Profiles of isoprene emission and photosynthetic parameters in hybrid poplars exposed to free-air CO 2 enrichment

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1 Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment Blackwell Science Ltd 2004? Original Article Isoprene emission and photosynthetic parameters of poplars exposed to FACE M. Centritto et al. Plant, Cell and Environment (2004) 27, Profiles of isoprene emission and photosynthetic parameters in hybrid poplars exposed to free-air CO 2 enrichment M. CENTRITTO 1, P. NASCETTI 2, L. PETRILLI 3, A. RASCHI 4 & F. LORETO 2 1 CNR Istituto sull Inquinamento Atmosferico, via Salaria km C.P. 10, Monterotondo Stazione (Roma), Italy, 2 CNR Istituto di Biologia Agroambientale e Forestale, via Salaria km , Monterotondo Scalo (Roma), Italy, 3 CNR Istituto di Struttura della Materia, via Salaria km , Monterotondo Scalo (Roma), Italy and 4 CNR Istituto di Biometeorologia, via Giovanni Caproni 8, Firenze, Italy. ABSTRACT Poplar (Populus euroamericana) saplings were grown in the field to study the changes of photosynthesis and isoprene emission with leaf ontogeny in response to free air carbon dioxide enrichment (FACE) and soil nutrient availability. Plants growing in elevated [CO 2 ] produced more leaves than those in ambient [CO 2 ]. The rate of leaf expansion was measured by comparing leaves along the plant profile. Leaf expansion and nitrogen concentration per unit of leaf area was similar between nutrient treatment, and this led to similar source sink functional balance. Consequently, soil nutrient availability did not cause downward acclimation of photosynthetic capacity in elevated [CO 2 ] and did not affect isoprene synthesis. Photosynthesis assessed in growth [CO 2 ] was higher in plants growing in elevated than in ambient [CO 2 ]. After normalizing for the different number of leaves over the profile, maximal photosynthesis was reached and started to decline earlier in elevated than in ambient [CO 2 ]. This may indicate a [CO 2 ]- driven acceleration of leaf maturity and senescence. Isoprene emission was adversely affected by elevated [CO 2 ]. When measured on the different leaves of the profile, isoprene peak emission was higher and was reached earlier in ambient than in elevated [CO 2 ]. However, a larger number of leaves was emitting isoprene in plant growing in elevated [CO 2 ]. When integrating over the plant profile, emissions in the two [CO 2 ] levels were not different. Normalization as for photosynthesis showed that profiles of isoprene emission were remarkably similar in the two [CO 2 ] levels, with peak emissions at the centre of the profile. Only the rate of increase of the emission of young leaves may have been faster in elevated than in ambient [CO 2 ]. Our results indicate that elevated [CO 2 ] may overall have a limited effect on isoprene emission from young seedlings and that plants generally regulate the emission to reach the maximum at Correspondence: Mauro Centritto, Fax: ; centritto@iia.cnr.it This work is dedicated to the memory of Dr Wolfgang Zimmer. the centre of the leaf profile, irrespective of the total leaf number. In comparison with leaf expansion and photosynthesis, isoprene showed marked and repeatable differences among leaves of the profile and may therefore be a useful trait to accurately monitor changes of leaf ontogeny as a consequence of elevated [CO 2 ]. Key-words: Populus euramericana; A C i curves; elevated CO 2 ; FACE; ontogeny; phytogenic isoprene. INTRODUCTION Carbon assimilation (A) and isoprene emission are generally closely linked in many forest and agriforest species (because they share common biochemical intermediates and because the source of reducing power for isoprene synthesis, which occurs in chloroplasts, is the photochemistry of A) (Sharkey & Yeh 2001), but they play a contrasting role in the biosphere atmosphere interactions. In fact, forest and agriforest vegetation is a dominant sink for the atmospheric CO 2 and account for about 90% of the globe s biomass carbon. Thus, it plays, by means of A, a key role in mitigating global change. In contrast, phytogenic isoprene may have an important, negative impact in atmospheric chemistry, because it affects the residence time of other greenhouse gases and, above all, has a large effect on the oxidizing potential of the atmosphere: in the presence of sufficient amounts of anthropogenic nitrogen oxides, isoprene can increase the concentration of tropospheric ozone thereby contributing to air pollution (Fehsenfeld et al. 1992). Given the importance of both A and isoprene emission for the biosphere atmosphere interactions, the consequences of rising [CO 2 ] for the biogeochemical carbon cycle and for atmospheric chemistry are potentially extremely large. Thus, there is great interest in determining how climate change might affect the processes of A and phytogenic volatile organic compound emissions at various stage of leaf ontogeny (Loreto et al. 2001). However, no data are available on the detailed spatial effects of elevated [CO 2 ] on photosynthetic capacity and isoprene emission, and on its interactive effects with soil nutrient availability Blackwell Publishing Ltd 403

2 404 M. Centritto et al. It is well known that the predicted changes in atmospheric [CO 2 ] are expected to increase photosynthetic rates in C 3 plants both by increasing the rate of carbon fixation and by reducing photorespiratory loss of carbon (Drake, Gonzàlez-Meler & Long 1997). However, in the long term, elevated [CO 2 ] may lead to a decline in the concentration of Rubisco and pigments of the light-harvesting system, resulting in reduction of the photosynthetic capacity (Drake et al. 1997; Moore et al. 1999). Downward acclimation of photosynthesis is thought to be caused by the disruption of the source sink functional balance; that is, N uptake does not keep pace with carbon uptake resulting in an increase in the sugar to amino acid ratio in leaves (Moore et al. 1999; Stitt & Krapp 1999). This is regarded as an optimization process which involves re-allocation of nitrogen away from non-limiting components into more limiting processes or organs (i.e. additional or larger sinks for the extra carbon assimilated), leading to increased nitrogen use efficiency (Drake et al. 1997; Centritto & Jarvis 1999). Despite, the debate on the mechanisms which underlie the observed source sink imbalance, and consequently the downward acclimation of A, it is still the subject of ongoing speculations (Woodward 2002), the literature contains increasing evidence that downward acclimation of A is an exception rather than the rule in plants rooted into the ground because these are able to keep their source sink functional balance by adjusting their growth rate to match N uptake. Unlike A, phytogenic isoprene emission per unit of leaf area is relatively insensitive to elevated [CO 2 ] (Sharkey & Yeh 2001). Sharkey, Loreto & Delwiche (1991) found that the basal isoprene emission in potted aspen (Populus tremuloides) seedlings grown in growth chambers was reduced by 30 40% in response to elevated [CO 2 ]. This result is similar to a recent report of ecosystem isoprene emission in a cottonwood (Populus deltoides) plantation exposed to elevated [CO 2 ] in the Biosphere 2 Center (i.e. a huge glassand-metal structure) located in Arizona (Rosenstiel et al. 2003). In this study, isoprene production, expressed per ecosystem area, was calculated hourly from three different mesocosms (with [CO 2 ] of 430, 800 and 1200 mmol mol -1, respectively) containing trees. When integrated over the whole growing season, ecosystem isoprene emission from the mesocosms with [CO 2 ] of 800 and 1200 mmol mol -1 was reduced by 21 and 41%, respectively, whereas plant above-ground biomass was increased by 60 and 82%, respectively. The authors concluded that elevated [CO 2 ], by uncoupling growth from isoprene emission, may partially reduce the negative air-quality impacts of proliferating agriforests. However, Boissad et al. (2001) measured volatile organic compound emission rates over the course of 1 year in gorse (Ulex europaeus) and showed that the isoprene emission factor could vary with seasons over a range of few orders of magnitude, leading to large discrepancies between measured and calculated emissions. Moreover, the impact of elevated [CO 2 ] on isoprene emission may also be mediated by its effects on plant ontogeny and leaf area expansion. In fact, many studies have shown that elevated [CO 2 ] accelerates ontogeny (Centritto, Lee & Jarvis 1999a; Centritto, Lucas & Jarvis 2002; Lewis et al. 2002) and promote leaf expansion in hybrid poplar (Taylor et al. 2001), although this effect declined dramatically after canopy closure in the second year of growth (Gielen et al. 2001). The aim of our research is to study the dynamics of isoprene emission and photosynthetic capacity in hybrid poplar (Populus euroamericana) saplings growing in the field in response to free air carbon dioxide enrichment (FACE) and soil nutrient availability. In the present study we focused on leaf ontogeny by measuring photosynthetic capacity and isoprene emission along plant profile. MATERIALS AND METHODS Poplar (Populus euroamericana) saplings were grown for a growing season in the FACE system located in Rapolano, Siena (Lat N, Long E). Poplar cuttings were planted before bud break at the end of April 1999 in three FACE (approximately 550 mmol mol -1 ) plots and three unenriched [CO 2 ] plots. Each emission array was formed by a rectangular plenum (10 m 3 m) positioned at the soil surface, connected with 22 vertical pipes. The whole system was assembled from polyvinyl chloride (PVC), using pipes with an internal diameter of 20 cm for the plenum and 2 m long pipes with an internal diameter of 4 cm for the vertical vents. Each vent pipe has four emission holes, 2 cm in diameter, placed in two groups of two holes each, at approximately 120 and 190 cm above the soil surface. High volume blowers (APEM, Firenze, Italy) were used to blow air into the plenum, through flexible pipes; pure carbon dioxide was mixed with ambient air by injecting it into the flexible pipes. The CO 2 injection rates were regulated using the proportional integral differential (PID) algorithm described by Lewin et al. (1994) and were controlled by motorized metering valves (Zonemaster; Satchwell Control Systems, Slough, Berks., UK). The algorithm makes use of both horizontal mass flow based on wind velocity and a PID component, based on CO 2 concentrations read in the centre of the array, to calculate the output voltage used to control the metering valves. The CO 2 concentration in the FACE array is measured by an infra-red gas analyser (WMA-2; PPS, Hitchin, Herts.,UK) having a mmol mol -1 range, with an accuracy of 20 mmol mol -1 full scale and a temporal resolution of 1 s. The FACE system used geologic CO 2 obtained from a local company (Geogas S.p.A, Rapolano Terme (SI), Italy) that extracts CO 2 for industrial and alimentary purposes. The distances between arrays was about 20 m to avoid unintended CO 2 enrichment of the controls. Further details on the performance of the fumigation system are given elsewhere (Bindi et al. 2001). There were 16 plants per plot, at a spacing of 1.5 m 1.5 m. Each plot was partitioned into two halves corresponding to two different fertilization treatments. Seven plants within one of the two subplots was fertilized once a week, with complete nutrient solution at low con-

3 Isoprene emission and photosynthetic parameters of poplars exposed to FACE 405 centration in amounts calculated to supply the unrestricted needs of the trees (HN), using the stock solutions described by Ingestad & Lund (1986), while the other subplot was not supplied with nutrient (LN). All subplots were supplied with ample irrigation water throughout the growing season. Short-term measurements of photosynthetic photon flux density (PPFD)-saturated 1000 mmol m -2 s -1 A in relation to leaf internal CO 2 concentration (C i ) were made at 30 C between 1000 and 1700 h at the beginning of July, over a range of CO 2 concentrations between 40 and 1000 mmol mol -1 using a portable gas exchange system (LI- 6400; Li-Cor, Lincoln, NE, USA) on four saplings per [CO 2 ]-nutrient treatment. A C i measurements were made on leaves at contrasting stages of development, namely the expanding leaves 2, 3 and 8 and the fully expanded leaf 15 (counting from the top of the plant). Values for the photosynthetic parameters V cmax (RuBP-saturated rate of Rubisco), J max (maximum rate of electron transport), and A max (the net CO 2 assimilation rate under conditions of PPFD and CO 2 saturation) were obtained by fitting the mechanistic model of CO 2 assimilation proposed by Farquhar, von Caemmerer & Berry (1980) to individual A C i response data using the method developed by de Pury & Farquhar (1997). The fitting model was run with the in vivo Rubisco kinetics parameters measured by Bernacchi et al. (2001). Fitting the model involved an optimization procedure in which the parameter values were optimized by adjusting them so as to minimize the sums of residuals between observed and modelled assimilation values over a range of C i. Simultaneously with A C i measurements, leaf discs of known area were collected from leaves 2, 3, 8 and 15 to determine nitrogen, soluble proteins, Rubisco, total chlorophyll and anthocyanin concentrations. These samples were first plunged into liquid nitrogen, subsequently kept in small plastic vials in a freezer at -80 C. Nitrogen concentration was measured following the quantitative dynamic flash combustion method (Pella & Colombo 1973), using an elemental analyser (EA 1108; Fisons, Milano, Italy). The frozen samples (leaf discs with a total area of cm 2 per CO 2 treatment) were first transferred to a freeze-drier (Minifast 1700; Edwards, Crawley, Sussex, UK), and then ground to fine powder using a ceramic grinding vessel. N was measured in ground tissue (ranging in mass 3.0 ± 0.1 mg) by quantitative combustion over a catalyst layer (WO 3 ), reducing over wired pure copper N X O Y to N 2, then separating and eluting the mixture through a chromatographic column, and finally detecting N by thermal conductivity measurement. The concentrations of soluble proteins were quantified in leaf discs, of area 2.5 cm 2, using the method of Bradford (1976) with bovine serum albumin (BSA) as a standard. For the determination of Rubisco concentration leaf discs of 2,5 cm 2 were ground in a chilled mortar containing 30 mg PVPP, quartz sand and 2 ml of extraction buffer (100 mm Bicine ph 7.8, 1 mm ethylenediaminetetraacetic acid, 5 mm MgCl 2, 5 mm dithiothreitol and 0.02% BSA (w/v). The homogenate was centrifuged for 30 s at g. The supernatant was denatured in sodium dodecyl sulphate (SDS) reducing buffer (20% SDS, 20% b-mercaptoethanol and 0.5 M Tris-HCl ph 6.8) at 95 C for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis using a 14% acrylamide gel. Gels were stained with Coomassie Brilliant Blue R-250, destained and scanned at 550 nm using a dualwavelenght flying spot scanner in the transmission mode (CS-9000; Shimadzu, Kyoto, Japan). The total concentration of chlorophylls was measured in intact leaf tissues by immersion in N,N-dimethylformamide (DMF) following the techniques described by Porra, Thompson & Kriedemann (1989). Leaf discs with a total area of 2.40 cm 2, were immersed in 5 cm 3 DMF and immediately placed in darkness for 4 to 5 d before absorbance of the solution was read on a spectrophotometer (LambdaBio20; Perkin Elmer, Boston, MA, USA) at 647, 664 and 750 nm, using DMF as a blank. Isoprene emission was measured at the beginning of July, in parallel with the A C i measurements. The isoprene measurements were made on four plants per [CO 2 ] and nutrient treatments. Leaves were sampled along the plant profile starting from top, last leaf emerged, to the plant bottom, first leaf expanded. Along the plant profile, there were an average of 22.5 ± 1.3 (mean ± SEM) and 32.2 ± 1.7 leaves per sapling in ambient and elevated [CO 2 ] treatments, respectively. In leaf 2, 3, 8, 15 and 21 in FACE and ambient [CO 2 ] treatment, isoprene emission was measured simultaneously with A measured at the growth [CO 2 ]. To measure isoprene emission, a single leaf was inserted in the Li-6400 gas-exchange cuvette, where it was exposed to a 0.5 dm 3 min -1 flow of synthetic air in which the ambient gas concentration was reconstituted but trace-gases were absent. The air leaving the cuvette was sent to the infrared gas analyser for measurements of CO 2 and H 2 O gas exchanges. Simultaneously, a small part of the flow (36 cm 3 ) was pumped into a gas chromatograph (GC Syntech 855; Syntech, Groningen, The Netherlands) for chromatographic determination of isoprene emissions. Measurements were performed after maintaining the leaf at a temperature of 30 C and at a light intensity of 1000 mmol photons m -2 s -1 for 1 h. These are the conditions used to measure the basal emission of isoprene (Guenther et al. 1993). The GC Syntech 855 is portable and equipped with a photo-ionization detector which enhances by about 50 times sensitivity to isoprenoids with respect to the flame ionization detector. Calibration against isoprene standards revealed the detection limit to be 0.1 p.p.b and were performed daily before measurements. The retention time for isoprene with an isothermal program was less than 3 min. This allowed measurements of transient changes of isoprene emissions in leaves exposed to different environments. To estimate the basal isoprene emission for the total number of leaves occurring along the plant profile, a regression curve was fitted through the set of data collected on different leaves in the profile, and then the curve integral was calculated for both ambient and elevated [CO 2 ] treatments. Leaf area (LA) of leaves 2, 3, 8 and 15 was measured using a leaf area meter (LI 3100; Li-Cor Inc.). Leaf devel-

4 406 M. Centritto et al. Figure 1. Leaf area of the leaves 2, 3 and 8 (from the plant apex) shown as percentage of the maximum leaf expansion in the poplar saplings grown in FACE and ambient [CO 2 ]. Data are means of four plants per treatment ±1 SEM; letters (a, b) indicate significant differences at P < 0.05; HN, high nitrogen; LN, low nitrogen. opment of the expanding leaves 2, 3, and 8 was then estimated as percentage of the fully expanded leaf 15. Data were tested using a simple factorial analysis of variance (ANOVA; two-way maximum interactions), and where appropriate, the treatment means were compared using Tukey s post-hoc test. RESULTS We measured the rate of leaf expansion by comparing four different leaves (i.e. leaf 2, 3, 8 and 15) along the plant profile. The saplings were growing rapidly, and leaf position along the plant profile was dependent on leaf age (i.e. leaf 1 from the plant apex was the youngest growing leaf, whereas the bottom leaf was the oldest). Leaf area was significantly increased by nutrient supply but it was not affected by elevated [CO 2 ] (data not shown). On the contrary, nutrient supply did not affect the relative rate of leaf expansion (Fig. 1). Only the development of leaf 2 was significantly accelerated in both nutrient treatments in response to elevated [CO 2 ]. Expansion of leaf 2 was on average 18 and 12% of the maximum in elevated and ambient [CO 2 ], respectively; whereas the expansion of leaf 3, averaged across the [CO 2 ] and nutrient treatments, was approximately 42% of the maximum. Expansion of leaf 8 was on average approximately 96% of the maximum, but its area was not statistically different from that of leaf 15. Neither growth in elevated [CO 2 ] nor nutrient supply affected the photosynthetic capacity of the poplar saplings: there were no differences in the A C i properties of photosynthesis (Fig. 2), soluble protein concentration, Rubisco concentration, and pigment concentrations (data not shown). Thus, there was no reduction in photosynthetic capacity in the saplings grown in elevated [CO 2 ]. However, all these parameters were significantly affected by leaf development. The shape of the A C i curves changed dramatically with leaf growth (Fig. 2). All the photosynthetic parameters obtained by fitting the Farquhar et al. (1980) model of C 3 leaf photosynthesis to individual A C i curves were significantly affected by the degree of leaf development (Table 1). V cmax (which is an estimate of the carboxylation efficiency of Rubisco and was determined from the slope of the A C i curve at [CO 2 ] of mmol mol -1, assuming that the resistance to CO 2 diffusion inside the leaf mesophyll is taken as zero, i.e. g m = ), A max and J max (both determined from the saturating portion of the curve at high [CO 2 ], i.e. in low photorespiratory conditions) were doubled from leaf 2 (less than 20% of maximum expansion) to leaves 8 (approximately 96% of maximum expansion) and 15 (fully expanded leaf). In parallel, the concentrations, per unit of leaf area, of soluble proteins, Rubisco, and total chlorophyll (Table 2) were also significantly increased with leaf development, in spite of leaf nitrogen concentration per unit of leaf area not being significantly affected by leaf growth (Table 2). Furthermore, leaf nitrogen concentration per unit of leaf area did not change in response to either growth in elevated [CO 2 ] or nutrient supply (data not shown). The increase in photosynthetic capacity with leaf development (Fig. 2) was reflected by the significant increase in the instantaneous rate of photosynthesis, A, measured at the growth [CO 2 ] (Table 3). A was almost Figure 2. CO 2 assimilation rate intercellular [CO 2 ] (A C i ) curves measured in the leaf 2 (2), leaf 3 (3), leaf 8 (8), and leaf 15 (15) from the apex in the poplar saplings grown in FACE and ambient [CO 2 ]. Four leaves in each treatment combination were measured at 30 C in saturating PPFD (1000 mmol m -2 s -1 ); HN, high nitrogen; LN, low nitrogen.

5 Isoprene emission and photosynthetic parameters of poplars exposed to FACE 407 Table 1. Combined photosynthetic parameters, averaged across the four [CO 2 ] and nutrient treatments, in the leaves 2, 3, 8 and 15 (from the plant apex) of the poplar saplings grown in FACE and ambient [CO 2 ] A max J max V cmax J max /V cmax C i /C a Leaf ± 1.00 a ± 7.1 a 45.1 ± 5.0 a 2.24 ± ± c Leaf ± 0.93 b ± 5.9 b 61.6 ± 4.1 b 2.13 ± ± b Leaf ± 1.33 c ± 11.9 c 95.1 ± 6.6 c 2.14 ± ± a Leaf ± 1.24 c ± 14.6 c ± 6.9 c 1.92 ± ± a A max (mmol m -2 s -1, maximum photosynthetic rate at saturating PPFD), J max (mmol m -2 s -1, potential rate of electron transport per unit leaf area), V cmax (mmol m -2 s -1, photosynthetic Rubisco capacity per unit leaf area), J max /V cmax ratio, and C i /C a (the intercellular [CO 2 ] to the growth [CO 2 ] ratio). All figures ± 1 SEM, n = 16. Letters (a, b, c, d) indicate significant differences at P < 0.05 in the same column. Proteins Rubisco Chlorophyll N Leaf ± 0.21 a 0.91 ± 0.10 a ± a 2.87 ± 0.19 Leaf ± 0.30 a 1.15 ± 0.08 b ± a 3.07 ± 0.25 Leaf ± 0.44 b 1.63 ± 0.17 c ± b 3.19 ± 0.20 Leaf ± 0.72 b 1.78 ± 0.21 c ± b 2.96 ± 0.13 All figures ± 1 SEM, n = 16. Letters (a, b, c) indicate significant differences at P < 0.05 in the same column. Table 2. Combined mean concentration per unit leaf area of soluble proteins (g m -2 ), Rubisco (g m -2 ), total chlorophyll (g m -2 ), and nitrogen (g m -2 ) averaged across the four [CO 2 ] and nutrient treatments, in the leaves 2, 3, 8 and 15 (from the plant apex) of the poplar saplings grown in FACE and ambient [CO 2 ] trebled from leaf 2 to leaves 8 to 15 in both [CO 2 ] treatments; but an ontogenetic decline of A was seen in leaf 21 in elevated [CO 2 ]. A measured in elevated [CO 2 ] was always significantly higher than in ambient [CO 2 ]. However, the extent of the stimulation of A in response to elevated [CO 2 ] treatment was strongly reduced with leaf expansion, as it was halved from leaf 2 (96%) to leaf 15 (49%). Despite these large differences in A, C i /C a (C a = external [CO 2 ]) was not affected by growth in elevated [CO 2 ] or nutrient supply (data not shown), but, as for A, it decreased significantly with leaf expansion (Table 1). Isoprene emission was not affected by nutrition (data not shown), but it responded significantly to [CO 2 ] treatment and was typically age-dependent (Fig. 3). In fact, young leaves did not emit isoprene, as it was not detectable in leaf 2 although this leaf was already photosynthetically competent. As leaf development proceeded, the rates of isoprene emission, and consequently also the fraction of photosynthetic carbon re-emitted, gradually increased in parallel to the increase of A measured at the growth [CO 2 ] (Table 3). However, diversely from A, the rate of isoprene emission per unit of leaf area was significantly reduced in elevated [CO 2 ]. The contrasting sensitivity of A and isoprene emission to elevated [CO 2 ] resulted also in lower fraction of photosynthetic carbon re-emitted in leaves 3, 8 and 15. Significant differences in isoprene emission were already evident in leaf 4, but they became larger as the rate of isoprene emission in ambient [CO 2 ] approached the maximum value (Fig. 3a). This was reached in leaves 11 and 12, whereas in elevated [CO 2 ] the maximum values of isoprene emission was reached in leaves 15 and 16 (Fig. 3b). These leaves were almost half of the total number of leaves of ambient and elevated [CO 2 ] saplings, respectively. Then, with leaf ageing, basal isoprene emission rate declined progressively. This decline was faster in ambient [CO 2 ], Table 3. Combined assimilation rate (A, mmol m -2 s -1 ) and percentage increase, isoprene emission (nmol m -2 s -1 ), and per mil of photosynthetic carbon lost as isoprene in the leaves 2, 3, 8 and 15 (from the plant apex) of the poplar sapling grown in FACE and ambient [CO 2 ] A Isoprene Carbon FACE Ambient % FACE Ambient FACE Ambient Leaf ± 0.36 A 5.4 ± 0.26 a*** 96 n.d. n.d. n.d. n.d. Leaf ± 0.35 B 8.8 ± 0.72 b*** ± 0.1 A 1.1 ± 0.2 a Leaf ± 0.33 C 18.3 ± 0.57 c*** ± 0.4 B 9.5 ± 1.0 b Leaf ± 0.43 C 18.7 ± 0.27 c*** ± 0.8 C 9.7 ± 1.2 b Leaf ± 1.12 D n.a. n.a. 6.9 ± 0.7 D 3.0 ± 0.2 c 1.82 n.a. The measurements were made at the growth CO 2 concentrations, with a leaf temperature of 30 C in saturating PPFD (1000 mmol m -2 s -1 ). Data are means of four to eight per [CO 2 ] treatment ± 1 SEM. Capital letters (A, B, C, D) and small letters (a, b, c) indicate significant differences at P < 0.05 in the same column for the FACE and Ambient treatments, respectively, whereas *** indicates significant differences at P < in the same line; n.d., non detectable; n.a., not available.

6 408 M. Centritto et al. Figure 3. Leaf isoprene emission (a) of the poplar sapling grown in FACE and ambient [CO 2 ] shown as emission rate along the plant profile (i.e. leaf number from the plant apex) and (b) regression lines fitted through the FACE (r 2 = 0.973) and ambient [CO 2 ] (r 2 = 0.951) data. Data are means of four plants per treatment; letters (a, b) indicate significant differences at P < because these plants had approximately 31% fewer leaves than plants in elevated [CO 2 ]. Consequently, isoprene emission became significantly higher in elevated [CO 2 ] than in ambient [CO 2 ] (Fig. 3). Thus, although the maximum isoprene emission per unit of leaf area was higher in ambient [CO 2 ], the faster decline and lower number of leaves resulted in similar isoprene emission per plant in elevated and ambient [CO 2 ] (Table 4). The effect of growth in elevated [CO 2 ] on leaf ontogeny was determined by comparing A and isoprene emission rate, normalized as a percentage of their maximum values, in leaves of similar physiological age, obtained by transforming the leaf number in relative leaf position in the stem (i.e. the leaf number to total plant leaf number ratio). Elevated [CO 2 ] brought forward the temporal shift of the normal ontogenetic decline of photosynthesis associated with leaf maturation (Fig. 4a), and the onset in the timing of isoprene emission by young leaves (Fig. 4b), indicating that leaf ontogeny was accelerated by FACE treatment. Despite this, the timing of the maximum emission rate of isoprene and its natural decline, associated with progressive leaf senescence, was remarkably similar during leaf development in both [CO 2 ] treatments. Table 4. Basal isoprene emission (nmol m -2 s -1 ) per leaf and per total number of leaves estimated from the integral of the regression curves in Fig. 3b Emission per leaf FACE Ambient Percentage Percentage = 100 FACE/Ambient. Emission total no. leaves Figure 4. Percentage of the combined maximum (a) assimilation rates (A, mmol m -2 s -1 ), showed in Table 3, and (b) leaf isoprene emission, in relation to the relative leaf position in the stem (i.e. the ratio of the leaf number counting from the apex of the plant to the total number of plant leaves)., FACE;, ambient.

7 Isoprene emission and photosynthetic parameters of poplars exposed to FACE 409 DISCUSSION This study, done on fast-growing poplar saplings exposed to FACE treatment, confirmed previous findings (Sharkey et al. 1991; Sharkey & Yeh 2001; Rosenstiel et al. 2003) that A and isoprene emission rate per unit of leaf area have an inverse sensitivity to elevated [CO 2 ] when measured at the growth [CO 2 ] (Table 3). However, the dynamic of both isoprene emission per unit of leaf area and A were strongly affected by ontogeny, which velocity was increased in response to elevated [CO 2 ] (Fig. 4). This is one of the most consistent effects of elevated [CO 2 ] (Centritto et al. 1999a). The poplar saplings were grown in two different nutrient conditions. Plants were either provided with a supply of mineral nutrients at free access, following the Ingestad approach (Ingestad & Ågren 1992) in order to maintain nutrient uptake proportional to plant growth, or were not supplied with nutrient. Drake et al. (1997) showed the importance of available nutrients in determining the extent of the stimulation of A in elevated [CO 2 ]: in a review of eight experiments the average stimulation dropped from 57% at high nitrogen supply to 23% with low availability of nitrogen. Such a large decrease in the stimulation of A could in turn have affected the rate of isoprene emission and plant ontogeny in response to elevated [CO 2 ]. However, the only parameter affected by nutrient treatment was leaf area. Leaf area was significantly decreased in plants not supplied with nutrient (data not shown). This reduction in leaf area allowed plants not supplied by nutrient to maintain nitrogen concentration per unit of leaf area similar to that of plant supplied with mineral nutrients at free access (Table 2), and this, in turn, led to similar source sink functional balance, because no significant differences were found in the physiology of plants exposed to the different nutrient treatments, as expressed in the relative rate of leaf expansion (Fig. 1), photosynthetic capacity (Fig. 2), concentrations of soluble proteins, Rubisco, and chlorophyll, and isoprene emission rate (data not shown). In our experiment, A max was reached at a much lower C i in expanded leaves than in the expanding leaves 2 and 3 (Fig. 2). This may indicate an age-dependent feedback limitation of photosynthesis, perhaps resulting from a limitation in the capacity for end-product synthesis. It should be noted, however, that this effect was independent of, and cannot be therefore attributed to growth [CO 2 ], as has been reported for bean leaves (Socias, Medrano & Sharkey 1993). Much literature suggests that elevated [CO 2 ] primarily affects photosynthesis in an indirect manner through secondary plant metabolic adjustments that modulate photosynthetic gene expression leading to downward acclimation of A (Moore et al. 1999). This process, however, is highly affected by the degree of nutrient and/or rooting volume limitation (Drake et al. 1997). Theoretically, plants grown in the field at optimum rates of nutrient supply (N primarily) should be able to accommodate increases in the rate of CO 2 uptake without recourse to feedback mechanisms, and should therefore show no downward acclimation. In contrast, downward acclimation will be most marked where there is a shortage of nutrients or where rooting is constrained by inadequate pot volume (Arp 1991). However, although there is often a larger downward acclimation of A in low compared with high N supply, results from studies in which both CO 2 concentration and N availability were varied have not been consistent (Stitt & Krapp 1999). Maroco et al. (2002) showed that photosynthetic capacity of cork oak was significantly decreased in plants grown with nitrogen deficiency in elevated [CO 2 ], whereas photosynthetic parameters derived from A C i curves did not reveal any significant effects of elevated [CO 2 ] on the photosynthetic capacity of well-fertilized plants. Similar results were found with Pinus taeda (Tissue, Thomas & Strain 1993; Thomas, Lewis & Strain 1994) and in a study with pea (Riviere- Rolland, Contard & Betsche 1996). In contrast, there was no evidence of downward acclimation in a study with wheat where both CO 2 concentration and N availability were varied (Mitchell et al. 1993). We have shown clearly that leaf nitrogen concentration per unit of leaf area was not affected in the poplar saplings exposed to elevated [CO 2 ] in the field. Consequently, soil nutrient availability did not cause downward acclimation of photosynthetic capacity in elevated [CO 2 ] (Fig. 2, Table 1). These results are similar to those reported by Farage, McKee & Long (1998), who showed that low rates of N supply do not necessarily result in downward acclimation of photosynthetic capacity in elevated [CO 2 ]. They hypothesize that in the natural environment, plants growing in the field are able to adjust their growth rate to match the N availability in the surroundings, so that N uptake keeps pace with carbon uptake and the source sink functional balance is not disrupted. Vice versa, photosynthesis acclimation occurs whenever nutrient uptake is less than the stimulation of carbon acquisition, because growth would be adjusted downward to a rate no longer determined by carbon assimilation but by nutrient acquisition (Lee et al. 2001). Recent findings seem to confirm this hypothesis that an increase in the leaf C/N ratio can trigger downward acclimation of photosynthesis (Paul & Driscoll 1997). Griffin et al. (2000) found a concomitant increase in total nonstructural carbohydrates and decrease in leaf nitrogen content in 1-year-old-needles that lead to photosynthesis acclimation in field-grown Pinus radiata after 4-year exposure to elevated [CO 2 ]. Rogers & Ellsworth (2002) observed downward acclimation of photosynthesis in 1-year-old needles of Pinus taeda exposed to FACE for approximately 2.5 years. Acclimation occurred in needles that had similar contents of nitrogen and soluble proteins, but about 30% more leaf sugar content than in ambient [CO 2 ]. Centritto & Jarvis (1999) showed, in four saplings of Picea sitchensis grown for 3 years in elevated [CO 2 ] and supplied with free access to nutrients, that acclimation of photosynthesis occurred in needles with similar sugar concentrations but lower nitrogen concentrations than in ambient [CO 2 ]. However, there have been signs of age-dependent downward acclimation of A in elevated [CO 2 ] (Fig. 4a).

8 410 M. Centritto et al. Downward acclimation of A to elevated [CO 2 ] generally varies with leaf age, with young leaves commonly showing no acclimation to elevated [CO 2 ] (Nie et al. 1995; Osborne et al. 1998; Moore et al. 1999). Miller et al. (1997), examining the role that leaf ontogeny plays in the acclimation of A, have proposed a temporal shift model in which the natural ontogenetic decline of A, which is normally associated with progressive leaf senescence, was shifted to an earlier transition in elevated [CO 2 ] and, thus, was altered in the timing but not in the magnitude. For now, however, the mechanisms involved in acclimation of the photosynthetic apparatus of mature leaves to elevated [CO 2 ] are not fully understood. It has been suggested that it may regulate source sink interactions by redistributing N from photosynthetic proteins of source leaves to sink tissues, allowing the optimization of whole-plant N use (Sheen 1994). In a study on clonal Sitka spruce saplings grown for three growing seasons in ambient and elevated [CO 2 ], Centritto & Jarvis (1999) clearly showed that reductions of Rubisco content (as inferred from activity measurements) and chlorophyll concentration lead to improved nitrogen use efficiency in elevated [CO 2 ]. In fact, leaf N concentration was lower when the plants were the same size in the two [CO 2 ] treatments, indicating that growth in elevated [CO 2 ] increased the dry mass produced per unit of nitrogen taken up, and, thus, increased the whole-plant N use efficiency. The early finding by Sharkey & Loreto (1993) that emission of isoprene was age-dependent in expanding leaves has been confirmed by our results (Fig. 3). The likely explanation of the delay of the onset of isoprene emission, is that the enzymes necessary to synthesize isoprene are not formed or are not active during the early stages of leaf development (Kuzma & Fall 1993). The distinct agedependent switch can make isoprene an ideal character to mark the passage between juvenility and maturity in ageing leaves. Isoprene emission peaked earlier in the profile in ambient than in elevated [CO 2 ] and rapidly decreased in older leaves (Fig. 3). However, after normalizing leaf position (i.e. making comparable the relative position in the stem of leaves growing in ambient and elevated [CO 2 ] and, thus, comparing leaves of similar physiological age), a distinct difference was observed in the patterns of A and isoprene emission along the profile (Fig. 4). In fact, A peaked earlier than isoprene, especially in elevated [CO 2 ], and maximal photosynthesis was maintained in several leaves along the profile. Isoprene maximal emission, on the other hand, was reached at almost half of the leaf profile in both [CO 2 ] levels and then rapidly decreased. We take this as a further indication of the validity of isoprene as a physiological trait indicating leaf ontogeny. It is interesting that normalization (i.e. comparing emission in leaves of similar physiological age) almost cancelled out the differences in isoprene emission in ambient and elevated [CO 2 ], clearly shown by Fig. 3. Isoprene emission seems to be determined by the leaf ranking in the profile, independently of the total number of leaves in the profile. In addition, the finding that FACE has accelerated the ontogenetic pattern of isoprene emission sustains the hypothesis that one of the main effects of elevated [CO 2 ] is to accelerate all aspects of plant ontogenetic development (Centritto et al. 1999a; Centritto 2000). Finally, the clear difference of isoprene emission among leaves of the profile, suggests that isoprene may also be a better trait to characterize leaf maturity and the onset of senescence in leaves, with respect to A in elevated [CO 2 ] (Fig. 4). The other confirmation of our study is that isoprene emission is reduced in elevated [CO 2 ], in terms of both maximum values of isoprene emission rate (Fig. 3) and isoprene emission per unit of leaf area averaged across the total number of leaves per plant (Table 4). Our data-set as well as that reported by Scholefield et al. (2003), in a companion experiment on Phragmites growing in a nearby CO 2 spring, mostly confirm that isoprene emission is inversely dependent on [CO 2 ] when this is above ambient, and suggest that a lower fraction of C will be re-emitted in the atmosphere as isoprene by single leaves in the future (Table 3). A progressive decrease of isoprene synthase activity at increasing level of [CO 2 ] (Scholefield et al. 2003) or a reduced substrate availability consequent to a metabolic competition for phosphoenolpyruvate (Rosenstiel et al. 2003), can be invoked as possible causes of the [CO 2 ] effect. Our experiments, on the other hand, have also shown that when isoprene emission is integrated over the whole plant profile, the emission of isoprene is not different in plants growing in ambient or elevated [CO 2 ] (Table 4), because an higher number of leaves was formed in elevated [CO 2 ] (Fig. 3). Thus, in our study the impact of elevated [CO 2 ] on the emission of this important phytogenic compound in the atmosphere is not as strong as that reported by Rosenstiel et al. (2003). There are many factors that can explain this discrepancy. This can be firstly explained by the lower [CO 2 ] used in our FACE treatment. Rosenstiel et al. (2003) exposed their poplar plantation to [CO 2 ] of 800 and 1200 mmol mol -1, and found that the higher the growth [CO 2 ], the lower the ecosystem isoprene emission. Thus, we can speculate that because our plants were grown at much lower [CO 2 ] (i.e. approximately 550 mmol mol -1 ), this may have resulted in a lower inhibition of isoprene emission. However, our plants were grown at a [CO 2 ] perhaps closer to what is expected to be the CO 2 concentration in the near future, and we think this should be taken into account when modelling the impact of isoprene emission on the atmospheric chemistry and airquality with the future scenario of global change. Secondly, we measured basal isoprene emission; that is, in standard light intensity (1000 mmol m -2 s -1 ) and temperature (30 C) conditions, whereas Rosenstiel et al. (2003) measured net ecosystem isoprene production at the growth light and temperature conditions. Another important trait that has to be considered in modelling future pattern of isoprene emission in forest and agriforest vegetation is the dynamic of leaf growth in response to elevated [CO 2 ]. We have shown that leaf number was increased in elevated [CO 2 ] in poplar sapling

9 Isoprene emission and photosynthetic parameters of poplars exposed to FACE 411 during their first year of growth, and that this in turn affected the amount of isoprene emission integrated over the whole plant profile. However, Centritto, Lee & Jarvis (1999b), in a study with potted cherry seedlings grown in open-top chambers, and Gielen et al. (2001), in a study with poplar saplings exposed to FACE, showed that the stimulation of total leaf area in response to elevated [CO 2 ] was a transient effect, because it occurred only during the first year of growth. Consequently, it may be expected that with similar levels of leaf area, the integrated emission of isoprene would have been much lower in elevated [CO 2 ]. The finding that isoprene emission per unit of leaf area was not affected by nutrient availability (data not shown) is apparently not in keeping with earlier studies showing that plants grown in high nitrogen increased the rate of isoprene emission (Harley et al. 1994). However, Harley et al. (1994) found that the high nitrogen treatment resulted in higher leaf nitrogen content whereas in our study nitrogen concentration per unit of leaf area was not affected by the rate of nutrient supply. This may also explain the lack of differences in the rate of isoprene emission. However, despite the result of our study, nitrogen nutrition remains one of the pivotal variables affecting plant responses to elevated [CO 2 ], and, in turn, isoprene emission rate. Finally, we suggest that if we are to accurately predict net ecosystem isoprene production in response to global climate change, and therefore its future impact on atmospheric pollution, there is the need for further investigations considering not only elevated [CO 2 ], but also the expected temperature increase. In fact, because isoprene emission is very sensitive to temperature [its Q 10 value ranges between 2 and 4 (Sharkey & Yeh 2001)], it can be expected that rising temperature will increase the rate of isoprene emission. Furthermore, respiration is very sensitive to temperature and will be increased in response to global change, although the extent of this increase is not known. However, isoprene emission is in competition with mitochondrial respiration in light for cytosolic phosphoenolpyruvate (PEP) (Rosenstiel et al. 2003). Isoprene synthesis is responsive to the intracellular availability of PEP, so that if mitochondrial respiration during the day is increased in response to elevated [CO 2 ], the intracellular concentration of PEP is reduced and this is likely to cause a decrease in isoprene emission (Rosenstiel et al. 2003). Consequently, increasing temperature by increasing respiration in light may indirectly decrease isoprene emission. 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