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Keywords:

  • biomass production;
  • modelling;
  • non-isoprene emitting;
  • outdoor conditions;
  • Populus × canescens

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Depending on the atmospheric composition, isoprene emissions from plants can have a severe impact on air quality and regional climate. For the plant itself, isoprene can enhance stress tolerance and also interfere with the attraction of herbivores and parasitoids.
  • Here, we tested the growth performance and fitness of Populus × canescens in which isoprene emission had been knocked down by RNA interference technology (PcISPS-RNAi plants) for two growing seasons under outdoor conditions.
  • Neither the growth nor biomass yield of the PcISPS-RNAi poplars was impaired, and they were even temporarily enhanced compared with control poplars. Modelling of the annual carbon balances revealed a reduced carbon loss of 2.2% of the total gross primary production by the absence of isoprene emission, and a 6.9% enhanced net growth of PcISPS-RNAi poplars. However, the knock down in isoprene emission resulted in reduced susceptibility to fungal infection, whereas the attractiveness for herbivores was enhanced.
  • The present study promises potential for the use of non- or low-isoprene-emitting poplars for more sustainable and environmentally friendly biomass production, as reducing isoprene emission will presumably have positive effects on regional climate and air quality.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Currently, poplar species are receiving enormous attention because of the increasing demand for renewable bioenergy. As a fast-growing pioneer tree with an easy generation of new hybrids and good regeneration from rootstocks, poplar allows for highly productive short-rotation coppice plantations (Laureysens et al., 2005; Aylott et al., 2008). Biomass from poplar is suitable for heat and power production, and is also a viable substitute for fossil fuels (Vande Walle et al., 2007; Aylott et al., 2008). In addition to the economic interest in bioenergy from biomass, the potential to reduce greenhouse gas (GHG) concentrations and to mitigate climate change is an additional incentive for bioenergy crop cultivation (Liberloo et al., 2010). Based on their economic and ecological benefits, a worldwide increase in large-scale tree plantations, accompanied by land use changes, is expected, mainly from the afforestation of marginal and apportioned agricultural lands (Beringer et al., 2011). Most of the species used for tree plantations across the globe emit volatile organic compounds (VOCs) in large quantities; in particular, the common bioenergy trees (poplar, willow, eucalypt and oil palm) and perennials (giant and common reed) are strong isoprene emitters (Kesselmeier & Staudt, 1999).

As a result of its high efflux from vegetation and its high reactivity with OH radicals, isoprene has a significant influence on photo-oxidative mechanisms in the atmosphere (for an overview, see Fuentes et al., 2000). Depending on the NOx concentration in the troposphere – high or low – isoprene causes either ozone formation or degradation, respectively. Its reaction with OH radicals also modulates the oxidation capacity of the atmosphere, and thus the lifetime of tropospheric methane, and it can contribute to secondary organic aerosol formation (summarized in Monks et al., 2009). However, with regard to the latter, recent investigations have indicated suppression of new particle formation by isoprene under specific conditions (Kiendler-Scharr et al., 2009; Kanawade et al., 2011). Overall, isoprene affects air quality at multiple scales with consequences on climate, ecosystems and even human health.

With a growing demand for bioenergy from tree plantations, these effects have become increasingly important. Wiedinmyer et al. (2006) developed expected land use changes in model-based estimates of future variations in global isoprene emissions. Their simulations revealed that the conversion of natural vegetation to plantations could substantially increase global isoprene flux by up to 37% compared with the current situation, which subsequently could cause O3 to increase regionally to potentially unhealthy concentrations. Hewitt et al. (2009) used measurements and models to evaluate, more specifically, the impact of tropical bioenergy oil palm plantations on O3 formation potential and local air quality in Borneo. They showed that this form of land use change would result in much greater emissions of isoprene, leading to severe ground-level O3 pollution depending on how human activities (industrialization and traffic) develop. However, the modelling of future land use changes and isoprene emissions is full of uncertainties, and great effort is needed by the scientific community to realistically assess the ‘environmental friendliness’ of growing bioenergy trees/crops (Beringer et al., 2011).

From an atmospheric perspective, low- or non-isoprene-emitting trees would avoid the above-discussed negative consequences of isoprene flux from plantations, and would therefore be highly desirable. Thus far, only transgenic Grey poplars (Populus × canescens) with extinguished isoprene emissions exist (Behnke et al., 2007). In these plants, isoprene synthase (ISPS) activity is effectively suppressed by RNA interference (RNAi) targeting ISPS gene expression (Behnke et al., 2007, 2009). Because isoprene emission is costly in terms of energy and carbon (Sharkey & Yeh, 2001), it can be assumed that isoprene-emitting species are most likely to gain some benefit from this emission. In most studies, isoprene is addressed as a thermoprotective molecule that stabilizes chloroplast membranes during short, high-temperature events caused by sunflecks or, more generally, isoprene is ascribed antioxidant properties (for an overview, see Sharkey et al., 2008; Loreto & Schnitzler, 2010). Laboratory studies using non-isoprene-emitting poplars have demonstrated the importance of isoprene for the protection of net CO2 assimilation and photosynthetic electron transport against heat stress (Behnke et al., 2007, 2010a; Way et al., 2011). From these results, however, it is not yet apparent whether isoprene plays a role under natural conditions. We therefore conducted a study under outdoor conditions over two growing seasons in which fitness, biomass growth and wood quality were analysed to assess whether a non-isoprene-emitting phenotype would be a potential benefit for biomass production in the field. For more comprehensive and conclusive estimates of the growth performance and biomass production of the non-isoprene-emitting poplars, we supplemented the physiological studies with the modelling of the annual carbon balances.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Cultivation of transgenic poplars

Transgenic Grey poplars (Populus × canescens (Aiton) Sm.) that had been knocked down with regard to isoprene emission were developed as described in Behnke et al. (2007). For the present study, two of these PcISPS-RNAi lines (RA1 and RA2) and a vector control line (C14) were selected and amplified by micropropagation (Loivamäki et al., 2007). The plants were acclimated to non-sterile conditions similarly to Behnke et al. (2007). After acclimation, the plants were potted (2.2-l pots) and further cultivated under glasshouse conditions for 2 months before being planted outdoors into replicated soil beds (box dimensions: length × width × height, 3050 mm × 3000 mm × 700 mm; macro- and micronutrient composition of the soil is summarized in Supporting Information Table S2), which had been installed between two glasshouses at the University of Göttingen (Germany). The experimental poplars in each soil bed were randomized and surrounded by a row of border trees, which were not used for analyses. For reasons of biological security, the area was fenced with coarse wire mesh (5 cm × 5 cm) at a height of c. 4 m. The trees were grown in the soil beds for two growing seasons (May–October 2007 and 2008). The plants were watered regularly. Weather conditions (photosynthetically active radiation (PAR), air temperature, air humidity) were recorded with a standard meteorological station (Hygro-thermo transmitter compact and sensor PAR 5.3; Thies Clima, Göttingen, Germany) as 30-min means throughout the experimental period (MeteoLOG TDL 14; Thies Clima). Recorded weather conditions for the two growing seasons are summarized in Fig. S1(a) and Table S1. In addition, Fig. S1(b) displays air quality parameters (ozone, nitric oxide and nitrogen dioxide concentrations) recorded by the Luftüberwachung Niedersachsen (http://www.umwelt.niedersachsen.de) close to the experimental site.

Harvesting and sampling

During the growing seasons, growth parameters (collar diameter, plant height and leaf formation as numbers of leaves per day) were monitored weekly. Gas exchange and isoprene emission data were recorded within 1-week-long measurement campaigns at four time points (September 2007, May 2008, July 2008 and September 2008; for details see next section). At the end of each measurement campaign, five trees of each line were harvested. The harvested trees were selected carefully to avoid deviations from the mean biometric data of each line. The harvested trees were separated into leaf, root and stem sections. Leaves selected for biochemical analyses were shock-frozen in liquid N2, and the remainder of the plant was oven-dried (60°C). As a result of the destructive harvesting of complete trees, the number of replicates for growth parameters varied: up to September 2007, nSept07 = 20; up to May 2008, nMay08 = 15; up to July 2008, nJuly08 = 10; and up to September 2008, nSept08 = 5.

Analysis of photosynthetic gas exchange and VOC emission

Photosynthetic gas exchange and online analysis of isoprene emission by proton transfer reaction mass spectroscopy (PTR-MS; for details see Tholl et al., 2006) were performed as described by Behnke et al. (2007). Before each leaf analysis, the cuvette was run empty for 20 min, during which background levels of VOCs were monitored and zero readings were taken for the CO2 and H2O channels of the infrared gas analyser. After that period, a mature leaf (leaf 9 or 10 below the apex, except for May 2008 where only leaf 5 was available because of the early date in the growing season) was inserted into the cuvette and analysed for 30 min in darkness with a leaf temperature of 30°C, followed by a light phase held constant at PAR = 1000 μmol photons m−2 s−1. Under these conditions, photosynthetic gas exchange and VOC emission were allowed to stabilize for an additional 45 min. Protonated masses of VOCs were monitored at masses of m33 (methanol), m45 (acetaldehyde), m69 (isoprene) and m137 (monoterpenes). Calibration of the instrument was performed using a mixture of 11 VOCs (1 ppmv) in N2 (Apel-Riemer Environmental, Denver, CO, USA).

The standard emission factor was calculated as an average of the 15 min of recording. To avoid bias in the standard emission factor caused by the diurnal variation, the diurnal sampling time points of the lines were randomized. As a result of the destructive harvesting of complete trees and the realizable measurements, the number of replicates for gas exchange and isoprene emission measurements varied for each measurement campaign: nSept07 ≥ 12, nMay08 ≥ 9, nJuly08 ≥ 9 and nSept08 ≥ 4.

Sample preparation for stem wood analyses

Stem sections were stripped of bark and pith, and oven-dried (60°C) for 2 days. The wood material was cut into small pieces with secateurs and ground to a fine powder (particle size < 20 μm) in a ball mill (MM2000; Retsch, Haan, Germany) for c. 4 min in a liquid N2 environment to prevent heating and to accelerate the milling process. A fine powder with a particle size of < 20 μm was achieved. This wood powder was used for Fourier transform infrared (FTIR) spectroscopy analyses, the determination of energy content and stable isotope analyses (δ13C). For further analyses, the milled wood was extracted four times in acetone, as described previously (Zhou et al., 2011). The extract-free wood powder was used for the determination of cellulose, holocellulose and total lignin content.

FTIR analyses of stem wood  FTIR-attenuated total reflection (FTIR-ATR) spectra of wood were recorded with an FTIR spectrometer (Equinox 55; Bruker Optics, Ettlingen, Germany) with a deuterium triglycine sulfate detector and an attached ATR unit (DuraSamplIR; SensIR Europe, Warrington, UK) at a resolution of 4 cm−1 in the range from 600 to 4000 cm−1. The wood powder was pressed against the diamond crystal of the ATR device; uniform pressure application was ensured using a torque knob. Individual analyses consisted of 32 scans, which were averaged to give one spectrum. From each sample, five technical replicates were measured, and the five spectra were averaged again, resulting in one mean spectrum per sample. Background scanning and correction were carried out regularly after 10–15 min. Mean spectra for individual plants were used for cluster analysis in the range from 1750 to 1200 cm−1 after vector normalization and calculation of the first derivatives with nine smoothing points using the analytical software OPUS version 6.5 (Bruker, Ettlingen, Germany). The compilation of a dendrogram was performed by implementing Ward’s algorithm.

Determination of cellulose and holocellulose content  Holocellulose and α-cellulose were determined using a modified microanalytical method developed by Yokoyama et al. (2002). Wood powder (10 mg) was weighed into a 2-ml tube and placed in a 90°C heating block. The reaction was initiated by the addition of 0.2 ml of NaClO2 solution (20 mg 80% NaClO2, dissolved in 0.2 ml of distilled H2O and 20 μl of acetic acid). After 2 h, the solution was cooled in a water bath. To remove lignin degradation products, 1.6 ml of distilled H2O was added, the solution was mixed, centrifuged (3000 g for 2 min) and the supernatant was removed with a Pasteur pipette. These steps were repeated at least three times. The samples were then dried overnight, and the amount of holocellulose was determined gravimetrically.

In addition, 5 mg of the dry holocellulose sample was weighed into a 2-ml tube, 400 μl of 17.5% NaOH solution was added, mixed and incubated for 30 min at 40°C in a heating block. Subsequently, 400 μl of distilled H2O was added and the mixture was incubated at 40°C for a further 30 min. The mixture was centrifuged (3000 g, 5 min) and the pellet was washed three times with 1 ml of distilled H2O. The pellet was soaked for 5 min at room temperature in 1.6 ml of 1.0 M acetic acid, subsequently washed five times with 2 ml of distilled H2O and dried overnight. The α-cellulose content was determined gravimetrically.

Determination of total lignin content  The total lignin content was determined using a modified acetyl bromide protocol (Brinkmann et al., 2002). One millilitre of freshly prepared 25% (w/w) acetyl bromide/glacial acetic acid solution was added to 1 mg of dried wood powder. The tube (2 ml) was sealed and placed under intermittent mixing at 70°C for 30 min in a water bath. The digestion was stopped by cooling the tube in an ice bath. After mixing, 100 μl were transferred to a new tube containing 200 μl of 2.0 M NaOH and filled with 1.7 ml of glacial acetic acid to a final volume of 2 ml. The absorbance of the solution at 280 nm was determined against a blank solution that was run in conjunction with the sample. The gram extinction coefficient of lignin treated with acetyl bromide is 20.09 l g−1 cm−1. All measurements were conducted with three technical replicates.

Determination of energy content  The calorific value of the wood was analysed with a calorimeter (IKA-Kalorimetersystem C 7000; IKA-Werke GmbH & Co. KG, Staufen, Germany). Approximately 500 mg of wood powder was weighed and pressed into pellets using a presser attached to the calorimeter. The pellet was combusted with O2 (30 mbar) using bomb calorimetry. The calorific value was determined as the increase in temperature of water with a direct measurement of the internal energy of the burning reaction in the calorimetric bomb. Using benzoic acid (pellets; IKA-Werke GmbH & Co. KG) as a standard (calorific value, 26457 ± 20 kJ g−1), the calorific values of the samples were calculated.

Stable isotope analyses  For stable isotope analyses, the stem material from the harvest in September 2008 was separated by a chisel into two parts, namely the young wood (wood 2008) and the old wood (2007). δ13C was analysed for wood samples from September 2007 and for young wood samples from September 2008 with an elemental analyser/isotope ratio mass spectrometer (EA-IRMS) system. A total of 0.2 mg of fine wood powder was transferred into a tin capsule (IVA Analysentechnik, Meerbusch, Germany) and combusted in an elemental analyser (Flash EA 1112; Thermo Fisher Scientific, Milan, Italy) with a Porapack QS 50/80 mesh GC column (Waters, Milford, MA, USA) coupled to a continuous-flow isotope-ratio mass spectrometer (DeltaPlusXP; Thermo Fisher Scientific, Bremen, Germany). The δ13C values were expressed in delta notation with respect to Vienna Peedee Belemnite (VPDB). IAEA-CH-6 (sucrose with a δ13CVPDB value of − 10.449‰; International Atomic Energy Agency (IAEA), Vienna, Austria) was used as an internal standard for the analysis.

Analysis of proanthocyanidins (condensed tannins)

Condensed tannins (proanthocyanidins) present in crude leaf extracts were hydrolysed according to Porter et al. (1986). Fifty milligrams of leaf powder were extracted with 1 ml of 70% (v/v) acetone for 5 min at room temperature. After centrifugation (3 min, 22 000 g, 4°C), the supernatant was removed and the pellet was washed again with 70% acetone. For hydrolysis, 100 μl of the combined supernatants were mixed with 400 μl of 70% acetone, 3 ml of butanol-HCl (95:5) and 0.1 ml of ferric reagent (2% (w/v) NH4Fe(SO4)2·12H2O in 2 M HCl). Blank value of absorption at 550 nm was recorded before incubating the mix in a test glass covered with a glass marble at 96°C for 1 h. Hydrolysis was stopped by cooling in an ice bath. The absorption of extracts was recorded at 550 nm. Proanthocyanidin concentrations were calculated assuming an effective E1%, 1 cm, 550 nm of leucocyanidin of 460. All measurements were conducted with three technical replicates.

Description of models and simulations

We applied the physiologically based vegetation model Physiological Simulation Model (PSIM), together with the ECM canopy model (Grote, 2007; Holst et al., 2010), the BIM2 VOC-emission model (Grote et al., 2006) and a modified dimensional growth model (Bossel, 1996; Miehle et al., 2010; Grote et al., 2011), within the Modular Biosphere simuLation Environment (MoBiLE; see, for example, Grote et al., 2009a,b) modelling framework. The PSIM model calculates primary production (Farquhar et al., 1980), plant respiration (Thornley & Cannell, 2000), litter fall (Lehning et al., 2001) and allocation (Grote, 1998), including increases in woody biomass. All of these processes depend directly or indirectly on the microclimatic environmental conditions. The supplies of water and nitrogen are assumed to be not limiting, although the physiological uptake rate allows for variations in tissue nitrogen concentrations (affecting the photosynthetic capacity). The parameterization of the physiological model in the present work follows literature recommendations for morphology and phenology (Calfapietra et al., 2005; Ryu et al., 2008), photosynthetic kinetics and temperature dependences (Amichev et al., 2010; Zhu et al., 2010) and enzyme kinetics for isoprene emission (Tholl et al., 2001; Schnitzler et al., 2005). Allometric relations and parameters for seasonal enzyme dynamics were derived directly from actual measurements.

The increase in woody biomass, which was diminished by a fraction attributed to branches and coarse roots, was used to calculate changes in stem height and diameter assuming a column shape for trees smaller than 1.3 m, a combination of a column (below 1.3 m) and a cone for trees smaller than 2.6 m, and using stem-form functions from the literature thereafter (Honer, 1967).

Microclimatic conditions, together with the assimilated carbon that is supplied by PSIM, determine VOC emissions. The model was run in 10-min time steps that were calculated from daily average temperature and radiation sums for the years 2007 and 2008 by assuming sinusoidal distribution schemes for temperature (De Wit et al., 1978) and radiation (Berninger, 1994). For the simulation of VOC emissions with BIM2, these data were further linearly extrapolated into time steps of 7 s. Anthropogenic and disease-induced biomass decreases were prescribed for specific dates and were considered at the start of the day. Spatially, microclimate and gas exchange processes were calculated in vertical layers with daily updated foliage biomass and area values and assuming a fairly homogeneous distribution (Gielen et al., 2003), represented by a parameter-sparse distribution function (Grote, 2007). In parallel, the number of layers was also updated according to the increasing height of the plants, starting from 6 (height, 0.4 m) and ending with 10 (height, 3.4 m).

Biomass harvests, causing a decrease in biomass in all compartments as well as in tree numbers, were considered for the day at which they were executed, and all trees were assumed to be of equal size. In acknowledgement of a considerable, but not precisely defined, fraction of foliage biomass consumption by insects, we introduced a loss term of 0.25% of foliage biomass per day throughout the period between the second and third harvests (days 130–200 in 2008). This loss term results in a total biomass loss of c. 5%, an amount corroborated by measurements of leaf area losses after each harvest. We decided in favour of a fixed percentage instead of a fixed or prescribed amount because this reflects the response of parasites to the availability of the substrate. This model was run with and without the VOC emission model to determine not only the direct losses from isoprene emission, but also the integrated loss throughout the year, which might involve follow-up impacts caused by, for example, a smaller amount of assimilates available for the building of productive tissue.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Growth rates and biomass yield of two growing seasons

We followed the growth of poplar mutants continuously over two growing seasons, measuring growth parameters such as collar diameter, plant height and leaf formation. Overall, no growth rate differences were observed between isoprene-emitting and non-isoprene-emitting poplars with respect to any of the three parameters. We found that growth rates increased rapidly shortly after planting in mid-June 2007. Maximal growth rates of 2.0–2.4 mm per day in collar diameter, 2.5–2.7 cm per day in height and 0.8–0.9 leaves per day occurred at the middle/end of July. Growth started to decrease at the seasonal break at the beginning of September (Fig. 1a–c). In 2008, leaf formation increased with the start of the growing season and dramatically peaked for 2–3 weeks just before the end of the growing season. Plant height and collar diameter increased moderately compared with the previous season, with maximum rates (≈2.5 cm per day in height, 0.15 mm per day in collar diameter) occurring in July 2008 and decreasing in growth thereafter. Compared with 2007, the growing season ended slightly earlier in August 2008.

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Figure 1. Relative growth rates of transgenic non-isoprene-emitting lines RA1 (light grey inverted triangle) and RA2 (dark grey square) and the isoprene-emitting vector control line C14 (black circle) of Grey poplar (Populus × canescens) grown for two seasons outdoors. Growth was continuously monitored by collar diameter (a), plant height (b) and leaf formation (number of leaves per day) (c) measurements.

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Collar diameter was chosen as a parameter most representative of absolute growth. After two growing seasons, plants reached maximum collar diameters of 23 mm (C14), 27 mm (RA1) and 29 mm (RA2) (Fig. 2a). The PcISPS-RNAi line RA2 developed larger collar diameters than the vector control (C14). The differences became significant shortly after planting at the end of June 2007 and remained so up to August 2008. Moreover, line RA1 showed larger collar diameters than the vector control line C14 at several time points at the end of the 2007 season and at the beginning of the 2008 season.

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Figure 2. Absolute growth and biomass yield of transgenic non-isoprene-emitting lines RA1 (light grey inverted triangle) and RA2 (dark grey square) and the isoprene-emitting vector control line C14 (black circle) of Grey poplar (Populus × canescens) grown for two seasons outdoors. (a) Growth was continuously monitored by collar diameter measurements. (b) Biomass yield was investigated four times within the experimental period (September 2007, May 2008, July 2008 and September 2008) by stem wood dry weight. For collar diameter, ANOVA (Holm–Sidak, > 0.05) was performed for each measurement point separately, and significant differences between the isoprene-emitting control line and either one or both non-isoprene-emitting lines are marked with one or two asterisks [*,**], respectively. Error bars represent the standard errors of the means. The collar diameter replicates decreased from = 20 > = 15 > = 10 > = 5 with harvests. In addition to ANOVA, repeated measures analysis was performed (http://www.ats.ucla.edu/stat/r/seminars/repeated_measures/repeated_measures.htm), validating highly significant differences between the three genotypes: < 2e−16. For stem wood dry weight, ANOVA (Holm–Sidak, > 0.05, = 5) was performed and significant differences between the isoprene-emitting control line and either one or both non-isoprene-emitting lines are marked with one or two asterisks [*,**], respectively. In addition to field site measurements, growth and biomass yield were simulated by applying the modelling framework MoBiLE (red line). Simulations were triggered with measured daily weather data and were driven with a 0.25% daily leaf decrease between day 130 and 200 in the second year without any emission.

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Biomass yield was determined as stem wood dry weight at four time points within the two growing seasons. After two growing seasons, the biomass yield ranged from 230 g (C14) to 280 g and 320 g for RA1 and RA2, respectively. The plants of line RA2 clearly yielded more overall biomass within the first growing season (Fig. 2b). At the next sampling time point in May 2008, both transgenic non-isoprene-emitting lines (RA1 and RA2) provided significantly higher biomass yields than the C14 plants. However, this difference in plant growth disappeared at the later samplings in July and September 2008.

Photosynthesis and VOC emission rates

Net CO2 assimilation, transpiration and isoprene emission rates were investigated under standard conditions (30°C leaf temperature and 1000 μmol photons m−2 s−1) four times within the two growing seasons. Both net CO2 assimilation and transpiration rates were higher in September 2007 than in the following year (Fig. 3a,b). We observed significant differences between genotypes in September 2007 for net CO2 assimilation, and in July 2008 for the transpiration rate. In both cases, the vector control line C14 showed higher gas exchange rates than the RA2 line.

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Figure 3. Net CO2 assimilation (a), transpiration (b) and isoprene emission (c) rates of transgenic non-isoprene-emitting lines RA1 (light grey columns) and RA2 (dark grey columns) and the isoprene-emitting vector control line C14 (black columns) of Grey poplar (Populus × canescens) grown for two seasons outdoors in a cage glasshouse. All parameters were investigated four times within the experimental period (September 2007, May 2008, July 2008 and September 2008). Significant differences between lines are marked with lower case letters; n.s., not significant. ANOVA (Holm–Sidak, > 0.05) was performed for each measurement point separately. Error bars represent the standards errors of the means: nSept07 ≥ 12, nMay08 ≥ 9, nJuly08 ≥ 9, nSept08 ≥ 4. In addition to field site measurements, these physiological parameters were simulated by applying the modelling framework MoBiLE (red circles).

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Isoprene emission by the C14 plants varied between 16 nmol m−2 s−1 in September 2007 and 71 nmol m−2 s−1 in July 2008 (Fig. 3c). Isoprene emission by both PcISPS-RNAi lines was constantly and stably repressed during the two growing seasons (Fig. 3c). RA1 and RA2 plants emitted negligible amounts of isoprene, ranging from 1% to 7% of the emission rates of C14 plants.

Within the measuring campaigns September 2007, May 2008 and July 2008, we also analysed the emission of methanol, acetaldehyde and monoterpenes (Fig. S3a–c). The emissions of these three VOCs were generally highly variable and showed no differences between vector control plants and the two PcISPS-RNAi lines. In May 2008 (Fig. S3b), methanol (80–100 nmol m−2 s−1) and monoterpene (0.6–1.7 nmol m−2 s1) emissions were highest, whereas the emission of acetaldehyde (27–47 nmol m−2 s−1) was at a maximum in July 2008 (Fig. S3c), parallel to the maximum of isoprene emission. Monoterpene emissions are part of the plant’s defence against herbivores and fungi (Keeling & Bohlmann, 2006; Eckhardt et al., 2009). Therefore, we compared the monoterpene emission of fungus-infected and non-infected leaves in September 2007 (Fig. S3d) and of leaves with feeding traces and undamaged leaves in May 2008 (Fig. S3e). However, this analysis revealed no difference caused by fungal infection or herbivory.

Wood composition and quality (FTIR, composition, carbon isotope ratio)

Wet chemical analyses of α-cellulose, hemicelluloses, lignin and soluble extractives in the stem wood of the 2-year-old poplars did not reveal differences in basic wood composition between poplar lines (Table 1). The stem wood of the three genotypes was composed of 45.8 ± 0.9%α-cellulose, 26.8 ± 0.5% hemicelluloses and 25.6 ± 0.3% lignin. The mean heating value of dry wood was 17 974 ± 70 J g−1. The FTIR spectra of wood, which provide a chemical fingerprint of wood composition, also confirmed that major compositional changes with regard to the amount of lignin (peak 4) or hemicelluloses (peak 1) did not occur (Fig. 4a). However, the data point to a decrease in the concentration of syringyl lignin in the PcISPS-RNAi lines (peak 9) compared with the vector control. Although the analysis of individual wood compounds did not show significant differences, cluster analyses of the FTIR spectra revealed that the wood of RA2 was distinguishable from that of controls (C14), whereas the wood of RA1 was intermingled with C14 and RA2 (Fig. 4b).

Table 1.   The contents of α-cellulose, hemicelluloses, lignin and soluble extractives of stem wood of transgenic isoprene-emitting vector control line (C14) and non-emitting lines (RA1 and RA2) of Grey poplar (Populus × canescens)
 α-CelluloseHemicelluloseLigninSoluble extractivesCalorific value
% (SE)% (SE)% (SE)% (SE)J g−1 (SE)
  1. SE, standard error. Lower case letters indicate results of ANOVA (Holm-Sidak, P > 0.05).

  2. Plants were grown for two seasons outdoors in a cage glasshouse. Wood samples after two growing seasons were analysed.

C1444.700a (0.9)27.206a (0.8)25.840a (0.4)1.505a (0.1)18052.4a (55.1)
RA146.259a (0.7)26.238a (0.7)25.702a (0.5)1.566a (0.1)17954.3a (145.8)
RA246.509a (0.6)26.897a (0.4)25.327a (0.6)1.106a (0.1)17916.5a (101.4)
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Figure 4. Chemical fingerprints of wood from transgenic non-isoprene-emitting lines (RA1 and RA2) and the isoprene-emitting control line (C14) of Grey poplar (Populus × canescens) grown for two seasons outdoors. (a) Mean baseline-corrected Fourier transform infrared (FTIR) spectra of wood powder in the region 1200–1750 cm−1 of C14 (black), RA1 (light grey) and RA2 (dark grey). (b) Cluster analysis of the fingerprint regions of the FTIR spectra. Spectra are means of five biological replicates. First derivates of spectra after baseline correction were employed for cluster analysis using Ward’s algorithm and the correlation coefficient as distance metric. Peak numbers in (a) refer to the following molecular components: 1, 1738 cm−1– C=O stretch in unconjugated ketones, carbonyls and ester groups in xylans (hemicellulose); 2, 1650 cm−1– absorbed O–H and conjugated C=O of proteins; 3, 1596 cm−1– aromatic skeletal vibrations in lignin plus C=O stretch; 4, 1505 cm−1– aromatic skeletal vibrations in lignin plus C=O stretch; 5, 1462 cm−1– C–H deformation; asymmetric in –CH3 and –CH2–; lignin and carbohydrates; 6, 1425 cm−1– aromatic skeletal vibrations combined with C–H plane deformation; lignin and carbohydrates; 7, 1375 cm−1– C–H deformation in cellulose and hemicelluloses; 8, 1330 cm−1– syringyl ring plus guaiacyl ring condensed; 9, 1235 cm−1– syringyl nuclei deformation combined with deformation of cellulose. Wavenumber assignments were taken from Rana et al. (2008).

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Further carbon isotope discrimination (δ13C) analyses were performed separately for wood from samples collected in September 2007 and only young wood from samples collected in September 2008 (Fig. 5). In 2007, both RA lines discriminated 13C significantly less strongly than the vector control line C14, as indicated by δ13C values of − 29.2‰ for RA1 and RA2 and − 29.7‰ for C14. At the end of the second growing season in September 2008, no difference in 13C discrimination was detectable.

image

Figure 5. Carbon isotope discrimination (δ13C) of transgenic non-isoprene-emitting lines RA1 (light grey columns) and RA2 (dark grey columns) and the isoprene-emitting control line C14 (black columns) of Grey poplar (Populus × canescens) grown for two seasons outdoors. Wood samples after first (September 2007) and second (September 2008) growing season were analysed. The δ13C values were expressed in delta notation with respect to Vienna Peedee Belemnite (VPDB). Significant differences between lines are marked with lower case letters; n.s., not significant. ANOVA (Holm–Sidak, > 0.05) was performed for each measurement point separately. Error bars represent the standard errors of the means, = 5.

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Susceptibility to pests and herbivores

In contrast with glasshouse or laboratory conditions, plants in natural or outdoor conditions are challenged by many – sometimes unexpected – environmental influences that cannot be simulated under controlled conditions. The climatic conditions in summer 2007 favoured the development of the pathogenic fungus Pollaccia radiosa (Lib.) Bald. Et Cif. (teleomorph: Venturiatremulae Aderh.). The poplar plants developed severe shoot blight disease, although with varying susceptibility, within a very short time period in July 2007. The degree of leaf infection by Pollaccia was significantly higher in C14 plants (c. 35%) than in the non-isoprene-emitting plants from mid-July to mid-August 2007 (Fig. 6a). In 2008, the climate in July and August did not favour the development of Pollaccia, and therefore only c. 4% of the leaves, regardless of the line, developed symptoms (data not shown). However, the local climate favoured the appearance of the herbivorous willow leaf beetle Phratora vitellinae (L.), which substantially attacked the plants. Host plant selection preferences for the different genotypes were monitored via the number of beetles per tree at three times (May 2008, July 2008 and September 2008; Fig. 6b). The beetles were generally found at the top of the branches, preferring the younger, newly unfolded leaves near the shoot top, as described in Urban (2006). The willow leaf beetles clearly selected the PcISPS-RNAi lines, most obviously in July when the overall amount of beetles was highest. As phenolic compounds are an important part of the plant’s direct defence against pests and pathogens (Keeling & Bohlmann, 2006; Eyles et al., 2009; Boeckler et al., 2011), we analysed the total proanthocyanidin concentration in this study, but no differences between genotypes were observed (Fig. S2). In addition, analyses of monoterpene emission in May 2008 showed no differences between genotypes (Fig. S3b).

image

Figure 6. Ecological studies of transgenic non-isoprene-emitting lines (RA1, RA2) and the isoprene-emitting vector control line (C14) of Grey poplar (Populus × canescens) grown for two seasons outdoors in a cage glasshouse. (a) Within the growing season 2007, poplars were strongly infected by the fungus Pollacia radiosa (teleomorph: Venturia macularis) causing shoot blight disease. Susceptibility to the fungus was estimated by determining the percentage of infected leaves in RA1 (light grey inverted triangles), RA2 (dark grey squares) and C14 (black circles). Significant differences between lines are marked with lower case letters; n.s., not significant. ANOVA (Holm–Sidak, > 0.05) was performed for each measurement point separately. Error bars represent the standard error of the mean, = 37. (b) Within the growing season 2008, poplars were strongly infested by the leaf beetle Phratora vitellinea. Host plant selection preferences for either isoprene-emitting control lines (C14, black columns) or non-isoprene-emitting lines (RA1, light grey columns; RA2, dark grey columns) were monitored by counting the number of beetles per tree at three times (May 2008, July 2008 and September 2008). Significant differences between lines are marked with lower case letters; n.s., not significant. ANOVA (Holm–Sidak, > 0.05) was performed for each measurement point separately. Error bars represent the standard errors of the means, n ≥ 20.

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Annual carbon balance

Because destructive harvesting and the analyses of photosynthetic gas exchange and isoprene emission rates were conducted at only four distinct time points in 2007 and 2008, we calculated the annual rates of gross primary production (GPP, estimated CO2 uptake) and net primary production (NPP, estimated net growth), together with carbon losses by respiration, litter fall, harvesting, insect damage and isoprene emission, using a mathematical approach, coupling together various models within the MoBiLE modelling framework.

As an example, with regard to the development of collar diameter and stem wood dry weight, the adapted model was well capable of calculating plant growth and biomass yield (Fig. 2a,b, red lines). The simulations conformed to the measured biometric data and were well within the range of the experimental uncertainties. This applied equally to the comparison of calculated and modelled net CO2 assimilation and isoprene emission rates (Fig. 3a,c, red dots). For transpiration rates, the model results for 2 months – based on average parameters – were lower than the measurements (Fig. 3b).

The detailed modelled annual carbon balances for all lines in 2008 are given in Table 2. Overall, the simulations revealed that isoprene emission used 2.2% of total GPP, whereas the overall effect of carbon loss by isoprene corresponded to a reduction in NPP of 6.9%. Compared with other losses, carbon loss via isoprene emission was of the same magnitude as insect-related carbon loss, but considerably less than carbon removal by harvest.

Table 2.   Simulated gross primary production (GPP, estimated CO2 uptake) and net primary production (NPP, estimated net growth) of the investigated Grey poplar (Populus × canescens) during the year 2008
 CO2 uptakeNet growth (%)Respiration (%)Litter loss (%)Harvest loss (%)Insect loss (%)VOC loss (%)
  1. VOC, volatile organic compound.

  2. Data shown take into account the carbon losses by respiration, litter fall, harvest, insect damage and sum of isoprene emission, considering isoprene emission (ISO+) or without isoprene emission (ISO−). Values in kg C m−2 yr−1; carbon proportion is in parentheses.

ISO−0.9020.117 (13.0)0.306 (33.9)0.257 (28.5)0.208 (23.1)0.013 (1.5)0.000 (0.0)
ISO+0.8790.109 (12.4)0.299 (34.0)0.253 (28.8)0.185 (21.0)0.013 (1.5)0.020 (2.2)
Isoprene effect (%)− 2.5− 6.9− 2.2− 1.6− 11.2− 0.6

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Isoprene is not essential for poplar under outdoor conditions in a humid, temperate climate

The functional loss of isoprene emission capacity in Grey poplar entailed no substantial growth impairment under outdoor conditions. The growth of PcISPS-RNAi poplars was even enhanced through a certain time period. This raises the question of whether poplars benefit from isoprene emission under realistic field conditions. Our previous laboratory studies with PcISPS-RNAi poplars confirmed the hypothesized roles of isoprene. We clearly showed earlier that isoprene protects photosynthesis during transient heat flecks (Behnke et al., 2007, 2010a; Way et al., 2011). However, this protective function was not apparent under the present conditions when growth performance was used as an integrative stress parameter, because both emitter types grew similarly. All laboratory studies on isoprene’s thermoprotective function have in common that the mechanism is effective at leaf temperatures above 35°C, and it specifically protects during heat flecks rather than during constant heat periods (Sharkey & Singsaas, 1995; Velikova & Loreto, 2005; Behnke et al., 2007). In the present work, we did not record leaf temperatures, but the climatic data showed no days with an air temperature above 35°C, and only 5 and 12 days with temperatures exceeding 30°C in 2007 and 2008, respectively (Table S1). Therefore, conditions favourable for the observation of a protective isoprene effect might have been rare, and the repression of isoprene emission was not relevant with regard to thermoprotection. In addition to specifically protecting against heat flecks, isoprene can also reduce oxidative stress caused by several conditions by acting as an antioxidant (summarized in Vickers et al., 2009a; Loreto & Schnitzler, 2010). In this more general mode of action, isoprene can contribute to the quenching of reactive oxygen species. However, isoprene’s efficiency might depend on the cause, degree and spatial localization of the oxidative stress. Furthermore, more specific antioxidants may have been produced under the investigated circumstances. The knock-down of isoprene emission results in the constitutive upregulation of ascorbate in poplar (Behnke et al., 2009), whereas its introduction into tobacco (Vickers et al., 2009b) downregulates ascorbate. The antioxidative systems of plants are known to be complex and overlapping (Noctor & Foyer, 1998; Foyer & Noctor, 2005); therefore, it is possible that certain other components might have substituted for isoprene as an antioxidant. Thus, isoprene is not indispensable for poplar viability, and its absence did not generally impair growth performance under the environmental conditions of our study.

Isoprene carbon is not reinvested in biomass production

Isoprene emission is costly in terms of energy and, with 1–10% of recently assimilated carbon ending up in isoprene under non-stressed conditions, it represents a significant loss of carbon for isoprene-emitting plants (Sharkey & Yeh, 2001). If isoprene is not needed because of the moderate climate and/or replacement by other less expensive antioxidants, the energy and carbon intended for isoprene production might be invested in better growth and a larger biomass. In particular, the higher growth and biomass production of PcISPS-RNAi poplars, observed during the first growing season (2007), support this hypothesis. However, these plants showed lower rates of net CO2 assimilation. A general reduction in photosynthesis is also supported by the reduced discrimination of 13C by the non-emitting lines in 2007, because 13C enrichment in leaf material can be mainly attributed to stomatal closure and higher uptake of the heavier 13C isotope during photosynthetic carbon acquisition (Brugnoli & Farquhar, 2000). It is therefore more likely that a negative feedback loop decreased photosynthesis rather than excess isoprene energy and carbon being redirected to growth and biomass production. Several studies have shown that the expression of photosynthesis-related genes (Pego et al., 2000) and photosynthetic activity (Goldschmidt & Huber, 1992; McCormick et al., 2008) negatively correlate with the concentration of carbohydrates. In PcISPS-RNAi poplars, the accumulation of the biosynthetic precursor dimethylallyl diphosphate (DMADP) is a definite consequence of repression of isoprene emission (Behnke et al., 2007, 2010b), and could possibly serve as a signal for the downregulation of photosynthesis and thus lower net CO2 assimilation rates. Transcriptomic and metabolomic analyses of PcISPS-RNAi poplars have demonstrated comprehensively altered carbohydrate metabolism because of the repression of isoprene emission (Behnke et al., 2010b). We do not yet understand the cross-links between the repression of isoprene emission, the accumulation of DMADP and the subsequent alterations of carbohydrate metabolism and photosynthesis. Further investigations are needed to determine whether the carbon and energy required to fuel isoprene production are balanced between carbon sinks and sources by carbohydrate metabolism and photosynthesis, or whether a portion of this carbon can be re-allocated to biomass.

The FTIR spectra-based analyses of stem wood of the 2-year-old poplars revealed certain differences between the three genotypes and, to some extent, clustering into groups. However, the wood constituents lignin, α-cellulose and hemicellulose and the energy content of the control and PcISPS-RNAi poplars were within the usual range for poplar (Lepléet al., 2007; Luo & Polle, 2009; Zhou et al., 2011), and no genotype effect was observed. Thus, we found no effects on basic wood composition or quality as a result of the repression of isoprene emission. The slight clustering observed with FTIR analyses could possibly be explained by the decrease in syringyl lignin in the PcISPS-RNAi lines.

The loss of isoprene emission alters the ecological performance of poplar

As a result of the complex interactions of abiotic and biotic stress factors under outdoor conditions, we cannot exclude factors other than a direct effect of isoprene loss on growth and fitness. As often observed in high-density poplar plantations, the plants were attacked by natural pests of poplar. Pollaccia radiosa is a common and destructive ascomycete causing so-called shoot blight disease (Dance, 1961; Newcombe, 1996; Kasanen et al., 2001). In 2007, spring in Göttingen was comparatively warm and wet (http://www.wetterstation-goettingen.de/klimadaten.htm) and thus favourable for Pollaccia infection (Newcombe, 1996). Pollaccia symptoms, such as brownish/black necrotic lesions, curling leaves and dead twisted young shoots (Newcombe, 1996), increased during the summer of 2007 in both control and PcISPS-RNAi poplars, but were more pronounced in vector control plants. In 2008, spring was comparatively warm but dry, and therefore favourable for the development of Phratora vitellinae (L.) (Urban, 2006). This willow leaf beetle clearly preferred PcISPS-RNAi poplars.

We can only speculate about the different susceptibilities to pathogens and herbivory of control and PcISPS-RNAi poplars. Generally, phenolic compounds are an important part of a plant’s direct defence against pests and pathogens (Keeling & Bohlmann, 2006; Eyles et al., 2009; Boeckler et al., 2011). Poplars defend themselves against herbivory and fungal infection with polyphenols (Gruppe et al., 1999; Urban, 2006; Miranda et al., 2007; Zhong et al., 2011). However, increased production of phenolic compounds and protection from herbivores did not result in a negative trade-off with biomass production (Kleemann et al., 2011). The comprehensive characterization of PcISPS-RNAi plants in Behnke et al. (2010b) revealed high-temperature-dependent transient alterations of phenolic biosynthesis, resulting in altered polyphenolic and proanthocyanidin concentrations in leaves. The analysis of total proanthocyanidins in this study showed no differences between genotypes, probably because the ambient air temperatures in the present study never reached very high values (Table S1). However, because susceptibility to herbivores, such as Coleoptera, can depend on a single compound (Urban, 2006), we cannot exclude compound-specific alterations in phenolic biosynthesis in the different lines as a cause for their divergent ecological behaviour.

In addition to the indirect pleiotropic effects of the repression of isoprene emission on secondary compound metabolism, recent investigations have demonstrated a direct role of isoprene in plant–insect interactions. Studies with transgenic isoprene-emitting tobacco (Laothawornkitkul et al., 2008) and Arabidopsis (Loivamäki et al., 2008) have demonstrated the ability of isoprene to repel both herbivores and parasitoids. A protective effect of isoprene against Phratora vitellinae could have led to their preference for PcISPS-RNAi poplars. Further ecological studies with PcISPS-RNAi poplars are essential to verify the role of isoprene as an orientation cue for insects. Monoterpenes are also important components of poplar–insect communication, which are constitutively emitted from young poplar leaves (Brilli et al., 2009; Ghirardo et al., 2011) or are part of the induced volatile blend (Brilli et al., 2009; Danner et al., 2011). In May 2008, the relatively young leaves emitted comparably large amounts of monoterpenes, but no differences between genotypes were observed, and therefore no side-effect of the repression of isoprene biosynthesis on the emission of other terpenes. In addition, fungus infection and beetle feeding did not result in an increase in monoterpene emission in September 2007 and May 2008. However, VOC emissions were not monitored directly after beetle infestation or fungal infection. Therefore, the induction of monoterpene emission as a result of herbivory or fungal infection could have been missed in our study.

Both herbivory and fungal infections influence growth (Kosola et al., 2001). Therefore, the initial head start of the non-isoprene-emitting poplars might have been lost under the pressure of naturally occurring pests.

Stand-level considerations and future prospects

With the modelling approach, the annual/biannual dynamics of plant growth, biomass and physiological parameters, such as isoprene emission, can be simulated very reasonably. This enabled us to quantify the annual overall carbon loss of poplar as a result of isoprene emission. In the short term, this loss has been estimated to be < 1% (Tingey et al., 1980), increasing with temperature to 2% or higher under extreme conditions when photosynthesis is severely impaired (Sharkey & Yeh, 2001). The calculated annual carbon loss as isoprene of 2.2% relative to GPP is similar to these observations. Net CO2 assimilation rates were slightly lower in non-isoprene-emitting leaves than in isoprene emitters. However, the annual calculation revealed that the non-isoprene emitters had higher CO2 uptake at the stand level. The higher 6.9% growth rate of PcISPS-RNAi poplars results in increased NPP at the stand level, which offsets the observations at the leaf level. Nevertheless, considerable uncertainties remain. For example, carbon and nitrogen losses may have occurred by root exudation, a factor not considered in the present study. Furthermore, the dependence of plant maintenance respiration and fine root turnover on site conditions implies that the use of specific literature-derived parameters (Pregitzer & Friend, 1996; Thornley & Cannell, 2000) may be cumbersome.

As a result of the debate over renewable resources, poplars have become more and more important as bioenergy trees. World-wide poplar plantations represent 5.3 million hectares with an increasingly positive trend in many countries (International Poplar Commission, Synthesis of Country Progress Reports 2008). In addition to being a renewable substitute for fossil fuels, bioenergy from biomass is seen as a carbon-neutral energy with carbon sequestration potential, and therefore is considered to mitigate against the greenhouse effect and climate change. Nevertheless, care must be taken to fulfil these hopes. Depending on the type of land use change, crop or tree species used, management system and application, the overall GHG balance can be positive (Deckmyn et al., 2004; Hill et al., 2006; Aylott et al., 2008; Liberloo et al., 2010) or negative (Crutzen et al., 2007; Fargione et al., 2008; Searchinger et al., 2008; Hillier et al., 2009). However, one aspect of the environmental friendliness of bioenergy plantations is considered only rarely: all plants emit VOCs, particularly the selected ‘biomass’ trees. Most importantly, with respect to climate change, isoprene contributes to tropospheric ozone formation and prolongs the lifetime of tropospheric methane (summarized in Monks et al., 2009). Laboratory studies (Kiendler-Scharr et al., 2009) and field observations (Kanawade et al., 2011) with mixed forests have provided new evidence that isoprene suppresses new particle formation, thus damping the negative radiative forcing effect of aerosols. Many plantation tree species that are cultivated throughout the globe are strong isoprene emitters (Kesselmeier & Staudt, 1999). Consequently, the growth of isoprene emitters in large-scale plantations might affect local climate and air quality (Wiedinmyer et al., 2006; Hewitt et al., 2009). The need for steps to control isoprene flux is evident (Hewitt et al., 2009), but, to date, they have barely been taken.

In summary, the present long-term outdoor study with non-isoprene-emitting poplars in the moderate climate of Central Europe revealed no remarkable differences with respect to plant growth and wood quality. The differences in sensitivity of the non-isoprene-emitting poplars to fungal disease and herbivory, however, show that the stress responses of these plants are affected and, indeed, require further combined molecular and ecological investigations under controlled and field conditions. In particular, more real-field trials under strongly contrasting climatic and soil conditions are needed to clarify conclusively whether isoprene-free poplars are an option for the second generation of biomass plants, either generated by genetic manipulation or selected by plant phenotyping.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to S. Wolfarth (University of Göttingen) and C. Kettner (University of Göttingen) for excellent technical assistance. We would like to thank G. Bahnweg (BIOP, Helmholtz Centre Munich) and P. Faubert (EUS, Helmholtz Centre Munich) for critical comments on the manuscript. This study was financially supported by the German Science Foundation (DFG; Schnitzler SCHN653/4 and Polle PO362/13) within the German joint research group ‘Poplar—A model to address tree-specific questions’ (FOR496) and by the European Commission within the Seventh Framework Programme for Research, Project Energypoplar (FP7-211917). G.W.Z. thanks the DAAD–CSC (German Academic Exchange Service – China Scholarship Council) Joint PhD scholarship programme and M.E. the University of Khartoum for providing PhD scholarships.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Weather conditions and air quality parameters of growing seasons 2007 and 2008.

Fig. S2 Proanthocyanidin concentration in leaves.

Fig. S3 Volatile organic compound (VOC) emissions.

Table S1 Number of days within growing seasons of years 2007 and 2008 with temperatures

Table S2 Macro- and micronutrient composition of the soil used for poplar growth

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