Environmental Pollution 157 (2009) 2629–2637

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Environmental Pollution

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BVOC emissions, photosynthetic characteristics and changes in chloroplast ultrastructure of orientalis L. exposed to 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 Physiology, Acad. G. Bonchev, Bl. 21, 1113 Sofia, Bulgaria b US Department of Agriculture, Agricultural Research Center, Arid-Land Agricultural Research Center (USDA, ARS) Maricopa, AZ, USA c Consiglio Nazionale delle Ricerche, Istituto di Biologia Agroambientale e Forestale, 00016 Monterotondo Scalo (RM), Italy d Sofia University, Faculty of Biology, 1000 Sofia, Bulgaria e Bulgarian Academy of Sciences, Institute of Biophysics, 1113 Sofia, Bulgaria Isoprene biosynthesis has a protective role on the photosynthetic machinery of Platanus exposed to changing environment

(i.e., interaction between rising [CO2] and heat wave). article info abstract

Article history: To investigate the interactive effects of increasing [CO2] and heat wave occurrence on isoprene (IE) and Received 27 January 2009 methanol (ME) emissions, Platanus orientalis was grown for one month in ambient (380 mmol mol1)or Received in revised form 1 elevated (800 mmol mol ) [CO2] and exposed to high temperature (HT) (38 C/4 h). In pre-existing 27 April 2009 , IE emissions were always higher but ME emissions lower as compared to newly-emerged leaves. Accepted 3 May 2009 They were both stimulated by HT. Elevated [CO2] significantly reduced IE in both types, whereas it increased ME in newly-emerged leaves only. In newly-emerged leaves, elevated [CO2] decreased Keywords: photosynthesis and altered the chloroplast ultrastructure and membrane integrity. These harmful effects Carbon dioxide Global climate change were amplified by HT. HT did not cause any unfavorable effects in pre-existing leaves, which were High temperature characterized by inherently higher IE rates. We conclude that: (1) these results further prove the iso- Isoprene emission prene’s putative thermo-protective role of membranes; (2) HT may likely outweigh the inhibitory effects Thermotolerance of elevated [CO2] on IE in the future. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction incidence of extreme climatic events (Ciais et al., 2005; IPCC, 2007), will affect BVOC emissions by plants. Isoprene, the most important biogenic volatile organic The effect of CO2 fertilization on primary physiological processes compound (BVOC) released by leaves into the atmosphere of C3 species, i.e. photosynthesis and stomata movement, is now (Guenther et al., 1995), is a key player in plant–environment firmly established (Curtis and Wang, 1998; Ainsworth and Long, interactions, as well as in atmospheric reactions. Because isoprene 2005). The stimulation of photosynthesis and the reduction in emission (IE) modulates plant tolerance to heat (Sharkey and stomata conductance, which result in increased water-use Singsaas, 1995), pollutant (Loreto and Velikova, 2001), and oxida- efficiency (Centritto et al., 2002) and delayed leaf senescence tive stresses (Loreto et al., 2001; Velikova et al., 2004), variations in (Ainsworth and Long, 2005), are the basis of the beneficial effects its emission fluxes caused by continuously changing environment of elevated [CO2]onC3 plant growth, and especially on forest may significantly alter the biological and physico-chemical that show the larger growth response to elevated [CO2](Ainsworth processes (Chameides et al., 1988; Grote and Niinemets, 2008). and Long, 2005). In addition, elevated [CO2] also causes a wide range Given the importance of BVOC in the biosphere–atmosphere of secondary responses, which includes isoprene biosynthesis. interactions, it is essential to understand whether current and In fact, while IE is closely linked to photosynthesis in ambient [CO2], future environmental changes, which include increased [CO2] and because 72–91% of the carbon in the isoprene molecule originates from recently fixed carbon (Delwiche and Sharkey, 1993; Ferrieri et al., 2005; Brilli et al., 2007) via the methylerythriol 4-phosphate

* Corresponding author. Tel.: þ35929792683; fax: þ35928739952. (MEP) chloroplastic pathway (Lichtenthaler, 1999), it has been E-mail address: [email protected] (V. Velikova). shown that, despite the stimulation in photosynthesis, rising [CO2]

0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.05.007 2630 V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637 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 of cytosolic phosphoenolpyruvate, necessary for the synthesis in the chloroplast of dimethylallyl-diphosphate, the immediate precursor in isoprene biosynthesis (Rosenstiel et al., 2003; Loreto et al., 2007). Temperature is another major factor controlling IE. The rate of IE increases exponentially with temperature, and the trend of this response depends on plant species and on the temperature at which 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 high temperature (HT) on IE, predicting plant IE and related changes in adaptive capacity to a changing environment becomes very difficult. The interest in studying IE changes is also enhanced because of its proposed protective role under different stress conditions. The mechanisms by which isoprene protects plants are still unknown. One of the most studied aspects of isoprene protection is the thermotolerance hypothesis introduced for the first time by Sharkey and Singsaas (1995), and isoprene thermoprotection was demonstrated in different species (Singsaas et al., 1997; Hanson et al., 1999; Velikova and Loreto, 2005). The objective of the present research was to investigate the impact of the simultaneous occurrence of rising CO2 and transient heat waves on IE in 2-year-old Platanus orientalis plants. We focused on the response of two leaf types developed and matured in either ambient or elevated [CO2]. We specifically examined the relationships between carbon assimilation, carbon metabolism and IE, and on indicators of ultrastructural damage to chloroplasts after the exposure to heat stress. It was hypothesized that (1) the exposure to elevated [CO2] might positively affect photosynthesis and that this could reduce the negative effect of heat on carbon metabolism; and (2) CO2 enrichment might inhibit isoprene Fig. 1. Photograph of 2-year-old Platanus orientalis seedling after 1-month growth at 1 biosynthesis, which in turn could reduce thermotolerance and 800 mmol mol CO2. Two types of leaves were used in the experiments. Leaves 1 worsen the heat stress for photosynthesis. developed and reached maturity during the fumigation with 800 mmol mol CO2 are termed as ‘‘newly-emerged’’; and leaves already reached their fully expanded state 1 2. Material and methods when the 800 mmol mol CO2 fumigation was implemented are termed as ‘‘pre- existing’’. 2.1. Plant material and growing conditions

3 Two-year-old P. orientalis L. plants were grown in 1.5 dm pots in a climate conductance (gm)toCO2 was calculated by assuming that the electron transport chamber under controlled conditions of 350 mmol m2 s1 photon flux density, rates calculated by gas exchange and fluorescence match in the absence of internal 25 C/20 C day/night temperature, 65% relative humidity, and 12 h photoperiod. resistances and photorespiration (Loreto et al., 1992). The actual [CO2] at the Plants were regularly watered to pot water capacity and fertilized once a week with chloroplast site (Cc) was then calculated from the gm value as shown elsewhere full-strength Hoagland solution. (Harley et al., 1992).

2.2. CO2 and high-temperature treatments 2.4. VOCs measurements

Plants were divided into two groups, ten saplings each, and transferred to two Isoprene and methanol (ME) emissions from leaves were measured in parallel 1 climatic chambers supplied with two different [CO2] amounts: 380 mmol mol with physiological parameters by diverting part of the cuvette outflow to a Proton 1 (ambient [CO2]) and 800 mmol mol (elevated [CO2]). To simulate exposure to Transfer Reaction-Mass Spectrometer (PTR-MS, Ionicon, Innsbruck, Austria), that a heat wave, plants were exposed once to HT (38 C) for 4 h at the end of 1 month of allows sensitive quantification of BVOC emissions (Lindinger et al., 1998). The PTR- treatment with ambient or elevated [CO2] by increasing the temperature in climatic MS was calibrated before measurements against an isoprene and methanol gas chambers. All analyses were done immediately after 4-h exposure to HT. Two types standards (60 nL L1) (Rivoira S.p.A. Milan, Italy). Isoprene and methanol were of leaves were used in the following experiments. Leaves that had already reached detected as parent ions at protonated m/z ¼ 69 and 33, respectively (see for details their fully expanded state before the onset of the CO2 fumigations are termed as Loreto et al., 2006; Filella et al., 2007). Then IE and ME rates (ER) were calculated as: ‘‘pre-existing’’ leaves, while leaves that developed during the CO2 fumigations are termed as ‘‘newly-emerged’’ leaves (Fig. 1). ER ¼ðE=26ÞðF=LAÞ where E is the gas concentration detected by PTR-MS (i.e. the ratio of count 2.3. Gas exchange and fluorescence measurements number s1 to gas standard), 26 is the gas volume (L) at 30 C, F is the air flow rate through the cuvette clamping a leaf of known area (LA). Steady-state photosynthesis and stomatal conductance (gs) were measured by a portable gas exchange system (LI-6400-40 LiCor, Lincoln, NE, USA) equipped with a fluorescence probe. Measurements were made at the [CO2] of 380 and 2.5. Phosphoenolpyruvate carboxylase (PEPc) activity measurements 800 mmol mol1 on individual leaves enclosed into a leaf cuvette at a rate of 0.3 L min1 air flow, relative humidity within the cuvette at 60–70% and growth- Flash frozen 0.2 g midrib-free leaf material was ground in a pre-chilled mortar intensity illumination. Gas exchange and chlorophyll fluorescence were measured in with 1.5 ml extraction buffer containing 100 mM HEPES–KOH (pH 7.2), 10 mM DTT, unstressed control plants for each [CO2] treatment and on the same plants after the 0.3% (w/v) Triton-X 100, 5 mM MgCl2 and PVP-40. Extracts were centrifuged at HT treatment. Gas exchange and fluorescence measurements were performed at 11 0 0 0 g for 5 min at 4 C. Then 20 ml of the supernatant was immediately used for 25 C for control plants and 38 C for temperature treated plants. Mesophyll PEPc activity in an assay mix containing 25 mM Tricine KOH (pH 8.1), 5 mM MgSO4, 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). The reaction was initiated by the addition of 2 mM PEP and NADH. Absorbance changes were registered for 5 min as 6A340 nm at room temperature, according to Rosenstiel et 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 TEM analyses. These segments were then prefixed in 3% glutaraldehyde (m/v) in 0.1 M sodium phosphate buffer (pH 7.4) for 12 h at 4 C and postfixed in 2% (m/v) OsO4 in the same buffer for 4 h at room temperature. After dehydration the material was embedded in Durcupan (Fluka). Ultra-thin sections were cut from palisade paren- chyma using a Reichert ultra-microtome and were examined using electron microscope (JEOL 1200EX, Japan). Leaf segments from five plants of each treatment were investigated and at least ten micrographs of different palisade cells were taken.

2.7. Isolation of thylakoid membranes and measurements of 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 MgCl2,10mM NaCl and 10 mM Tris (pH 7.8). Low temperature (77 K) chlorophyll fluorescence emission spectra were recorded with a Jobin Yvon JY3 spectrofluorimeter (Jobin Yvon ISA, Longjumeau, France) supplied with a low-temperature device. Additional methodology can be found in Apostolova et al. (2006). To determine the relative contribution of specific chlorophyll–protein complexes to the overall fluorescence pattern, 0.5 mM fluorescein (sodium salt) was added as an internal standard to the medium. The fluorescence spectra were normalized to the same area (100) under the spectrum (Andreeva et al., 2003). The chlorophyll content of the samples was adjusted to 10 mgml1 (Lichtenthaler, 1987).

2.8. Statistics

All data shown in the figures are means derived from replicates on different plants. 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, are Fig. 2. Photosynthesis (a, b) and electron transport rate (c, d) in newly-emerged and shown by different letters among groups. pre-existing Platanus orientalis leaves, before (white bars) and after a 4-h heat stress (38 C) (hatched bars). Measurements were performed at 350 mmol m2 s1 PPFD and 1 growing CO2 concentrations (380 or 800 mmol mol CO2). Each data point represents 3. Results and discussion the mean (SE) of 6–8 replicates. Means were separated by Tukey’s test and different letters indicate means that are statistically different with P < 0.05. 3.1. Impact of high temperature on photosynthesis and emission of volatiles in elevated [CO2] cause loss of photosynthetic capacity in C3 species in elevated Photosynthesis and the electron transport rate (ETR) were not [CO2]. significantly different in newly-emerged and pre-existing leaves in Moderate heat stress was shown to induce photochemical ambient [CO2](Fig. 2). Growth in elevated [CO2] often stimulates damage, stimulating the reduction of the plastoquinone pool in the net photosynthetic rate in C3 plants due to enhanced supply of dark and increasing cyclic electron flow around PSI in the light substrate to the CO2 fixing enzymes and depression of photores- (Pastenes and Horton, 1996), and inducing changes in thylakoid piration (Moore et al., 1999; Ainsworth and Long, 2005). However, structure that increase membrane leakiness (Havaux et al., 1996). In we did not observe a significant stimulation of photosynthesis in our experiments, a 4-h exposure to HT caused a significant decline pre-existing leaves, whereas photosynthesis of newly-emerged of photosynthesis and especially in photosynthetic (linear) ETR leaves grown in elevated [CO2] was even reduced in comparison to only in newly-emerged leaves under elevated [CO2], further indi- that measured in ambient [CO2](Fig. 2a,b). ETR of both types of cating a downward acclimation of Rubisco amount and/or activa- leaves was significantly lower in elevated [CO2](Fig. 2d), due to the tion state in elevated [CO2], while it did not significantly affect these reduction of photorespiratory electron transport. CO2 insensitivity parameters in all other cases (Fig. 2c,d). We hypothesized that or reversed sensitivity can be due to a downward acclimation of photosynthesis stimulation in elevated [CO2] could reduce the photosynthetic capacity (Curtis and Wang, 1998; Moore et al., negative effect of HT on the photochemical apparatus, mainly by 1999). This process, which is likely related to unbalanced source– keeping a high linear ETR. Inefficient photosynthetic ETR contrib- sink relations, often occurs when plants exposed to elevated [CO2] utes to direct oxygen photoreduction and to the accumulation of are grown in small pots. In our study, the rather small pot volume in dangerous reactive oxygen species in the mesophyll (Sharkey, which the 2-year-old saplings were grown have likely affected the 2005). However, photosynthesis was not stimulated by elevated sink size by restricting root growth. This may have induced [CO2]inPlatanus plants and our hypothesis could not be success- a decrease in the amount of the photosynthetic pigments and fully tested. enzymes, for instance amount and activation state of Rubisco and, We verified whether reduction of photosynthesis of newly- thus, acclimation of the photosynthetic apparatus (Centritto and emerged leaves in elevated [CO2] after the HT treatment was due to Jarvis, 1999; Moore et al., 1999; Stitt and Krapp, 1999; Ainsworth diffusive limitations, i.e. to reduced CO2 availability for photosyn- and Long, 2005). However, negative feedback on enzymes involved thesis. As expected (Ainsworth and Long, 2005), gs, which was in sucrose synthesis and transport (Micallef et al., 1996), or direct clearly higher in newly-emerged than in pre-existing leaves in damage to chloroplast membranes (Cave et al., 1981) may also ambient [CO2], was decreased in both type of leaves in response to 2632 V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637 elevated [CO2](Fig. 3a,b). After the HT treatment, gs of newly- constraints limited the use of CO2 acquired by the leaves (Flexas emerged leaves grown in ambient [CO2] decreased significantly et al., 2004). Numerous experiments have shown that CO2 draw- while no changes were observed in pre-existing leaves grown in down in the mesophyll can largely reduce CO2 concentration the same CO2 level (Fig. 3a). A slight but not significant increase of available at chloroplasts. The mesophyll therefore constitutes an gs was observed in both leaf types grown in elevated [CO2] after the additional diffusive resistance that should be accounted for, espe- HT treatment (Fig. 3b). However, stomatal limitation to photosyn- cially when limiting photosynthesis, such as in species (Loreto thesis did not increase during heat stress as revealed by the et al., 1992), and under stress conditions (Centritto et al., 2003; generally steady intercellular CO2 concentration (Ci)(Fig. 3c,d). In Flexas et al., 2004). the case of newly-emerged leaves, in particular, a higher Ci was Mesophyll conductance was indeed strongly reduced in leaves observed, suggesting that biochemical or photochemical growing in elevated [CO2](Fig. 3e,f). The reduction of gm upon short-term exposure to elevated [CO2] was observed first in Quercus rubra leaves by Harley et al. (1992) and then repeatedly, e.g. by Centritto 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]ongm in almost all species studied. They also found a linear relationship between gm and photosynthetic capacity. Our finding reveals that gm reduction in high [CO2] can be an acclimatory response when plants are grown under enhanced [CO2]. In addition, we also showed that the effect of elevated [CO2]ongm was much higher in newly-emerged leaves, suggesting that gm is more influenced by changes occurring during leaf development. The anatomical features are indeed the main determinants of gm (Evans, 1999) and the impact of CO2 on mesophyll assembly and packaging, and associated gaseous and liquid phase resistances to CO2 diffusion may 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. While the heat stress slightly reversed the CO2-induced reduction of gm in pre-existing leaves, gm of newly-emerged leaves was further reduced. Cc was not affected in ambient [CO2](Fig. 3g), whereas Cc of pre-existing leaves was higher than in newly-emerged leaves in elevated [CO2](Fig. 3h). However, irrespective of the impact of HT on the leaf types, Cc increased in leaves grown in elevated [CO2] and after the HT treatment (Fig. 3h). This further confirms that diffusive limitations are not responsible for photosynthesis inhibition caused by 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 on photosynthesis. 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 and in both CO2 treatments, but this stimulation was much lower in elevated [CO2](Fig. 4b) than in ambient [CO2](Fig. 4a). Indeed our data show that newly-emerged leaves in elevated [CO2], which are characterized by the lowest IE, were the most sensitive leaves to HT (compare Figs. 2 and 4). Thus, isoprene thermo-protective function remains once more, though indirectly, confirmed. It is known that young leaves become slowly competent to emit isoprene and emit less than mature leaves (Sharkey and Loreto, 1993; Centritto et al., 2004; Loreto et al., 2007). The lower capacity of young leaves to emit isoprene was attributed to isoprene synthase activity and quantity as the enzyme features developed similarly to the emis- sion rates (Kuzma and Fall, 1993), and it has been suggested that the biosynthesis of isoprene is developmentally regulated at the level of gene expression (Mayrhofer et al., 2005; Wiberley et al., 2005). Growth in elevated [CO2] also caused the expected decrease in IE in both newly-emerged and pre-existing P. orientalis leaves (Fig. 4b). In elevated [CO2] a substantial decline in IE is commonly Fig. 3. Stomatal conductance (a, b), intercellular CO2 concentration (c, d), mesophyll observed (Rosenstiel et al., 2003; Centritto et al., 2004; Scholefield conductance (e, f), and chloroplast CO concentration (g, h) in newly-emerged and pre- 2 et al., 2004), even though photosynthesis, which provides the bulk 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 in of the carbon needed for isoprene biosynthesis (Sharkey and Yeh, Fig. 2. 2001), is often stimulated, as previously commented. Such V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637 2633

[CO2] was also associated with a lower activity of PEPc (Fig. 4b,d), thus confirming the hypothesis by Rosenstiel et al. (2003) that isoprene inhibition in elevated [CO2] may be due to insufficient pyruvate supply. However, in leaves exposed to HT treatment the relationship between IE and PEPc was not straight. In particular, in elevated [CO2] PEPc activity, which was largely stimulated by the HT treatment (Fig. 4d), did not sustain high rates of IE. In contrast, in ambient [CO2], the large increase of IE after the HT treatment was associated with reduced PEPc activity (Fig. 4c) with respect to that measured before heat stress. It is therefore suggested that PEPc- generated cytosolic pyruvate may not limit IE or regulate isoprene stimulation 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 in both [CO2] levels, is attributed to higher isoprene synthase activity. It should be noted, however, that the stimulation of IE was lower in elevated [CO2], and especially in newly-emerged leaves. As we discussed before, the low stimulation of IE may be due to consti- tutively low levels of this enzyme in newly-emerged leaves and/or to inhibition of enzyme activity in elevated [CO2]. It is suggested that a down-regulation of isoprene synthase, often occurring upon prolonged exposure to HT (Singsaas and Sharkey, 2000; Loreto et al., 2006) might be also responsible for the loss of temperature stimulation of IE and could therefore indirectly influence leaf sensitivity to HT. Methanol is a volatile product of pectin demethylation during cell wall formation (Fall, 2003) in rapidly expanding leaves which is associated 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 maturity before being exposed to elevated [CO2], was consistently lower than in 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 in growing leaves than in adult leaves. In our study ME was signifi- cantly higher in newly-emerged leaves in elevated [CO2] compared to those grown in ambient [CO2]. The high rate of pectin deme- thylation and cellular break-down and formation may indeed confirm that at least in potted plants, elevated [CO2] induces an accelerated maturation process in leaves, and that newly-emerged leaves in our experimental conditions might have experienced an

Fig. 4. Isoprene emission (a, b), PEPc activity (c, d) and methanol emission (e, f) in ontogenic shift in elevated [CO2]. Thus, the higher emission rates newly-emerged and pre-existing Platanus orientalis leaves, before (white bars) and observed in newly-emerged leaves in elevated [CO2] could in fact after a 4-h heat stress (38 C) (hatched bars). Bar assignment, treatments, replications be lower if leaves were of comparable age with those sampled in and statistical treatment as in Fig. 2. ambient [CO2]. High-temperature exposure under both growth [CO2] resulted in significantly enhanced ME, this stimulation being more signifi- a surprising effect of elevated [CO2] was attributed to either cant in newly-emerged leaves (Fig. 4e,f) despite the lower gs a decrease of isoprene synthase activity (Scholefield et al., 2004), or (Fig. 3a,b) in elevated [CO2]. This is further evidence that ME is only the metabolic competition for phosphoenol pyruvate (PEP), the partially controlled by gs, being also related to methanol biosyn- common substrate of isoprene biosynthesis and mitochondrial thesis rate (Hu¨ ve et al., 2007). We therefore attributed the increase respiration (Rosenstiel et al., 2003). Rosenstiel et al. (2003, 2004) of ME at HT to its enhanced formation, perhaps due to a high rate of showed that inhibition of the activity of phosphoenolpyruvate damage in cell wall structures. The very high emission of methanol carboxylase (PEPc), the enzyme that provides substrate for respi- in heat stressed newly-emerged leaves grown in elevated [CO2] and ration and nitrate assimilation, stimulated IE. In our experimental emitting low levels of isoprene may therefore additionally indicate conditions, PEPc activity was almost two-fold higher in pre-existing that in the absence of isoprene more damage to cellular structure leaves compared to newly-emerged leaves in ambient [CO2] occurs. (Fig. 4c) and, consequently, a direct relationship was generally found between IE and PEPc activity when comparing newly- 3.2. Impact of high temperature on chloroplast ultrastructure emerged and pre-existing leaves grown in ambient [CO2] (compare and functions Fig. 4a,c). This suggests that the lower emission of newly-emerged leaves can also be caused by a low contribution of cytosolic pyru- Confirmation of the thermo-protective effect of isoprene on vate. The lower emission of isoprene in leaves grown in elevated photosynthesis may be associated to more preserved chloroplast 2634 V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637 ultrastructure and functionality. Well-differentiated chloroplasts characterized by an unusually large peristromium located toward characterized the mesophyll of pre-existing (Fig. 5a) and newly- the cell wall without destruction of the inner membrane system emerged leaves (Fig. 5b) at ambient [CO2], containing a well- (Fig. 5c). These chloroplasts were also in close structural contact developed inner membrane system, composed of grana of different with mitochondria through relatively large typically structured sizes (from 5–10 up to 30 thylakoids) and long stromal thylakoids. associative regions. Changes in the chloroplast structure organi- Single midsize starch grains were rarely discerned. Investigation of zation of newly-emerged leaves in elevated [CO2] are unlikely chloroplast ultrastructure, however, revealed that it was affected by stress-induced traits and rather represent adaptive changes at the [CO2] treatment, especially in newly-emerged leaves. Chloro- subcellular level to our experimental conditions (Mostowska, plasts of newly-emerged leaves in elevated [CO2]were 1997). The increase of stroma volume and the reduction of part of

Fig. 5. Electron micrographs of Platanus orientalis leaves, illustrating chloroplast structural changes in leaf mesophyll cells in pre-existing (A, D) and newly-emerged (B, C, E) leaves 1 under two regimes of CO2 (380 and 800 mmol mol ) and after exposure to high temperature (38 C) for 4 h, applied alone and in combination. Scale bars ¼ 500 nm. CH, chloroplast; G, grana; ST, stroma thylakoid; S, starch grain; P, peristromium; T, tannins. (A) Intact chloroplast of pre-existing leaves at ambient CO2 and 25 C; (B) intact chloroplast of newly- emerged leaves at ambient CO2 and 25 C; (C) elevated-CO2-induced structural changes in newly-emerged leaves at 25 C; (D) combined effect of elevated CO2 and 38 C/4 h on chloroplast structure of pre-existing leaves; (E) combined effect of elevated CO2 and 38 C/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 thylakoid wavelike and parts of stroma thylakoids were fragmented. Several electron density and increased number of plastoglobules, have studies showed that heat stress and elevated [CO2] can cause been previously observed after treatments with elevated [CO2] thylakoid destruction and starch accumulation in woody and (Kutı´k et al., 1995). It has been hypothesized that these changes in herbaceous plants (Mostowska, 1997; Oksanen et al., 2001; Sam chloroplast ultrastructure in plants grown in elevated [CO2]maybe et al., 2001; Lambreva et al., 2005). needed to meet the higher energy demand required for faster Starch did not accumulate in the chloroplasts of newly-emerged growth (Griffin et al., 2001). In fact, similar changes were only leaves, probably because photosynthesis of these leaves was observed in newly-emerged, fast-growing leaves, and not in pre- impaired before starch synthesis. In fact, when newly-emerged existing leaves in our experiment. It should be noted, however, that leaves grown in elevated [CO2] were exposed to HT, a destruction of these ultrastructural changes did not cause a stimulation of part of the peripheral thylakoid membranes was observed (Fig. 5e). photosynthesis in Platanus leaves. The unusually large peristromium was filled with membrane TEM analysis also showed that exposure to elevated [CO2] substance from fragmented peripheral thylakoids with swollen caused the accumulation of tannin-like vacuolar deposition in both intrathylacoidal space (Fig. 5e, arrows). This is a typical ultra- types (Fig. 5c,d). This was observed both in newly-emerged and structural damage induced by HT. Kislyuk et al. (2004) also found pre-existing leaves and therefore is not associated to growth that a large volume of the stroma was occupied by sinuous thyla- requirements. Increased tannin depositions were also observed in koids and fragments of thylakoid membranes in response to 30 min aspen plants exposed to increasing [CO2], suggesting a [CO2]- heating at 42 C under high light. We speculate that the negative induced activation of the phenylpropanoid pathway (Oksanen impact of HT on membrane structure of newly-emerged leaves that et al., 2001), with possible but yet largely unknown protective emit the lowest level of isoprene indicates that isoprene is indeed functions at cellular level. involved in a mechanism of thylakoid membrane protection, as Four-hour HT treatment did not cause any structural changes in previously hypothesized (Sharkey and Singsaas, 1995; Singsaas the chloroplasts of both types of leaves grown in ambient [CO2] et al.,1997) and theoretically recently predicted (Siwko et al., 2007). (data not shown). However, in leaves grown in elevated [CO2] the Isoprene lipophilic properties may actually allow easy insertion of HT exposure affected chloroplast ultrastructure of both leaf types. the molecule in thylakoidal membranes that are strengthened by HT treatment of pre-existing leaves grown in elevated [CO2] caused such an insertion. However, localization of isoprene into chloro- the accumulation of starch and structural changes of thylakoid plast membranes is needed to conclusively prove the proposed membranes that became wavy (Fig. 5d). A large part of chloroplasts mechanism of action. was occupied by 1–2 big starch grains, in which zones of saccha- 77 K chlorophyll fluorescence emission spectra were measured rides and capsule, containing enzymes, were not typically struc- in order to understand if isoprene presence can modulate the tured. Starch accumulation in elevated [CO2] occurs when the integrity and energy transfer between both photosystems and production of new assimilates is larger than the capacity to handle whether it can protect photosynthetic apparatus against HT them (Stitt and Krapp, 1999). Normally, starch accumulation in injuries. These analyses revealed that lower IE in newly-emerged leaves, by maintaining the Pi cycling, allows photosynthesis to leaves also considerably affected the functionality and integrity of continue (Stitt and Krapp, 1999). However, when the source–sink pigment–protein complexes even under control conditions. Pre- relation is disrupted, starch accumulation may become excessive existing and newly-emerged leaves grown at both [CO2] regimes and Pi cycling results progressively inhibited; this is, in turn, usually had well-defined F685 and F735 peaks (Fig. 6). The differences related to feedback inhibition of photosynthesis (Pritchard et al., occurred in amplitude ratio F735/F685 (Table 1), used to charac- 1997). The thylakoid membranes of the same chloroplast were terize the direct energy transfer from PSII to PSI complexes

Fig. 6. 77 K emission spectra of thylakoids isolated from: (a) newly-emerged leaves at ambient [CO2]; (b) pre-existing leaves at ambient [CO2]; (c) newly-emerged leaves at elevated [CO2]; (d) pre-existing leaves at elevated [CO2]. 2636 V. Velikova et al. / Environmental Pollution 157 (2009) 2629–2637

Table 1 emissions. We believe that these findings may be useful for a better 77 K fluorescence ratio (F735/F685) of thylakoids from newly-emerged and pre- parameterization of large-scale emission models on the impact of existing leaves of P. orientalis grown at ambient or elevated [CO ]at25C and after 2 BVOC emissions on air quality on regional and global scales. exposure to 38 C for 4 h. Statistically significant differences (P < 0.05) among groups are shown by different letters. Treatments 25 C38C Acknowledgments Newly-emerged Pre-existing Newly-emerged Pre-existing b a b a This research was funded by a NATO Reintegration Grant (No Ambient [CO2] 1.03 0.02 1.43 0.03 1.05 0.05 1.56 0.07 d d b c Elevated [CO2] 0.74 0.02 0.78 0.02 1.04 0.09 0.86 0.03 981279), 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- (Strasser and Butler, 1977; Andreeva et al., 2003; Apostolova et al., ment between Italian National Research Council and Bulgarian 2006) or arrangement of chlorophyll–protein complexes, i.e. PSI/ Academy of Sciences. PSII stoichiometry (Andreeva et al., 2003). In ambient [CO2] the PSI/ PSII ratio was around 1.43 (typical for higher plants) in pre-existing References leaves, while it was significantly lower (1.03) in newly-emerged leaves (Fig. 6a,b; Table 1). The lower PSI/PSII ratio demonstrated Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air CO2 a reduction in energy transfer to PSI. This observation could be enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, related to differences in maturity of chloroplast protein structure canopy properties and plant production to rising CO2. New Phytologist 165, 351–372. between both types of leaves (Kutı´k, 1998). When plants were Andreeva, A., Stoitchkova, K., Busheva, M., Apostolova, E., 2003. Changes in the exposed to elevated [CO2], which in turn caused a significant energy distribution between chlorophyll–protein complexes of thylakoid reduction of IE (Fig. 4b), the ratio F735/F685 was also largely membranes from pea mutants with modiWed pigment content I. Changes due to the modiWed pigment content. Journal of Photochemistry and Photobiology reduced (Fig. 6c,d; Table 1). 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