process was captured on a second soda-lime trap placed near a well-shielded sodium iodide scintillation detector (Canberra, Inc., Meriden, CT, USA), configured for single 511 keV photon counting and sensitive to less than 1 µCi of radioactivity. All data were decay-corrected for display and analysis.

We verified in one experiment that radioactivity retained on the second trap reflected only [11C]isoprene emission by passing the outflow from the first soda-lime trap through a small glass cartridge packed with Tenex GR (graphitized carbon molecular sieve; Alltech). This material has a high retention for VOCs and was preloaded with unlabelled isoprene. The trapped radioactivity was quantified, and then the contents desorbed into a packed column radio gas chromatograph (6 ft. × 1/8 in. o.d. stainless steel column packed with 80–100 mesh Porapak T; Analabs, Inc., North Haven, CT, USA) equipped with thermal conductivity and radiation detectors connected in series. We observed one major radioactive peak that co-eluted with unlabelled isoprene. The integral of the radioactivity peak matched the starting amount of radioactivity on the cartridge. A trace amount of [11C]ethylene was also observed (< 0.01% of total trapped [11C]isoprene) which is ignored in this work.

Quantification of leaf isoprene emissions

We defined the transit time as the time for tracer to diffuse to the chloroplast, via the initial stages of the Calvin cycle, enter isoprenoid pathway, diffuse in mesophyll cells as [11C]isoprene for final release through the stomata, and subsequently be transported to the analytical system's isoprene trap. To infer a transit time, the time of half-maximum activity in the trap was estimated from the time of the maximum of a transform of the data using Eqn 1 (see inset in Fig. 4):

image

Figure 4. Integrated [11C]isoprene emissions from birch (a low isoprene emitter) and aspen (a high isoprene emitter). The data was decay corrected and normalized to 11C fixation. The insert figure illustrates the half maximum transformation of data using 1, and can be used to measure the transit time (to half maximum) for tracer to move through the leaf.

Download figure to PowerPoint

  • Atransformed = Ahalfmax − ABS(Ahalfmax − At)(1)

Isoprene activity was presented as percent of fixed 11C relying on the PIN diode detector to measure tissue fixation, and calibrated response of the NaI detector. The half-maximum activity (Ahalfmax) was determined by dividing the maximum activity by two. The maximum activity was an average of several values at the emission plateau. Activity at specific time points (At) were the sum of [11C]isoprene emissions that accumulated within the trap over time. The transit time was measured as the time from the detector's indication of the initial tracer pulse (seen through the shielding from the leaf chamber and gas lines) to the half-maximum time in [11C]isoprene emission, and reflected a combination of leaf physico-chemical processes as well as the delay due to gas flow (which was less than 1 min for the system's plumbing). We made no attempt to disentangle these aspects, but assumed that the system's features were constant since the same flow rates were used throughout all the studies.

Integrated [11C]isoprene emission after treatment was expressed as a fraction of the emission from that plant before treatment (baseline). Similarly, total carbon isoprene emission after treatment was expressed as a fraction of the emission rate before treatment for each plant, averaging 10 independent GC measurements of isoprene.

Plant material

There were four experiments. In experiment 1 we compared isoprene emissions from both aspen and birch seedlings representing a high isoprene emitter and a low isoprene emitter, respectively. In experiment 2 we measured isoprene emissions from a single aspen plant in a series of consecutive tracer runs spanning 4.5 h with slightly increasing leaf temperature. In experiment 3 we measured ‘local’ isoprene emissions from intact aspen leaves of several plants, each treated with JA. In experiment 4 we measured ‘systemic’ emissions from several intact hybrid poplar leaves, to determine the response to JA treatment of another leaf. Seeds of aspen (Populus tremuloides) and of birch (Betula papyrifera) were sown 2 years prior to experiments. The seedlings were coppiced in the winter prior to experiments, and the stems that sprouted were used in experiments 1, 2 and 3. For experiment 4, hybrid poplar (Populus nigra clone NC5271) cuttings were dipped in 0.1% indole-3-butyric acid (TakeRoot; Schultz Co., Bridgeton, MO, USA) and planted in fafards ♯2 potting soil in 2.83 L ‘Tall-one’ tree-pots (Stuewe & Sons, Corvallis, OR, USA). NC5271 clones were used for experiments approximately 45–60 d after sprouting, when stems were approximately 0.4 m in height.

JA treatments

Baseline measurements of [11C]isoprene and total carbon isoprene emission were taken on the day prior to treatment. In the evening of that day, either a JA treatment comprised of 1 m m JA dissolved in 1% aq. acetone, or a control treatment comprised of 1% aq. acetone (without JA), was sprayed onto a single mature leaf. During this process the entire plant was contained in a plastic bag with the exception of the treatment leaf. The bag was removed after the spray had dried. Treatments were coordinated so that all plants were tested at the same time of day (within an hour) to avoid any diurnal effects. Additionally, leaf temperature was constant (± 0.5 °C) throughout these measurements.

Statistical analyses

We used single factor analysis of variance (anova) to test whether treatment means within an experiment were equal. Data reflecting ratios of changes relative to baseline were subjected to log-transformation prior to statistical analysis.

RESULTS

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

Validation of the approach: comparison of a high isoprene emitter and a low isoprene emitter

Integrated time-activity data reflecting [11C]isoprene emission (at 27 °C) showed striking differences in emissions levels between seedlings of aspen, a high emitter, and birch, a low-emitter (Fig. 4). The birch data served as a system check to ensure that pulse remnants, or photorespired 11CO2 that may have passed through the CO2 trap, were not contaminating the [11C]isoprene trap. Birch does not emit appreciable levels of isoprene (Hakola, Rinne & Laurila 1998). Indeed, no [11C]isoprene emission was observed from birch.

Gas exchange (IRGA) data on the aspen in Fig. 4 revealed a net carbon exchange rate of 7 µmol m−2 s−1. Multiplying the integrated [11C]isoprene emission value (equal to 0.22% fixed 11C) by the carbon exchange rate, the emission rate for isoprene derived from recently fixed carbon was calculated as 15.6 nmol m−2 s−1. This was 81% of the total isoprene emission rate of 19.3 nmol m−2 s−1.

The time to half-maximum (as inset to Fig. 4), determined from the data transformed by Eqn 1, defined a time for tracer transit through the DOXP pathway. Using the first peak (background of initial pulse) as a time marker gave a transit time to half-maximum for this aspen of 13.5 min.

Validation of the approach: influence of diurnal variation and leaf temperature on recent and total carbon contributions to isoprene emission

Three consecutive tracer runs were carried out by administering 11CO2 to the same leaf of an aspen at 90 min intervals beginning at 1100 h. Leaf temperature increased from 27 to 29 °C over the course of these runs. Both [11C]isoprene (recent carbon) and total carbon isoprene emission rates increased, from 15.6 to 25.0 nmol m−2 s−1 and 19.3 to 32.5 nmol m−2 s−1, respectively, from the first to the third run. The [11C]isoprene data increased from 0.22 to 0.37% of fixed 11C in leaf tissue for the series. Composition of recently fixed carbon in emitted isoprene remained constant over the series with a mean value of 80%. Tracer transit time decreased by 13% (from 13.5 to 11.6 min) between the first and the third run in the series. Gas exchange data did not show a significant change in the carbon exchange rate over this period of time, or for the small temperature change.

Effect of exogenous JA treatment on recent and total carbon contributions to isoprene emissions

Both local and systemic recent carbon and total carbon isoprene responses to exogenous JA or control treatments were measured in Populus species 12 h after treatment, and compared against prior baseline emissions from the same plant at the same time of day.

Studies on the local response of isoprene emission after control and JA treatments of aspen (Fig. 5a) revealed that recent carbon isoprene emission increased to 2.19 ± 0.25 times the baseline emission (i.e. before treatment) while total carbon isoprene emission increased to 1.5 ± 0.1 times the baseline emission. This reflects a change in the recent carbon isoprene emission rate from 27.2 ± 1.8 to 59.6 ± 7.9 nmol m−2 s−1, and a change in the total isoprene emission rate from 35.4 ± 2.2 to 53.1 ± 4.8 nmol m−2 s−1. The increase in [11C]isoprene emissions after JA treatment reflected a change from 0.31 to 0.68% of fixed 11C in the leaf tissue. Control treatments had no effect on recent or total carbon isoprene emissions. An experiment with a single hybrid poplar plant gave a similar response (see below, Fig. 6).

image

Figure 5. JA effects on isoprene emission from leaves of Populus tremuloides and hybrid poplar, showing changes in the relative contributions from multiple carbon sources. (a) Changes in local aspen leaf isoprene emissions (n = 5) derived from recently fixed carbon and total carbon sources, and measured 12 h after treatment of the load leaf with 0 or 1 m m JA, relative to emissions before treatment (baseline). Absolute mean baseline emission rates were 27.2 ± 1.8 and 35.4 ± 2.2 nmol m−2 s−1 for recent and total isoprene emissions, respectively. (b) Changes in systemic hybrid poplar leaf isoprene emissions from a younger leaf that was orthostichous to the target leaf after 0 or 1 m m JA treatment. Absolute mean baseline emission rates were 55.7 ± 6.9 and 64.8 ± 7.2 nmol m−2 s−1 for recent carbon and total carbon isoprene emissions, respectively. (The hybrid poplar had higher isoprene emissions than the aspen for age-matched leaves.) Plots show mean, standard deviation error bars, and P-values from anova using log-transformed data.

Download figure to PowerPoint

image

Figure 6. The recent carbon and total carbon isoprene emissions are presented for 9 d after JA treatment, as the emission relative to the initial state (baseline) before treatment. Jasmonic acid was administered at Day 0 on the timeline.

Download figure to PowerPoint

JA also increased the systemic production of isoprene (Fig. 5b) from hybrid poplar. This species gave higher isoprene emissions from aged-matched leaves than aspen. Twelve hours after treating one leaf, recent carbon isoprene emission from a younger leaf increased slightly to 1.28 ± 0.05 times the baseline emission while total carbon isoprene emission showed even less response increasing to 1.12 ± 0.08 times the baseline emission. Control treatments had no effect on either recent or total carbon isoprene emissions.

Acclimation after exogenous JA treatment

One hybrid poplar plant was tested for baseline emissions at 1100 and 1500 h, and subjected to a JA treatment at 2300 h. Measurement of the response to treatment was started 12 h later beginning at 1100 h on the next day, and emissions were measured twice per day for 3 d. Data reported in Fig. 6 show integrated recent carbon isoprene and total carbon isoprene presented as the emissions relative to baseline. The greatest changes in recent and total carbon isoprene emissions, in response to treatment, occurred within 12–16 h with increases to 2.3 and 1.7 times the baseline responses, respectively. Local response of this single poplar was similar to the response in local emissions of the aspen plants tested in replicate (Fig. 5a). Similarly, emissions of recent carbon isoprene, as well as total carbon isoprene, increased in response to JA, but not by the same amount, indicating a relative shift in carbon sources used in isoprene production (Fig. 6). However, this shift was transient as the relative contributions of the different carbon sources to isoprene biosynthesis returned to their pre-treatment levels within 24 h after JA application. Even so, total isoprene emissions remained slightly elevated over initial state from 28 h to 9 d.

DISCUSSION

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

System validation

The approach described in this paper for pulse labelling with 11CO2 appears well suited for observing the dynamics of tracer passage through the physical and biochemical pathways leading to isoprene emission. Several advantages of the short-lived radiotracer 11C over its counterparts, 13C or 14C, include: (1) a high sensitivity for detecting minute emissions and their response to treatments; (2) the ability to disentangle the impacts of treatments on recently acquired carbon and older established carbon sources for isoprene synthesis; and (3) no buildup of tracer with time allowing repeat measurements of dynamics on the same plant.

Our initial test of the system compared a high isoprene emitter (aspen) to a low isoprene emitter (birch). We showed that [11C]isoprene emissions were not compromised by remnants of the tracer input pulse, nor from photorespired 11CO2. We also noted that [11C]isoprene emissions had reduced to undetectable amounts within 40 min, supporting earlier findings that there are little or no storage pools within the leaf tissue for isoprene or its biochemical precursors (Fall & Monson 1992).

Diurnal and temperature effects on recent and total contributions to isoprene emission

Previous work has shown that significant increases in isoprene emission occur when leaf temperature is increased (Sharkey & Singaas 1995; Singaas et al. 1997; Wang & Fuentes 2003; Funk, Mak & Lerdau 2004). Additionally, isoprene emissions are strongly linked to diurnal cycling of leaf carbon storage, increasing with time of day, but eventually turning downward later in the day (Funk et al. 2003). In agreement with these findings, our results also showed an increase isoprene emissions which was probably due more to the diurnal variation of leaf carbon storage than to the slight rise in leaf temperature, although it is impossible to disentangle the two effects. Our results also showed that roughly 80% of the emitted isoprene derived from recently fixed carbon, in agreement with previous 13C tracer work (Delwiche & Sharkey 1993; Karl et al. 2002; Affek & Yakir 2003; Funk et al. 2004).

This tracer technique afforded a unique opportunity to measure transit time for recently fixed carbon to pass through the DOXP pathway, with values ranging from 13.5 to 11.6 min in the series. Interestingly, these values correlate well with the rapid time course for the inhibition of isoprene emission after feeding leaves with fosmidomycin, a powerful and specific inhibitor of the DOXP pathway (Zeidler et al. 1998; Sharkey et al. 2001; Loreto & Velikova 2001). Further, our observation of decreased transit times from the first to the third run in the time series suggests there may have been effects on physical transport processes (e.g. diffusion), as well as biochemical processes linked with the DOXP pathway. These observations need further testing, both with more data, and by relating biochemistry to transit time through enzyme profiling coupled with chemical treatments such as fosmidomycin.

Regulatory effects of exogenous JA treatment on carbon re-allocation

One of the most striking observations of this work on Populus is the increase – both local and systemic – in isoprene emission in response to JA treatment. These results contrast with earlier work on mechanical damage and partial defoliation, which reported decreased systemic isoprene emissions after intervention (Loreto & Sharkey 1993; Funk et al. 1999). Mechanical damage and defoliation elicit a host of molecular, biochemical, and physiological responses, including changes in carbon availability as well as effects on endogenous JA, salicylic acid and other plant hormones associated with defence (Karban & Baldwin 1997). In some instances these signal molecules (e.g. JA and salicylic acid) may interfere with each other (Glazebrook 2001). The net effect of these resource and hormonal changes may be to lower isoprene emission. It would be interesting to test whether individual treatments of other plant hormones have negative effects on isoprene emission, sufficient to offset the increase that would arise if endogenous JA biosynthesis were stimulated. Additionally, we need to measure the dose–response to determine whether the JA effect on isoprene emission is monotonic. Perhaps at higher doses of JA, a decrease in net isoprene emission owing to a decreased photosynthetic rate explains the different effects of JA and other treatments on isoprene emission. Indeed, past reports have indicated that high levels of JA can induce leaf senescence (Parthier 1990). Our present results, however, did not show a decline in the photosynthetic rate after JA treatment for either Populus species, nor was there an indication of leaf senescence 3–6 weeks after treatment. These observations suggest that our treatment stimulated a focused response to the JA signal and not a general stressor.

It is not surprising that there was a systemic response in isoprene emission to a localized JA treatment. Recent work using [2-14C]JA in Nicotiana sylvestris combined with autoradiography indicated that JA could be transported from one leaf to other younger leaves and throughout the root system (Zhang & Baldwin 1997). We observed a smaller increase in the systemic isoprene emission after treatment than in the local response. However, since we observed similar increased local emissions in the two species (although this observation draws on a comparison of a single poplar hybrid plant with several aspen), the much smaller systemic response may likely be explained by dilution of the initial JA dose on dispersal throughout the whole plant.

The results of our local response measurements strongly suggest that there was a drastic and rapid change in plant utilization of new carbon pools after JA treatment, since the increase in recent carbon emission was not matched by the change in total carbon isoprene emission. The change in the absolute emission rates for new and total carbon suggest that essentially all of the isoprene emission post-JA treatment derived from newly acquired carbon, although the magnitude of error in this work does not allow us to conclusively argue that old carbon sources were reduced by JA. It is clear, however, that JA did stimulate the isoprene pathway, and that this stimulation derived largely from increases in new carbon contributions.

Isoprene is derived from multiple carbon sources (Karl et al. 2002; Affek & Yakir 2003). Recent work has also shown that the relative contributions of these sources can vary with environmental stress (Funk et al. 2004). Herbivory or manual defoliation can cause a re-allocation of older carbon pools within the plant within weeks of treatment from storage organs to shoots for compensatory growth (Karban & Baldwin 1997; Strauss & Agrawal 1999). This action is far too slow to compare with the short timescales for response we observed in the present work. The increased supply of recent carbon for isoprene biosynthesis may be explained by an up-regulation of enzymes involved in the DOXP pathway. However, we noted that the transit time for the tracer to pass through this pathway was unchanged by JA treatment (12.6 ± 0.5 min for n = 5 of the ‘local’ response studies using aspen). This suggests that JA did not elicit a direct effect impacting enzyme kinetics in this pathway, but a general regulatory response, perhaps through a change in substrate supply. This conclusion is supported by recent findings (Arimura, Huber & Bohlmann 2004) showing increased volatile terpenoid emissions when hybrid poplar (Populus trichocarpa × deltoids) were exposed to forest tent caterpillar feeding, mechanical wounding or exogenous methyl jasmonate, but no change was found in the transcript levels of 1-deoxy- d-xylulose 5-phosphate reductoisomerase nor of isoprene synthase. Similarly, application of methyl jasmonate increased terpene biosynthesis in Norway spruce, resulting in two-fold increase in stored monoterpene and sesquiterpene levels in needles, and five-fold increase in total terpene emissions (Martin, Gershenzon & Bohlmann 2003). Finally, studies using exogenous dideuterated deoxyxylulose treatment showed evidence for decreased endogenous carbon feeding the DOXP pathway without affecting the overall emission rate of isoprene (Wolfertz et al. 2004). It was concluded from this work that the isoprene pathway is subject to substrate regulation, in part, through feedback regulation of deoxyxylulose-5-phosophate synthase.

Two possible explanations for the noted change in recent carbon allocation are that JA either causes a remobilization of starch stores, or shunts recent carbon away from starch synthesis to begin with. Indeed, starch pools in P. tremuloides decreased by 18% after JA treatment, net photosynthesis was unchanged, but the rate of export of newly fixed carbon from the leaf (reflected by 11C export) increased (Babst et al. unpublished). Since the supply of carbon to the leaf was unchanged with treatment, but the rate of export increased, it stands to reason that at any given time there should be a higher proportion of G3P (both in the chloroplast and cytosol) relative to the starch pool. The increased amount of new carbon being directed into isoprene biosynthesis probably reflects this change in the underlying carbon substrate pool.

Acclimation after exogenous JA treatment

Our protocol gave a first measurement of isoprene emission 12 h after JA treatment, showing a maximum response between 12 and 16 h, and a recovery (although not complete) within 28 h. It is impossible to say whether acclimation was due to a change in metabolism or redistribution of JA. The latter seems more plausible since [2-14C]JA has been observed to redistribute between treated leaves and roots within 12 h (Zhang & Baldwin 1997).

Although new carbon sources feeding the DOXP pathway were rapidly altered, the multiple sources of carbon appeared to recouple before the return of total isoprene emissions to pre-treatment levels. This observation suggests that in addition to the rapid responses we noted earlier, there may be some longer-lasting effects for JA. A recent study demonstrated that altering phosphoenolpyruvate (PEP) by differential induction of cytosolic nitrate reductase and PEP carboxylase resulted in long-lasting affects on isoprene biosynthesis from a supply of extrachloroplastic PEP to the chloroplast (Rosenstiel et al. 2004). Earlier findings using 13C as a tracer also support multiple subcellular origins of leaf isoprene precursors (Karl et al. 2002; Affek & Yakir 2003), and even suggest xylem-transported glucose as an additional carbon source (Kreuzwieser et al. 2002).

The question remains whether the increase we observed in new carbon being allocated to isoprene biosynthesis is just a consequence of changes in recent carbon metabolism that happens to affect substrate supply to the DOXP pathway, or whether the increase actually reflects a more active role of JA in metabolic regulation of isoprene as a protective agent (Fineblum & Rausher 1995; Stowe 1998). Recent studies have demonstrated that isoprene and volatile terpenoids, particularly α-pinene, β-pinene and sabinene, may play active protective roles as antioxidants (Loreto et al. 2001, 2004). Isoprenoid emissions increased upon exposure of Quercus ilex (L.) leaves to ozone, and plant tolerance to ozone was enhanced through their application. Furthermore, treatment with exogenous jasmonate increased tobacco tolerance to ozone (Örvar, Mcpherson & Ellis 1997) implicating it, as well, with the antioxidant defence train. Indeed, its application elicited increased expression of mRNA encoding ascorbate peroxidase (Örvar et al. 1997; García-Pineda, Castro-Mercado & Lozoya-Gloria 2004) leading to increased ascorbate, another well-recognized antioxidant (Howard et al. 2000).

Without a doubt, the biochemical pathways contributing to and/or regulating the flow of carbon to isoprene biosynthesis are complex. The present work using carbon-11 as a tracer provided clear evidence that JA impacted key pathways within a short timescale that affected the flow of at least new carbon for isoprene biosynthesis, but it did not have a direct effect on the enzyme kinetics within the DOXP pathway. We have yet to determine whether the response seen here is a reflection of an integration of several overlapping effects. It may be possible to disentangle certain biochemical effects of JA by measuring responses on even shorter timescales. Additionally, responses of these diverse metabolic and/or regulatory pathways controlling the flow of multiple carbon sources to isoprene may have different thresholds to jasmonate concentration. It would be interesting to test whether or not similar responses are observed across a range of jasmonate concentrations.

ACKNOWLEDGMENTS

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

This work was supported by a Laboratory Directed Research and Development grant awarded by BNL (to R.A.F.), and in part by the US Department of Energy, Office of Biological and Environmental Research under contract DE-ACO2–98CH10886, a Sarah Blaffer Hardy Visiting Professorship (to M.T.L.), and The Andrew Mellon Foundation (to C.M.O.). The authors would like to thank Ken Raffa (U. Wisconsin) for supplying cuttings of hybrid poplar and Alistair Rogers (BNL Environmental Division), and Missy Holbrook (Harvard U) for their insightful comments.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  • Affek H.P. & Yakir D. (2002) Protection by isoprene against singlet oxygen in leaves. Plant Physiology 129, 269277.
  • Affek H.P. & Yakir D. (2003) Natural abundance carbon isotope composition of isoprene reflects incomplete coupling between isoprene synthesis and photosynthetic carbon flow. Plant Physiology 131, 17271736.
  • Arimura G., Huber D.P.W. & Bohlmann J. (2004) Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emission of terpenoid volatiles in hybrid poplar (Populus trichocarpa×deltoids): cDNA cloning, functional characterization, and patterns of gene expression of (–) germacrene D synthase, Ptdtps1. Plant Journal 37, 603616.
  • Brasseur G.P. & Chatfeld R.B. (1991) The fate of biogenic trace gases in the atmosphere. In Trace Gas Emissions by Plants (eds T.D.Sharkey, E.A.Holland & H.A. Mooney), pp. 127. Academic Press, San Diego, California, USA.
  • Chameides W.L., Lindsay R.W., Richardson J. & Liang C.S. (1988) The role of biogenic hydrocarbons in urban photochemical smog: Atlanta as a case study. Science 241, 14731475.
  • Creelman R.A. & Mullet J.E. (1997) Biosynthesis and action of jasmonates in plants. Annual Review of Plant Physiology and Plant Molecular Biology 48, 355381.
  • Delwiche C. & Sharkey T.D. (1993) Rapid appearance of 13C in biogenic isoprene when 13CO2 is fed to intact leaves. Plant, Cell and Environment 16, 587591.
  • Fall R. & Monson R.K. (1992) Isoprene emission rate and intercellular isoprene concentration as influenced by stomatal distribution and conductance. Plant Physiology 100, 987992.
  • Fall R. & Wildermuth M.C. (1998) Isoprene synthase: from biochemical mechanism to emission algorithm. Journal of Geophysical Research – Atmospheres 103, 2559925609.
  • Fehsenfeld F., Calvert J., Fall R., et al.. (1992) Emissions of volatile organic compounds from vegetation and the implications for atmospheric chemistry. Global Biogeochemical Cycles 6, 389430.
  • Ferrieri R.A. & Wolf A.P. (1983) The chemistry of positron emitting nucleogenic atoms with regard to preparation of labeled compounds of practical utility. Radiochimica Acta 34, 6983.
  • Fineblum T.R. & Rausher M.D. (1995) Tradeoff between resistance and tolerance to herbivore damage in a morning glory. Nature 377, 517520.
  • Fisher A.J., Baker B.M., Greenberg J.P. & Fall R. (2000) Enzymatic synthesis of methylbutenol from dimethylallyl diphosphate in needles of Pinus sabiniana. Archives of Biochemistry and Biophysics 383, 128134.
  • Fuentes J.D., Lerdau M., Atkinson R., et al. (2000) Biogenic hydrocarbons in the atmospheric boundary layer: a review. Bulletin of the American Meteorological Society 81, 15371575.
  • Funk J.L., Jones C.G., Baker C.J., Fuller H.M., Giardina C.P. & Lerdau M.T. (2003) Diurnal variation in the basal emission rate of isoprene. Ecological Applications 13, 269278.
  • Funk J.L., Jones C.G. & Lerdau M.T. (1999) Defoliation effects on isoprene emission from Populus deltoides. Oecologia 118, 333339.
  • Funk J.L., Mak J.E. & Lerdau M.T. (2004) Stress-induced changes in carbon sources for isoprene production in Populus deltoids. Plant, Cell and Environment 27, 747755.
  • García-Pineda E., Castro-Mercado E. & Lozoya-Gloria E. (2004) Gene expression and enzyme activity of pepper (Capsicum annuum L.) ascorbate oxidase during elicitor and wounding stress. Plant Science 166, 237243.
  • Glazebrook J. (2001) Genes controlling expression of defense responses in Arabidopsis – 2001 status. Current Opinion in Plant Biology 4, 301308.
  • 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.
  • Guenther A.B., Zimmerman P.R., Harley P.C., Monson R.K. & Fall R. (1993) Isoprene and monoterpene emission rate variability: model evaluations and sensitivity analyses. Journal of Geophysical Research 98, 1260912617.
  • Hakola H., Rinne J. & Laurila T. (1998) The hydrocarbon emission rates of tea-leafed willow (Salix phylicifolia), silver birch (Betula pendula) and European Aspen (Populus tremula). Atmospheric Environment 32, 18251833.
  • Harley P.C., Monson R.K. & Lerdau M.T. (1999) Ecological and evolutionary aspects of isoprene emission from plants. Oecologia 118, 109123.
  • Holdren M.W., Westberg H.H. & Zimmerman P.R. (1979) Analysis of monoterpene hydrocarbons in rural atmospheres. Journal of Geophysical Research-Oceans and Atmospheres 84, 50835088.
  • Howard L.R., Talcott S.T., Brenes C.H. & Villalon B. (2000) Changes in phytochemical and antioxidant activity of selected pepper cultivars (Capsicum species) as influenced by maturity. Journal of Agricultural Food Chemistry 48, 17131720.
  • Jacob D.J. & Wofsy S.C. (1988) Photochemistry of biogenic emissions over Amazon forest. Journal of Geophysical Research 93, 14771486.
  • Karban R. & Baldwin I.T. (1997) Induced Responses to Herbivory. University of Chicago Press, Chicago, IL, USA.
  • Karl T., Fall R., Rosentiel T.N., Prazeller P., Larsen B., Seufert G. & Lindinger W. (2002) On-line analysis of the 13CO2 labeling of leaf isoprene suggests multiple subcellular origins of isoprene precursors. Planta 215, 894905.
  • Kesselmeier J. & Staudt M. (1999) Biogenic volatile organic compounds (VOC): an overview on emission, physiology and ecology. Journal of Atmospheric Chemistry 33, 2388.
  • Kreuzwieser J., Graus M., Wisthaler A., Hansel A., Rennenberg H. & Schnitzler J.-P. (2002) Xylem-transported glucose as an additional carbon source for leaf isoprene formation in Quercus robur. New Physiologist 156, 171178.
  • Kreuzwieser J., Schnitzler J.P. & Steinbrecher R. (1999) Biosynthesis of organic compounds emitted by plants. Plant Biology 1, 149159.
  • Lamb B., Gay D. & Westberg H. (1993) A biogenic hydrocarbon emission inventory for the USA using a simple forest canopy model. Atmospheric Environment 27, 16731690.
  • Lerdau M.T., Guenther A. & Monson R. (1997) Plant production and emission of volatile organic compounds. Bioscience 47, 373383.
  • Lerdau M. & Keller M. (1997) Controls over isoprene emission from trees in a sub- tropical dry forest. Plant, Cell, and Environment 20, 569578.
  • Lichtenthaler H.K. (1999) The-1-deoxy- d-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annual Review of Plant Physiology and Plant Molecular Biology 50, 4765.
  • Loreto F., Mannozzi M., Maris C., Nasecetti P., Ferranti F. & Pasqualini S. (2001) Ozone quenching properties of isoprene and its antioxidant role in leaves. Plant Physiology 126, 9931000.
  • Loreto F., Pinelli P., Manes F. & Kollist H. (2004) Impact of ozone on monoterpene emissions and evidence for an isoprene-like antioxidant action of monoterpenes emitted by Quercus ilex leaves. Tree Physiology 24, 361367.
  • Loreto F. & Sharkey T.D. (1993) Isoprene emission by plants is affected by transmissible wound signals. Plant Cell and 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.
  • Martin D.M., Gershenzon J. & Bohlmann J. (2003) Induction of volatile terpene biosynthesis and diurnal emission by methyl jasmonate in foliage of Norway spruce. Plant Physiology 132, 15861599.
  • Minchin P.E.H. & Thorpe M.R. (2003) Using the short-lived isotope 11C in mechanistic studies of photosynthate transport. Functional Plant Biology 30, 831841.
  • Monson R.K., Lerdau M.T., Sharkey T.D., Schimel D.S. & Fall R. (1995) Biological aspects of constructing volatile organic-compound emission inventories. Atmospheric Environment 29, 29893002.
  • Niinemets U., Tenhunen J.D., Harley P.C. & Steinbrecher R. (1999) A model of isoprene emission based on energetic requirements for isoprene synthesis and leaf photosynthetic properties for Liquidambar and Quercus. Plant, Cell and Environment 22, 13191335.
  • Örvar B.L., Mcpherson J. & Ellis B.E. (1997) Pre-activating wounding response in tobacco prior to high-level ozone exposure prevents necrotic injury. Plant Journal 11, 203212.
  • Pare P.W. & Tumlinson J.H. (1999) Plant volatiles as a defense against insect herbivores. Plant Physiology 121, 325331.
  • Parthier B. (1990) Jasmonates – hormonal regulators or stress factors in leaf senescence. Journal of Plant Growth Regulation 9, 5763.
  • Ping L.Y., Shen Y.B., Jin Y.J. & Hao J.H. (2001) Leaf volatiles induced by mechanical damage from diverse taxonomic tree species. Acta Botanica Sinica 43, 261266.
  • Rohdich F., Wungsintaweekul J., Luttgen H., Fisher M., Eisenreich W., Schuhr C.A., Fellermeir M., Schramek N., Zenk M.H. & Bacher A. (2000) Biosynthesis of terpenoids: 4-diphosphocytidyl-2C-methyl- d-erythritolsynthase of Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the USA 97, 64516456.
  • Rohmer M., Knani M., Simonin P., Sutter B. & Sahm H. (1993) Isoprenoid biosynthesis in bacteria-a novel pathway for the early steps leading to isopentyl diphosphate. Biochemical Journal 295, 517524.
  • Rose U.S.R. & Tumlinson J.H. (2004) Volatiles released from cotton plants in response to Helicoverpa zea feeding damage on cotton flower buds. Planta 218, 824832.
  • Rosenstiel T.N., Ebbets A.L., Khatri W.C., Fall R. & Monson R.K. (2004) Induction of poplar leaf nitrate reductase: a test of extrachloroplastic control of isoprene emission rate. Plant Biology 6, 1221.
  • Rosenstiel T.N., Potosnak M.J., Griffin K.L., Fall R. & Monson R.K. (2003) Increased CO2 uncouples growth from isoprene emission in an agriforest ecosystem. Nature 421, 256259.
  • Schwender J., Seemann M., Lichtenthaler H.K. & Rohmer M. (1996) Biosynthesis of isoprenoids (carotenoids, sterols, prenyl side-chains of chorophylls and plastoquinone) via a novel pyruvate/glyceraldehydes 3-phosphate non-mevalonate pathway in the green algae Scenedesmus obliquus. Journal of Biochemistry 316, 7380.
  • Schwender J., Zeidler J., Groner R., Muller C., Focke M., Braun S., Lichtenthaler F.W. & Lichtenthaler H.K. (1997) Incorporation of 1-deoxy- d-xylulose into isoprene and phytol by higher plants and algae. FEBS Letters 414, 129134.
  • Sharkey T.D., Chen X. & Yeh S.S. (2001) Isoprene increases thermotolerance of fosmidomycin-fed leaves. Plant Physiology 125, 20012006.
  • Sharkey T.D. & Singaas E.L. (1995) Why plants emit isoprene. Nature 374, 769.
  • Silver G.M. & Fall R. (1995) Characterization of aspen isoprene synthase, an enzyme responsible for leaf isoprene emission to the atmosphere. Journal of Biology Chemistry 270, 1301013016.
  • Simpson D., Guenther A., Hewitt C.N. & Steinbrecher R. (1995) Biogenic emissions in Europe. 1. Estimates and uncertainties. Journal of Geophysical Research-Atmospheres 100, 2287522890.
  • Singaas E.L., Laporte M.M., Shi J.-Z., Monson R.K., Bowling D.R., Johnson K., Lerdau M., Jasentuliytana A. & Sharkey T.D. (1999) Kinetics of leaf temperature fluctuation affect isoprene emission from red oak (Quercus rubra) leaves. Tree Physiology 19, 917924.
  • Singaas E.L., Lerdau M.T., Winter K. & Sharkey T.D. (1997) Isoprene increases thermotolerance of isoprene-emitting species. Plant Physiology 115, 14131420.
  • Stowe K.A. (1998) Experimental evolution of resistance in Braassica rapa: correlated response of tolerance in lines selected for glucosinolate content. Evolution 52, 703712.
  • Strauss S.Y. & Agrawal A.A. (1999) The ecology and evolution of plant tolerance to herbivory. Trends in Ecology and Evolution 14, 179185.
  • Trainer M., Williams E.J., Parish D.D., Buhr M.P., Allwine E.J., Westberg H.H., Fehsenfeld F.C. & Liu S.C. (1987) Models and observations of the impacts of natural hydrocarbons on rural zone. Nature 329, 705707.
  • Wang D. & Fuentes J.D. (2003) In situ isoprene measurements from foliage using a fast-response hydrocarbon instrument. Agricultural and Forest Meteorology 116, 3748.
  • Wasternack C. & Parthier B. (1997) Jasmonate-signalled plant gene expression. Trends in Plant Science 2, 302307.
  • Wolfertz M., Sharkey T.D., Boland W. & Kühnemann F. (2004) Rapid regulation of the methylerythritol 4-phosphate pathway during isoprene synthesis. Plant Physiology 135, 19391945.
  • Wuebbles D., Grant K., Connell P. & Penner J. (1989) The role of atmospheric chemistry in climate change. Journal of the Air Pollution Control Association 39, 2228.
  • Zeidler J.G., Lichtenthaler H.K., May H.U. & Lichtenthaler F.W. (1997) Is isoprene emitted by plants synthesized via the novel isopentyl pyrophosphate pathway? Zeitschrift für Naturforschung – Journal of Biosciences 52c, 1523.
  • Zeidler J., Schwender J., Muller C., Wiesner J., Weidemeyer C., Beck E., Jomaa H. & Lichtenthaler H.K. (1998) Inhibition of the non-mevalonate 1-deoxy- d-xylulose-5- phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Zeitschrift für Naturforschung – Journal of Biosciences 53c, 980986.
  • Zhang Z.-P. & Baldwin I.T. (1997) Transport of [2–14C]jasmonic acid from leaves to roots mimics wound-induced changes in endogenous jasmonic acid pools in Nicotiana sylvestris. Planta 203, 436441.
  • Zimmer W., Bruggemann N., Emeis S., Giersch C., Lehning A., Steinbrecher R. & Schnitzler J.P. (2000) Process-based modelling of isoprene emission by oak leaves. Plant, Cell and Environment 23, 585559.

Received 22 June 2004; received in revised form 17 September 2004; accepted for publication 3 November 2004