Isoprene emission-free poplars – a chance to reduce the impactfrom poplar plantations on the atmosphere

Katja Behnke1, Rudiger Grote2, Nicolas Bruggemann3, Ina Zimmer1, Guanwu Zhou4, Mudawi Elobeid4,

Dennis Janz4, Andrea Polle4 and Jorg-Peter Schnitzler1

1Helmholtz Zentrum Munchen, Institute of Biochemical Plant Pathology, Research Unit Environmental Simulation (EUS), Ingolstadter Landstraße 1, D-85764 Neuherberg, Germany;

2Karlsruhe Institute of Technology, Institute of Meteorology and Climate Research (IMK-IFU), Kreuzeckbahnstraße 19, 82467 Garmisch-Partenkirchen, Germany; 3Forschungszentrum Julich,

Agrosphere (IBG-3), 52425 Julich, Germany; 4Forest Botany and Tree Physiology, Busgen-Institute, Georg-August-University Gottingen, Busgenweg 2, 37077 Gottingen, Germany

Author for correspondence:Jorg-Peter Schnitzler

Tel: +49 89 3187 2413E-mail: jp.schnitzler@helmholtz-

muenchen.de

Received: 25 August 2011Accepted: 14 October 2011

New Phytologist (2012) 194: 70–82doi: 10.1111/j.1469-8137.2011.03979.x

Key words: biomass production, modelling,non-isoprene emitting, outdoor conditions,Populus · canescens.

Summary

• 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 iso-

prene 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

Currently, poplar species are receiving enormous attentionbecause of the increasing demand for renewable bioenergy. As afast-growing pioneer tree with an easy generation of new hybridsand good regeneration from rootstocks, poplar allows for highlyproductive short-rotation coppice plantations (Laureysens et al.,2005; Aylott et al., 2008). Biomass from poplar is suitable forheat and power production, and is also a viable substitute forfossil fuels (Vande Walle et al., 2007; Aylott et al., 2008). Inaddition to the economic interest in bioenergy from biomass, thepotential to reduce greenhouse gas (GHG) concentrations and tomitigate climate change is an additional incentive for bioenergycrop cultivation (Liberloo et al., 2010). Based on their economicand ecological benefits, a worldwide increase in large-scale treeplantations, accompanied by land use changes, is expected,mainly from the afforestation of marginal and apportioned agri-cultural lands (Beringer et al., 2011). Most of the species used fortree plantations across the globe emit volatile organic compounds

(VOCs) in large quantities; in particular, the common bioenergytrees (poplar, willow, eucalypt and oil palm) and perennials (giantand common reed) are strong isoprene emitters (Kesselmeier &Staudt, 1999).

As a result of its high efflux from vegetation and its high reac-tivity with OH radicals, isoprene has a significant influence onphoto-oxidative mechanisms in the atmosphere (for an overview,see Fuentes et al., 2000). Depending on the NOx concentrationin the troposphere – high or low – isoprene causes either ozoneformation or degradation, respectively. Its reaction with OHradicals also modulates the oxidation capacity of the atmosphere,and thus the lifetime of tropospheric methane, and it can contrib-ute to secondary organic aerosol formation (summarized inMonks et al., 2009). However, with regard to the latter, recentinvestigations have indicated suppression of new particle forma-tion by isoprene under specific conditions (Kiendler-Scharr et al.,2009; Kanawade et al., 2011). Overall, isoprene affects air qualityat multiple scales with consequences on climate, ecosystems andeven human health.

With a growing demand for bioenergy from tree plantations,these effects have become increasingly important. Wiedinmyeret al. (2006) developed expected land use changes in model-based

This work is dedicated to the memory of Hanns Ulrich Seitz who died on 17August 2011.

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estimates of future variations in global isoprene emissions. Theirsimulations revealed that the conversion of natural vegetation toplantations could substantially increase global isoprene flux by upto 37% compared with the current situation, which subsequentlycould cause O3 to increase regionally to potentially unhealthyconcentrations. Hewitt et al. (2009) used measurements andmodels to evaluate, more specifically, the impact of tropical bio-energy oil palm plantations on O3 formation potential and localair quality in Borneo. They showed that this form of land usechange would result in much greater emissions of isoprene, lead-ing to severe ground-level O3 pollution depending on howhuman activities (industrialization and traffic) develop. However,the modelling of future land use changes and isoprene emissionsis full of uncertainties, and great effort is needed by the scientificcommunity to realistically assess the ‘environmental friendliness’of growing bioenergy trees ⁄ crops (Beringer et al., 2011).

From an atmospheric perspective, low- or non-isoprene-emittingtrees would avoid the above-discussed negative consequencesof isoprene flux from plantations, and would therefore be highlydesirable. Thus far, only transgenic Grey poplars (Populus ·canescens) with extinguished isoprene emissions exist (Behnkeet al., 2007). In these plants, isoprene synthase (ISPS) activity iseffectively suppressed by RNA interference (RNAi) targeting ISPSgene expression (Behnke et al., 2007, 2009). Because isopreneemission is costly in terms of energy and carbon (Sharkey & Yeh,2001), it can be assumed that isoprene-emitting species are mostlikely to gain some benefit from this emission. In most studies,isoprene is addressed as a thermoprotective molecule that stabi-lizes chloroplast membranes during short, high-temperatureevents caused by sunflecks or, more generally, isoprene is ascribedantioxidant properties (for an overview, see Sharkey et al., 2008;Loreto & Schnitzler, 2010). Laboratory studies using non-isoprene-emitting poplars have demonstrated the importance ofisoprene for the protection of net CO2 assimilation and photo-synthetic electron transport against heat stress (Behnke et al.,2007, 2010a; Way et al., 2011). From these results, however, it isnot yet apparent whether isoprene plays a role under naturalconditions. We therefore conducted a study under outdoor con-ditions over two growing seasons in which fitness, biomassgrowth and wood quality were analysed to assess whether a non-isoprene-emitting phenotype would be a potential benefit forbiomass production in the field. For more comprehensive andconclusive estimates of the growth performance and biomassproduction of the non-isoprene-emitting poplars, we supple-mented the physiological studies with the modelling of theannual carbon balances.

Materials and Methods

Cultivation of transgenic poplars

Transgenic Grey poplars (Populus · canescens (Aiton) Sm.) thathad been knocked down with regard to isoprene emission weredeveloped as described in Behnke et al. (2007). For the presentstudy, two of these PcISPS-RNAi lines (RA1 and RA2) and avector control line (C14) were selected and amplified by

micropropagation (Loivamaki et al., 2007). The plants wereacclimated 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 monthsbefore being planted outdoors into replicated soil beds (boxdimensions: length · width · height, 3050 mm · 3000 mm ·700 mm; macro- and micronutrient composition of the soil issummarized in Supporting Information Table S2), which hadbeen installed between two glasshouses at the University ofGottingen (Germany). The experimental poplars in each soilbed were randomized and surrounded by a row of border trees,which were not used for analyses. For reasons of biological secu-rity, 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 fortwo growing seasons (May–October 2007 and 2008). Theplants were watered regularly. Weather conditions (photosyn-thetically 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,Gottingen, Germany) as 30-min means throughout the experi-mental period (MeteoLOG TDL 14; Thies Clima). Recordedweather conditions for the two growing seasons are summarizedin Fig. S1(a) and Table S1. In addition, Fig. S1(b) displays airquality parameters (ozone, nitric oxide and nitrogen dioxideconcentrations) recorded by the Luftuberwachung 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 datawere recorded within 1-week-long measurement campaigns atfour time points (September 2007, May 2008, July 2008 andSeptember 2008; for details see next section). At the end of eachmeasurement campaign, five trees of each line were harvested.The harvested trees were selected carefully to avoid deviationsfrom the mean biometric data of each line. The harvested treeswere separated into leaf, root and stem sections. Leaves selectedfor biochemical analyses were shock-frozen in liquid N2, and theremainder of the plant was oven-dried (60�C). As a result of thedestructive harvesting of complete trees, the number of replicatesfor 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 VOCemission

Photosynthetic gas exchange and online analysis of isoprene emis-sion by proton transfer reaction mass spectroscopy (PTR-MS; fordetails see Tholl et al., 2006) were performed as described byBehnke et al. (2007). Before each leaf analysis, the cuvette wasrun empty for 20 min, during which background levels of VOCswere monitored and zero readings were taken for the CO2 andH2O channels of the infrared gas analyser. After that period, a

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mature leaf (leaf 9 or 10 below the apex, except for May 2008where only leaf 5 was available because of the early date in thegrowing season) was inserted into the cuvette and analysed for30 min in darkness with a leaf temperature of 30�C, followed bya light phase held constant at PAR = 1000 lmol photonsm)2 s)1. Under these conditions, photosynthetic gas exchangeand VOC emission were allowed to stabilize for an additional45 min. Protonated masses of VOCs were monitored at massesof m33 (methanol), m45 (acetaldehyde), m69 (isoprene) andm137 (monoterpenes). Calibration of the instrument wasperformed 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 ofthe 15 min of recording. To avoid bias in the standard emissionfactor caused by the diurnal variation, the diurnal sampling timepoints of the lines were randomized. As a result of the destructiveharvesting of complete trees and the realizable measurements, thenumber of replicates for gas exchange and isoprene emission mea-surements 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 pieceswith secateurs and ground to a fine powder (particle size < 20lm) in a ball mill (MM2000; Retsch, Haan, Germany) forc. 4 min in a liquid N2 environment to prevent heating and toaccelerate the milling process. A fine powder with a particle sizeof < 20 lm was achieved. This wood powder was used forFourier transform infrared (FTIR) spectroscopy analyses, thedetermination of energy content and stable isotope analyses(d13C). For further analyses, the milled wood was extracted fourtimes in acetone, as described previously (Zhou et al., 2011).The extract-free wood powder was used for the determination ofcellulose, holocellulose and total lignin content.

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

Determination of cellulose and holocellulose content Holo-cellulose and a-cellulose were determined using a modified micro-analytical method developed by Yokoyama et al. (2002). Woodpowder (10 mg) was weighed into a 2-ml tube and placed in a90�C heating block. The reaction was initiated by the addition of0.2 ml of NaClO2 solution (20 mg 80% NaClO2, dissolved in0.2 ml of distilled H2O and 20 ll of acetic acid). After 2 h, thesolution was cooled in a water bath. To remove lignin degradationproducts, 1.6 ml of distilled H2O was added, the solution wasmixed, centrifuged (3000 g for 2 min) and the supernatant wasremoved with a Pasteur pipette. These steps were repeated at leastthree times. The samples were then dried overnight, and theamount of holocellulose was determined gravimetrically.

In addition, 5 mg of the dry holocellulose sample was weighedinto a 2-ml tube, 400 ll of 17.5% NaOH solution was added,mixed and incubated for 30 min at 40�C in a heating block. Sub-sequently, 400 ll of distilled H2O was added and the mixturewas incubated at 40�C for a further 30 min. The mixture wascentrifuged (3000 g, 5 min) and the pellet was washed threetimes with 1 ml of distilled H2O. The pellet was soaked for5 min at room temperature in 1.6 ml of 1.0 M acetic acid, sub-sequently washed five times with 2 ml of distilled H2O and driedovernight. The a-cellulose content was determined gravimetrically.

Determination of total lignin content The total lignin contentwas determined using a modified acetyl bromide protocol (Brinkmannet al., 2002). One millilitre of freshly prepared 25% (w ⁄ w) acetylbromide ⁄ glacial acetic acid solution was added to 1 mg of driedwood powder. The tube (2 ml) was sealed and placed under inter-mittent mixing at 70�C for 30 min in a water bath. The digestionwas stopped by cooling the tube in an ice bath. After mixing,100 ll were transferred to a new tube containing 200 ll of 2.0 MNaOH and filled with 1.7 ml of glacial acetic acid to a finalvolume of 2 ml. The absorbance of the solution at 280 nm wasdetermined against a blank solution that was run in conjunctionwith the sample. The gram extinction coefficient of lignin treatedwith acetyl bromide is 20.09 l g)1 cm)1. All measurements wereconducted with three technical replicates.

Determination of energy content The calorific value of thewood was analysed with a calorimeter (IKA-Kalorimetersystem C7000; IKA-Werke GmbH & Co. KG, Staufen, Germany).Approximately 500 mg of wood powder was weighed andpressed into pellets using a presser attached to the calorimeter.The pellet was combusted with O2 (30 mbar) using bomb calo-rimetry. The calorific value was determined as the increase intemperature of water with a direct measurement of the internalenergy of the burning reaction in the calorimetric bomb. Usingbenzoic acid (pellets; IKA-Werke GmbH & Co. KG) as a stan-dard (calorific value, 26457 ± 20 kJ g)1), the calorific values ofthe samples were calculated.

Stable isotope analyses For stable isotope analyses, the stemmaterial from the harvest in September 2008 was separated by achisel into two parts, namely the young wood (wood 2008) andthe old wood (2007). d13C was analysed for wood samples from

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September 2007 and for young wood samples from September2008 with an elemental analyser ⁄ isotope ratio mass spectrometer(EA-IRMS) system. A total of 0.2 mg of fine wood powder wastransferred into a tin capsule (IVA Analysentechnik, Meerbusch,Germany) and combusted in an elemental analyser (Flash EA1112; Thermo Fisher Scientific, Milan, Italy) with a PorapackQS 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 d13C values were expressed in delta notation with respect toVienna Peedee Belemnite (VPDB). IAEA-CH-6 (sucrose with ad13CVPDB value of ) 10.449&; International Atomic EnergyAgency (IAEA), Vienna, Austria) was used as an internal standardfor the analysis.

Analysis of proanthocyanidins (condensed tannins)

Condensed tannins (proanthocyanidins) present in crude leafextracts were hydrolysed according to Porter et al. (1986). Fiftymilligrams 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 thepellet was washed again with 70% acetone. For hydrolysis, 100 llof the combined supernatants were mixed with 400 ll of 70%acetone, 3 ml of butanol-HCl (95:5) and 0.1 ml of ferric reagent(2% (w ⁄ v) NH4Fe(SO4)2Æ12H2O in 2 M HCl). Blank value ofabsorption at 550 nm was recorded before incubating the mix ina test glass covered with a glass marble at 96�C for 1 h. Hydrolysiswas stopped by cooling in an ice bath. The absorption of extractswas recorded at 550 nm. Proanthocyanidin concentrations werecalculated assuming an effective E1%, 1 cm, 550 nm of leucocyanidinof 460. All measurements were conducted with three technicalreplicates.

Description of models and simulations

We applied the physiologically based vegetation model Physio-logical Simulation Model (PSIM), together with the ECMcanopy model (Grote, 2007; Holst et al., 2010), the BIM2VOC-emission model (Grote et al., 2006) and a modifieddimensional growth model (Bossel, 1996; Miehle et al., 2010;Grote et al., 2011), within the Modular Biosphere simuLationEnvironment (MoBiLE; see, for example, Grote et al., 2009a,b)modelling framework. The PSIM model calculates primary pro-duction (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 theseprocesses depend directly or indirectly on the microclimaticenvironmental conditions. The supplies of water and nitrogenare assumed to be not limiting, although the physiological uptakerate allows for variations in tissue nitrogen concentrations (affect-ing the photosynthetic capacity). The parameterization of thephysiological model in the present work follows literature recom-mendations for morphology and phenology (Calfapietra et al.,2005; Ryu et al., 2008), photosynthetic kinetics and temperaturedependences (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 forseasonal enzyme dynamics were derived directly from actualmeasurements.

The increase in woody biomass, which was diminished bya fraction attributed to branches and coarse roots, was used tocalculate changes in stem height and diameter assuming a columnshape 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 usingstem-form functions from the literature thereafter (Honer, 1967).

Microclimatic conditions, together with the assimilated carbonthat is supplied by PSIM, determine VOC emissions. The modelwas run in 10-min time steps that were calculated from dailyaverage temperature and radiation sums for the years 2007 and2008 by assuming sinusoidal distribution schemes for tempera-ture (De Wit et al., 1978) and radiation (Berninger, 1994). Forthe simulation of VOC emissions with BIM2, these data werefurther linearly extrapolated into time steps of 7 s. Anthro-pogenic and disease-induced biomass decreases were prescribedfor specific dates and were considered at the start of the day.Spatially, microclimate and gas exchange processes were calcu-lated in vertical layers with daily updated foliage biomass and areavalues and assuming a fairly homogeneous distribution (Gielenet al., 2003), represented by a parameter-sparse distribution func-tion (Grote, 2007). In parallel, the number of layers was alsoupdated according to the increasing height of the plants, startingfrom 6 (height, 0.4 m) and ending with 10 (height, 3.4 m).

Biomass harvests, causing a decrease in biomass in all compart-ments as well as in tree numbers, were considered for the day atwhich they were executed, and all trees were assumed to be ofequal size. In acknowledgement of a considerable, but not pre-cisely defined, fraction of foliage biomass consumption by insects,we introduced a loss term of 0.25% of foliage biomass per daythroughout the period between the second and third harvests (days130–200 in 2008). This loss term results in a total biomass loss ofc. 5%, an amount corroborated by measurements of leaf area lossesafter each harvest. We decided in favour of a fixed percentageinstead of a fixed or prescribed amount because this reflects theresponse of parasites to the availability of the substrate. Thismodel was run with and without the VOC emission model todetermine not only the direct losses from isoprene emission, butalso the integrated loss throughout the year, which might involvefollow-up impacts caused by, for example, a smaller amount ofassimilates available for the building of productive tissue.

Results

Growth rates and biomass yield of two growing seasons

We followed the growth of poplar mutants continuously overtwo growing seasons, measuring growth parameters such as collardiameter, plant height and leaf formation. Overall, no growthrate differences were observed between isoprene-emitting andnon-isoprene-emitting poplars with respect to any of the threeparameters. We found that growth rates increased rapidly shortlyafter planting in mid-June 2007. Maximal growth rates of

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2.0–2.4 mm per day in collar diameter, 2.5–2.7 cm per day inheight and 0.8–0.9 leaves per day occurred at the middle ⁄ end ofJuly. Growth started to decrease at the seasonal break at thebeginning of September (Fig. 1a–c). In 2008, leaf formationincreased with the start of the growing season and dramaticallypeaked for 2–3 weeks just before the end of the growing season.Plant height and collar diameter increased moderately comparedwith the previous season, with maximum rates (�2.5 cm per dayin height, 0.15 mm per day in collar diameter) occurring in July2008 and decreasing in growth thereafter. Compared with 2007,the growing season ended slightly earlier in August 2008.

Collar diameter was chosen as a parameter most representativeof absolute growth. After two growing seasons, plants reachedmaximum collar diameters of 23 mm (C14), 27 mm (RA1) and29 mm (RA2) (Fig. 2a). The PcISPS-RNAi line RA2 developedlarger collar diameters than the vector control (C14). The differ-ences became significant shortly after planting at the end of June2007 and remained so up to August 2008. Moreover, line RA1

showed larger collar diameters than the vector control line C14 atseveral time points at the end of the 2007 season and at thebeginning of the 2008 season.

Biomass yield was determined as stem wood dry weight at fourtime points within the two growing seasons. After two growingseasons, the biomass yield ranged from 230 g (C14) to 280 gand 320 g for RA1 and RA2, respectively. The plants of lineRA2 clearly yielded more overall biomass within the first growingseason (Fig. 2b). At the next sampling time point in May 2008,both transgenic non-isoprene-emitting lines (RA1 and RA2) pro-vided significantly higher biomass yields than the C14 plants.

(a)

(b)

(c)

Date (day/month/year)

Fig. 1 Relative growth rates of transgenic non-isoprene-emitting linesRA1 (light grey inverted triangle) and RA2 (dark grey square) and theisoprene-emitting vector control line C14 (black circle) of Grey poplar(Populus · canescens) grown for two seasons outdoors. Growth wascontinuously monitored by collar diameter (a), plant height (b) and leafformation (number of leaves per day) (c) measurements.

(a)

(b)

Date (day/month/year)

Fig. 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 Greypoplar (Populus · canescens) grown for two seasons outdoors. (a) Growthwas continuously monitored by collar diameter measurements. (b) Biomassyield was investigated four times within the experimental period (September2007, May 2008, July 2008 and September 2008) by stem wood dryweight. For collar diameter, ANOVA (Holm–Sidak, P > 0.05) was per-formed for each measurement point separately, and significant differencesbetween the isoprene-emitting control line and either one or both non-iso-prene-emitting lines are marked with one or two asterisks [*,**], respec-tively. Error bars represent the standard errors of the means. The collardiameter replicates decreased from n = 20 > n = 15 > n = 10 > n = 5with harvests. In addition to ANOVA, repeated measures analysis was per-formed (http://www.ats.ucla.edu/stat/r/seminars/repeated_measures/repeated_measures.htm), validating highly significant differences betweenthe three genotypes: P < 2e)16. For stem wood dry weight, ANOVA(Holm–Sidak, P > 0.05, n = 5) was performed and significant differencesbetween the isoprene-emitting control line and either one or both non-iso-prene-emitting lines are marked with one or two asterisks [*,**], respec-tively. In addition to field site measurements, growth and biomass yieldwere simulated by applying the modelling framework MoBiLE (red line).Simulations were triggered with measured daily weather data and weredriven with a 0.25% daily leaf decrease between day 130 and 200 in thesecond year without any emission.

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However, this difference in plant growth disappeared at the latersamplings in July and September 2008.

Photosynthesis and VOC emission rates

Net CO2 assimilation, transpiration and isoprene emission rateswere investigated under standard conditions (30�C leaf tempera-ture and 1000 lmol photons m)2 s)1) four times within the twogrowing seasons. Both net CO2 assimilation and transpirationrates were higher in September 2007 than in the following year

(Fig. 3a,b). We observed significant differences between geno-types in September 2007 for net CO2 assimilation, and in July2008 for the transpiration rate. In both cases, the vector controlline C14 showed higher gas exchange rates than the RA2 line.

Isoprene emission by the C14 plants varied between 16 nmolm)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 con-stantly and stably repressed during the two growing seasons(Fig. 3c). RA1 and RA2 plants emitted negligible amounts of iso-prene, ranging from 1% to 7% of the emission rates of C14 plants.

Within the measuring campaigns September 2007, May 2008and July 2008, we also analysed the emission of methanol, acetal-dehyde and monoterpenes (Fig. S3a–c). The emissions of thesethree VOCs were generally highly variable and showed no differ-ences between vector control plants and the two PcISPS-RNAilines. In May 2008 (Fig. S3b), methanol (80–100 nmol m)2

s)1) and monoterpene (0.6–1.7 nmol m)2 s)1) emissions werehighest, whereas the emission of acetaldehyde (27–47 nmol m)2

s)1) was at a maximum in July 2008 (Fig. S3c), parallel to themaximum of isoprene emission. Monoterpene emissions are partof the plant’s defence against herbivores and fungi (Keeling &Bohlmann, 2006; Eckhardt et al., 2009). Therefore, wecompared the monoterpene emission of fungus-infected andnon-infected leaves in September 2007 (Fig. S3d) and of leaveswith feeding traces and undamaged leaves in May 2008(Fig. S3e). However, this analysis revealed no difference causedby fungal infection or herbivory.

Wood composition and quality (FTIR, composition, carbonisotope ratio)

Wet chemical analyses of a-cellulose, hemicelluloses, lignin andsoluble extractives in the stem wood of the 2-year-old poplars didnot reveal differences in basic wood composition between poplarlines (Table 1). The stem wood of the three genotypes was com-posed of 45.8 ± 0.9% a-cellulose, 26.8 ± 0.5% hemicellulosesand 25.6 ± 0.3% lignin. The mean heating value of dry woodwas 17 974 ± 70 J g)1. The FTIR spectra of wood, which pro-vide a chemical fingerprint of wood composition, also confirmedthat major compositional changes with regard to the amount oflignin (peak 4) or hemicelluloses (peak 1) did not occur (Fig. 4a).However, the data point to a decrease in the concentration of sy-ringyl lignin in the PcISPS-RNAi lines (peak 9) compared withthe vector control. Although the analysis of individual woodcompounds did not show significant differences, cluster analysesof the FTIR spectra revealed that the wood of RA2 was distin-guishable from that of controls (C14), whereas the wood of RA1was intermingled with C14 and RA2 (Fig. 4b).

Further carbon isotope discrimination (d13C) analyses wereperformed separately for wood from samples collected in Septem-ber 2007 and only young wood from samples collected in Septem-ber 2008 (Fig. 5). In 2007, both RA lines discriminated 13Csignificantly less strongly than the vector control line C14, as indi-cated by d13C values of ) 29.2& for RA1 and RA2 and ) 29.7&

for C14. At the end of the second growing season in September2008, no difference in 13C discrimination was detectable.

(a)

(b)

(c)

Date

Fig. 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 lineC14 (black columns) of Grey poplar (Populus · canescens) grown for twoseasons outdoors in a cage glasshouse. All parameters were investigatedfour times within the experimental period (September 2007, May 2008,July 2008 and September 2008). Significant differences between lines aremarked with lower case letters; n.s., not significant. ANOVA (Holm–Sidak,P > 0.05) was performed for each measurement point separately. Errorbars represent the standards errors of the means: nSept07 ‡ 12, nMay08 ‡ 9,nJuly08 ‡ 9, nSept08 ‡ 4. In addition to field site measurements, thesephysiological parameters were simulated by applying the modelling frame-work MoBiLE (red circles).

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Susceptibility to pests and herbivores

In contrast with glasshouse or laboratory conditions, plants innatural or outdoor conditions are challenged by many – some-times unexpected – environmental influences that cannot besimulated under controlled conditions. The climatic conditionsin summer 2007 favoured the development of the pathogenicfungus Pollaccia radiosa (Lib.) Bald. Et Cif. (teleomorph: Venturiatremulae Aderh.). The poplar plants developed severe shootblight disease, although with varying susceptibility, within a veryshort time period in July 2007. The degree of leaf infection byPollaccia was significantly higher in C14 plants (c. 35%) than inthe non-isoprene-emitting plants from mid-July to mid-August2007 (Fig. 6a). In 2008, the climate in July and August did notfavour the development of Pollaccia, and therefore only c. 4% ofthe leaves, regardless of the line, developed symptoms (data notshown). However, the local climate favoured the appearance ofthe herbivorous willow leaf beetle Phratora vitellinae (L.), whichsubstantially attacked the plants. Host plant selection preferencesfor the different genotypes were monitored via the number ofbeetles per tree at three times (May 2008, July 2008 and

September 2008; Fig. 6b). The beetles were generally found atthe top of the branches, preferring the younger, newly unfoldedleaves near the shoot top, as described in Urban (2006). Thewillow leaf beetles clearly selected the PcISPS-RNAi lines, mostobviously in July when the overall amount of beetles was highest.As phenolic compounds are an important part of the plant’sdirect defence against pests and pathogens (Keeling &Bohlmann, 2006; Eyles et al., 2009; Boeckler et al., 2011), weanalysed the total proanthocyanidin concentration in this study,but no differences between genotypes were observed (Fig. S2). Inaddition, analyses of monoterpene emission in May 2008 showedno differences between genotypes (Fig. S3b).

Annual carbon balance

Because destructive harvesting and the analyses of photosyntheticgas exchange and isoprene emission rates were conducted at onlyfour distinct time points in 2007 and 2008, we calculated theannual rates of gross primary production (GPP, estimated CO2

uptake) and net primary production (NPP, estimated netgrowth), together with carbon losses by respiration, litter fall,

Table 1 The contents of a-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)

a-Cellulose Hemicellulose Lignin Soluble extractives Calorific value

% (SE) % (SE) % (SE) % (SE) J g)1 (SE)

C14 44.700a (0.9) 27.206a (0.8) 25.840a (0.4) 1.505a (0.1) 18052.4a (55.1)RA1 46.259a (0.7) 26.238a (0.7) 25.702a (0.5) 1.566a (0.1) 17954.3a (145.8)RA2 46.509a (0.6) 26.897a (0.4) 25.327a (0.6) 1.106a (0.1) 17916.5a (101.4)

SE, standard error. Lower case letters indicate results of ANOVA (Holm-Sidak, P > 0.05).Plants were grown for two seasons outdoors in a cage glasshouse. Wood samples after two growing seasons were analysed.

(a) (b)

Fig. 4 Chemical fingerprints of wood from transgenic non-isoprene-emitting lines (RA1 and RA2) and the isoprene-emitting control line (C14) of Greypoplar (Populus · canescens) grown for two seasons outdoors. (a) Mean baseline-corrected Fourier transform infrared (FTIR) spectra of wood powder inthe 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. Spectraare means of five biological replicates. First derivates of spectra after baseline correction were employed for cluster analysis using Ward’s algorithm and thecorrelation coefficient as distance metric. Peak numbers in (a) refer to the following molecular components: 1, 1738 cm)1 – C=O stretch in unconjugatedketones, carbonyls and ester groups in xylans (hemicellulose); 2, 1650 cm)1 – absorbed O–H and conjugated C=O of proteins; 3, 1596 cm)1 – aromaticskeletal 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 andcarbohydrates; 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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harvesting, insect damage and isoprene emission, using a mathe-matical approach, coupling together various models within theMoBiLE modelling framework.

As an example, with regard to the development of collar dia-meter and stem wood dry weight, the adapted model was wellcapable of calculating plant growth and biomass yield (Fig. 2a,b,red lines). The simulations conformed to the measured biometricdata and were well within the range of the experimental uncer-tainties. This applied equally to the comparison of calculated andmodelled net CO2 assimilation and isoprene emission rates(Fig. 3a,c, red dots). For transpiration rates, the model results for2 months – based on average parameters – were lower than themeasurements (Fig. 3b).

The detailed modelled annual carbon balances for all lines in2008 are given in Table 2. Overall, the simulations revealedthat isoprene emission used 2.2% of total GPP, whereas theoverall effect of carbon loss by isoprene corresponded to areduction in NPP of 6.9%. Compared with other losses, carbonloss via isoprene emission was of the same magnitude as insect-related carbon loss, but considerably less than carbon removalby harvest.

Discussion

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

The functional loss of isoprene emission capacity in Grey poplarentailed no substantial growth impairment under outdoor condi-tions. The growth of PcISPS-RNAi poplars was even enhancedthrough a certain time period. This raises the question of whetherpoplars benefit from isoprene emission under realistic field condi-tions. Our previous laboratory studies with PcISPS-RNAi poplarsconfirmed the hypothesized roles of isoprene. We clearly showedearlier that isoprene protects photosynthesis during transient heatflecks (Behnke et al., 2007, 2010a; Way et al., 2011). However,this protective function was not apparent under the present con-ditions when growth performance was used as an integrativestress parameter, because both emitter types grew similarly. Alllaboratory studies on isoprene’s thermoprotective function havein common that the mechanism is effective at leaf temperaturesabove 35�C, and it specifically protects during heat flecks ratherthan during constant heat periods (Sharkey & Singsaas, 1995;Velikova & Loreto, 2005; Behnke et al., 2007). In the presentwork, we did not record leaf temperatures, but the climatic datashowed no days with an air temperature above 35�C, and only 5and 12 days with temperatures exceeding 30�C in 2007 and2008, respectively (Table S1). Therefore, conditions favourablefor the observation of a protective isoprene effect might havebeen rare, and the repression of isoprene emission was not rele-vant with regard to thermoprotection. In addition to specificallyprotecting against heat flecks, isoprene can also reduce oxidativestress 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 tothe quenching of reactive oxygen species. However, isoprene’sefficiency might depend on the cause, degree and spatial localiza-tion of the oxidative stress. Furthermore, more specific antioxi-dants may have been produced under the investigatedcircumstances. The knock-down of isoprene emission results inthe constitutive upregulation of ascorbate in poplar (Behnkeet al., 2009), whereas its introduction into tobacco (Vickerset al., 2009b) downregulates ascorbate. The antioxidative systemsof plants are known to be complex and overlapping (Noctor &Foyer, 1998; Foyer & Noctor, 2005); therefore, it is possible thatcertain other components might have substituted for isoprene asan antioxidant. Thus, isoprene is not indispensable for poplar

Table 2 Simulated gross primary production (GPP, estimated CO2 uptake) and net primary production (NPP, estimated net growth) of the investigatedGrey poplar (Populus · canescens) during the year 2008

CO2 uptake Net growth (%) Respiration (%) Litter loss (%) Harvest loss (%) Insect loss (%) VOC loss (%)

ISO) 0.902 0.117 (13.0) 0.306 (33.9) 0.257 (28.5) 0.208 (23.1) 0.013 (1.5) 0.000 (0.0)ISO+ 0.879 0.109 (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 –

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

Date

Fig. 5 Carbon isotope discrimination (d13C) of transgenic non-isoprene-emitting lines RA1 (light grey columns) and RA2 (dark grey columns) andthe isoprene-emitting control line C14 (black columns) of Grey poplar(Populus · canescens) grown for two seasons outdoors. Wood samplesafter first (September 2007) and second (September 2008) growing seasonwere analysed. The d13C values were expressed in delta notation withrespect to Vienna Peedee Belemnite (VPDB). Significant differencesbetween lines are marked with lower case letters; n.s., not significant.ANOVA (Holm–Sidak, P > 0.05) was performed for each measurementpoint separately. Error bars represent the standard errors of the means,n = 5.

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viability, and its absence did not generally impair growth perfor-mance 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 forisoprene-emitting plants (Sharkey & Yeh, 2001). If isoprene isnot needed because of the moderate climate and ⁄ or replacementby other less expensive antioxidants, the energy and carbonintended for isoprene production might be invested in bettergrowth and a larger biomass. In particular, the higher growth andbiomass production of PcISPS-RNAi poplars, observed duringthe first growing season (2007), support this hypothesis. How-ever, these plants showed lower rates of net CO2 assimilation. Ageneral reduction in photosynthesis is also supported by thereduced discrimination of 13C by the non-emitting lines in 2007,because 13C enrichment in leaf material can be mainly attributedto stomatal closure and higher uptake of the heavier 13C isotopeduring photosynthetic carbon acquisition (Brugnoli & Farquhar,2000). It is therefore more likely that a negative feedback loopdecreased photosynthesis rather than excess isoprene energy andcarbon 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) nega-tively correlate with the concentration of carbohydrates. InPcISPS-RNAi poplars, the accumulation of the biosyntheticprecursor dimethylallyl diphosphate (DMADP) is a definite con-sequence of repression of isoprene emission (Behnke et al., 2007,2010b), and could possibly serve as a signal for the downregula-tion of photosynthesis and thus lower net CO2 assimilation rates.Transcriptomic and metabolomic analyses of PcISPS-RNAi pop-lars have demonstrated comprehensively altered carbohydratemetabolism because of the repression of isoprene emission(Behnke et al., 2010b). We do not yet understand the cross-linksbetween the repression of isoprene emission, the accumulation ofDMADP and the subsequent alterations of carbohydrate metabo-lism and photosynthesis. Further investigations are needed todetermine whether the carbon and energy required to fuel iso-prene production are balanced between carbon sinks and sourcesby carbohydrate metabolism and photosynthesis, or whether aportion 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 geno-types and, to some extent, clustering into groups. However, thewood constituents lignin, a-cellulose and hemicellulose and theenergy content of the control and PcISPS-RNAi poplars werewithin the usual range for poplar (Leple et al., 2007; Luo &Polle, 2009; Zhou et al., 2011), and no genotype effect wasobserved. Thus, we found no effects on basic wood composition

(c)(d)

(a) (b)

(c)

Date (day/month/year)

Fig. 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 thefungus Pollacia radiosa (teleomorph: Venturia macularis) causing shoot blight disease. Susceptibility to the fungus was estimated by determining thepercentage of infected leaves in RA1 (light grey inverted triangles), RA2 (dark grey squares) and C14 (black circles). Significant differences between linesare marked with lower case letters; n.s., not significant. ANOVA (Holm–Sidak, P > 0.05) was performed for each measurement point separately. Error barsrepresent the standard error of the mean, n = 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, P > 0.05) was performed for eachmeasurement point separately. Error bars represent the standard errors of the means, n ‡ 20.

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or quality as a result of the repression of isoprene emission. Theslight clustering observed with FTIR analyses could possibly beexplained by the decrease in syringyl lignin in the PcISPS-RNAilines.

The loss of isoprene emission alters the ecologicalperformance of poplar

As a result of the complex interactions of abiotic and biotic stressfactors under outdoor conditions, we cannot exclude factors otherthan a direct effect of isoprene loss on growth and fitness. As oftenobserved in high-density poplar plantations, the plants wereattacked by natural pests of poplar. Pollaccia radiosa is a commonand destructive ascomycete causing so-called shoot blight disease(Dance, 1961; Newcombe, 1996; Kasanen et al., 2001). In 2007,spring in Gottingen was comparatively warm and wet (http://www.wetterstation-goettingen.de/klimadaten.htm) and thusfavourable for Pollaccia infection (Newcombe, 1996). Pollacciasymptoms, such as brownish ⁄ black necrotic lesions, curling leavesand dead twisted young shoots (Newcombe, 1996), increasedduring the summer of 2007 in both control and PcISPS-RNAipoplars, but were more pronounced in vector control plants. In2008, springwas comparativelywarmbutdry, and therefore favour-able 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 topathogens and herbivory of control and PcISPS-RNAi poplars.Generally, phenolic compounds are an important part of a plant’sdirect defence against pests and pathogens (Keeling & Bohl-mann, 2006; Eyles et al., 2009; Boeckler et al., 2011). Poplarsdefend themselves against herbivory and fungal infection withpolyphenols (Gruppe et al., 1999; Urban, 2006; Miranda et al.,2007; Zhong et al., 2011). However, increased production ofphenolic compounds and protection from herbivores did notresult in a negative trade-off with biomass production (Kleemannet al., 2011). The comprehensive characterization of PcISPS-RNAi plants in Behnke et al. (2010b) revealed high-temperature-dependent transient alterations of phenolic biosynthesis, resultingin altered polyphenolic and proanthocyanidin concentrations inleaves. The analysis of total proanthocyanidins in this studyshowed no differences between genotypes, probably because theambient air temperatures in the present study never reached veryhigh values (Table S1). However, because susceptibility to herbi-vores, such as Coleoptera, can depend on a single compound(Urban, 2006), we cannot exclude compound-specific alterationsin phenolic biosynthesis in the different lines as a cause for theirdivergent ecological behaviour.

In addition to the indirect pleiotropic effects of the repressionof isoprene emission on secondary compound metabolism, recentinvestigations have demonstrated a direct role of isoprene inplant–insect interactions. Studies with transgenic isoprene-emitting tobacco (Laothawornkitkul et al., 2008) and Arabidopsis(Loivamaki et al., 2008) have demonstrated the ability of iso-prene to repel both herbivores and parasitoids. A protective effectof isoprene against Phratora vitellinae could have led to their pref-erence for PcISPS-RNAi poplars. Further ecological studies with

PcISPS-RNAi poplars are essential to verify the role of isopreneas an orientation cue for insects. Monoterpenes are also impor-tant components of poplar–insect communication, which areconstitutively emitted from young poplar leaves (Brilli et al.,2009; Ghirardo et al., 2011) or are part of the induced volatileblend (Brilli et al., 2009; Danner et al., 2011). In May 2008, therelatively young leaves emitted comparably large amounts ofmonoterpenes, but no differences between genotypes wereobserved, and therefore no side-effect of the repression of iso-prene biosynthesis on the emission of other terpenes. In addition,fungus infection and beetle feeding did not result in an increasein monoterpene emission in September 2007 and May 2008.However, VOC emissions were not monitored directly afterbeetle infestation or fungal infection. Therefore, the induction ofmonoterpene emission as a result of herbivory or fungal infectioncould have been missed in our study.

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

Stand-level considerations and future prospects

With the modelling approach, the annual ⁄ biannual dynamics ofplant growth, biomass and physiological parameters, such as iso-prene emission, can be simulated very reasonably. This enabledus to quantify the annual overall carbon loss of poplar as a resultof isoprene emission. In the short term, this loss has beenestimated to be < 1% (Tingey et al., 1980), increasing withtemperature to 2% or higher under extreme conditions whenphotosynthesis is severely impaired (Sharkey & Yeh, 2001). Thecalculated annual carbon loss as isoprene of 2.2% relative to GPPis similar to these observations. Net CO2 assimilation rates wereslightly lower in non-isoprene-emitting leaves than in isopreneemitters. However, the annual calculation revealed that the non-isoprene emitters had higher CO2 uptake at the stand level. Thehigher 6.9% growth rate of PcISPS-RNAi poplars results inincreased NPP at the stand level, which offsets the observations atthe leaf level. Nevertheless, considerable uncertainties remain.For example, carbon and nitrogen losses may have occurred byroot exudation, a factor not considered in the present study.Furthermore, the dependence of plant maintenance respirationand fine root turnover on site conditions implies that the use ofspecific literature-derived parameters (Pregitzer & Friend, 1996;Thornley & Cannell, 2000) may be cumbersome.

As a result of the debate over renewable resources, poplars havebecome more and more important as bioenergy trees. World-widepoplar plantations represent 5.3 million hectares with an increas-ingly positive trend in many countries (International Poplar Com-mission, Synthesis of Country Progress Reports 2008). Inaddition to being a renewable substitute for fossil fuels, bioenergyfrom biomass is seen as a carbon-neutral energy with carbonsequestration potential, and therefore is considered to mitigateagainst the greenhouse effect and climate change. Nevertheless,care must be taken to fulfil these hopes. Depending on the type ofland use change, crop or tree species used, management system

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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; Fargioneet al., 2008; Searchinger et al., 2008; Hillier et al., 2009). How-ever, one aspect of the environmental friendliness of bioenergyplantations is considered only rarely: all plants emit VOCs, partic-ularly the selected ‘biomass’ trees. Most importantly, with respectto climate change, isoprene contributes to tropospheric ozoneformation and prolongs the lifetime of tropospheric methane(summarized in Monks et al., 2009). Laboratory studies(Kiendler-Scharr et al., 2009) and field observations (Kanawadeet al., 2011) with mixed forests have provided new evidence thatisoprene suppresses new particle formation, thus damping thenegative radiative forcing effect of aerosols. Many plantation treespecies that are cultivated throughout the globe are strong isopreneemitters (Kesselmeier & Staudt, 1999). Consequently, the growthof isoprene emitters in large-scale plantations might affect localclimate 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 CentralEurope revealed no remarkable differences with respect to plantgrowth and wood quality. The differences in sensitivity of thenon-isoprene-emitting poplars to fungal disease and herbivory,however, show that the stress responses of these plants areaffected and, indeed, require further combined molecular andecological investigations under controlled and field conditions. Inparticular, more real-field trials under strongly contrastingclimatic and soil conditions are needed to clarify conclusivelywhether isoprene-free poplars are an option for the secondgeneration of biomass plants, either generated by geneticmanipulation or selected by plant phenotyping.

Acknowledgements

We are grateful to S. Wolfarth (University of Gottingen) and C.Kettner (University of Gottingen) for excellent technical assis-tance. We would like to thank G. Bahnweg (BIOP, HelmholtzCentre Munich) and P. Faubert (EUS, Helmholtz CentreMunich) for critical comments on the manuscript. This studywas financially supported by the German Science Foundation(DFG; Schnitzler SCHN653 ⁄ 4 and Polle PO362 ⁄ 13) within theGerman joint research group ‘Poplar—A model to address tree-specific questions’ (FOR496) and by the European Commissionwithin the Seventh Framework Programme for Research, ProjectEnergypoplar (FP7-211917). G.W.Z. thanks the DAAD–CSC(German Academic Exchange Service – China ScholarshipCouncil) Joint PhD scholarship programme and M.E. theUniversity of Khartoum for providing PhD scholarships.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Weather conditions and air quality parameters of grow-ing 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 2007and 2008 with temperatures

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

Please note: Wiley-Blackwell are not responsible for the contentor functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the New Phytologist Central Office.

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