Isoprene function in two contrasting poplars under salt and sunflecks

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Tree Physiology 33, 562–578doi:10.1093/treephys/tpt018

Isoprene function in two contrasting poplars under salt and sunflecks

K. Behnke1†, A. Ghirardo1†, D. Janz2, B. Kanawati3, J. Esperschütz4, I. Zimmer1, P. Schmitt-Kopplin3, Ü. Niinemets5, A. Polle2, J. P. Schnitzler1 and M. Rosenkranz1,6

1Research Unit Environmental Simulation, Institute of Biochemical Plant Pathology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; 2Forest Botany and Tree Physiology, Büsgen-Institute, Georg-August-Universität Göttingen, 37077 Göttingen, Germany; 3Research Unit Biogeochemistry and Analytics, Helmholtz Zentrum München, 85764 Neuherberg, Germany; 4Institute of Soil Ecology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; 5Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Tartu 51014, Estonia; 6Corresponding author ([email protected]) †These authors contributed equally to this work.

Received November 14, 2012; accepted February 12, 2013; published online March 26, 2013; handling Editor Heinz Rennenberg

In the present study, biogenic volatile organic compound (BVOC) emissions and photosynthetic gas exchange of salt-sensitive (Populus x canescens (Aiton) Sm.) and salt-tolerant (Populus euphratica Oliv.) isoprene-emitting and non-isoprene-emitting poplars were examined under controlled high-salinity and high-temperature and -light episode (‘sunfleck’) treatments. Combined treatment with salt and sunflecks led to an increased isoprene emission capacity in both poplar species, although the photosynthetic performance of P. × canescens was reduced. Indeed, different allocations of isoprene precursors between the cytosol and the chloroplast in the two species were uncovered by means of 13CO2 labeling. Populus × canescens leaves, moreover, increased their use of ‘alternative’ carbon (C) sources in comparison with recently fixed C for isoprene biosynthesis under salinity. Our studies show, however, that isoprene itself does not have a function in poplar survival under salt stress: the non-isoprene-emitting leaves showed only a slightly decreased photosynthetic performance compared with wild type under salt treatment. Lipid composition analysis revealed differences in the double bond index between the isoprene-emit-ting and non-isoprene-emitting poplars. Four clear metabolomics patterns were recognized, reflecting systemic changes in flavonoids, sterols and C fixation metabolites due to the lack/presence of isoprene and the absence/presence of salt stress. The studies were complemented by long-term temperature stress experiments, which revealed the thermotolerance role of isoprene as the non-isoprene-emitting leaves collapsed under high temperature, releasing a burst of BVOCs. Engineered plants with a low isoprene emission potential might therefore not be capable of resisting high-temperature episodes.

Keywords: 13C labeling, isoprene, lipids, metabolomics, Populus euphratica, Populus × canescens, salt, thermotolerance.

Introduction

Bioenergy production is achieved to an increasing extent by poplar biomass production systems in Europe, USA and Asia (European Commission 2005, Aylott et al. 2008, Zalesny et al. 2009). One of the challenges for such biomass systems is the competition of land use with agriculture. As drought and soil salinization are expected to increase in the future (Chen and

Polle 2010, Munns 2011, FAO Soils Bulletin 39), compatible biomass production could be achieved by utilizing marginal lands that are no longer suitable for traditional agriculture.

Poplar biomass production can be considered to be close to ‘carbon neutral’, at least when compared with the fossil energy sources that release carbon (C) from fossil reserves to the atmosphere (Sims et al. 2006). However, when planning poplar

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biomass production systems, one should remember that pop-lars emit high amounts of isoprene. Isoprene (C5), which glob-ally dominates biogenic hydrocarbon emissions (Guenther et al. 1995), plays an important role in modifying atmospheric chemistry (Fuentes et al. 2000), particularly in altering the lifespan of the greenhouse gas methane (Archibald et al. 2011) and the formation of secondary organic aerosols (Claeys et al. 2004, Kiendler-Scharr et al. 2012). By affecting the regional production of tropospheric ozone, isoprene may also indirectly pose a risk to human health (Fuentes et al. 2000, Pacifico et al. 2009). Thus, for sustainable poplar biomass production, it is not just C fixation that is important; it is also important to elucidate why, under which circumstances and how much iso-prene is emitted. By now, it has been demonstrated that the physiological significance of isoprene emission is related to plant thermotolerance (Monson et al. 1992, Sharkey and Singsaas 1995, Sharkey et al. 2001, Behnke et al. 2007) and tolerance to oxidative stress (Loreto and Velikova 2001, Affek and Yakir 2002, Velikova et al. 2005). The mechanisms of iso-prene action are still not completely understood. Isoprene is suggested to physically stabilize thylakoid membrane struc-tures (Sharkey and Singsaas 1995, Sharkey et al. 2001, Siwko et al. 2007) or to react directly with reactive oxidative species (ROS) (Loreto and Velikova 2001, Affek and Yakir 2002).

Some poplar species are more salt-tolerant (e.g., Populus euphratica) than others (e.g., Populus × canescens) (Chang et al. 2006, Wang et al. 2007, Chen and Polle 2010). The abil-ity of P. euphratica to tolerate salt is due to several different mechanisms that help the plant maintain ionic homeostasis and avoid cellular toxification (for a review, see Chen and Polle 2010). A transcriptomic and metabolomic analysis revealed that P. euphratica does not rely on adjusting of the stress-related pathways to environmental cues as P. canescens appar-ently does, but has actually permanently activated its stress control mechanism to tolerate salinity (Janz et al. 2010). So far, no studies have been conducted to compare the isoprene emission capacity of salt-sensitive and salt-tolerant poplar spe-cies. Furthermore, there is only scarce data about the role of isoprene in the responses to salinity (Loreto and Delfine 2000, Teuber et al. 2008). Because photosynthesis is the main C source for isoprene and many other biogenic volatile organic compounds (BVOCs) (Schnitzler et al. 2004, Ghirardo et al. 2011) and because salinity is known to decrease photosyn-thetic capacity (Sudhir and Murthy 2004, Parida and Das 2005, Teuber et al. 2008, Chaves et al. 2009), it is expected that BVOC emissions are affected by salinity. However, Loreto and Delfine (2000) and Teuber et al. (2008) both showed that isoprene emission in salt-treated Eucalytus globulus Labill. or in P. × canescens was not altered, although the net CO2 assimila-tion rate strongly decreased, up to almost complete inhibition. It is unclear how and from which sources the substrate for isoprene biosynthesis is provided under such conditions. In

general, the capacity for isoprene emission is controlled by enzyme activities (Eisenreich et al. 2001, Brüggemann and Schnitzler 2002a), while short-term modifications to environmental conditions are driven by isoprene synthase activity, the photosynthetic rate (Schnitzler et al. 2004), the substrate dimethylallyl diphosphate (DMADP) and the pool size (Brüggemann and Schnitzler 2002b, Rasulov et al. 2009, 2010, Sun et al. 2012). However, under stress conditions, when the C input from photosynthesis seriously declines, alter-native C sources such as sugar and starch pools can be remo-bilized (Ghirardo et al. 2011).

Regarding many abiotic stresses, no consensus exists about their influence on BVOC emissions and how BVOC emissions may protect from different abiotic stresses (for a review see Loreto and Schnitzler 2010). A unified mechanism of plant abi-otic stress resistance due to the antioxidative properties of iso-prene has been proposed (Vickers et al. 2009), but this mechanism is apparently not valid for flooding stress (Copolovici and Niinemets 2010). It is also unclear whether this mechanism can be applied to salinity tolerance. Furthermore, plant responses to multiple sequential and simultaneous stresses are hard to predict from single stress responses, and the under-standing of interactive responses is still very limited (Niinemets 2010a, 2010b). Plants growing in infertile saline soils may fre-quently encounter reductions in stomatal conductance, reduc-ing their capacity for transpiratory cooling and thus increasing the probability of heat stress. Only a few studies have so far investigated multiple stress interactions, such as simultaneous heat and high-light stress (Behnke et al. 2010b, Way et al. 2011). In the present study, we combined salinity with sunflecks (i.e., transient exposure to high temperature in combination with high light). Sunflecks are frequent in tree canopies (Singsaas et al. 1999, Behnke et al. 2010b), and isoprene has been shown to effectively protect the photosynthetic apparatus from photo-inhibition and heat stress resulting from sunfleck exposure (Sharkey et al. 2001, Behnke et al. 2010b), but it is unclear how salinity can modify this protective capacity.

Therefore, to gain insight into poplar performance in one of the most common stress interactions, salinity and high tem-peratures, the present study addressed three key questions. First, we investigated how photosynthetically fixed C is allo-cated within isoprene and its precursor DMADP. This question was tackled by 13C-labeling experiments in salt-treated poplar trees under constant light and temperature. Second, we sought to answer whether salt stress can influence isoprene emission and to determine the function of isoprene in salt-tolerant P. euphratica or in salt-sensitive P. × canescens. For this task, the performances of P. euphratica and the two poplar geno-types of P. × canescens (isoprene-emitting and non-isoprene-emitting) under combined salinity and sunflecks were examined. To get deeper insight into isoprene function in the salt- and sunfleck-exposed leaves of isoprene-emitting and

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non-isoprene-emitting P. × canescens, metabolomics and lipid content were analyzed. Finally, we sought to gain new knowl-edge of the proposed function of isoprene as a membrane sta-bilization agent under thermal stress. Here, wild-type (WT) and non-isoprene-emitting transgenic P. × canescens genotypes were exposed to severe thermal stress, and stress-elicited vol-atile compounds were investigated.

Materials and methods

Plant material and experimental setup

To determine whether isoprene can protect plants from ther-mal stress under salt exposure, two experiments were designed in which the salt treatment was combined with high tempera-ture and high light intensities. In both experiments, the plant growth procedure and the treatments were similar: WT P. × canescens (syn. Populus tremula × P. alba), P. euphratica and transgenic PcISPS-RNAi lines (RA1 and RA22, Behnke et al 2007, 2010b) of P. × canescens were propagated by tis-sue culture methods and kept in aerated hydroponics using the Long Ashton nutrient solution, which was changed on a weekly basis. Plants were grown for 12 weeks in an acclimation cham-ber with a 16 h/8 h dark/light rhythm at 26 °C, a relative air humidity of 60% and photosynthetic active radiation (PAR) of 150 µmol m−2 s−1. For salt treatment, the plants were exposed first to 25 mM NaCl at the age of 8 weeks, followed by 100 mM NaCl at the age of 10 weeks.

In the 13C-labeling experiment (first experiment) with control and salt-treated P. × canescens (only WT) and P. euphratica, the gas exchange and isoprene emission rates of the plants were analyzed under standard conditions (30 °C leaf tempera-ture and 1000 µmol m−2 s−1 PAR). In the second experiment, salt exposure and six cycles of high-temperature (ca. 40 °C) and high-light (1000 µmol m−2 s−1 PAR) events were combined (‘sunflecks’). This experiment was performed with WT geno-types of both species and with the non-isoprene-emitting P. × canescens line RA1.

For the long-term exposure to a leaf temperature of 40 °C, WT P. × canescens and the non-isoprene-emitting PcISPS-RNAi line RA22 (Behnke et al. 2007) were cultivated under green-house conditions as described earlier (Behnke et al. 2010b). One month before the experiment started, plants were accli-mated in a separate room at 20 °C, 150 µmol m−2 s−1 PAR and a relative humidity of ~60%.

For all three experiments (13C-labeling, sunfleck treatment and long-term high temperature), the 9th or 10th leaf below the apex was chosen.

Photosynthetic gas exchange and BVOC emission measurements

To study the gas exchange of the salt-treated poplar plants under constant temperature and light, the Walz GFS-3000 gas-exchange

system (Heinz Walz GmbH, Effeltrich, Germany) with an 8 cm2 clip-on-type cuvette was used. The measurements were carried out at 30 °C and with a light intensity of PAR 1000 µmol m−2 s−1. The cuvette was flushed with 0.81 l min−1 synthetic air with 385 ppmv CO2 (either 12CO2 or 13CO2; Air Liquide, Griesheim, Germany) that was humidified to 10,000 ppmv. 13C labeling was initiated by replacing the 12CO2 (natural 13C abundance) with 99% of 13CO2 385 ± 7.7 ppmv (Air Liquide) when the net CO2 assimilation and isoprene emission were stable (ca. 40 min). 13CO2 was fed for 60 min. For dynamic online monitoring of photo synthetic gas exchange and isoprene emission, the air stream was divided between the proton-transfer-reaction quad-rupol mass spectrometer (PTR-QMS, Ionicon Analytik GmbH, Innsbruck, Austria), drawing 100 ml min−1 (detailed description in Behnke et al. 2007) and the Walz system. Calibrations were per-formed regularly in both experiments (see Behnke et al. 2007). After the experiments, the 13C-incubated leaf parts were cut out and immediately shock-frozen in liquid N2.

The combined cycles of high temperature and light intensity (‘sunflecks’) started with 10 min darkness, which was followed by 60 min of stabilization time in 26 °C and PAR of 100 µmol m−2 s−1, after which six sunfleck episodes (heat and light cycles in which the temperature was switched every 10 min from 26 to 40 °C and back, and the light intensity was simultaneously raised from 100 to 1000 µmol m−2 s−1 PAR and back) were performed. The chlorophyll fluorescence measure-ments (GFS-3000) were always taken just before the switch to the next high-temperature/high-light or low-temperature/low-light period. The Fv/Fm value was measured in the dark just before the start of the stabilization period. At the end of the last high-temperature/high-light cycle, the exposed leaf part was cut out and immediately shock-frozen in liquid N2.

In the long-term high-temperature experiments, WT P. × canescens and the non-isoprene-emitting PcISPS-RNAi line RA22 (Behnke et al. 2007) were examined. One poplar leaf (the 9th–10th leaf of each individual tree) was enclosed in a home-made cuvette (for details, see Ghirardo et al. 2011) and flushed with 1 l min−1 (a mix of synthetic air 20%/80% O2/N2, 390 ppm CO2, Lurgi GmbH, Tartu, Estonia). The light intensity was set to PAR 1000 µmol m−2 s−1. Starting from plants adapted to 20 °C, the leaf temperature was brought to 30 °C in <4 min, kept at 30 °C for 45 min and then brought to 40 °C at a rate of 2 °C min−1 and held at 40 °C for the next 90 min. This was per-formed to simulate exposure to high shifts in temperature changes that are typical of certain regions and seasons (e.g., the beginning of summer in southern Europe). The released BVOCs were quantified by PTR-QMS. Masses were recorded similarly as described in Ghirardo et al. (2011). The BVOCs produced within the octadecanoid pathway (the so-called ‘LOX products’) were measured at m/z 99 for LOX1 = [(Z)-3-hexenal + (E)-2-hexenal], m/z 101 for LOX2 = [(Z)-3-hexenol + (E)-3-hexenol + (E)-2-hexenol + hexanal] and m/z 85 for LOX3 = hexanol.

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Chlorophyll fluorescence measurements by using imaging PAM

Imaging PAM from Walz (IMAG-MAX/L with the camera DX4-205 FW, Walz, Germany) was used for additional chlorophyll fluorescence measurements in salt-exposed plants. These mea-surements were recorded at the same time as cuvette measure-ments for the sunfleck treatment, but used the next neighboring leaf below the sunfleck-treated leaf (10th/11th leaf), assuming a comparable physiology. Additionally, the 3rd and 16th/17th leaves were taken to analyze different leaf age classes. The recordings of light curves by imaging PAM consisted of a num-ber of illumination steps (according to the standard settings with actinic light intensities of 0, 1, 36, 111, 231, 396, 611, 926 µmol m−2 s−1 PAR) taken every 5 min, at the end of which various parameters were determined with the help of a satura-tion pulse. For the calculations of effective photosystem (PS) II quantum yield (Y = (Fm′ − F)/Fm′), non-photochemical quench-ing (NPQ = (Fm − Fm′)/Fm′) and electron transport rate (ETR = 0.5 × yield × PAR × 0.84 µ equivalents m−2 s−1), the whole leaf was defined as the area of interest.

Biochemical analysis of the isoprene substrate DMADP

Cytosolic and chloroplastidic DMADP levels were measured and calculated as described by Ghirardo et al. (2010) with the following changes: 5 mg of freeze-dried leaf powder was solu-bilized with 200 µl of 8.5% H3PO4 in a 2 ml gas-tight glass vial and incubated at 70 °C for 90 min to catalyze the reaction from DMADP to isoprene. Adding 100 ml of 9.2 N NaOH stopped the reaction. Dimethylallyl diphosphate was measured as iso-prene after hydrolysis by measuring the protonated masses of isoprene and its isotopes at m/z 69–74 with PTR-QMS.

Metabolomic and cluster analyses

The non-targeted metabolome analysis was performed with the leaf sections cut out at the end of the last high-temperature high-light cycle using a modification of the protocol of Cho et al. (1992). Twenty-five milligrams of frozen leaf material was extracted two times with 1 ml of CHCl3 : CH3OH : HCl(1N) (40 : 80 : 1, v/v/v). After centrifugation at 2000 rpm, 1.7 ml of the extract was used for fatty acid analysis (described in the next section) and the remaining 0.3 ml was used for further metabolome analysis. The metabolome analysis continued as described by Behnke et al. (2010a). High-resolution mass spec-tra for molecular formula assignment were acquired on a Fourier-transform ion-cyclotron-resonance mass-spectrometer (Solarix, Bruker, Bremen, Germany) equipped with a 12-T superconduct-ing magnet and an Apollo II electrospray (ESI) source. Data were uploaded into MarVis Filter, which calculates a list of compounds significantly affected by the treatments (analysis of variance (ANOVA) with a threshold of 5 × e5; X = log2(X) where X is the peak intensity), applying a false discovery rate of 5% according to the Benjamini–Hochberg correction (Benjamini and Hochberg

1995); the data were subsequently analyzed using the MarVis Cluster (http://marvis.gobics.de/, last accessed 14 March 2013), which produces clusters of compounds with similar responses to treatments in a self-organizing map (Kaever et al. 2009). Annotation was achieved by either the MassTRIX3 platform (http://masstrix3.helmholtz-muenchen.de/masstrix3/, last accessed 14 March 2013; Waegele et al. 2012) or the KEGG/API (http://www.genome.jp/kegg/soap/, last accessed 14 March 2013) database for Populus trichocarpa Torr. & A. Gray with expanded lipids (KEGG/HMDB/LipidMaps with isotopes; v. 06-2011). The maximum error accepted was 2 ppm.

Fatty acid extraction

For lipid analysis, the same extracts as for non-targeted metabo-lome analysis were used. The initial leaf material was again washed with water, centrifuged and the supernatant was com-bined with the 1.7 ml extract gained previously (see the section above). The extract was washed twice with 0.75 ml CHCl3 and centrifuged, after which the lower phases were combined for fur-ther analysis. The combined fractions were washed with a 1.5 ml washing solution (1 N HCl : CH3OH, 1 : 1, v/v), centrifuged and the lower phase was used for analyses. The samples were fur-ther analyzed as described by Zelles et al. (1995). The separa-tion of phospholipids, neutral lipids and glycolipids was obtained with a silica-bonded phase column (SPE-SI 2 g 12 ml−1; Bond Elut, Analytical Chem International, CA, USA). Fatty acid methyl esters (FAMEs) were obtained after mild alkaline hydrolysis. Un-substituted FAMEs were prepared for gas chromatography (GC) separation using myristic FAME as an internal standard. The FAMEs were measured using a GC/mass spectrometry (MS) sys-tem (5973MSD GC/MS, Agilent Technologies, Palo Alto, CA, USA) linked via a combustion unit to an isotope ratio mass spec-trometer (DeltaPlus, Thermo Electron Cooperation, Bremen, Germany) and identified via established fatty acid libraries and characteristic retention times. Fatty acids are designated as the total number of C atoms followed by the number of double bonds and their location (ω) after the colon. Saturated straight-chain fatty acids are indicated by ‘n’. The percentage of double bonds in the fatty acids was presented as the double bond index (DBI), calculated according to Knowles and Knowles (1989).

Statistical analyses

Before statistical analyses, all data were tested for normality (the Shapiro–Wilk test). The differences among treatments were tested using paired Student’s t-tests, one-way ANOVA and/or Kruskal–Wallis ANOVA on ranks, combined with Tukey’s honestly significant difference (HSD) post hoc test. To determine the over-all significances between the genotypes and treatments, two-way ANOVA was applied. In the case of gas exchange and dynamic isoprene emission data, the mean values of each 10 min stable period (before ‘sunfleck’) or the 1 min mean of the highest or lowest stable periods (during ‘sunfleck’) were used to test the




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statistical differences between species/genotypes. In the chloro-phyll fluorescence data, the individual measurement points were compared in order to find differences between/within species/genotypes. All statistical analyses were performed in SigmaPlot version 11.0 (Systat Software, San Jose, CA).

Results

Populus × canescens uses ‘alternative’ C sources for isoprene emission under salt exposure

Salinity stress strongly reduced the net CO2 assimilation rate and transpiration rate, particularly in salt-sensitive P. × canescens

(Figure 1b, P < 0.05, one-way ANOVA and Tukey’s HSD). Salinity also tended to reduce isoprene emission, but the effects were not statistically significant (Figure 1a). To study the rate at which recently fixed C is supplied to isoprene biosynthesis, 13CO2 labeling of P. euphratica and P. × canescens was per-formed. Labeling with 13CO2 demonstrated that incorporation of 13C was similar in isoprene emission (~85–90%) and DMADP (~70–85%) within species and within treatments (Figure 1c).

In general, P. × canescens exhibited a higher fraction of cyto-solic DMADP (~65–84% of the total) than P. euphratica (37–47%) (Figure 1d). When P. × canescens was treated by salt, the total DMADP content decreased by ~25%. The decrease in

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Figure 1. Isoprene emission (a) and net CO2 assimilation (b) of the control (white bars) and salt-treated (black bars) P. × canescens and P. euphratica leaves (leaf no. 9 below the apex) measured at 38 °C and 1000 µmol m−2 s−1. (c–f) DMADP and isoprene 13C labeling studies in the control and salt-treated P. × canescens and P. euphratica plants fed with 13CO2. Incorporation of 13C into chloroplastidic DMADP (c, black bars) and isoprene (c, gray bars), chloroplastidic (chl, gray) and cytosolic (cyt, white) DMADP pool sizes (d), isotopic distributions of total DMADP (e) and isoprene (f) both given as the distribution of protonated forms (Fx1 m69, Fx2 m70, Fx3 m71, Fx4 m72, Fx5 m73, Fx6 m74). The results shown are the means of four individual plants. Error bars in (a), (b) and (c) show +SE, and statistical significances (P < 0.05; Tukey’s HSD) are marked by different letters.



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total DMADP was primarily due to a decrease in the chloro-plastidic DMADP content. Approximately 16% of the total DMADP was of chloroplastidic origin in salt-treated plants whereas ~35% was of chloroplastidic origin in control plants of P. × canescens (Figure 1d).

The isotopologue masses of isoprene m/z 70, m/z 71, m/z 72, m/z 73 and m/z 74 summed together equaled ~50% of the entire measured DMADP content in P. euphratica, whereas in untreated P. × canescens, these masses equaled up to ~30% only, and in the salt-treated P. × canescens, up to ~10% (Figure 1e). This is in accordance with the isotope ratio of iso-prene, which revealed an equal proportion of appearance of the different detected masses in the salt-treated P. canescens than in the other plants. In P. euphratica and in untreated P. × canescens, mass 74 comprised most (~ 60%) of the whole isoprene emission, whereas in salt-treated P. × canes-cens, the corresponding proportion was only ~30% (Figure 1f). Consistently, salt-treated P. × canescens contained a higher proportion of the other masses (m/z 72, m/z 71 and m/z 70).

Induction of isoprene emission by salt and sunflecks

In contrast to the steady-state conditions in which no differ-ence in isoprene emission was found (Figure 1a), the dynamic isoprene emission measurements revealed increases in emis-sion due to the salt exposure in both species when the sun-flecks were applied. In both P. euphratica and P. × canescens, more isoprene was released from the salt-treated plants than from the controls when exposed to sunflecks (P < 0.01, t-test, Figure 2a and b).

Photosynthesis of non-isoprene-emitting P. × canescens is slightly more sensitive than that of the WT under combined salt and sunfleck treatment

At the beginning of the gas exchange under low light and 26 °C, salt-treated WT and the non-isoprene-emitting RA lines behaved similarly, with both displaying lower net CO2 assimilation than the control (P < 0.05, ANOVA and Tukey’s HSD). The differ-ences increased during the sunflecks, as the salt-treated plants were less able to recover from each episode than the untreated plants (P < 0.05 for the last sunfleck episode, ANOVA and Tukey’s HSD). Between the non-isoprene-emitting RA line and WT, no significant differences could be detected, but overall the assimilation of the RA line tended to decrease over the sun-flecks whereas that of WT remained unaffected (Figure 2c).

In transpiration rates, no clear differences between the P. × canescens WT and RA line were found. In general, transpiration rates decreased in both of the genotypes due to salt exposure before and during the sunflecks (P < 0.05, ANOVA and Tukey’s HSD, Figure 2e).

Chlorophyll fluorescence was measured regularly during the online gas analysis under both the high and low conditions for the sunflecks. Figure 2g and h shows the ETR rates in the

recovery phase of each sunfleck (at 26 °C and 100 µmol m−2 s−1 PAR). In salt-stressed leaves of P. × canescens, a decrease in ETR was observed after the third exposure to sunflecks (P < 0.05; Kruskal–Wallis and Tukey’s HSD, Figure 2g). In gen-eral, the ETR of the salt-treated plants decreased over time (P < 0.05, paired t-test). In accordance with ETR, a clear increase in NPQ rates was measured in both salt-stressed WT and RA P. × canescens after the first sunfleck, but the increase remained significant only in salt-treated non-isoprene-emitting plants after the fourth and fifth sunfleck (P < 0.05, ANOVA or Kruskal–Wallis and Tukey’s HSD, Figure 2i). In general, the NPQ increased further during each sunfleck and was signifi-cant between the first and last sunfleck in all the treatments (P < 0.05, paired t-test).

When the ETR and NPQ were measured at the maximum values of temperature and light intensity, in general, the results obtained were similar to those obtained when the measure-ments were carried out during the recovery phases (see Figure S1 available as Supplementary Data at Tree Physiology Online). However, salt-affected non-isoprene-emitting P. × cane-scens individuals displayed lower ETR during the last sunfleck compared with the control (P < 0.05, ANOVA and Tukey’s HSD; Figure S1a available as Supplementary Data at Tree Physiology Online). For NPQ, no differences between WT and RA plants or salt treatments were found, but the NPQ increased over time in both genotypes and treatments (P < 0.01, paired t-test; Figure S1c available as Supplementary Data at Tree Physiology Online).

The NPQ of salt-treated P. euphratica is slightly lower under exposure

In P. euphratica, no differences between salt-treated and control plants were detected in net CO2 assimilation rates (Figure 2d), and only a slight decrease in transpiration (Figure 2f) and sto-matal conductance (results not shown) due to the salt treat-ment was observed. In accordance with our gas exchange analyses, no differences in ETR were detected (Figure 2h and Figure S1b available as Supplementary Data at Tree Physiology Online). Additionally, no difference in NPQ between the treat-ments was found; however, significant differences (P < 0.05, paired t-test) were detected between the first and the last measurements of both treatments (Figure 2j and Figure S1d available as Supplementary Data at Tree Physiology Online), testifying to a slight increase in stress due to each sunfleck episode. Interestingly, this increase in NPQ was slightly less pronounced in the salt-treated plants.

Chlorophyll fluorescence imaging displays leaf age- and species-dependent salt stress under increasing light

In addition to the online fluorescence analysis that parallels the gas exchange measurements, we applied imaging PAM to obtain deeper insight into the photosynthetic performance of




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different leaf age classes of salt-stressed plants. The effective PS II quantum yield showed lower values in the non-isoprene-emitting RA line compared with the isoprene-emitting WT P. × canescens (Figure 3a, the two upper rows, representative

pictures of the 10th or 11th leaf, are shown). Salt-tolerant leaves of P. euphratica, in contrast to P. × canescens, showed similar quantum yields as the controls (Figure 3a, the two lower rows, representative pictures of the 10th or 11th leaf are

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Figure 2. Isoprene emission (a, b), net CO2 assimilation (c, d), transpiration (e, f), electron transport rate (g, h) and non-photochemical quenching (i, j) of P. × canescens (left), P. euphratica (right) leaves (9th or 10th leaf below the apex) during the simulation of sunflecks. For P. × canescens, the results are shown for the control (filled circle) and salt-treated (filled square) isoprene-emitting WT plants (black) and for non-isoprene-emit-ting PcISPS-RNAi plants (red); for P. euphratica the results are shown for the control (filled circle, black) and salt-treated (Fx1, green). After 40 min stabilization at 26 °C and 100 µmol m−2 s−1, six high-temperature/light cycles (temperature increased in 10 min from 26 to 38 °C and back; light increased simultaneously from 100 to 1000 µmol m−2 s−1 and back) were performed. The stabilization period is indicated in grey in the schema on the top of the figure, whereas the highs and lows of the sunflecks are indicated in white and gray, respectively; black indicates darkness. The fluo-rescence measurements were carried out always at the end of the low-light/temperature phase, the maximum phase, of each sunfleck focusing the stress phase of each sunfleck. The given values for gas exchange are 60 s means + SE of four individual plants and for the fluorescence measure-ment the means of four individual plants +SE.



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shown). The quantum yield calculation in salt-treated leaves relative to the control revealed a better salt-coping capacity in young leaves than in older leaves in both P. × canescens and P. euphratica (Figure 3b and c). The salt-sensitive WT and non-isoprene-emitting P. × canescens lines, however, behaved simi-larly (Figure S2a, b, d, e, g and h, respectively, available as Supplementary Data at Tree Physiology Online). Additionally, P. euphratica displayed only a small variation between the treatments, testifying to non-stressful conditions (Figure S2c, f and i available as Supplementary Data at Tree Physiology Online).

Metabolomic analysis reveals systemic differences within P. × canescens genotypes

To further elucidate the possible impact of salt and sunfleck exposures in the different P. × canescens genotypes, a

non-target metabolomic analysis of the WT and RA lines was performed. Overall, we found 432 masses that were signifi-cantly different across treatments. These masses could be sorted into 9 clearly distinguishable clusters (Figure 4a) con-taining between 20 and 90 masses each (Figure 4b) that rep-resented metabolic changes between the WT and RA lines and between the treatments. Salt treatment largely affected the metabolome of both P. × canescens genotypes, with 126 decreased (Clusters 1–2; Figure 4a and b) and 183 up-regu-lated (Clusters 7–9; Figure 4a and b) masses in salt-treated plants relative to controls. Metabolites of the primary metabo-lism and, in particular, those related to C fixation and sugar metabolism were strongly decreased under salt treatment, whereas steroid and flavonoid biosyntheses were increased (Table 1). Within the main genotype effect, the exposure to salt resulted in a few specific increases of 22 masses in only the


Figure 3. Examples of spatial variability of effective PS II quantum yield of salt-treated and untreated P. × canescens (WT) and P. euphratica leaves (a) and relative values of effective PS II quantum yield of P. × canescens (b) and P. euphratica (c) measured by imaging PAM and performing light curves. In (a), middle aged leaves are chosen as examples and the false color scale is given on the right. The symbols in (b) and (c) are as follows: (filled circle) 3rd leaf below the apex, (filled inverted triangle) 10th or 11th leaf below the apex and (filled square) 16th or 17th leaf below the apex; the black color indicates WT and the red color the PcISPS-RNAi line in (b). The given values are the means + SE of four individual plants.

Figure 4. Heat map (a) and the number of masses (b) from cluster analysis of metabolomic data from isoprene-emitting (WT) and non-isoprene-emitting (RA) plants under control conditions (WT and RA) and salt stress (WTS and RAS). Blue and red colors refer to, respectively, low and high metabolite concentrations. Masses were grouped into nine clusters containing metabolites which decreased (Clusters 1, 2) or increased (Clusters 7, 8, 9) in response to salt, high regulated only in WT (Cluster 3) or only in RA (Cluster 4) under control conditions, responding more strongly in WTS (Cluster 5) or in RAS (Cluster 6) to salt treatment.



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RA line but not in the WT (Cluster 6; Figure 4a and b), and 21 masses only in the WT but not in RA plants (Cluster 5; Figure 4a and b). Metabolites related to flavonoids and steroids increased more in the RA than in the WT under salt treatment. Under unstressed conditions, the transgenic line contained a higher amount of compounds related to pyruvate and glyoxylate/dicarboxylate metabolism compared with the WT (Cluster 4; Table 1, Figure 4a and b).

High-temperature treatment led to different BVOC emissions from non-isoprene-emitting P. × canescens genotype compared with WT, implying dying of the non-isoprene-emitting leaves

The combined salt and sunfleck treatment did not result in changes in ethanol, acetaldehyde and LOX emissions (data not shown), indicating that no destruction of membranes occurred (i.e., as observed in poplars after ozone damage; Behnke et al. 2009). Thus, we searched for stress thresholds that led to the appearance of stress volatiles in WT and RA P. × canescens genotypes. As one option to further analyze the possible roles of isoprene in the thermotolerance of leaves under more severe conditions, we exposed P. × canescens leaves to a constant high leaf temperature of 40 °C. Starting from a temperate leaf temperature of 20 °C (data not shown), followed by an

acclimation at 30 °C (45 min), we then exposed WT and RA leaves to a temperature of 40 °C and monitored the behavior of photosynthetic gas exchange and BVOC emissions for the other 90 min (Figure 5).

The absence of isoprene in the RA line had a serious conse-quence for photosynthetic gas exchange (Figure 5a and b), fol-lowed by a coordinated large release of many BVOCs (Figure 5c–j): in RA leaves, the net CO2 assimilation was already very low after 45 min at 30 °C, and after a few minutes of treat-ment at 40 °C, leaves were no longer assimilating (Figure 5a). RA leaves began with a very strong but transient respiration with a maximum peak after ca. 10 min exposure to 40 °C, which decreased over time to zero as the leaves dried out and began necrosis. At the end of the 40 °C treatment, neither net CO2 assimilation nor respiration was detected (Figure 5a), reflecting death of the RA leaves, which became extensively necrotic (see Figure S3 available as Supplementary Data at Tree Physiology Online) by the end of the experiment. Wild-type leaves main-tained their net CO2 assimilation longer than RA, although the temperature stress induced increasing respiration, annulling the net CO2 assimilation after ca. 30 min exposure to 40 °C and becoming constant 20 min later until the end of the experiment. Transpiration of WT leaves decreased in parallel with the net CO2 assimilation, whereas in RA leaves, an increase in transpiration

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Table 1. List of annotated compounds for clusters (according to Figure 4). For each annotated mass the possible compound name, class name, related pathway, measured mass, chemical formula, error and P value of ANOVA from cluster analysis are given.

Cluster no.

Compound no.

Possible compound name

Class name Related pathways Measured mass

Chemical formula

Error (ppm)

ANOVA P value

1 1 Xanthylic acid Purine nucleotides Biosynthesis of alkaloids derived from purine metabolism

364.041436 C10H13N4O9P −1.59 1.42E-04

1 2 Acetyl-maltose Carbohydrate derivative

Carbohydrate metabolism 384.126676 C14H24O12 −0.26 3.41E-02

2 3 N-Acetyl-L-glutamate Amino acid and derivatives

Amino acid metabolism 189.063757 C7H11NO5 0.19 3.91E-02

2 4 1,3-Bisphospho-D-glycerate

Organic phosphoric acids and derivatives

Glycolysis/gluconeogenesis 265.959574 C3H8O10P2 1.15 8.33E-04

2 5 Sedoheptulose-7P Disaccharides Pentose phosphate pathway 290.040301 C7H15O10P 0.06 3.26E-032 6 Salicin-6P Glycoside

compoundsGlycolysis/gluconeogenesis 366.072280 C13H19O10P 1.91 1.82E-06

2 7 Unknown isomer Flavonoids Flavonoid biosynthesis 366.074015 C20H14O7 0.17 1.82E-063 8 (9Z)-Octadecenoic

acidFatty acids and conjugates

Biosynthesis of unsaturated fatty acids

282.255714 C18H34O2 −0.59 1.19E-02

4 9 Citrate Fatty acids and conjugates

Glyoxylate and dicarboxylate metabolism

192.026996 C6H8O7 −0.03 4.15E-03

4 10 Homocitric acid Carboxylic acids and derivatives

Pyruvate metabolism 206.042655 C7H10O7 0.01 2.50E-03

6 11 Unknown isomer Flavonoids Flavonoid biosynthesis 334.120878 C21H18O4 1.11 7.17E-036 12 Unknown isomer Hopanoids Steroid biosynthesis 528.454280 C35H60O3 0.07 7.17E-037 13 Unknown isomer Sterols Steroid biosynthesis 388.297755 C25H40O3 0.03 4.57E-027 14 Vitamin D3 derivative Sterols Steroid biosynthesis 412.297689 C27H40O3 −0.14 2.53E-027 15 Unknown isomer Flavonoids Flavonoid biosynthesis 432.157622 C26H24O6 0.77 6.59E-047 16 Unknown isomer Sterols Steroid biosynthesis 432.323914 C27H44O4 −0.11 3.72E-028 17 Unknown isomer Flavonoids Flavonoid biosynthesis 462.168133 C27H26O7 0.61 1.75E-04



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was recorded in parallel with changes in the temperature of the leaves (Figure 5b). Congruently with the breakdown of net CO2 assimilation in RA, we observed a transient burst of methanol, ethanol, acetaldehyde, isoprene, monoterpenes and GLV emis-sions, whereas emissions from WT plants remained constant or only changed slowly (Figure 5c–j). Methanol and acetaldehyde emissions (Figure 5c and d) in RA plants reached a very high emission rate of 200–350 nmol m−2 s−1, which is 50–1000 times higher than that for the respective WT leaves. For mono-terpene emission, the difference between WT and RA was pri-marily in the quantity of the emission and not in its transient profile (Figure 5i), indicating a temperature dependence rather than membrane breakdown.

This burst of emissions varied slightly according to the com-pounds, and the maximum values were observed after 30–50 min of exposure to 40 °C. The emission of the LOX products 1 and 2 from the RA plants most likely reflected the oxidation time course of α-linolenic acid (n18 : 3) into the C6 aldehydes (Z)-3-hexenal and (E)-2-hexenal followed by pro-duction of the corresponding alcohols (Z)-3-hexenol, (E)-3-hexenol and (E)-2-hexenol (Figure 5f and h). The emission LOX3 (n-hexanol) originates from the oxidation of linoleic acid (n18 : 2) (Fall et al. 1999) and was detected only in non-iso-prene-emitting leaves, while WT plants showed no hexanol emission under high temperature (Figure 5j). LOX emissions achieved their maximum within ~45 min of exposure to 40 °C


Figure 5. Net CO2 assimilation (a) and transpiration (b) rates, BVOC emissions of methanol (c), acetaldehyde (d), ethanol (e), LOX1 = [(Z)-3-hexenal + (E)-2-hexenal] (f), isoprene (g), LOX2 = [(Z)-3-hexenol + (E)-3-hexenol + (E)-2-hexenol + hexanal] (h), monoterpenes (i), LOX3 = hexanol (j) emission of trans-genic non-isoprene-emitting (red) and WT plants (black). Leaves were adapted at 20 °C and PAR 150 µmol m−2 s−1, then brought to 30 °C and PAR 1000 µml m−2 s−1 for 45 min and then to 40 °C and PAR 1000 µmol m−2 s−1 for 120 min. The start of the treatment in 40 °C is indicated by the black line and the red, dashed line highlights the beginning of the stress response in the form of a high emission phase in the non-isoprene-emitting lines. The given values are 5 min means + SE of four individual plants. The measurements were carried out with PTR-QMS and the isomers could not be separated.



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and then decreased in a similar manner to almost zero again, probably reflecting a death of RA leaves (see also CO2 exchange in Figure 5b). In comparison, the emission of stress-induced BVOCs in the WT did not decay after the initial increase (Figure 5f, h and j), indicating a continuing oxidation process of dam-aged, but not yet dead leaves. Astonishingly, isoprene emission from the non-isoprene-emitting line transiently reached levels similar to those from isoprene-emitting plants (Figure 5g).

Both salt and genotype affect lipid composition

To gain insight into the function of isoprene as a possible mem-brane stabilizing agent, the fatty acid composition of different lipid classes from salt- and sunfleck-treated WT and RA P. × canescens was analyzed. We restricted this analysis to P. × canescens because no transgenic non-isoprene-emitting P. euphratica was available and the suppression of isoprene biosynthesis by fosmidomycin had side effects (data not shown), hindering a conclusive comparison.

In terms of the total fatty acid content, only glycolipids were affected by the salt treatment, which manifested as slightly lower total fatty acid content in the salt-treated RA and WT plants (Figure 6a, b and c). Even if no differences were observed in phospho- and neutral lipid levels within the total amount of lip-ids, all lipid contents revealed differences in unsaturated lipid levels when salt was applied (Figure 6d, e and f). Phospho- and glycolipid levels became slightly decreased, whereas the amount of neutral lipids was enhanced in salt-treated plants. Furthermore,

glycolipids and neutral lipids exhibited significant general differ-ences between treatments in the individual fatty acids n22 : 0 (behenic acid) and n18 : 3 (α-linolenic acid), and phospholipids showed differences in n16 : 1 (palmitoleic or sapienic acid) (Table S1 available as Supplementary Data at Tree Physiology Online). Double bond index analysis did not reveal differences in the unsaturation of phospholipids (Figure 6g); however, the glyco- and neutral lipid levels showed differences in DBI, both due to treatment and due to genotype: the DBI of glycolipids was significantly lower in the salt-treated RA line compared with the RA or WT control lines (Figure 6h), and the DBI of neutral lipids was significantly higher in salt-treated leaves than in the control leaves. Furthermore, the DBI of salt-treated RA plants was higher than that of salt-treated WT plants (Figure 6i).

Discussion

Allocation of recently fixed C to isoprene biosynthesis differs in the two contrasting poplar species

Our results revealed differences in the allocation of photosyn-thetically fixed C for isoprene biosynthesis between the two species without treatments. The 13CO2 feeding revealed differ-ent activities of DMADP biosynthesis in the two species; our results show high DMADP percentages from the cytosol of P. × canescens on the one hand and from chloroplasts of P. euphratica on the other hand. In P. euphratica the high

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Figure 6. Total phospholipids (a), glycolipids (b) and neutral lipids (c), unsaturated phospholipids (d), glycolipids (e) and neutral lipids (f) and DBI of phospholipids (g), glycolipids (h) and neutral lipids (i) measured in the control (white bars) and salt-treated (black bars) P. × canescens that had faced a sequence of six sunflecks. The tested genotypes are denoted below the graph and the given values are the means of three or four indi-vidual samples +SE. RA = P. × canescens PcISPS-RNAi line repressed in isoprene emission. The asterisks indicate significant treatment effects across the genotypes (P < 0.05; two-way ANOVA), while different letters indicate significant differences between the treatments or genotypes (P < 0.05; Tukey’s HSD); n.s. = non-significant differences.



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DMADP content in chloroplasts remained unaffected under salt exposure and constant light, emphasizing the salt tolerance of the species in the form of stable C fixation. In contrast, in salt-sensitive P. × canescens, salt treatment caused a further decrease in the chloroplastidic DMADP content, suggesting a decreased availability of photosynthetically fixed C for the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. It, how-ever, did not lead to decreased isoprene emission rates: the isotope ratios of DMADP and isoprene showed a higher pro-portion of unlabeled DMADP and isoprene compounds in the salt-treated P. × canescens plants than in the control or in P. euphratica. Thus, P. × canescens replaced the photosyntheti-cally fixed C with other sources to sustain isoprene biosynthe-sis, similar to its behavior under CO2 limitation in a low-CO2-enriched atmosphere (Trowbridge et al. 2012). It is likely that in P. × canescens, the proportion of C used for iso-prene biosynthesis from existing reserves, such as sugars and starch, was higher in the salt-treated than in the control trees. Moreover, several previous studies have proposed that in P. × canescens, cross-talk between the mevalonate and the MEP pathway occurs (Lichtenthaler 1999, Laule et al. 2003, Rodríguez-Concepción et al. 2004; Paetzold et al. 2010).

Isoprene function as a thermoprotectant under salinity

In global warming, salinity will be more often combined with other stresses, such as higher temperatures. We combined salt stress with simulated short-term increases of temperature and light (sunflecks) to analyze whether isoprene emission or iso-prene function is affected in salt-tolerant or salt-sensitive pop-lar species. The results revealed an increased isoprene emission capacity under salinity in P. euphratica and in P. × canescens. Considering that isoprene emission decreased in concert with decreasing temperature, the emission is expected to be under enzymatic control (Loreto and Delfine 2000, Teuber et al. 2008), and the high isoprene emission does not imply irreversible changes.

Similar to our results, Loreto and Delfine (2000) and Teuber et al. (2008) found that in isoprene-emitting species, isoprene emission remained unaffected, even if net CO2 assimilation was suppressed under salinity. Indeed, Loreto and Delfine observed in Eucalyptus an increase in isoprene emission under salt exposure, especially when combined with high light and temperature so that the maximum isoprene emission capacity was found at 5 °C higher than in control plants; however, this was true only when photosynthesis and photorespiration of the plants were impaired. In accordance with our results, this study let us suggest that the transiently increased isoprene emission under salinity is possible also under decreased net CO2 assimi-lation. A common observation with all studies investigating the responses to salinity is that salinity results in a reduction in stomatal conductance and typically also results in reductions in the intercellular CO2 concentration (Ci). Thus, from a broader

perspective, our finding of enhanced or invariable isoprene emission under salinity concurs with past observations of enhanced or invariable isoprene emissions in plants with reduced stomatal conductance due to drought or altered ambi-ent CO2 concentration (for a review, see Niinemets et al. 2010). In fact, the optimum CO2 concentration for isoprene emission is reached at a very low Ci, between 90 and 150 µmol mol−1, depending on the DMADP concentration (Sun et al. 2012), and such low Ci values were not reached in our study. Especially in salt-tolerant P. euphratica, the effect of salinity on stomatal conductance and net CO2 assimilation was minor, which might have been caused by reduced salt transport in P. euphratica compared with P. × canescens (Janz et al. 2012). This small change in Ci was also associated with a moderate change in isoprene emissions.

How long-lasting are such high periodic isoprene emissions remains to be elucidated. Furthermore, the C source of this emission remains unclear. If the isoprene emission is still under enzymatic control, the thylakoid-bound isoprene synthase (Schnitzler et al. 2004) could be involved in the higher emis-sion capacity, as was suggested by Loreto and Delfine (2000). Although uncontrolled, it is also possible that isoprene pro-duced in biomembranes (Sharkey and Singsaas 1995, Singsaas et al. 1997, Sharkey et al. 2001) or as a result of the non-enzymatic degradation of DMADP (Ward et al. 2011) was released at higher rates from salt-treated than from control plants when sunflecks were applied.

In the context of plant volatiles and osmotic stresses, drought has so far been studied more often than salinity. Drought is shown to cause somewhat similar (and, thus far, incompletely understood) increases in BVOC emissions under suppressed assimilation as salt stress: for example, Sharkey and Loreto (1993) showed increased isoprene emission rates from kudzu (Pueraria lobata (Willd.) Ohwi) under drought stress. In the herbs rosemary (Rosmarinus officinalis L.) and spearmint (Mentha spi-cata L.), mono- and sesquiterpene emissions increased per leaf area when drought stress was applied (Delfine et al. 2005). Knowing that salt and drought stress are often combined in nature (FAO Soils Bulletin 39), more studies should be per-formed on salt alone and in combination with other connected environmental constraints to increase our knowledge of plant physiology and emission rates and to make more accurate pre-dictions of regional or even global BVOC fluxes.

Despite the slightly increased isoprene emission under salin-ity, isoprene does not play an important role in coping with salt: both WT and transgenic non-isoprene-emitting plants showed similar decreased net CO2 assimilation and transpira-tion rates when treated with salt and additional sunflecks. The photosynthetic performance of the non-isoprene-emitting plants was, however, affected negatively by the end of the treatment as proven by the slightly higher increase in NPQ and decrease in ETR. Apart from these results, the salt-treated WT




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and RA lines performed astonishing similarly, possibly due to metabolic shifts that compensate for the lack of isoprene. Our metabolomics analysis indicated that different metabolites are involved, but unfortunately, only a few of these masses could be reliably annotated by MassTRIX3. In addition, a lipophilic extraction of metabolites was used to identify fatty acids from the same sunfleck-treated samples. The treatment of the plants as well as the extraction method differed from that used by Behnke et al. (2010b), which renders the comparison of the annotated metabolites between the two works difficult. In gen-eral, the scarce annotation of masses in metabolites indicates that many metabolites are currently not yet included in data-bases or have not yet been discovered (Baker 2011, Patti et al. 2012). Annotated metabolites that are regulated by salt primar-ily display changes in C metabolism (e.g., glycolysis/gluconeo-genesis, C fixation and the pentose phosphate pathway), possibly indicating changes in primary energy metabolism to compensate for the increased energy demand under stress (Janz et al. 2012). Furthermore, carbohydrates play roles in salt tolerance by counteracting osmotic stress (Janz and Polle 2012). Additionally, phenolic compounds, well known to have antioxidant properties, were affected by salt and might have been regulated in response to salt stress (Rice-Evans et al. 1997). Changes in carbohydrate and phenolic compound metabolism were also detected when salt-tolerant and salt-sensitive poplar species were compared (Janz et al. 2012). Differences in flavonoids and anthocyanin were also found between the non-isoprene-emitting and WT plants, as observed earlier by Behnke et al. (2010a). The recurrence of this geno-type effect supports a consistent alteration of the phenolic compound metabolism due to missing isoprene, which is enhanced under salty conditions.

Taken together, our results confirm that isoprene plays an important role when poplar overcomes sunflecks, a feature faced regularly by the plant canopy in nature; however, when it comes to coping with salt, isoprene does not offer an impor-tant advantage.

Isoprene as a membrane stabilization agent

Scientists have discussed the possibility that isoprene might function as a stabilizing agent in the hydrophobic interactions between membrane lipid bilayer structures (Sharkey and Singsaas 1995, Singsaas et al. 1997, Garab 2000, Sharkey et al. 2001, Siwko et al. 2007, Velikova et al. 2011). Sharkey and colleagues showed that such stabilization could be achieved with various double bonds, including compounds such as butenes or ethylene (Sharkey et al. 2001). In the present study, the emission of different compounds increased suddenly and drastically when leaves of the non-isoprene-emitting plants began to die upon long-term leaf incubation at 40 °C. Our result supports this hypothesis because such a burst of stress-induced BVOCs can only be explained by

membrane breakage, comparable, but not identical, to the membrane breakage observed after wounding (Fall et al. 1999, Loreto et al. 2006, Brilli et al. 2011, Ghirardo et al. 2012). At the end of 40 °C exposure, dying leaves of non-isoprene-emit-ting plants showed almost no BVOC emissions, whereas LOX emissions from isoprene-emitting plants were still high, prob-ably due to the biochemical activity of lipoxygenases in still living cells. It is interesting to note that emission of LOX prod-ucts between WT and non-isoprene-emitting lines differed also in LOX product composition during the BVOC burst, as the emission from WT lacked the alcohol hexanol (LOX3). The alcohol dehydrogenases (ADH) responsible for converting hexanal into its alcohol hexanol are ubiquitous enzymes in the cell and therefore a deficiency of ADH activity seems unlikely. The different emission patterns might be explained by the dif-ferent fatty acid precursors and the activity of the enzymes responsible for their oxidation. Indeed, the LOX3 (hexanal fam-ily) emissions have a different precursor (linoleic acid) than the LOX1 and LOX2 emissions (originate mostly from α-linolenic acid) (Fall et al. 1999). Moreover, in potato (Solanum tuberosum L.) it has been shown that 13-lipoxygenase prefers α-linolenic acid as the substrate, whereas 9-lipoxygenase rather oxidizes lin-oleic acid (Royo et al. 1999). Whether similar preferences could be found in poplar remains to be examined in future. However, α-linolenic acid is a precursor in several different plants’ stress-induced responses (Feussner and Wasternack 2002) and, thus, it is no surprise that its oxidation products were released from both, high-temperature-treated WT and RA plants. In general, the plant response to acute temperature stress involves membrane dysfunctions due to breakage of fatty acid hydroperoxides and, thus, the release of various compounds such as aldehydes or ethane (Nanaiah and Anderson 1992). Aldehydes, C6 volatiles and their acetates are also typically released due to mechanical damage (Engelberth et al. 2004, Ruther and Furstenau 2005, Hu et al. 2008). Thus, the individual volatiles detected in our study suggest the physical damage of membranes in the non-isoprene-emitting plants under severe temperature stress. The fact that the vola-tile burst was not detected or was lower and occurred later in the isoprene-emitting plants implies a protective role of iso-prene in stabilizing membrane structures. Although an increase of isoprene and LOX products from the hexenal family (Fall et al. 1999) was measured in WT plants, the released volatile level remained high upon the start of emission, indicating dif-ferential regulation of the contributing enzyme activities. Although the source of acetaldehyde emission remains unclear (Graus et al. 2004), the extremely high amount of acetaldehyde produced might originate from fatty acid peroxidation reactions initiated by the accumulation of ROS (Jardine et al. 2009) in the damaged tissues. Wounding-related emissions of isoprene were also observed earlier (e.g., Fall et al. 1999, Brilli et al. 2011); however, the source of such a high emission in

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non- isoprene-emitting lines remains to be investigated further. A possibility could be a non-enzymatic, acid-catalyzed conver-sion of the isoprene precursors DMADP (Brüggemann et al. 2002b) due to the rapid decrease of pH in the chloroplasts’ stroma. Thus, our data collectively allowed us to suggest that the actual stress level sensed by WT plants was lower than that in the non-isoprene-emitting plants, corroborating the role of isoprene emissions in plant stress resistance. It has been pre-viously demonstrated that the stress-elicited emission of LOX products is greater and induced earlier for more severe stress (Beauchamp et al. 2005) and in more stress-vulnerable geno-types (Niinemets 2010a).

The data from Knowles and Knowles (1989) showed an inverse correlation between the DBI and the maximum electro-lyte leakage in polar lipid fatty acids from aged potatoes (S. tuberosum). Such a greater resistance to stress-induced electrolyte leakages could be due to a similar basis to that sug-gested for isoprene, e.g., mechanical membrane stabilization ability or an antioxidative activity of the unsaturated fatty acids. With a modeling approach, Siwko et al. (2007) demonstrated that higher concentrations of isoprene could indeed physically maintain a constant level of membrane cohesiveness. More support for isoprene function in membrane stabilization came from the recent results of Velikova et al. (2011). They show biophysical evidence that isoprene can indeed protect thyla-koid membranes under high temperatures. Velikova and co-workers proved higher integrity and functionality in the thylakoid membranes of isoprene-emitting species compared with that in which isoprene emission was inhibited under high temperatures (Velikova et al. 2011).

To gain deeper insight into the membrane composition of salt- and sunfleck-stressed WT and non-isoprene-emitting P. × canescens, we analyzed the lipid composition of leaves. The results revealed differences in the saturation degree of membrane-bound phospholipids and glycolipids between the salt-stressed and control plants. In the salt-treated plants, the membrane structures involved more saturated fatty acids, implying a decreased plasticity of the membrane structures. The fact that the DBI of glycolipids decreased only in salt-treated RA lines suggests that the changes in membrane struc-tures were more pronounced when no isoprene was present. Interestingly, between the isoprene-emitting and non-isoprene-emitting plants, significant differences were found in the DBI of neutral lipids. Connected to that, in a previous study, we have shown that the malondialdehyde (MDA) concentration in the transgenic lines could be initially elevated compared with the WT (Behnke et al. 2010b). As the lipid saturation is connected to MDA concentration in the leaves (Weber et al. 2004), we suggest that the membrane structures have principally changed due to a lack of isoprene. Furthermore, Mène-Saffrané and col-leagues showed in Arabidopsis that unsaturated fatty acids, i.e., α-linolenic acid (n18 : 3) can have antioxidative capacities

(Mène-Saffrané et al. 2008). Unsaturated lipids could partly take over the suggested role of isoprene as an antioxidant in the transgenic non-isoprene-emitting line. Indeed, when indi-vidual fatty acid levels were analyzed, significant differences were found for α-linolenic acid (n18 : 3) in glycolipids and neu-tral lipids. Although significant differences were found only between salt-treated and control plants, these differences tend to be higher in the non-isoprene-emitting plants. α-Linolenic acid is a direct precursor of jasmonic acid synthesis, and jas-monic acid is involved, among others, in the defense against pathogens (Thomma et al. 1998, Vijayan et al. 1998) and her-bivores (for a review, see, e.g., Heil 2008; Smith et al. 2009). Indeed, Behnke and colleagues showed recently that the non-isoprene-emitting lines have higher resistance to the fungal pathogen Pollaccia radiosa (Lib.) E. Bald & Cif. 1939 but higher susceptibility to the leaf beetle Phratora vitellinae (Linnaeus 1758) (Behnke et al. 2011). Whether changes in lipid composi-tion, the concentration of other antioxidants or ROS are the underlying cause remains to be elucidated.

Conclusions/outlook

The present results, together with our previous results (Behnke et al. 2010a, 2010b, 2011, Way et al. 2011), imply that iso-prene has well-established functions in a narrow window of environmental conditions that are (partly) replaced by other means when no isoprene synthase is present. Overall, isoprene reduces the level of plant stress under high leaf temperatures. Complete recovery of isoprene functions in poplars is, how-ever, not possible, as demonstrated by our transient and long-term high-temperature treatments. The enhanced isoprene emission detected in both poplar species under salinity and sunflecks does not seem to play an important role in the poplar salt tolerance under the given conditions. The high isoprene emission would, however, add more than previously thought to tropospheric chemistry when marginal salty lands are changed to poplar biomass production systems.

Supplementary data

Supplementary data for this article are available at Tree Physiology Online.

Acknowledgments

We would like to thank Violeta Velikova for a critical reading of the manuscript and Christine Kettner (University of Göttingen) for the maintenance and propagation of the plants.

Conflict of interest

None declared.




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Funding

This work was supported by grants from the Estonian Science Agency (grants SF1090065s07 and SF0180045s08), Deutsche Forschungsgemeinschaft (DFG) grants to Poplar Research Group Germany (Schnitzler SCHN653/4 and Polle PO362/12 and PO362/13) and the European Commission through European Regional Fund (Center of Excellence in Environmental Adaptation), as well as by the Human Frontier of Science Program (HFSP; AG, JPS, ÜN).

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Isoprene function in two contrasting poplars under salt and sunflecks