lable at ScienceDirect

Environmental Pollution 157 (2009) 2629–2637

Contents lists avai

Environmental Pollution

journal homepage: www.elsevier .com/locate/envpol

BVOC emissions, photosynthetic characteristics and changesin chloroplast ultrastructure of Platanus orientalis L. exposedto elevated CO2 and high temperature

Violeta Velikova a,*, Tsonko Tsonev a, Csengele Barta b, Mauro Centritto c, Dimitrina Koleva d,Miroslava Stefanova d, Mira Busheva e, Francesco Loreto c

a Bulgarian Academy of Sciences, Institute of Plant Physiology, Acad. G. Bonchev, Bl. 21, 1113 Sofia, Bulgariab US Department of Agriculture, Agricultural Research Center, Arid-Land Agricultural Research Center (USDA, ARS) Maricopa, AZ, USAc Consiglio Nazionale delle Ricerche, Istituto di Biologia Agroambientale e Forestale, 00016 Monterotondo Scalo (RM), Italyd Sofia University, Faculty of Biology, 1000 Sofia, Bulgariae Bulgarian Academy of Sciences, Institute of Biophysics, 1113 Sofia, Bulgaria

Isoprene biosynthesis has a protective role on the photosynthetic mac(i.e., interaction between rising [CO2] and heat wave).

hinery of Platanus plants exposed to changing environment

a r t i c l e i n f o

Article history:Received 27 January 2009Received in revised form27 April 2009Accepted 3 May 2009

Keywords:Carbon dioxideGlobal climate changeHigh temperatureIsoprene emissionThermotolerance

* Corresponding author. Tel.: þ35929792683; fax:E-mail address: violet@bio21.bas.bg (V. Velikova).

0269-7491/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.envpol.2009.05.007

a b s t r a c t

To investigate the interactive effects of increasing [CO2] and heat wave occurrence on isoprene (IE) andmethanol (ME) emissions, Platanus orientalis was grown for one month in ambient (380 mmol mol�1) orelevated (800 mmol mol�1) [CO2] and exposed to high temperature (HT) (38 �C/4 h). In pre-existingleaves, IE emissions were always higher but ME emissions lower as compared to newly-emerged leaves.They were both stimulated by HT. Elevated [CO2] significantly reduced IE in both leaf types, whereas itincreased ME in newly-emerged leaves only. In newly-emerged leaves, elevated [CO2] decreasedphotosynthesis and altered the chloroplast ultrastructure and membrane integrity. These harmful effectswere amplified by HT. HT did not cause any unfavorable effects in pre-existing leaves, which werecharacterized by inherently higher IE rates. We conclude that: (1) these results further prove the iso-prene’s putative thermo-protective role of membranes; (2) HT may likely outweigh the inhibitory effectsof elevated [CO2] on IE in the future.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Isoprene, the most important biogenic volatile organiccompound (BVOC) released by leaves into the atmosphere(Guenther et al., 1995), is a key player in plant–environmentinteractions, as well as in atmospheric reactions. Because isopreneemission (IE) modulates plant tolerance to heat (Sharkey andSingsaas, 1995), pollutant (Loreto and Velikova, 2001), and oxida-tive stresses (Loreto et al., 2001; Velikova et al., 2004), variations inits emission fluxes caused by continuously changing environmentmay significantly alter the biological and physico-chemicalprocesses (Chameides et al., 1988; Grote and Niinemets, 2008).Given the importance of BVOC in the biosphere–atmosphereinteractions, it is essential to understand whether current andfuture environmental changes, which include increased [CO2] and

þ35928739952.

All rights reserved.

incidence of extreme climatic events (Ciais et al., 2005; IPCC, 2007),will affect BVOC emissions by plants.

The effect of CO2 fertilization on primary physiological processesof C3 species, i.e. photosynthesis and stomata movement, is nowfirmly established (Curtis and Wang, 1998; Ainsworth and Long,2005). The stimulation of photosynthesis and the reduction instomata conductance, which result in increased water-useefficiency (Centritto et al., 2002) and delayed leaf senescence(Ainsworth and Long, 2005), are the basis of the beneficial effectsof elevated [CO2] on C3 plant growth, and especially on forest treesthat show the larger growth response to elevated [CO2] (Ainsworthand Long, 2005). In addition, elevated [CO2] also causes a wide rangeof secondary responses, which includes isoprene biosynthesis.In fact, while IE is closely linked to photosynthesis in ambient [CO2],because 72–91% of the carbon in the isoprene molecule originatesfrom recently fixed carbon (Delwiche and Sharkey, 1993; Ferrieriet al., 2005; Brilli et al., 2007) via the methylerythriol 4-phosphate(MEP) chloroplastic pathway (Lichtenthaler, 1999), it has beenshown that, despite the stimulation in photosynthesis, rising [CO2]

Fig. 1. Photograph of 2-year-old Platanus orientalis seedling after 1-month growth at800 mmol mol�1 CO2. Two types of leaves were used in the experiments. Leavesdeveloped and reached maturity during the fumigation with 800 mmol mol�1 CO2 aretermed as ‘‘newly-emerged’’; and leaves already reached their fully expanded statewhen the 800 mmol mol�1 CO2 fumigation was implemented are termed as ‘‘pre-existing’’.

V. Velikova et al. / Environmental Pollution 157 (2009) 2629–26372630

negatively affects IE (Monson and Fall, 1989; Sharkey et al., 1991;Rosenstiel et al., 2003; Centritto et al., 2004; Scholefield et al.,2004). This reduction can be due to restricted availability ofcytosolic phosphoenolpyruvate, necessary for the synthesis in thechloroplast of dimethylallyl-diphosphate, the immediate precursorin isoprene biosynthesis (Rosenstiel et al., 2003; Loreto et al., 2007).

Temperature is another major factor controlling IE. The rate of IEincreases exponentially with temperature, and the trend of thisresponse depends on plant species and on the temperature atwhich plants were grown or exposed (Monson and Fall, 1989;Monson et al., 1992; Sharkey and Loreto, 1993; Filella et al., 2007).Thus, because of the contrasting effects of elevated [CO2] and hightemperature (HT) on IE, predicting plant IE and related changes inadaptive capacity to a changing environment becomes verydifficult.

The interest in studying IE changes is also enhanced because ofits proposed protective role under different stress conditions. Themechanisms by which isoprene protects plants are still unknown.One of the most studied aspects of isoprene protection is thethermotolerance hypothesis introduced for the first time bySharkey and Singsaas (1995), and isoprene thermoprotection wasdemonstrated in different species (Singsaas et al., 1997; Hansonet al., 1999; Velikova and Loreto, 2005).

The objective of the present research was to investigate theimpact of the simultaneous occurrence of rising CO2 and transientheat waves on IE in 2-year-old Platanus orientalis plants. Wefocused on the response of two leaf types developed and maturedin either ambient or elevated [CO2]. We specifically examined therelationships between carbon assimilation, carbon metabolism andIE, and on indicators of ultrastructural damage to chloroplasts afterthe exposure to heat stress. It was hypothesized that (1) theexposure to elevated [CO2] might positively affect photosynthesisand that this could reduce the negative effect of heat on carbonmetabolism; and (2) CO2 enrichment might inhibit isoprenebiosynthesis, which in turn could reduce thermotolerance andworsen the heat stress for photosynthesis.

2. Material and methods

2.1. Plant material and growing conditions

Two-year-old P. orientalis L. plants were grown in 1.5 dm3 pots in a climatechamber under controlled conditions of 350 mmol m�2 s�1 photon flux density,25�C/20 �C day/night temperature, 65% relative humidity, and 12 h photoperiod.Plants were regularly watered to pot water capacity and fertilized once a week withfull-strength Hoagland solution.

2.2. CO2 and high-temperature treatments

Plants were divided into two groups, ten saplings each, and transferred to twoclimatic chambers supplied with two different [CO2] amounts: 380 mmol mol�1

(ambient [CO2]) and 800 mmol mol�1 (elevated [CO2]). To simulate exposure toa heat wave, plants were exposed once to HT (38 �C) for 4 h at the end of 1 month oftreatment with ambient or elevated [CO2] by increasing the temperature in climaticchambers. All analyses were done immediately after 4-h exposure to HT. Two typesof leaves were used in the following experiments. Leaves that had already reachedtheir fully expanded state before the onset of the CO2 fumigations are termed as‘‘pre-existing’’ leaves, while leaves that developed during the CO2 fumigations aretermed as ‘‘newly-emerged’’ leaves (Fig. 1).

2.3. Gas exchange and fluorescence measurements

Steady-state photosynthesis and stomatal conductance (gs) were measured bya portable gas exchange system (LI-6400-40 LiCor, Lincoln, NE, USA) equipped witha fluorescence probe. Measurements were made at the [CO2] of 380 and800 mmol mol�1 on individual leaves enclosed into a leaf cuvette at a rate of0.3 L min�1 air flow, relative humidity within the cuvette at 60–70% and growth-intensity illumination. Gas exchange and chlorophyll fluorescence were measured inunstressed control plants for each [CO2] treatment and on the same plants after theHT treatment. Gas exchange and fluorescence measurements were performed at25 �C for control plants and 38 �C for temperature treated plants. Mesophyll

conductance (gm) to CO2 was calculated by assuming that the electron transportrates calculated by gas exchange and fluorescence match in the absence of internalresistances and photorespiration (Loreto et al., 1992). The actual [CO2] at thechloroplast site (Cc) was then calculated from the gm value as shown elsewhere(Harley et al., 1992).

2.4. VOCs measurements

Isoprene and methanol (ME) emissions from leaves were measured in parallelwith physiological parameters by diverting part of the cuvette outflow to a ProtonTransfer Reaction-Mass Spectrometer (PTR-MS, Ionicon, Innsbruck, Austria), thatallows sensitive quantification of BVOC emissions (Lindinger et al., 1998). The PTR-MS was calibrated before measurements against an isoprene and methanol gasstandards (60 nL L�1) (Rivoira S.p.A. Milan, Italy). Isoprene and methanol weredetected as parent ions at protonated m/z ¼ 69 and 33, respectively (see for detailsLoreto et al., 2006; Filella et al., 2007). Then IE and ME rates (ER) were calculated as:

ER ¼ ðE=26ÞðF=LAÞ

where E is the gas concentration detected by PTR-MS (i.e. the ratio of countnumber s�1 to gas standard), 26 is the gas volume (L) at 30 �C, F is the air flow ratethrough the cuvette clamping a leaf of known area (LA).

2.5. Phosphoenolpyruvate carboxylase (PEPc) activity measurements

Flash frozen 0.2 g midrib-free leaf material was ground in a pre-chilled mortarwith 1.5 ml extraction buffer containing 100 mM HEPES–KOH (pH 7.2), 10 mM DTT,0.3% (w/v) Triton-X 100, 5 mM MgCl2 and PVP-40. Extracts were centrifuged at11 000� g for 5 min at 4 �C. Then 20 ml of the supernatant was immediately used forPEPc activity in an assay mix containing 25 mM Tricine KOH (pH 8.1), 5 mM MgSO4,

Fig. 2. Photosynthesis (a, b) and electron transport rate (c, d) in newly-emerged andpre-existing Platanus orientalis leaves, before (white bars) and after a 4-h heat stress

V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637 2631

5 mM NaHCO3, 5 mM DTT, 0.2 mM NADH and 5 U MDH (malic dehydrogenase). Thereaction was initiated by the addition of 2 mM PEP and NADH. Absorbance changeswere registered for 5 min as 6A340 nm at room temperature, according to Rosenstielet al. (2004). Total sample protein content was determined according to Bradford(1976). Assays were performed on triplicate samples per leaf type and treatment.

2.6. Transmission electron microscopy (TEM)

Leaf segments (1 mm2) were cut from the middle part of the leaves for TEManalyses. These segments were then prefixed in 3% glutaraldehyde (m/v) in 0.1 Msodium phosphate buffer (pH 7.4) for 12 h at 4 �C and postfixed in 2% (m/v) OsO4 inthe same buffer for 4 h at room temperature. After dehydration the material wasembedded in Durcupan (Fluka). Ultra-thin sections were cut from palisade paren-chyma using a Reichert ultra-microtome and were examined using electronmicroscope (JEOL 1200EX, Japan). Leaf segments from five plants of each treatmentwere investigated and at least ten micrographs of different palisade cells were taken.

2.7. Isolation of thylakoid membranes and measurementsof low-temperature fluorescence

Thylakoid membranes were isolated as described by Harrison and Melis (1992)and were suspended in a medium containing 350 mM sorbitol, 5 mM MgCl2, 10 mMNaCl and 10 mM Tris (pH 7.8). Low temperature (77 K) chlorophyll fluorescenceemission spectra were recorded with a Jobin Yvon JY3 spectrofluorimeter (JobinYvon ISA, Longjumeau, France) supplied with a low-temperature device. Additionalmethodology can be found in Apostolova et al. (2006). To determine the relativecontribution of specific chlorophyll–protein complexes to the overall fluorescencepattern, 0.5 mM fluorescein (sodium salt) was added as an internal standard to themedium. The fluorescence spectra were normalized to the same area (100) underthe spectrum (Andreeva et al., 2003). The chlorophyll content of the samples wasadjusted to 10 mg ml�1 (Lichtenthaler, 1987).

2.8. Statistics

All data shown in the figures are means derived from replicates on differentplants. The treatment means were statistically compared by Tukey’s test. Signifi-cantly different means (P < 0.05), derived from 6 to 8 plants per treatment, areshown by different letters among groups.

(38 �C) (hatched bars). Measurements were performed at 350 mmol m�2 s�1 PPFD andgrowing CO2 concentrations (380 or 800 mmol mol�1 CO2). Each data point representsthe mean (�SE) of 6–8 replicates. Means were separated by Tukey’s test and differentletters indicate means that are statistically different with P < 0.05.

3. Results and discussion

3.1. Impact of high temperature on photosynthesis and emissionof volatiles in elevated [CO2]

Photosynthesis and the electron transport rate (ETR) were notsignificantly different in newly-emerged and pre-existing leaves inambient [CO2] (Fig. 2). Growth in elevated [CO2] often stimulatesnet photosynthetic rate in C3 plants due to enhanced supply ofsubstrate to the CO2 fixing enzymes and depression of photores-piration (Moore et al., 1999; Ainsworth and Long, 2005). However,we did not observe a significant stimulation of photosynthesis inpre-existing leaves, whereas photosynthesis of newly-emergedleaves grown in elevated [CO2] was even reduced in comparison tothat measured in ambient [CO2] (Fig. 2a,b). ETR of both types ofleaves was significantly lower in elevated [CO2] (Fig. 2d), due to thereduction of photorespiratory electron transport. CO2 insensitivityor reversed sensitivity can be due to a downward acclimation ofphotosynthetic capacity (Curtis and Wang, 1998; Moore et al.,1999). This process, which is likely related to unbalanced source–sink relations, often occurs when plants exposed to elevated [CO2]are grown in small pots. In our study, the rather small pot volume inwhich the 2-year-old saplings were grown have likely affected thesink size by restricting root growth. This may have induceda decrease in the amount of the photosynthetic pigments andenzymes, for instance amount and activation state of Rubisco and,thus, acclimation of the photosynthetic apparatus (Centritto andJarvis, 1999; Moore et al., 1999; Stitt and Krapp, 1999; Ainsworthand Long, 2005). However, negative feedback on enzymes involvedin sucrose synthesis and transport (Micallef et al., 1996), or directdamage to chloroplast membranes (Cave et al., 1981) may also

cause loss of photosynthetic capacity in C3 species in elevated[CO2].

Moderate heat stress was shown to induce photochemicaldamage, stimulating the reduction of the plastoquinone pool in thedark and increasing cyclic electron flow around PSI in the light(Pastenes and Horton, 1996), and inducing changes in thylakoidstructure that increase membrane leakiness (Havaux et al., 1996). Inour experiments, a 4-h exposure to HT caused a significant declineof photosynthesis and especially in photosynthetic (linear) ETRonly in newly-emerged leaves under elevated [CO2], further indi-cating a downward acclimation of Rubisco amount and/or activa-tion state in elevated [CO2], while it did not significantly affect theseparameters in all other cases (Fig. 2c,d). We hypothesized thatphotosynthesis stimulation in elevated [CO2] could reduce thenegative effect of HT on the photochemical apparatus, mainly bykeeping a high linear ETR. Inefficient photosynthetic ETR contrib-utes to direct oxygen photoreduction and to the accumulation ofdangerous reactive oxygen species in the mesophyll (Sharkey,2005). However, photosynthesis was not stimulated by elevated[CO2] in Platanus plants and our hypothesis could not be success-fully tested.

We verified whether reduction of photosynthesis of newly-emerged leaves in elevated [CO2] after the HT treatment was due todiffusive limitations, i.e. to reduced CO2 availability for photosyn-thesis. As expected (Ainsworth and Long, 2005), gs, which wasclearly higher in newly-emerged than in pre-existing leaves inambient [CO2], was decreased in both type of leaves in response to

V. Velikova et al. / Environmental Pollution 157 (2009) 2629–26372632

elevated [CO2] (Fig. 3a,b). After the HT treatment, gs of newly-emerged leaves grown in ambient [CO2] decreased significantlywhile no changes were observed in pre-existing leaves grown inthe same CO2 level (Fig. 3a). A slight but not significant increase ofgs was observed in both leaf types grown in elevated [CO2] after theHT treatment (Fig. 3b). However, stomatal limitation to photosyn-thesis did not increase during heat stress as revealed by thegenerally steady intercellular CO2 concentration (Ci) (Fig. 3c,d). Inthe case of newly-emerged leaves, in particular, a higher Ci wasobserved, suggesting that biochemical or photochemical

Fig. 3. Stomatal conductance (a, b), intercellular CO2 concentration (c, d), mesophyllconductance (e, f), and chloroplast CO2 concentration (g, h) in newly-emerged and pre-existing Platanus orientalis leaves, before (white bars) and after a 4-h heat stress (38 �C)(hatched bars). Bar assignment, treatments, replications and statistical treatment as inFig. 2.

constraints limited the use of CO2 acquired by the leaves (Flexaset al., 2004). Numerous experiments have shown that CO2 draw-down in the mesophyll can largely reduce CO2 concentrationavailable at chloroplasts. The mesophyll therefore constitutes anadditional diffusive resistance that should be accounted for, espe-cially when limiting photosynthesis, such as in tree species (Loretoet al., 1992), and under stress conditions (Centritto et al., 2003;Flexas et al., 2004).

Mesophyll conductance was indeed strongly reduced in leavesgrowing in elevated [CO2] (Fig. 3e,f). The reduction of gm uponshort-term exposure to elevated [CO2] was observed first in Quercusrubra leaves by Harley et al. (1992) and then repeatedly, e.g. byCentritto et al. (2003) and Flexas et al. (2007). However, acclama-tory responses to high [CO2] showed controversial results.Bernacchi et al. (2005) did not find any significant difference in gm

in soybean under FACE conditions, whereas Singsaas et al. (2003)found a small, but significant effect of elevated [CO2] on gm inalmost all species studied. They also found a linear relationshipbetween gm and photosynthetic capacity. Our finding reveals thatgm reduction in high [CO2] can be an acclimatory response whenplants are grown under enhanced [CO2]. In addition, we alsoshowed that the effect of elevated [CO2] on gm was much higher innewly-emerged leaves, suggesting that gm is more influenced bychanges occurring during leaf development. The anatomicalfeatures are indeed the main determinants of gm (Evans, 1999) andthe impact of CO2 on mesophyll assembly and packaging, andassociated gaseous and liquid phase resistances to CO2 diffusionmay be responsible for the observed acclimatory response. Inter-estingly, the HT treatment associated with growth in elevated [CO2]had contrasting consequences for gm of the two leaf types. Whilethe heat stress slightly reversed the CO2-induced reduction of gm inpre-existing leaves, gm of newly-emerged leaves was furtherreduced. Cc was not affected in ambient [CO2] (Fig. 3g), whereas Cc

of pre-existing leaves was higher than in newly-emerged leaves inelevated [CO2] (Fig. 3h). However, irrespective of the impact of HTon the leaf types, Cc increased in leaves grown in elevated [CO2] andafter the HT treatment (Fig. 3h). This further confirms that diffusivelimitations are not responsible for photosynthesis inhibition causedby HT in the newly-emerged leaves in elevated [CO2].

The second question that we asked was whether CO2-depen-dent inhibition of IE could make worse the impact of HT onphotosynthesis. Isoprene emission was inherently higher in pre-existing than newly-emerged leaves in both CO2 treatments(Fig. 4a,b). IE was stimulated by HT exposure in both leaf types andin both CO2 treatments, but this stimulation was much lower inelevated [CO2] (Fig. 4b) than in ambient [CO2] (Fig. 4a). Indeed ourdata show that newly-emerged leaves in elevated [CO2], which arecharacterized by the lowest IE, were the most sensitive leaves to HT(compare Figs. 2 and 4). Thus, isoprene thermo-protective functionremains once more, though indirectly, confirmed. It is known thatyoung leaves become slowly competent to emit isoprene and emitless than mature leaves (Sharkey and Loreto, 1993; Centritto et al.,2004; Loreto et al., 2007). The lower capacity of young leaves toemit isoprene was attributed to isoprene synthase activity andquantity as the enzyme features developed similarly to the emis-sion rates (Kuzma and Fall, 1993), and it has been suggested that thebiosynthesis of isoprene is developmentally regulated at the levelof gene expression (Mayrhofer et al., 2005; Wiberley et al., 2005).

Growth in elevated [CO2] also caused the expected decrease in IEin both newly-emerged and pre-existing P. orientalis leaves(Fig. 4b). In elevated [CO2] a substantial decline in IE is commonlyobserved (Rosenstiel et al., 2003; Centritto et al., 2004; Scholefieldet al., 2004), even though photosynthesis, which provides the bulkof the carbon needed for isoprene biosynthesis (Sharkey and Yeh,2001), is often stimulated, as previously commented. Such

Fig. 4. Isoprene emission (a, b), PEPc activity (c, d) and methanol emission (e, f) innewly-emerged and pre-existing Platanus orientalis leaves, before (white bars) andafter a 4-h heat stress (38 �C) (hatched bars). Bar assignment, treatments, replicationsand statistical treatment as in Fig. 2.

V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637 2633

a surprising effect of elevated [CO2] was attributed to eithera decrease of isoprene synthase activity (Scholefield et al., 2004), orthe metabolic competition for phosphoenol pyruvate (PEP), thecommon substrate of isoprene biosynthesis and mitochondrialrespiration (Rosenstiel et al., 2003). Rosenstiel et al. (2003, 2004)showed that inhibition of the activity of phosphoenolpyruvatecarboxylase (PEPc), the enzyme that provides substrate for respi-ration and nitrate assimilation, stimulated IE. In our experimentalconditions, PEPc activity was almost two-fold higher in pre-existingleaves compared to newly-emerged leaves in ambient [CO2](Fig. 4c) and, consequently, a direct relationship was generallyfound between IE and PEPc activity when comparing newly-emerged and pre-existing leaves grown in ambient [CO2] (compareFig. 4a,c). This suggests that the lower emission of newly-emergedleaves can also be caused by a low contribution of cytosolic pyru-vate. The lower emission of isoprene in leaves grown in elevated

[CO2] was also associated with a lower activity of PEPc (Fig. 4b,d),thus confirming the hypothesis by Rosenstiel et al. (2003) thatisoprene inhibition in elevated [CO2] may be due to insufficientpyruvate supply. However, in leaves exposed to HT treatment therelationship between IE and PEPc was not straight. In particular, inelevated [CO2] PEPc activity, which was largely stimulated by theHT treatment (Fig. 4d), did not sustain high rates of IE. In contrast,in ambient [CO2], the large increase of IE after the HT treatment wasassociated with reduced PEPc activity (Fig. 4c) with respect to thatmeasured before heat stress. It is therefore suggested that PEPc-generated cytosolic pyruvate may not limit IE or regulate isoprenestimulation upon HT treatments.

The activity of isoprene synthase and IE are temperature-dependent processes (Loreto and Sharkey, 1990; Monson et al.,1992). Consistently, the stimulation of IE after the HT treatment,observed in both newly-emerged and pre-existing leaves, and inboth [CO2] levels, is attributed to higher isoprene synthase activity.It should be noted, however, that the stimulation of IE was lower inelevated [CO2], and especially in newly-emerged leaves. As wediscussed before, the low stimulation of IE may be due to consti-tutively low levels of this enzyme in newly-emerged leaves and/orto inhibition of enzyme activity in elevated [CO2]. It is suggestedthat a down-regulation of isoprene synthase, often occurring uponprolonged exposure to HT (Singsaas and Sharkey, 2000; Loretoet al., 2006) might be also responsible for the loss of temperaturestimulation of IE and could therefore indirectly influence leafsensitivity to HT.

Methanol is a volatile product of pectin demethylation duringcell wall formation (Fall, 2003) in rapidly expanding leaves which isassociated with the expansion of intercellular spaces (Knox et al.,1990), and can be used as a possible marker of leaf development.ME of pre-existing leaves, which already reached their maturitybefore being exposed to elevated [CO2], was consistently lower thanin newly-emerged leaves and was not stimulated by rising [CO2],confirming that leaves were fully developed in both cases (Fig. 4e,f).Nemecek-Marshall et al. (1995) showed that ME is higher ingrowing leaves than in adult leaves. In our study ME was signifi-cantly higher in newly-emerged leaves in elevated [CO2] comparedto those grown in ambient [CO2]. The high rate of pectin deme-thylation and cellular break-down and formation may indeedconfirm that at least in potted plants, elevated [CO2] induces anaccelerated maturation process in leaves, and that newly-emergedleaves in our experimental conditions might have experienced anontogenic shift in elevated [CO2]. Thus, the higher emission ratesobserved in newly-emerged leaves in elevated [CO2] could in factbe lower if leaves were of comparable age with those sampled inambient [CO2].

High-temperature exposure under both growth [CO2] resultedin significantly enhanced ME, this stimulation being more signifi-cant in newly-emerged leaves (Fig. 4e,f) despite the lower gs

(Fig. 3a,b) in elevated [CO2]. This is further evidence that ME is onlypartially controlled by gs, being also related to methanol biosyn-thesis rate (Huve et al., 2007). We therefore attributed the increaseof ME at HT to its enhanced formation, perhaps due to a high rate ofdamage in cell wall structures. The very high emission of methanolin heat stressed newly-emerged leaves grown in elevated [CO2] andemitting low levels of isoprene may therefore additionally indicatethat in the absence of isoprene more damage to cellular structureoccurs.

3.2. Impact of high temperature on chloroplast ultrastructureand functions

Confirmation of the thermo-protective effect of isoprene onphotosynthesis may be associated to more preserved chloroplast

V. Velikova et al. / Environmental Pollution 157 (2009) 2629–26372634

ultrastructure and functionality. Well-differentiated chloroplastscharacterized the mesophyll of pre-existing (Fig. 5a) and newly-emerged leaves (Fig. 5b) at ambient [CO2], containing a well-developed inner membrane system, composed of grana of differentsizes (from 5–10 up to 30 thylakoids) and long stromal thylakoids.Single midsize starch grains were rarely discerned. Investigation ofchloroplast ultrastructure, however, revealed that it was affected bythe [CO2] treatment, especially in newly-emerged leaves. Chloro-plasts of newly-emerged leaves in elevated [CO2] were

Fig. 5. Electron micrographs of Platanus orientalis leaves, illustrating chloroplast structural cunder two regimes of CO2 (380 and 800 mmol mol�1) and after exposure to high temperatureG, grana; ST, stroma thylakoid; S, starch grain; P, peristromium; T, tannins. (A) Intact chloroemerged leaves at ambient CO2 and 25 �C; (C) elevated-CO2-induced structural changes inchloroplast structure of pre-existing leaves; (E) combined effect of elevated CO2 and 38 �C

characterized by an unusually large peristromium located towardthe cell wall without destruction of the inner membrane system(Fig. 5c). These chloroplasts were also in close structural contactwith mitochondria through relatively large typically structuredassociative regions. Changes in the chloroplast structure organi-zation of newly-emerged leaves in elevated [CO2] are unlikelystress-induced traits and rather represent adaptive changes atsubcellular level to our experimental conditions (Mostowska,1997). The increase of stroma volume and the reduction of part of

hanges in leaf mesophyll cells in pre-existing (A, D) and newly-emerged (B, C, E) leaves(38 �C) for 4 h, applied alone and in combination. Scale bars ¼ 500 nm. CH, chloroplast;plast of pre-existing leaves at ambient CO2 and 25 �C; (B) intact chloroplast of newly-newly-emerged leaves at 25 �C; (D) combined effect of elevated CO2 and 38 �C/4 h on/4 h on chloroplast structure of newly-emerged leaves.

V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637 2635

the thylakoid system, accompanied by a reduction of thylakoidelectron density and increased number of plastoglobules, havebeen previously observed after treatments with elevated [CO2](Kutık et al., 1995). It has been hypothesized that these changes inchloroplast ultrastructure in plants grown in elevated [CO2] may beneeded to meet the higher energy demand required for fastergrowth (Griffin et al., 2001). In fact, similar changes were onlyobserved in newly-emerged, fast-growing leaves, and not in pre-existing leaves in our experiment. It should be noted, however, thatthese ultrastructural changes did not cause a stimulation ofphotosynthesis in Platanus leaves.

TEM analysis also showed that exposure to elevated [CO2]caused the accumulation of tannin-like vacuolar deposition in bothtypes (Fig. 5c,d). This was observed both in newly-emerged andpre-existing leaves and therefore is not associated to growthrequirements. Increased tannin depositions were also observed inaspen plants exposed to increasing [CO2], suggesting a [CO2]-induced activation of the phenylpropanoid pathway (Oksanenet al., 2001), with possible but yet largely unknown protectivefunctions at cellular level.

Four-hour HT treatment did not cause any structural changes inthe chloroplasts of both types of leaves grown in ambient [CO2](data not shown). However, in leaves grown in elevated [CO2] theHT exposure affected chloroplast ultrastructure of both leaf types.HT treatment of pre-existing leaves grown in elevated [CO2] causedthe accumulation of starch and structural changes of thylakoidmembranes that became wavy (Fig. 5d). A large part of chloroplastswas occupied by 1–2 big starch grains, in which zones of saccha-rides and capsule, containing enzymes, were not typically struc-tured. Starch accumulation in elevated [CO2] occurs when theproduction of new assimilates is larger than the capacity to handlethem (Stitt and Krapp, 1999). Normally, starch accumulation inleaves, by maintaining the Pi cycling, allows photosynthesis tocontinue (Stitt and Krapp, 1999). However, when the source–sinkrelation is disrupted, starch accumulation may become excessiveand Pi cycling results progressively inhibited; this is, in turn, usuallyrelated to feedback inhibition of photosynthesis (Pritchard et al.,1997). The thylakoid membranes of the same chloroplast were

Fig. 6. 77 K emission spectra of thylakoids isolated from: (a) newly-emerged leaves at ambie[CO2]; (d) pre-existing leaves at elevated [CO2].

wavelike and parts of stroma thylakoids were fragmented. Severalstudies showed that heat stress and elevated [CO2] can causethylakoid destruction and starch accumulation in woody andherbaceous plants (Mostowska, 1997; Oksanen et al., 2001; Samet al., 2001; Lambreva et al., 2005).

Starch did not accumulate in the chloroplasts of newly-emergedleaves, probably because photosynthesis of these leaves wasimpaired before starch synthesis. In fact, when newly-emergedleaves grown in elevated [CO2] were exposed to HT, a destruction ofpart of the peripheral thylakoid membranes was observed (Fig. 5e).The unusually large peristromium was filled with membranesubstance from fragmented peripheral thylakoids with swollenintrathylacoidal space (Fig. 5e, arrows). This is a typical ultra-structural damage induced by HT. Kislyuk et al. (2004) also foundthat a large volume of the stroma was occupied by sinuous thyla-koids and fragments of thylakoid membranes in response to 30 minheating at 42 �C under high light. We speculate that the negativeimpact of HT on membrane structure of newly-emerged leaves thatemit the lowest level of isoprene indicates that isoprene is indeedinvolved in a mechanism of thylakoid membrane protection, aspreviously hypothesized (Sharkey and Singsaas, 1995; Singsaaset al., 1997) and theoretically recently predicted (Siwko et al., 2007).Isoprene lipophilic properties may actually allow easy insertion ofthe molecule in thylakoidal membranes that are strengthened bysuch an insertion. However, localization of isoprene into chloro-plast membranes is needed to conclusively prove the proposedmechanism of action.

77 K chlorophyll fluorescence emission spectra were measuredin order to understand if isoprene presence can modulate theintegrity and energy transfer between both photosystems andwhether it can protect photosynthetic apparatus against HTinjuries. These analyses revealed that lower IE in newly-emergedleaves also considerably affected the functionality and integrity ofpigment–protein complexes even under control conditions. Pre-existing and newly-emerged leaves grown at both [CO2] regimeshad well-defined F685 and F735 peaks (Fig. 6). The differencesoccurred in amplitude ratio F735/F685 (Table 1), used to charac-terize the direct energy transfer from PSII to PSI complexes

nt [CO2]; (b) pre-existing leaves at ambient [CO2]; (c) newly-emerged leaves at elevated

Table 177 K fluorescence ratio (F735/F685) of thylakoids from newly-emerged and pre-existing leaves of P. orientalis grown at ambient or elevated [CO2] at 25 �C and afterexposure to 38 �C for 4 h. Statistically significant differences (P < 0.05) amonggroups are shown by different letters.

Treatments 25 �C 38 �C

Newly-emerged Pre-existing Newly-emerged Pre-existing

Ambient [CO2] 1.03 � 0.02b 1.43 � 0.03a 1.05 � 0.05b 1.56 � 0.07a

Elevated [CO2] 0.74 � 0.02d 0.78 � 0.02d 1.04 � 0.09b 0.86 � 0.03c

V. Velikova et al. / Environmental Pollution 157 (2009) 2629–26372636

(Strasser and Butler, 1977; Andreeva et al., 2003; Apostolova et al.,2006) or arrangement of chlorophyll–protein complexes, i.e. PSI/PSII stoichiometry (Andreeva et al., 2003). In ambient [CO2] the PSI/PSII ratio was around 1.43 (typical for higher plants) in pre-existingleaves, while it was significantly lower (1.03) in newly-emergedleaves (Fig. 6a,b; Table 1). The lower PSI/PSII ratio demonstrateda reduction in energy transfer to PSI. This observation could berelated to differences in maturity of chloroplast protein structurebetween both types of leaves (Kutık, 1998). When plants wereexposed to elevated [CO2], which in turn caused a significantreduction of IE (Fig. 4b), the ratio F735/F685 was also largelyreduced (Fig. 6c,d; Table 1). Such destructive changes could be dueto loss of PSI pigment–protein complexes or strong aggregation ofLHCs. It was shown (Tang et al., 2005) that the total chlorophyllfluorescence yields of PSI and PSII decreased significantly withsenescence progressing and the decrease in the yield of PSI wasgreater than the yield of PSII, suggesting that the rate of degrada-tion in chlorophyll–protein complexes of PSI was greater than inchlorophyll–protein complexes of PSII. Indeed, in both types ofleaves exposure to elevated [CO2] caused an increase of the relativeemission from PSII at about 685 nm which was accompanied bya simultaneous decrease of emission from PSI complex (Fig. 6c,d).Isoprene inhibition at elevated [CO2] combined with HT exposureconsiderably enhanced the energy distribution in favor of PSI(Fig. 6c,d), and the ratio F735/F685 significantly increased, espe-cially in newly-emerged leaves (Table 1). At HT the relative amountof emitted PSII fluorescence declined while the contribution fromPSI emission increased (Fig. 6c,d). Changes in PSII and PSI overallemission and the electron transfer between them are attributed tothe differential status of membrane stacking and redistribution ofprotein–pigment complexes (Stoichkova et al., 2006).

4. Conclusions

Heat treatment alone did not cause any unfavorable effects onphotosynthesis and chloroplast ultrastructure of pre-existingleaves, which are characterized by significantly higher isopreneemission. The growth-promoting effect of elevated [CO2] was not asclear as expected, but in agreement with our first hypothesis,elevated [CO2] was able to counteract the negative impact of HTstress in pre-existing leaves. However, HT induced a considerabledecrease of photosynthesis and ETR, increase of ME and changes ofthe chloroplast ultrastructure and membrane integrity in newly-emerged leaves, especially in elevated [CO2] environment. Thissupports our second hypothesis that leaves with inhibited IE aremore sensitive to heat stress. Thus, in a warmer and elevated [CO2]environment, Platanus orientalis is likely be more sensitive toclimate-induced damage than in the present climate. This investi-gation also confirms that IE rate is uncoupled from photosynthesisand it is likely to be reduced under future elevated [CO2] levels. Onthe other hand, elevated [CO2] by stimulating leaf growth mayincrease ME. Moreover, HT not only increases ME, but is likely tocontrast the inhibitory effects of elevated [CO2] on IE, confirmingthat temperature is the main factor affecting the rate of BVOC

emissions. We believe that these findings may be useful for a betterparameterization of large-scale emission models on the impact ofBVOC emissions on air quality on regional and global scales.

Acknowledgments

This research was funded by a NATO Reintegration Grant (No981279), by the National Science Fund (contract TKB-1604/2006),by the Network of Excellence for Atmospheric composition Change‘‘ACCENT’’, and by a bilateral project within the framework agree-ment between Italian National Research Council and BulgarianAcademy of Sciences.

References

Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air CO2enrichment (FACE)? A meta-analytic review of the responses of photosynthesis,canopy properties and plant production to rising CO2. New Phytologist 165,351–372.

Andreeva, A., Stoitchkova, K., Busheva, M., Apostolova, E., 2003. Changes in theenergy distribution between chlorophyll–protein complexes of thylakoidmembranes from pea mutants with modiWed pigment content I. Changes dueto the modiWed pigment content. Journal of Photochemistry and PhotobiologyB: Biology 70, 153–162.

Apostolova, E.L., Dobrikova, A.G., Ivanova, P.I., Petkanchin, I.B., Taneva, S.G., 2006.Relationship between the organization of the PSII supercomplex and thefunctions of the photosynthetic apparatus. Journal of Photochemistry andPhotobiology B: Biology 83, 114–122.

Bernacchi, C.J., Morgan, P.B., Ort, D.R., Long, S.P., 2005. The growth of soybean underfree air [CO2] enrichment (FACE) stimulates photosynthesis while decreasing invivo Rubisco capacity. Planta 220, 434–446.

Bradford, M.B., 1976. A rapid and sensitive method for the quantification ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry 72, 248–254.

Brilli, F., Barta, C., Fortunati, A., Lerdau, M., Loreto, F., Centritto, M., 2007. Response ofisoprene emission and carbon metabolism to drought in white poplar (Populusalba) saplings. New Phytologist 175, 244–254.

Cave, G., Tolley, L.C., Strain, B.R., 1981. Effect of carbon dioxide enrichment onchlorophyll content, starch content and starch grain structure in Trifoliumsubterraneum leaves. Physiologia Plantarum 51, 171–174.

Centritto, M., Lucas, M.E., Jarvis, P.G., 2002. Gas exchange, biomass, whole-plantwater-use efficiency and water uptake of peach (Prunus persica) seedlings inresponse to elevated carbon dioxide concentration and water availability. TreePhysiology 22, 699–706.

Centritto, M., Loreto, F., Chartzoulakis, K., 2003. The use of low [CO2] to estimatediffusional and non-diffusional limitations of photosynthetic capacity of salt-stressed olive saplings. Plant, Cell and Environment 26, 585–594.

Centritto, M., Nascetti, P., Petrilli, L., Raschi, A., Loreto, F., 2004. Profiles of isopreneemission and photosynthetic parameters in hybrid poplars exposed to free-airCO2 enrichment. Plant, Cell and Environment 27, 403–412.

Centritto, M., Jarvis, P.G., 1999. Long-term effects of elevated carbon dioxideconcentration and provenance on four clones of Sitka spruce (Picea sitchensis) II.Photosynthetic capacity and nitrogen use efficiency. Tree Physiology 19,807–814.

Chameides, W.L., Lindsay, R.W., Richardson, J., Kiang, C.S., 1988. The role of biogenichydrocarbons in urban photochemical smog: Atlanta as a case study. Science241, 1473–1475.

Ciais, P.H., Reichstein, M., Viovy, N., Granier, A., Ogee, J., Allard, V., Aubinet, M.,Buchmann, N., Bernhofer, Chr., Carrara, A., Chevallier, F., De Noblet, N.,Friend, A.D., Friedlingstein, P., Grunwald, T., Heinesch, B., Keronen, P., Knohl, A.,Krinner, G., Loustau, D., Manca, G., Matteucci, G., Miglietta, F., Ourcival, J.M.,Papale, D., Pilegaard, K., Rambal, S., Seufert, G., Soussana, J.F., Sanz, M.J.,Schulze, E.D., Vesala, T., Valentini, R., 2005. Europe-wide reduction in primaryproductivity caused by the heat and drought in 2003. Nature 437, 529–533.

Curtis, P.S., Wang, X., 1998. A meta-analysis of elevated CO2 effects on woody plantmass, form and physiology. Oecologia 113, 299–313.

Delwiche, C.F., Sharkey, T.D., 1993. Rapid appearance of 13C in biogenic isoprenewhen 13CO2 is fed to intact leaves. Plant, Cell and Environment 16, 587–591.

Evans, J.R., 1999. Leaf anatomy enables more equal access to light and CO2 betweenchloroplasts. New Phytologist 143, 93–104.

Fall, R., 2003. Abundant oxygenates in the atmosphere: a biochemical perspective.Chemical Reviews 103, 4941–4951.

Ferrieri, R.A., Gray, D.W., Babst, B.A., Schueller, M.J., Schlyer, D.J., Thorpe, M.R.,Orians, C.M., Lerdau, M., 2005. Use of carbon11 in Populus shows that exogenousjasmonic acid increases biosynthesis of isoprene from recently fixed carbon.Plant, Cell and Environment 28, 591–602.

Filella, I., Wilkinson, M.J., Llusia, J., Hewitt, N., Penuelas, J., 2007. Volatile organiccompounds emissions in Norway spruce (Picea abies) in response to tempera-ture changes. Physiologia Plantarum 130, 58–66.

V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637 2637

Flexas, J., Bota, J., Loreto, F., Cornic, G., Sharkey, T.D., 2004. Diffusive and metaboliclimitations to photosynthesis under drought and salinity in C-3 plants. PlantBiology 6, 269–279.

Flexas, J., Diaz-Espejo, A., Galmes, J., Kaldenhoff, R., Medrano, H., Ribas-Carbo, M.,2007. Rapid variations of mesophyll conductance in response to changes in CO2concentration around leaves. Plant, Cell and Environment 30, 1284–1298.

Griffin, K.L., Anderson, O.R., Gastrich, M.D., Lewis, J.D., Lin, G., Schuster, W.,Seemann, J.R., Tissue, D.T., Turnbull, M.H., Whitehead, D., 2001. Plant growth inelevated CO2 alters mitochondrial number and chloroplast fine structure.Proceedings of the National Academy of Sciences of USA 98, 2473–2478.

Grote, R., Niinemets, U, 2008. Modeling volatile isoprenoid emissions – a story withsplit ends. Plant Biology 10, 8–28.

Guenther, A., Hewitt, C.N., Erickson, D., Fall, R., Geron, C., Graedel, T., Harley, P.,Klinger, L., Lerdau, M., McKay, W., Pierce, T., Scholes, B., Steinbrecher, R.,Tallamraju, R., Taylor, J., Zimmerman, P., 1995. A global model of natural volatileorganic compound emissions. Journal Geophysical Research 100, 8873–8892.

Hanson, D.T., Swanson, S., Graham, L.E., Sharkey, T.D., 1999. Evolutionary signifi-cance of isoprene emission from mosses. American Journal of Botany 86,634–639.

Harley, P.C., Loreto, F., Di Marco, G., Sharkey, T.D., 1992. Theoretical considerationswhen estimating the mesophyll conductance to CO2 flux by analysis of theresponse of photosynthesis to CO2. Plant Physiology 98, 1429–1436.

Harrison, M.A., Melis, A., 1992. Organization and stability of polypeptides associatedwith chlorophyll a–b light-harvesting complex of photosystem II. Plant and CellPhysiology 33, 627–637.

Havaux, M., Tardy, F., Ravenel, J., Chanu, D., Parot, P., 1996. Thylakoid membranestability to heat stress studied by flash spectroscopic measurements of theelectrochromic shift in intact potato leaves: influence of the xanthophyllcontent. Plant, Cell and Environment 19, 1359–1368.

Huve, K., Christ, M.M., Kleist, E., Uerlings, R., Niinemets, U., Walter, A., Wild, J., 2007.Simultaneous growth and emission measurements demonstrate an interactivecontrol of methanol release by leaf expansion and stomata. Journal of Experi-mental Botany 58, 1783–1793.

IPCC, 2007. Climate change 2007: the physical science basis. In: Solomon, S., Qin, D.,Manning, M., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.),Contribution of Working Group I to the Fourth Assessment Report of theIntergovernmental Panel on Climate Change. Cambridge University Press,Cambridge, United Kingdom and New York, NY, USA.

Kislyuk, I.M., Bubolo, L.S., Paleeva, T.V., Sherstneva, O.A., 2004. Heat-inducedincrease in the tolerance of the wheat photosynthetic apparatus to combinedaction of high temperature and visible light: CO2 fixation, photosyntheticpigments and chloroplast ultrastructure. Russian Journal of Plant Physiology 51,455–463.

Knox, J.P., Linstead, P.J., King, J., Cooper, C., Roberts, K., 1990. Pectin esterification isspatially regulated both within cell walls and between developing tissues ofroot apices. Planta 181, 512–521.

Kutık, J., 1998. The development of chloroplast structure during leaf ontogeny.Photosynthetica 35, 481–505.

Kutık, J., Natr, L., Demmers-Derks, H.H., Lawlor, D.W., 1995. Chloroplast ultrastruc-ture of sugar beet (Beta vulgaris L.) cultivated in normal and elevated CO2concentrations with two contrasted nitrogen supplies. Journal of ExperimentalBotany 46, 1797–1802.

Kuzma, J., Fall, R., 1993. Leaf isoprene emission rate is dependent on leaf develop-ment and the level of isoprene synthase. Plant Physiology 101, 435–440.

Lambreva, M., Stoyanova-Koleva, D., Baldjiev, G., Tsonev, T., 2005. Early acclimationchanges in the photosynthetic apparatus of bean plants during short-termexposure to elevated CO2 concentration under high temperature and lightintensity. Agriculture, Ecosystems and Environment 106, 219–232.

Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids – pigments of photosyn-thetic membranes. Methods of Enzymology 148, 350–382.

Lichtenthaler, H.K., 1999. The 1-deoxy-D-xylulose-5-phosphate pathway of iso-prenoid biosynthesis in plants. Annual Reviews Plant Physiology Plant Molec-ular Biology 50, 47–65.

Lindinger, W., Hansel, A., Jordan, A., 1998. Proton-transfer-reaction mass spec-trometry (PTR-MS): on-line monitoring of volatile organic compounds at pptvlevels. Chemical Society Reviews 27, 347–354.

Loreto, F., Harley, P.C., Di Marco, G., Sharkey, T.D., 1992. Estimation of mesophyllconductance to CO2 flux by different methods. Plant Physiology 98, 1437–1443.

Loreto, F., Mannozzi, M., Maris, C., Nascetti, P., Ferranti, F., Pasqualini, S., 2001. Ozonequenching properties of isoprene and its antioxidant role in plants. PlantPhysiology 126, 993–1000.

Loreto, F., Barta, C., Brilli, F., Nogues, I., 2006. On the induction of volatile organiccompound emissions by plants as consequence of wounding or fluctuations oflight and temperature. Plant, Cell and Environment 29, 1820–1828.

Loreto, F., Centritto, M., Barta, C., Calfapietra, C., Fares, S., Monson, R.K., 2007. Therelationship between isoprene emission rate and dark respiration rate in whitepoplar (Populus alba L.) leaves. Plant, Cell and Environment 30, 662–669.

Loreto, F., Sharkey, T.D., 1990. A gas-exchange study of photosynthesis and isopreneemission in Quercus rubra L. Planta 182, 523–531.

Loreto, F., Velikova, V., 2001. Isoprene produced by leaves protects the photosyn-thetic apparatus against ozone damage, quenches ozone products, and reduceslipid peroxidation of cellular membranes. Plant Physiology 127, 1781–1787.

Mayrhofer, S., Teuber, M., Zimmer, I., Louis, S., Fischbach, R.J., Schnitzler, J.-P., 2005.Diurnal and seasonal variation of isoprene biosynthesis-related genes in greypoplar leaves. Plant Physiology 139, 474–484.

Micallef, B.J., Vanderveer, P.J., Sharkey, T.D., 1996. Responses to elevated CO2 ofFlaveria linearis plants having a reduced activity of cytosolic fructose-1,6-bisphosphatase. Plant, Cell and Environment 19, 10–16.

Monson, R.K., Jaeger, C.H., Adams III, W.W., Driggers, E.M., Silver, G.M., Fall, R., 1992.Relationships among isoprene emission rate, photosynthesis, and isoprenesynthase activity as influenced by temperature. Plant Physiology 98, 1175–1180.

Monson, R.K., Fall, R.R., 1989. Isoprene emission from aspen leaves – influence ofenvironment and relation to photosynthesis and photorespiration. Plant Phys-iology 90, 267–274.

Moore, B.D., Cheng, S.-H., Sims, D., Seemann, J.R., 1999. The biochemical andmolecular basis for photosynthetic acclimation to elevated atmospheric CO2.Plant, Cell and Environment 22, 567–582.

Mostowska, A., 1997. Environmental factors affecting chloroplasts. In: Pessarakli, M.(Ed.), Handbook of Photosynthesis. Marcel Dekker, New York, pp. 407–426.

Nemecek-Marshall, M., MacDonald, R.C., Franzen, J.J., Wojciechowski, C.L., Fal, R.,1995. Methanol emission from leaves. Enzymatic detection of gas-phasemethanol and relation of methanol fluxes to stomatal conductance and leafdevelopment. Plant Physiology 108, 1359–1361.

Oksanen, E., Sober, J., Karnosky, D.F., 2001. Impact of elevated CO2 and/or O3 on leafultrastructure of aspen (Populus tremuloides) and birch (Betula papyrifera) in theAspen FACE experiment. Environmental Pollution 115, 437–446.

Pastenes, C., Horton, P.,1996. Effect of high temperature on photosynthesis in beans. I.Oxygen evolution and chlorophyll fluorescence. Plant Physiology 112,1245–1251.

Pritchard, S.G., Peterson, C.M., Prior, S.A., Rogers, H.H., 1997. Elevated atmosphericCO2 differentially affects needle chloroplast ultrastructure and phloem anatomyin Pinus palustris: interactions with soil resource availability. Plant, Cell andEnvironment 20, 461–471.

Rosenstiel, T.N., Potosnak, M.J., Griffin, K.L., Fall, R., Monson, R.K., 2003. IncreasedCO2 uncouples growth from isoprene emission in an agriforest ecosystem.Nature 421, 256–259.

Rosenstiel, T.N., Ebbets, A.L., Khatri, W.C., Fall, R., Monson, R.K., 2004. Induction ofpoplar leaf nitrate reductase: a test of extrachloroplastic control of isopreneemission rate. Plant Biology 6, 12–21.

Sam, O., Nunez, M., Ruiz-Sanchez, M.C., Dell’Amico, J., Falcon, V., de la Rosa, M.C.,Seoane, J., 2001. Effect of brassinosteroid analogue and high temperature stresson leaf ultrastructure of Lycopersicon esculentum. Biologia Plantarum 44 (2),213–218.

Scholefield, P.A., Doick, K.J., Herbert, B.M.J., Hewitt, C.N.S., Schnitzler, J.-P., Pinelli, P.,Loreto, F., 2004. Impact of rising CO2 on emissions of volatile organiccompounds: isoprene emission from Phragmites australis growing at elevatedCO2 in a natural carbon dioxide spring. Plant, Cell and Environment 27, 393–401.

Sharkey, T.D., 2005. Effects of moderate heat stress on photosynthesis: importanceof thylakoid reactions, rubisco deactivation, reactive oxygen species, and ther-motolerance provided by isoprene. Plant, Cell and Environment 28, 269–277.

Sharkey, T.D., Loreto, F., Delwiche, C.F., 1991. High carbon dioxide and sun/shadeeffects on isoprene emission from oak and aspen tree leaves. Plant, Cell andEnvironment 14, 333–338.

Sharkey, T.D., Loreto, F., 1993. Water stress, temperature, and light effects on thecapacity for isoprene emission and photosynthesis of kudzu leaves. Oecologia95, 328–333.

Sharkey, T.D., Singsaas, E.L., 1995. Why plants emit isoprene. Nature 374, 769.Sharkey, T.D., Yeh, S., 2001. Isoprene emission from plants. Annual Review Plant

Physiology Plant Molecular Biology 52, 407–436.Singsaas, E.L., Lerdau, M., Winter, K., Sharkey, T.D., 1997. Isoprene increases ther-

motolerance of isoprene-emitting species. Plant Physiology 115, 1413–1420.Singsaas, E.L., Ort, D., DeLucia, E., 2003. Elevated CO2 effects on mesophyll

conductance and its consequences for interpreting photosynthetic physiology.Plant, Cell and Environment 27, 41–50.

Singsaas, E.L., Sharkey, T.D., 2000. The effects of high temperature on isoprenesynthesis in oak leaves. Plant, Cell and Environment 23, 751–757.

Siwko, M.E., Marrink, S.J., de Vries, A.H., Kozubek, A., Uiterkamp, A.J.M.S., Mark, A.E.,2007. Does isoprene protect plant membranes from thermal shock? A molec-ular dynamics study. Biochimica et Biophysica Acta 1768, 198–206.

Stitt, M., Krapp, A., 1999. The interaction between elevated carbon dioxide andnitrogen nutrition: the physiological and molecular background. Plant, Cell andEnvironment 22, 583–621.

Stoichkova, K., Busheva, M., Apostolova, E., Andreeva, A., 2006. Changes in energydistribution between chlorophyll–protein complexes of thylakoid membranesfrom pea mutants with modified pigment content. II. Changes due to magne-sium ions concentration. Journal Photochechemistry and Photobiology B:Biology 83, 11–20.

Strasser, R.J., Butler, W.L., 1977. Fluorescence emission spectra of photosystem I,photosystem II and the light-harvesting chlorophyll a/b complex of higherplants. Biochimica Biophysica Acta 462, 307–313.

Tang, Y., Wen, X., Lu, C., 2005. Differential changes in degradation of chlorophyll–protein complexes of photosystem I and photosystem II during flag leafsenescence of rice. Plant Physiology Biochemistry 43, 193–201.

Velikova, V., Edreva, A., Loreto, F., 2004. Endogenous isoprene protects Phragmitesaustralis leaves against singlet oxygen. Physiologia Plantarum 122, 219–225.

Velikova, V., Loreto, F., 2005. On the relationship between isoprene emission andthermotolerance in Phragmites australis leaves exposed to high temperatures andduring the recovery from a heat stress. Plant, Cell and Environment 28, 318–327.

Wiberley, A.E., Linskey, A.R., Falbel, T.G., Sharkey, T.D., 2005. Development of thecapacity for isoprene emission in kudzu. Plant, Cell and Environment 28,898–905.