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Environmental and Experimental Botany 95 (2013) 50– 58

Contents lists available at ScienceDirect

Environmental and Experimental Botany

j o ur nal hom epa ge: www.elsev ier .com/ locate /e nvexpbot

soprenoid emissions, photosynthesis and mesophyll diffusiononductance in response to blue light

manuele Pallozzia,b, Tsonko Tsonevc, Giovanni Marinoa,d, Lucian Copolovici e,lo Niinemetse, Francesco Loreto f, Mauro Centritto f,∗

Institute of Agroenvironmental and Forest Biology, National Research Council, Via Salaria km 29.300, 00015 Monterotondo Scalo, RM, ItalyDepartment of Forest Environment and Resources, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo, ItalyInstitute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, BulgariaDipartimento di Bioscienze e Territorio, Università degli Studi del Molise, Contrada Fonte Lappone, 86090 Pesche, IS, ItalyInstitute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, EstoniaInstitute for Plant Protection, National Research Council, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy

r t i c l e i n f o

rticle history:eceived 1 February 2013eceived in revised form 24 May 2013ccepted 1 June 2013

eywords:iffusive limitations to photosynthesis

soprenoidsight qualityandarinakoplar

a b s t r a c t

The effects of blue light (BL) on leaf gas exchange of Populus × canadensis, a strong isoprene emitter,and Quercus ilex and Citrus reticulata, two monoterpene emitters with respectively small and large stor-age pools for monoterpenes, were studied. Leaves were initially exposed to a saturating photosyntheticphoton flux density (PPFD) of white light (WL), which was then progressively reduced to perform WL-response curves. Leaves acclimated to saturating WL were then quickly exposed to equivalent BL levelsto perform BL-response curves. Blue light did not significantly affect photosynthetic parameters in thelight-limited portion of the PPFD-response curves in both P. × canadensis and Q. ilex. Whereas photosyn-thesis (A), stomatal conductance (gs), and mesophyll conductance (gm) were significantly decreased athigh PPFDs of BL. A was similarly inhibited by BL in C. reticulata, but there was no significant effect of lightquality on gs. Overall these results show that the negative effect of BL on photosynthesis is widespreadin tree species with different leaf characteristics, and that this involves coordinated reductions in gs and

gm. BL negatively affected isoprene emission and, to a lesser extent monoterpene emissions, in concertwith photosynthetic inhibition. Interesting, both isoprene and monoterpene emissions were shown tobe inversely dependent upon intercellular [CO2]. These results indicate that a change in light spectralquality, which can vary during the day, between days and within seasons, can alter photosynthesis andisoprenoid emissions, depending on the PPFD intensity. Such effects should be strongly considered in

ile iso

photosynthesis and volat

. Introduction

A wide range of plant processes such as phototropism, photo-

orphogenesis, and stomatal opening are influenced by blue light

BL). Positional movements of chloroplasts are also induced by BLn vascular terrestrial plants (Banas et al., 2012). In the mesophyll of

Abbreviations: A, photosynthesis; Asat, PPFD-saturated photosynthesis; BL, blueight; Ci , intercellular CO2 partial pressure; gm, mesophyll conductance; gm,sat, PPFD-aturated mesophyll conductance; gs, stomatal conductance; gs,sat, PPFD-saturatedtomatal conductance; gt , total conductance; IEsat, PPFD-saturated isoprenoidmission; J, photosynthetic electron transport; LED, light-emitting diode; MEP,ethylerythritol 4-phosphate; PEP, phospho-enolpyruvate; PPFD, photosynthetic

hoton flux density; Rn, dark respiration; WL, white light; �, quantum yield of CO2

ssimilation.∗ Corresponding author at: CNR-IPP, Via Madonna del Piano 10, 50019 Sesto

iorentino, Firenze, Italy. Tel.: +39 0555225585; fax: +39 0555225666.E-mail address: mauro.centritto@cnr.it (M. Centritto).

098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.envexpbot.2013.06.001

prenoid emission models.© 2013 Elsevier B.V. All rights reserved.

these plants, chloroplasts align perpendicularly to the light direc-tion along cell walls (periclinal walls) in the weakly illuminatedparts of the cell, thus increasing photosynthetic light absorption. Incontrast, when exposed to high light intensities, chloroplasts moveaway from BL, appressing themselves to the cell wall parallel to thelight beams (anticlinal walls) (Kasahara et al., 2002; Wada et al.,2003; Banas et al., 2012).

Chloroplast redistribution in response to BL influences photo-synthesis (A) because the avoidance response in the upper layersof a leaf exposed to strong light may reduce the absorbance of theincident photosynthetic photon flux density (PPFD). This canaliza-tion of light into the deeper layers of a leaf, is characteristic ofleaves with a high photosynthetic capacity (Evans and Vogelmann,2003; Brodersen et al., 2008). Nevertheless, BL is still very strongly

absorbed by chlorophyll and thus, even after chloroplast move-ments, BL tends to be distributed less uniformly through leaf layersthan white, green or red light (Evans and Vogelmann, 2006), reduc-ing the contribution of chloroplasts in leaf interior (Terashima et al.,

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011). In fact, McCree (1972) and Evans (1987) demonstrated atrong reduction of both action spectrum and quantum yield of leafhotosynthesis (i.e., spectral quantum yield) under BL comparedo red light regardless of the directional movement of chloroplasts.n addition to a strongly skewed distribution of light within theeaves observed in early studies, these BL-driven effects have alsoeen related to inefficient energy transfer from carotenoids tohlorophylls (Duysens, 1952) because of increased absorption bypidermal cells (Inada, 1976), and unbalanced excitation of the twohotosystems (Evans, 1987). Finally, Oguchi et al. (2009) showedhat BL has a larger impact than white, green and red light on lossf photochemical efficiency, likely indicating the enhanced absorp-ion and photoinhibition within the upper layers of leaves with highhotosynthetic capacity.

Blue light has a well-known positive influence on stomatal con-uctance (gs) (Sharkey and Raschke, 1981), whereas the reducedurface area of chloroplasts exposed to intercellular airspaceswhen positioned along anticlinal walls) (Tholen et al., 2008) mayffect the CO2 diffusion path to Rubisco carboxylation sites. Loretot al. (2009) performed a study on the effects of BL on diffus-onal limitations to A in leaves of Nicotiana tabacum and Platanusrientalis. They showed that when leaves were exposed to BL frac-ions increasing from 0 to 80% of incident light intensity (fixedt 300 �mol photons m−2 s−1), A was quickly inhibited despitencreased stomatal conductance (gs). Photosynthetic inhibition wasnly partially explained by chloroplast movements, because thereas a concomitant, fast decrease in mesophyll conductance to CO2

gm). A 50% reduction in gm occurred within ∼2 min of BL expo-ure, and was thus faster than any possible chloroplast movementrom periclinal to anticlinal cell walls (Wada et al., 2003; Tholent al., 2008). Kaldenhoff (2012) recently hypothesized that thisapid decline in gm might be related to the role of aquaporins,hich are significant components facilitating CO2 diffusion in theesophyll.Isoprenoid emissions, in particular isoprene and monoterpenes,

ave a putative defensive role against abiotic and biotic stressSharkey and Yeh, 2001; Loreto and Centritto, 2008). It has beenemonstrated that isoprenoid emissions modulate plant toler-nce to heat, pollutant and oxidative stress, and are involvedn plant defense reactions against pathogens, parasites or herbi-ores (Loreto and Schnitzler, 2010). Numerous early studies haveemonstrated a link between photosynthesis and the instanta-eous emission of isoprene (Sanadze, 1969). Brilli et al. (2007)emonstrated that in non-stressed poplar about 90% of the carbonxed into isoprene originates from recently assimilated photosyn-hetic intermediates. The light dependence of isoprene emissionas been attributed to control by photosynthetic electron trans-ort (Niinemets et al., 1999), in addition to direct light activationf isoprene synthase and/or light-dependent supply of the sub-trate dimethylallyldiphosphate (Rasulov et al., 2009). Regulationf monoterpene emission is more controversial, as both physiolog-cal and physico-chemical factors may play a role. In plants withouttorage pools, monoterpene emission is partially controlled by non-pecific monoterpene storage in leaf lipid and aqueous phasesNiinemets et al., 2010), with stomatal regulation of only the water-oluble monoterpenes such as linalool (Noe et al., 2006). However,dentical to isoprene, most of the control on monoterpene emissions carried out by photochemically driven reactions (Loreto et al.,996). Thus, any reduction of photosynthesis is expected to lead toeduced carbon and energy supply for the synthesis of isoprenoidsNiinemets et al., 1999; Brilli et al., 2007; Rasulov et al., 2009), ateast at ambient CO2 concentration (Loreto and Schnitzler, 2010).

n turn, reduced emissions of isoprenoids could predispose plantso be more susceptible to abiotic and biotic stress.

Despite substantial accumulation of information about theffects of light intensity on photosynthesis and isoprenoid

erimental Botany 95 (2013) 50– 58 51

emissions, the influence of light spectral composition on theseprocesses has not yet been studied. Blue light has signal func-tions regulating many processes in plants (such as chloroplastrelocation, stomatal opening, expression of genes that encode keyenzymes in the Calvin cycle and respiration) that may in turnaffect isoprenoid emission. We hypothesize that a negative effectof highly energetic light wavelengths (namely BL) on photosyn-thesis would also affect volatile isoprenoids formed in chloroplastsby reducing the availability of freshly fixed carbon. Comparison ofisoprenoid emission and photosynthesis under different light qual-ities could contribute to further clarification of the relationshipsbetween these two processes and the balance between energeticand carbon requirements for biosynthesis and emission of iso-prenoids. In order to understand whether the quantity of emittedisoprenoids will change under BL, and how these changes will berelated to photosynthetic characteristics, we performed a study ontree species characterized by different emissions of isoprenoids:Populus × canadensis, a strong isoprene emitter, and Quercus ilexand Citrus reticulata, two monoterpene emitters with small andlarge storage pools for monoterpenes, respectively. Nevertheless,even in species with large storage pools, it has been demonstratedthat a significant component of the emissions relies upon light-dependent de novo synthesized emissions that may occasionallycontribute 30–90% of total emissions (Staudt et al., 1997; Komendaand Koppmann, 2002). As light spectral quality can vary during theday, between days and within seasons, depending upon conditionssuch as cloudiness, solar angle and atmospheric clarity (Olesen,1992; Grant and Heisler, 2001), an understanding of the plant pho-tosynthetic and isoprenoid emission responses to light quality canenhance large-scale mathematical models of carbon and isoprenoidfluxes.

2. Materials and methods

Gas exchange measurements were performed in laboratory con-ditions on single leaves of potted plants exposed to either whitelight (WL) or blue light (BL). To measure photosynthesis (A) vs. WLresponse curves, leaves were first exposed to a saturating photo-synthetic photon flux density (PPFD) and then were exposed toprogressively reduced intensities. Before performing the A vs. BLresponse curves, leaves were exposed to saturating PPFD of WLuntil steady-state values of the photosynthetic parameters wereobtained. Afterward leaves were exposed to equivalent PPFD val-ues of BL, by quickly switching from white to blue wavelengths,and then to progressively reduced intensities.

2.1. Laboratory experiments on P. × canadensis and Q. ilex

One-year-old P. × canadensis saplings and three-year-old Q. ilexsaplings were potted into 4 dm3 and 10 dm3 pots, respectively,containing commercial soil. All saplings were grown outdoor inMonterotondo Scalo (RM), Italy (42◦04′ N; 12◦36′ E) under natu-ral sunlight conditions, regularly watered to pot water capacity,and fertilized once a week following Ingestad principles in order tosupply mineral nutrients at free-access rates (Magnani et al., 1996;Sun et al., 2008).

Measurements of gas exchange, chlorophyll fluorescence andisoprenoid emissions were made on the central section of a newlyexpanded leaf exposed to either BL or WL (five saplings per plantspecies per treatment). Isoprenoid emissions were detected con-tinuously, whereas gas exchange and fluorescence were recorded

when at a light-induced steady-state. A (�mol m−2 s−1) and gs

(mol m−2 s−1) were measured using a portable infrared gas ana-lyzer system LI-6400 (LI-Cor, Lincoln, NE, USA) by clamping aportion of fully expanded leaf into a standard 2 cm × 3 cm chamber

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2 E. Pallozzi et al. / Environmental an

uvette, with Propafilm® top window, holding an internal quantumensor (spectrally sensitive almost entirely only in photosyntheticctive spectral region) placed close to the leaf lamina. An optic fiberf a pulse-amplitude-modulated fluorimeter (MINI-PAM, Heinzalz GmbH, Effeltrich, Germany) was mounted to the top of the

uvette at an angle of 45◦. The optic fiber was placed about 1 cmrom the leaf without shading it.

Leaves were exposed to a synthetic air flux, free of contami-ants and pollutants, comprising N2, O2 and CO2 in atmosphericoncentrations (80%, 20% and 385 ppmv, respectively). Leaf tem-erature was set at 30 ◦C, and the relative humidity in the cuvetteanged between 40 and 50%. To reduce diffusion leaks throughhe chamber gaskets, the CO2 and H2O gradients between then-chamber air and pre-chamber air were minimized by enclos-ng the whole leaf chamber in a polyvinyl fluoride bag supplied

ith the exhaust air from the IRGA as described by Rodeghierot al. (2007). Light was provided by a computer controlled lightystem that combines four lamps with 4 cm2 arrays of RGBAred, green, blue, amber) LEDs (light-emitting diodes) (ENFIS Ltd.,wansea, UK), capable of generating different monochromatic lightithin the visible spectrum in addition to WL. The blue LEDs

425–495 nm) had a peak emission at 460 nm. To enable mea-urements of BL-saturated photosynthetic rates, illumination ofhe leaf cuvette by RGBA LEDs was supplemented with lightrovided by an auxiliary BL led lamp (450–475 nm) (Vinci Fine

nstruments, Monterotondo, Italy). Then, gas exchange parametersnd chlorophyll fluorescence yield were measured simultaneously.he PPFD values used to generate A/light response curvesanged between 2000 and 0 �mol photons m−2 s−1, with both WLnd BL.

The quantum yield of PSII in the light (�F/F ′m, where �F is

he difference between the maximum F ′m and minimum fluores-

ence yield in the light-adapted state) was measured following aaturating pulse (10,000 �mol m−2 s−1) of WL. Mesophyll conduc-ance (gm, mol m−2 s−1) was calculated by the variable J method,s described by Harley et al. (1992). Jf (rate of electron flow,mol e− m−2 s−1): Jf = �F/F ′

m · PPFD · · ˇ. In our samples, ˛, theotal leaf absorbance (equal to 0.851 for WL and 0.917 for BL in. × canadensis, and 0.89 for WL and 0.924 for BL in Q. ilex) waseasured using the LI-1800 spectroradiometer (LI-Cor, Lincoln, NE,SA) with an integrating sphere in which the leaves were exposed

o WL, and (the distribution of light between the two photosys-ems) was estimated using the Laisk plot (Laisk and Loreto, 1996) asescribed by Loreto et al. (2009). Measurements of dark respirationRn, �mol m−2 s−1) were made after maintaining leaves in dark-ess for 10 min. Although diffusion leaks were minimized, it was

mpossible to rule out the occurrence of other measurement errorsRodeghiero et al., 2007). However, Centritto et al. (2009) have cal-ulated gm by using both the variable J method and carbon isotopeiscrimination in recently synthesized sugars in water-stressed riceenotypes, and found that the two methods yielded congruent esti-ations of gm, confirming the reliability of the variable J method.

hen, the total conductance (gt, mol m−2 s−1) was calculated as:t = gsgm/(gs + gm).

Isoprenoid emissions were detected on-line, after recordinghotosynthetic and fluorescence data, by connecting the cuvetteutflow to a proton transfer reaction mass spectrometer (PTR-MS;onicon, Innsbruck, Austria) with short Teflon polytetrafluoroethy-ene (PTFE) tubes, limiting the potential adhesion of isoprenoids toubing surfaces. Isoprenoids measurements were recorded whenhe emissions reached steady-state values for each light intensity;he time taken to reach this state was approximately 5 min. TheTR-MS was set in single ion mode (SIM) to record traces of pro-

onated isoprene, detected as parent ion at protonated mass m/z 69,nd total monoterpenes, measured by the signals of the protonatedasses m/z 81 and m/z 137. Details on the theory and practice of

rimental Botany 95 (2013) 50– 58

the PTR-MS technique are reported by Lindinger et al. (1998). ThePTR-MS was calibrated daily before measurements using isopreneand monoterpene gaseous standards (100 nL L−1) (Rivoira, Milano,Italy).

2.2. Laboratory experiments on C. reticulata

Three-year-old C. reticulata saplings were grown in 6 dm3 potscontaining garden soil mixed with peat (1:1) in a controlled-condition plant growth room under 600 �mol m−2 s−1 photon fluxdensity, 25/20 ◦C of day/night temperature, 65% relative humidity,and 12 h of photoperiod. Pots were regularly watered to field capac-ity when needed and fertilized with a slow release NPK mineralfertilizer with microelements.

Measurements of gas exchange and isoprenoid emissions werecarried out on newly expanded leaves exposed to either WL orBL (four saplings per plant species per treatment) using an opentwo-channel gas-exchange system described in detail in Copoloviciand Niinemets (2010). Net assimilation rate (A) and stomatalconductance (gs) were measured on whole leaves enclosed in athermostated 1.2 dm3 glass chamber. The gas flow rate through thesystem was 1.4 L min−1, and CO2 and H2O exchange was monitoredwith an infrared dual-channel gas analyzer operated in differen-tial mode (CIRAS II, PP-systems, Amesbury, MA, USA). The CO2concentration inside the chamber was 385 ± 10 �mol mol−1, leaftemperature was set to 28 ◦C and relative humidity at 60%. PPFDwas varied between 0 and 900 �mol m−2 s−1. The WL was providedby four 50 W Osram wide-beam halogen lamps, and BL providedby an IMAGING-PAM M-Series fluorimeter blue 300 W LED actiniclight source consisting of 44 blue Luxeon LEDs (460 nm) (HeinzWalz GmbH, Effeltrich, Germany). Light intensity at the leaf surfacewas measured for both light sources individually by a SA-190 quan-tum sensor (LI-Cor, Inc., Lincoln, NE, USA). Leaves were exposedto a synthetic air flux, free of contaminants and pollutants, com-prising of N2, O2 and CO2 in atmospheric concentrations (80%, 20%and 385 ppmv, respectively) mixed by three Bronkhorst mass flowcontrollers. To measure isoprenoid emissions, the chamber outflowwas diverted into a silcosteel cartridge packed with 200 mg of Car-botrap C 20/40 mesh, 100 mg Carbopack C 40/60 mesh and 100 mgCarbotrap X 20/40 mesh (Supelco, Belefonte, USA). A volume of 2 Lof air was pumped through the trap at a rate of 200 mL min−1. Themeasurements were replicated four times.

The stainless steel cartridges were analyzed by a Shimadzu 2010plus gas chromatograph–mass spectrometer (GC–MS) instrumentwith TD-20 automated cartridge thermodesorber unit (ShimadzuCorporation, Kyoto, Japan). The GC was equipped with a splitlessinjector and a ZB-624 capillary column (60 m in length, 320 �mi.d. and 1.8 �m film thickness) (Zebron, Phenomenex, Torrance, CA,USA). Helium was used as the carrier gas. The GC–MS was calibratedusing authentic standards (GC purity, Sigma–Aldrich, St. Louis, MO,USA). The compound identification was made using the NIST spec-tral library and based on retention time identity with the authenticstandards. GC peak retention time was substantiated by analysis ofparent ions and main fragments on the spectra. The absolute con-centrations of volatile isoprenoids were calculated from the peakareas.

2.3. Curve fitting

The quantum yield of CO2 assimilation (�, mol mol−1), darkrespiration (Rn, �mol m−2 s−1) and PPFD-saturated photosynthesis(Asat, �mol m−2 s−1) were estimated by fitting a non-rectangular

PPFD-saturated stomatal conductance (gs,sat, mol m−2 s−1), PPFD-saturated mesophyll conductance (gm,sat, mol m−2 s−1), andPPFD-saturated isoprenoid emission (IEsat, nmol m−2 s−1) were

d Experimental Botany 95 (2013) 50– 58 53

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Fig. 1. Average responses of (a) photosynthesis (A), (b) stomatal conductance (gs),(c) mesophyll conductance (gm), (d) leaf intercellular CO2 concentration (Ci) and (e)isoprene emission to photosynthetic photon flux density (PPFD) in P. × canadensissaplings. The measurements were made in white (©) and blue (�) lights. Values aremeans ± SE of five plants per treatment. The values with a star (*) indicate significantdifferences between treatments at given PPFD (P < 0.05). Non-rectangular hyperbolafunctions, generated by the Sigma Plot 11.0 software, were fitted to the responsesof A (r2 = 0.994, P < 0.0001 and r2 = 0.999, P < 0.0001 for WL and BL, respectively),gs (r2 = 0.998, P < 0.0001 and r2 = 0.985, P < 0.0001 for WL and BL, respectively), gm

(r2 = 0.997, P < 0.0001 and r2 = 0.997, P < 0.0001 for WL and BL, respectively), andisoprene emission (r2 = 0.990, P < 0.0001 and r2 = 0.997, P < 0.0001 for WL and BL,respectively) to PPFD.

E. Pallozzi et al. / Environmental an

stimated by fitting non-rectangular hyperbola functions to thendividual gs/PPFD, gm/PPFD and isoprenoid (i.e., isoprene or

onoterpene)/PPFD response curves, respectively.

.4. Statistical analysis

All measurements were performed on four replicates. Resultsre presented as means ± SE, unless otherwise specified. Beforeerforming the ANOVA test, the Shapiro–Wilk test of normality wasndertaken to determine if the data in each group was normally dis-ributed. All statistical analyses were conducted using Sigma Plot1.0 software (Systat Inc., San Jose, CA, USA). Asterisks (*) indicateignificant differences (Tukey’s test, P < 0.05) between treatmenteans measured at the same fractions of PPFD.

. Results

.1. Light response curves of P. × canadensis

There were no significant differences in Rn, light compensa-ion point (the light intensity at which A is zero), and maximumuantum yield (�, i.e., the slope of A/PPFD response when this

s linear) between the two light quality treatments (Table 1). AsPFD increased, and no longer limited A, a strong effect of lightuality was observed. BL strongly reduced A from approximately00 �mol m−2 s−1 of PPFD, and light-saturated photosynthesis washifted to higher PPFD (Table 1 and Fig. 1a). Asat, estimated from/PPFD curves, was inhibited by approximately 30% under BL. Sim-

larly to A, gs (Fig. 1b) and gm (Fig. 1c), were not significantly affectedy BL in the light-limited portion of the curves, but were signif-

cantly reduced by BL at higher PPFDs. In a similar fashion to A,m was inhibited at PPFD higher than 600 �mol m−2 s−1 (Fig. 1c).stimated gm,sat was inhibited by ∼46% and saturated at higherPFD under BL (Table 1). Stomatal conductance was affected byL at slightly higher PPFDs than gm (around 800 �mol m−2 s−1)Fig. 1b), and was reduced by ∼25% under saturating PPFD. A exhib-ted higher inhibition than gs at BL above 1400 �mol m−2 s−1, asndicated by the significant increase of the intercellular CO2 partialressure (Ci) with respect to that measured at similar intensitiesf WL (Fig. 1d). The response of isoprene emission to BL followedhe response of A, as the emission was unaffected at low lightntensities, and significantly inhibited by BL at PPFDs higher than600 �mol m−2 s−1 (Fig. 1e). Saturated isoprene emission (IEsat,mol m−2 s−1), estimated from isoprene/PPFD curves, reached sat-ration at about 3200 �mol m−2 s−1 under both light treatmentsnd was ∼24% lower under BL (Table 1).

.2. Light response curves of Q. ilex

The gas exchange responses to WL or BL of Q. ilex, a sclerophyl-ous species with no permanent pools for monoterpenes (Staudtnd Bertin, 1998), are shown in Fig. 2. As seen in P. × canadensis, theight-limited portion of the A/PPFD curve (Fig. 2a) was not affectedy the light quality treatments and, consequently, there wereo significant differences in Rn, light compensation point and �Table 1). However, as PPFD increased above ∼800 �mol m−2 s−1, Aas significantly reduced by BL (Fig. 2a). Asat decreased by ∼30% andhotosynthesis was saturated at higher PPFD (Table 1) in responseo BL. Stomatal conductance (Fig. 2b) and gm (Fig. 2c) were also onlyignificantly affected when leaves were exposed at high PPFDs of BL.L strongly decreased gs,sat (∼−43%), which also saturated at lowerPFD as compared to WL intensities (Table 1). Whereas, gm was

nhibited at lower PPFD (∼600 �mol m−2 s−1) than A and gs, andaturated at PPFDs ∼1900 �mol m−2 s−1 under BL (Table 1). Under

L, it was not possible to estimate gm,sat from the PPFD responseurves within a range of realistic radiation intensities. Ci dropped

54 E. Pallozzi et al. / Environmental and Experimental Botany 95 (2013) 50– 58

Table 1Values of quantum yield of CO2 assimilation (�, mol mol−1), dark respiration (Rn, �mol m−2 s−1), PPFD-saturated photosynthesis (Asat, �mol m−2 s−1), PPFD-saturated stomatalconductance (gs,sat, mol m−2 s−1), PPFD-saturated mesophyll conductance (gm,sat, mol m−2 s−1), and PPFD-saturated isoprenoid (either isoprene or monoterpene) emission(IEsat, nmol m−2 s−1) obtained by fitting a non-rectangular hyperbola function to the individual PPFD response curves shown in Figs. 1–3. Average values of PPFD saturationrelatives to Asat, gs,sat, gm,sat, and IEsat are also shown. N.A. = data not available, N.D. = data not determined within realistic PPFD values. Asterisks indicate significantly differentvalues between white and blue light within each species (P < 0.05).

P. × canadensis � Q. ilex � C. reticulata �

White light Blue light White light Blue light White light Blue light

� 0.035 ± 0.003 0.034 ± 0.001 0.018 ± 0.001 0.018 ± 0.001 0.034 ± 0.002* 0.009 ± 0.002Rn −2.70 ± 0.27 −2.49 ± 0.32 −0.89 ± 0.21 −0.99 ± 0.07 −0.28 ± 0.05 −0.26 ± 0.02Asat 21.65 ± 0.74* 15.24 ± 0.60 6.45 ± 0.54* 4.54 ± 0.32 3.48 ± 0.50 2.77 ± 0.27Asat-PPFD saturation 2100 2350 1560 1710 990 990gs,sat 0.536 ± 0.065 0.401 ± 0.040 0.058 ± 0.006* 0.033 ± 0.005 N.D. N.D.gs,sat-PPFD saturation 2340 2350 2100 1620 N.D. N.D.gm,sat 0.207 ± 0.030* 0.112 ± 0.015 N.D. 0.058 ± 0.008 N.A. N.A.

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IEsat 43.65 ± 1.11* 33.38 ± 1.63

IEsat-PPFD saturation 3180 3240

apidly at increasing PPFD, but this response was not affected byight quality (Fig. 2d), indicating that the relative effects of BL on

and gs were similar. Monoterpene emission was higher underL than under BL, but statistical differences were evident only

t intermediate PPFD values, between 800 and 1000 �mol m−2 s−1

Fig. 2e). Consequently, there were no significant differences in IEsat

etween the light quality treatments, despite the estimated sat-rating PPFD being much higher under BL in comparison to WLTable 1).

.3. Diffusional limitations in P. × canadensis and Q. ilex

Photosynthesis was significantly correlated to diffusional con-uctances in both P. × canadensis and Q. ilex (Fig. 3). Typicalyperbolic relationships were found between A and gs (Fig. 3a and), gm (Fig. 3b and e), and the total conductance gt (Fig. 3c and), after pooling together data collected under both light qualityreatments.

.4. Light response curves of C. reticulata

In leaves of C. reticulata, a sclerophyllous species with a signif-cant storage pool for monoterpenes, A was lower under BL thannder WL (Fig. 4a). This effect was already observed at low PPFDnd, consequently, the maximum quantum yield � was stronglynhibited under BL (Table 1). In contrast, no significant differences

ere found in Rn in response to the light quality treatments. Thenhibition of A by BL was again significant at high PPFD. At the sat-rating PPFD of our experiment (Fig. 4a), Asat decreased by ∼20%

n response to BL (Table 1), but this effect was not statisticallyignificant. There was no significant effect of light quality on gs

Fig. 4b); gs scaled linearly as PPFD increased both under WL andL and, thus, it was not possible to estimate gs,sat within a real-

stic range of radiation intensities (Table 1). Furthermore, it waslso not possible to estimate gm because the experimental set-upid not allow the simultaneous measurements of gas exchangend chlorophyll fluorescence. Total monoterpene emission wasight-dependent and decreased to very low levels (∼7% of maxi-

um) in darkness (Fig. 4c). Monoterpene emissions were generallyower under BL than under WL, but statistically significant dif-erences were only evident at intermediate light intensities (i.e.,etween 150 and 450 �mol m−2 s−1). Nevertheless, total monoter-

ene emission was apparently light-saturated only under WL, as

t was not possible to estimate IEsat under BL (Table 1). The blendf monoterpenes emitted by C. reticulata leaves, �-pinene (∼63%),amphene (∼23%), �3-carene (∼5%), limonene (∼5%) and �-pinene∼4%) was not affected by light quality (data not shown).

1910 N.A. N.A. 0.80 5.27 ± 0.49 1.93 ± 0.20 N.D.

2105 940 N.D.

3.5. Isoprene and monoterpene sensitivity to Ci

The emission of isoprene from P. × canadensis and monoter-penes from Q. ilex showed a strong dependence upon Ci, but wereindependent of light quality treatment. After pooling togetherdata collected under BL and WL, significant inverse relationshipswere found between Ci and both isoprene (Fig. 5a; r2 = 0.946)and monoterpene (Fig. 5b; r2 = 0.938) emissions. Monoterpeneemissions from C. reticulata (Fig. 5c) also showed an inverse Cidependence, but this relationship was weaker than in the othertwo species (r2 = 0.283).

4. Discussion

4.1. Blue light effects on photosynthesis and mesophyll diffusion

The negative effect of BL on photosynthesis is well known(McCree, 1972), although some responses may be specific to partic-ular tree species (Sarala et al., 2009). BL mainly affects the quantumyield of photosynthesis, perhaps altering the distribution of lightbetween photosystems (Evans, 1987). We therefore expected BLto negatively impact on photosynthesis when light is limiting. Themajority of the previous studies could not be conducted at lightintensities above those where photosynthesis is constrained (e.g.max 300 �mol m−2 s−1 in Loreto et al., 2009), a technical limitationthat we have now overcome. However, in our study, �, calculated onan incident light basis, was significantly reduced only in C. reticulataunder BL (Fig. 4a), whereas in the other two species the negativeimpact of BL was only significant when photosynthesis was nolonger light-dependent (i.e. at higher PPFDs) or experiencing otherphotosynthetic limitations (Table 1).

Loreto et al. (2009) found a strong effect of BL on diffusive limita-tions to photosynthesis. The first, and most important, componentof these diffusive limitations is stomatal resistance. However, BL isknown to induce stomatal opening (Sharkey and Raschke, 1981);and therefore increasing limitation of photosynthesis due to stoma-tal conductance is considered unlikely under BL. In P. × canadensis(Fig. 1) and Q. ilex (Fig. 2), gs was inhibited by high PPFD of BL, asurprising result that could not be observed at lower PPFD. How-ever, Ci was either unaffected (Q. ilex) or increased (P. × canadensis)under high intensities of BL, indicating that stomatal limitations toCO2 diffusion was unlikely the major limiting factor to A in theseleaves.

The resistance to CO2 diffusion inside the leaf mesophyll (gm)

might also increase under BL, thus limiting A, however, approx-imately half of the observed effect of BL on gm may be due toexperimental artifacts, as recently demonstrated by Loreto et al.(2009). In this study, consideration was given to the possibility of

E. Pallozzi et al. / Environmental and Exp

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O2]

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(nm

ol

m-2

s-1)

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2

4

6 **

(a)

(b)

(c)

(d)

(e)

*

Fig. 2. Average responses of (a) photosynthesis (A), (b) stomatal conductance (gs),(c) mesophyll conductance (gm), (d) leaf intercellular CO2 concentration (Ci) and(e) total monoterpene emission to photosynthetic photon flux density (PPFD) inQ. ilex saplings. The measurements were made in white (©) and blue (�) light.Values are means ± SE for five plants per treatment. Significance values as in Fig. 1.Non-rectangular hyperbola functions, generated by the Sigma Plot 11.0 software,were fitted to the responses of A (r2 = 0.997, P < 0.0001 and r2 = 0.999, P < 0.0001 forWL and BL, respectively), gs (r2 = 0.991, P < 0.0001 and r2 = 0.992, P < 0.0001 for WLand BL, respectively), gm (r2 = 0.977, P < 0.0001 and r2 = 0.992, P < 0.0001 for WL andBL, respectively), and monoterpene emission (r2 = 0.993, P < 0.0001 and r2 = 0.990,P < 0.0001 for WL and BL, respectively) to PPFD.

erimental Botany 95 (2013) 50– 58 55

alternative electron transport (Laisk and Loreto, 1996) and strongabsorption of BL by carotenoids that may reduce the efficiency ofenergy transfer to chlorophylls (Duysens, 1952). Therefore, our gm

data should not be affected by such artifacts, other than possibleerrors caused by light distribution changes (Loreto et al., 2009)in measurements performed at high light intensity. On the otherhand, because gm calculation is fairly sensitive at low light inten-sity (Harley et al., 1992; Flexas et al., 2008), the gm values obtainedat low PPFD intensities (Figs. 1c and 2c) should be viewed withcaution. We found that the reduction of gm under BL was strongin both poplar and oak species (Figs. 1c and 2c). It is also note-worthy that the reduction of A, gs and gm of the two species wasextremely rapid, and occurred within 3 min of switching from sat-urating PPFD of WL to BL. These results confirm earlier findings(Loreto et al., 2009) that A and gm declined within 2 min in Nico-tiana tabacum and Platanus orientalis leaves upon exposure to BL.Due to its rapid occurrence, it is unlikely that gm inhibition by BL iscaused by chloroplast movements from periclinal to anticlinal walls(Tholen et al., 2008), whereas it might have been elicited by as yetunknown factors affecting aquaporin-facilitated CO2 diffusion inthe mesophyll (Kaldenhoff, 2012).

Under PPFD-saturating WL, gs was ∼2.8 times higher than gm inP. × canadensis, whereas in Q. ilex gs was significantly smaller (∼0.4)than gm. In general, these data confirm that photosynthetic limita-tion by gm is in the same order of magnitude as gs (Centritto et al.,2003; Loreto and Centritto, 2008; Flexas et al., 2008; Terashimaet al., 2011; Brilli et al., 2013). After pooling together data fromboth light treatments, there was a hyperbolic relationship betweenA and leaf conductance in both species (Fig. 3), indicating a coordi-nated reduction of A with increasing internal diffusive limitationsinduced by different levels of BL. Although artifacts in estimationof gm (e.g. caused by sampling mainly the uppermost population ofchloroplasts under BL) cannot be ruled out, these findings confirmthe importance of the internal diffusion of CO2, from intercellularspaces to the active sites of Rubisco inside chloroplasts, in limitingphotosynthesis in plants with different growth forms and leaf struc-tures (Shi et al., 2006; Flexas et al., 2008; Aganchich et al., 2009;Niinemets et al., 2009; Centritto et al., 2011; Terashima et al., 2011).

4.2. Blue light effects on isoprene and monoterpene emissions

The reduction in the emissions of both isoprene, inP. × canadensis (Fig. 1e), and of total monoterpenes, in Q. ilex(Fig. 2e), was significant only at moderate to high light intensities.The quantum yield of isoprenoid emission (the linear slope atlow light intensity) was similar under WL and BL, thus inferringa similar use efficiency of WL and BL for isoprenoid biosyn-thesis. However, the absorbed BL was clearly not sufficient tosustain efficiently high rates of isoprenoid biosynthesis. Isoprenebiosynthesis is costly in terms of energy and reducing powerrequirement, as 20 ATP and 14 NADPH are requested to form eachmolecule of isoprene through the methylerythritol 4-phosphate(MEP) pathway in chloroplasts (Sharkey and Yeh, 2001). Thus,insufficient ATP and NADPH production through light reactionsmight be the cause of reduced isoprenoid emissions under BL.However, monoterpenes that are formed through the same MEPpathway as isoprene (Loreto et al., 1996) should require at leastdouble the amount of ATP and NADPH in their formation. How-ever, monoterpene emission is 5–10 times lower than isopreneemission, which may suggest that overall the energy requestedto sustain their biosynthesis is lower than for isoprene. However,monoterpenes are less volatile than isoprene and therefore can

be found inside leaves in larger pools than isoprene (Loreto et al.,1996). If the estimate of energy requirements for isoprenoidformation is not made on the basis of emissions but accordingto actual biosynthesis, the energy requirement for monoterpene

56 E. Pallozzi et al. / Environmental and Experimental Botany 95 (2013) 50– 58

gs (mol m-2 s-1)

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r2

r089.0=2

r899.0=2 = 0.999

r2

r299.0=2

r189.0=2 = 0.990

(a) )c()b(

(d) )f()e(

F ynthes( Data a

bnitiaWai

umcIsmKoHeilptleto

m(e2fi

ig. 3. Effect of white (©) and blue (�) light on relationships between (a, d) photosc, f) between A and total conductance (gt) in (a–c) P. × canadensis and (d–f) Q. ilex.

iosynthesis is probably comparable or even higher than thateeded for isoprene biosynthesis. Consequently, the negative

mpact of BL should be similar or even higher for monoterpeneshan for isoprene, which was not the case. Our finding mayndicate that isoprenoid biosynthesis is instead limited by carbonvailability. As described above, photosynthesis is lower under BL.e surmise that this may also feedback on isoprenoid production

nd emission, reducing the availability of the photosyntheticntermediates required to start the MEP pathway.

Interestingly, a similar emission pattern was observed in C. retic-lata in response to BL, a species with a significant storage pool foronoterpenes that would be expected to sustain emission of these

ompounds in the absence of fresh carbon (Staudt et al., 1997).ndeed, C. reticulata emitted ∼7% of maximum monoterpene emis-ion in darkness, which is in agreement with previous studies ofonoterpene storing species (Staudt et al., 1997; Komenda and

oppmann, 2002), and in contrast to the other two species relyingn freshly fixed photosynthetic carbon where emissions stopped.owever, in C. reticulata both photosynthesis and monoterpenemission were stimulated by high levels of BL, reaching rates sim-lar to those observed under comparable WL levels. Therefore, inine with the experimental hypothesis, it seems that availability ofhotosynthetic carbon is the main limitation to isoprenoid biosyn-hesis under BL. We conclude that this limitation is apparent at aarge range of PPFD in isoprene emitters, whereas in monoterpenemitters emissions can be restored at high levels of BL because pho-osynthesis also recovers or through the use of additional sourcesf carbon.

Ci negatively influenced emissions of isoprene and totalonoterpenes, and these relationships were not affected by BL

Fig. 5). The negative impact of rising atmospheric CO2 on isoprene

mission has been reported (Rosenstiel et al., 2003; Centritto et al.,004). The impact of increasing [CO2] on monoterpene emission isar more controversial. Our results add evidence to the idea that Cis the critical factor regulating isoprenoid biosynthesis. An inverse

is (A) and stomatal conductance (gs), (b, e) A and mesophyll conductance (gm), andre means ± SE of five plants per treatment.

relationship between isoprene emission and Ci has been observedin P. alba (Loreto et al., 2007), hybrid aspen (Rasulov et al., 2009)and poplar genotypes of different species (Guidolotti et al., 2011).Rosenstiel et al. (2004) suggested that emission of isoprene com-petes with light respiration for cytosolic phospho-enolpyruvate(PEP), and as light respiration is stimulated by rising [CO2], thismetabolic competition for PEP results in inhibition of isopreneemission. This hypothesis was supported by Loreto et al. (2007),though competition for PEP seems to be restricted to oxaloacetateproduction. Clearly, BL does not affect PEP formation and respi-ratory metabolism, which in turn does not further feedback onisoprenoid metabolism.

More interestingly, we show that Ci may also regulate monoter-pene emissions. This effect is particularly clear in species withsmall storage capacity such as Q. ilex, but it is also apparent inspecies with large monoterpene storage capacity such as C. retic-ulata. Our data indicate that the Ci at which the effect starts tobecome apparent in all species is below current atmospheric lev-els. This finding suggests, therefore, that Ci is an important driverof isoprenoid emission under current [CO2] (Loreto et al., 2007;Guidolotti et al., 2011), whereas a much smoother effect of Ci isexpected under further rising [CO2]. As many previous studieshave been carried out at [CO2] levels higher than current ambi-ent, the impact of [CO2] on monoterpene emissions may have beenmissed or under-estimated. We conclude that Ci may be a valuableproxy of emission for all isoprenoids, and an important controllingagent for isoprenoid biosynthesis, possibly regulating PEP avail-ability for the MEP pathway when [CO2] levels are low. Manyenvironmental stresses that are exacerbated by climate change (e.g.high temperatures, water stress and salinity) are known to reducestomatal conductance and Ci, which according to our results and

those of Guidolotti et al. (2011) will positively feedback on iso-prenoid emission. Moreover, isoprenoid biosynthesis is known tobe elicited under stress conditions as these compounds may protectchloroplast membranes (Loreto and Schnitzler, 2010). It may

E. Pallozzi et al. / Environmental and Experimental Botany 95 (2013) 50– 58 57

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Fig. 4. Average responses of (a) photosynthesis (A), (b) stomatal conductance (gs)and (c) total monoterpene emission to photosynthetic photon flux density (PPFD) inC. reticulata saplings. The measurements were made in white (©) and blue (�) light.Values are means ± SE of four plants. Significant difference indicators as in Fig. 1.Non-rectangular hyperbola functions, generated by the Sigma Plot 11.0 software,were fitted to the responses of A (r2 = 0.994, P < 0.0001 and r2 = 0.996, P < 0.0001f 2 2

aP

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Fig. 5. Effect of white (©) and blue (�) light on the relationships betweenisoprenoids and leaf intercellular CO2 concentration (Ci) in (a) P. × canadensis(r2 = 0.946, P < 0.01), (b) Q. ilex (r2 = 0.938, P < 0.01) and C. reticulata (r2 = 0.283,P < 0.05). Data are means ± SE of four C. reticulata plants and five P. × canadensis andQ. ilex plants per treatment.

or WL and BL, respectively), gs (r = 0.954, P < 0.001 and r = 0.990, P < 0.002 for WLnd BL, respectively), and monoterpene emission (r2 = 0.954, P < 0.001 and r2 = 0.990,

< 0.0001 for WL and BL, respectively) to PPFD.

herefore be speculated that climate change will have contrastingffects on the emission of isoprenoids dependent upon the respec-ive prevalence of elevated [CO2] or environmental stresses.

In conclusion, our results show that the negative effect of blueight on photosynthesis (Loreto et al., 2009) (i) is widespread inree species with different leaf characteristics, being particularlyvident in species with more mesophytic leaves and higher pho-osynthetic rates; (ii) is sensed especially at light intensities abovehose for which photosynthesis is PPFD-limited, and responds lin-arly to increasing PPFD; (iii) involves coordinate reductions intomatal and mesophyll conductance to CO2; (iv) negatively affectssoprenoid emissions, in a synchronized fashion with photosynthe-is inhibition. Our results indicate that spectral changes in light (e.g.nhanced blue light fraction in diffuse light and reduced blue frac-ion in morning and evening hours) can alter the photosynthetic

ctivity and isoprenoid emission rate, depending on the PPFD inten-ity. Such effects have so far not been considered in carbon andsoprenoid flux models, but can significantly alter fluxes of carbonnd the biosynthesis of metabolites both during and between days.

Acknowledgements

The authors would like to extend their sincere thanks to MarcoGiorgetti for his technical assistance in the field. This work hasbeen funded by the Italian National Research Council (project RSTL-DG.RSTL.010.003), Italian National Research Council and BulgarianAcademy scientific and technologic agreement, Italian Ministry ofForeign Affairs and Estonian Ministry of Education and Researchexecutive program of cultural, educational, scientific and techno-logical co-operation, Estonian Ministry of Science and Education

(Grant no. SF1090065s07), Estonian Science Foundation (Grantno. 9253) and the European Commission through the EuropeanRegional Fund (the Center of Excellence in Environmental Adap-tation).

5 d Expe

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eferences

ganchich, B., Wahbi, S., Loreto, F., Centritto, M., 2009. Partial root zone drying:regulation of photosynthetic limitations and antioxidant enzymatic activities inyoung olive (Olea europaea) saplings. Tree Physiology 29, 685–696.

anas, A.K., Aggarwal, C., Łabuz, J., Sztatelman, O., Gabrys, H., 2012. Blue lightsignalling in chloroplast movements. Journal of Experimental Botany 63,1559–1574.

rilli, 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.

rilli, F., Tsonev, T., Mahmood, T., Velikova, V., Loreto, F., Centritto, M., 2013. Ultra-dian variation of isoprene emission, photosynthesis, mesophyll conductanceand optimum temperature sensitivity for isoprene emission in water-stressedEucalyptus citriodora saplings. Journal of Experimental Botany 64, 519–528.

rodersen, C.R., Vogelmann, T.C., Williams, W.E., Gorton, H.L., 2008. A new paradigmin leaf-level photosynthesis: direct and diffuse lights are not equal. Plant, Celland Environment 31, 159–164.

entritto, M., Lauteri, M., Monteverdi, M.C., Serraj, R., 2009. Leaf gas exchange, carbonisotope discrimination, and grain yield in contrasting rice genotypes subjectedto water deficits during the reproductive stage. Journal of Experimental Botany60, 2325–2339.

entritto, 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.

entritto, 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.

entritto, M., Tognetti, R., Leitgeb, E., Strelcová, K., Cohen, S., 2011. Above groundprocesses – anticipating climate change influences. In: Bredemeier, M., Cohen,S., Godbold, D.L., Lode, E., Pichler, V., Schleppi, P. (Eds.), Forest Management andthe Water Cycle: An Ecosystem-Based Approach. Ecological Studies, vol. 212.Springer, New York/Dordrecht, pp. 31–64.

opolovici, L., Niinemets, Ü., 2010. Flooding induced emissions of volatile signallingcompounds in three tree species with differing waterlogging tolerance. Plant,Cell and Environment 33, 1582–1594.

uysens, L.N.M., 1952. Transfer of Excitation Energy in Photosynthesis. Universityof Utrecht, the Netherlands (Ph.D. Thesis).

vans, J.R., 1987. The dependence of quantum yield on wavelength and growthirradiance. Australian Journal of Plant Physiology 14, 69–79.

vans, J.R., Vogelmann, T.C., 2003. Profiles of 14C fixation through spinach leaves inrelation to light absorption and photosynthetic capacity. Plant, Cell and Envi-ronment 26, 547–560.

vans, J.R., Vogelmann, T.C., 2006. Photosynthesis within isobilateral Eucalyptus pau-ciflora leaves. New Phytologist 171, 171–182.

lexas, J., Ribas-Carbó, M., Diaz-Espejo, A., Galmés, J., Medrano, H., 2008. Mesophyllconductance to CO2: current knowledge and future prospects. Plant, Cell andEnvironment 31, 602–621.

rant, R.H., Heisler, G.M., 2001. Multi-spectral solar irradiance on tree-shaded verti-cal and horizontal surfaces: cloud-free and partly cloudy skies. Photochemistryand Photobiology 73, 24–31.

uidolotti, G., Calfapietra, C., Loreto, F., 2011. The relationship between isopreneemission, CO2 assimilation and water use efficiency across a range of poplargenotypes. Physiologia Plantarum 142, 297–304.

arley, 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.

nada, K., 1976. Action spectra for photosynthesis in higher plants. Plant and CellPhysiology 17, 355–365.

aldenhoff, R., 2012. Mechanisms underlying CO2 diffusion in leaves. Current Opin-ion in Plant Biology 15, 1–6.

asahara, M., Kagawa, T., Oikawa, K., Suetsugu, N., Miyao, M., Wada, M., 2002.Chloroplast avoidance movement reduces photodamage in plants. Nature 420,829–832.

omenda, M., Koppmann, R., 2002. Monoterpene emissions from Scots pine (Pinussylvestris): field studies of emission rate variabilities. Journal of GeophysicalResearch 107, 4161, http://dx.doi.org/10.1029/2001JD000691.

aisk, A., Loreto, F., 1996. Determining photosynthetic parameters from leaf CO2

exchange and chlorophyll fluorescence: rubisco specificity factor, dark respi-ration in the light, excitation distribution between photosystems, alternativeelectron transport rate and mesophyll diffusion resistance. Plant Physiology 110,903–912.

indinger, W., Hansel, A., Jordan, A., 1998. Proton-transfer-reaction mass spectrom-etry (PTR-MS): on-line monitoring of volatile organic compounds at pptv level.Chemical Society Reviews 27, 347–354.

oreto, F., Centritto, M., 2008. Leaf carbon assimilation in a water-limited world.Plant Biosystems 142, 154–161.

rimental Botany 95 (2013) 50– 58

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., Cecinato, A., Brancaleoni, E., Frattoni, M., Sharkey, T.D., 1996. Differentsources of reduced carbon contribute to form three classes of terpenoids emittedby Quercus ilex L. leaves. Proceedings of the National Academy of Sciences of theUnited States of America 93, 9966–9969.

Loreto, F., Schnitzler, J.-P., 2010. Abiotic stresses and induced BVOCs. Trends in PlantScience 15, 154–166.

Loreto, F., Tsonev, T., Centritto, M., 2009. The impact of blue light on leaf mesophyllconductance. Journal of Experimental Botany 60, 2283–2290.

Magnani, F., Centritto, M., Grace, J., 1996. Measurement of apoplasmic and cell-to-cell components of root hydraulic conductance by a pressure-clamp technique.Planta 199, 296–306.

McCree, K.J., 1972. The action spectrum, absorptance and quantum yield of photo-synthesis in crop plants. Agricultural Meteorology 9, 191–216.

Niinemets, Ü., Monson, R.K., Arneth, A., Ciccioli, P., Kesselmeier, J., Kuhn, U., Noe,S.M., Penuelas, J., Staudt, M., 2010. The leaf-level emission factor of volatileisoprenoids: caveats, model algorithms, response shapes and scaling. Biogeo-sciences 7, 1809–1832.

Niinemets, Ü., Tenhunen, J.D., Harley, P.C., Steinbrecher, R., 1999. A model of isopreneemission based on energetic requirements for isoprene synthesis and leaf pho-tosynthetic properties for Liquidambar and Quercus. Plant, Cell and Environment22, 1319–1335.

Niinemets, Ü., Wright, I.J., Evans, J.R., 2009. Leaf mesophyll diffusion conductancein 35 Australian sclerophylls covering a broad range of foliage structural andphysiological variation. Journal of Experimental Botany 60, 2433–2449.

Noe, S.M., Ciccioli, P., Brancaleoni, E., Loreto, F., Niinemets, Ü., 2006. Emissionsof monoterpenes linalool and ocimene respond differently to environmentalchanges due to differences in physico-chemical characteristics. AtmosphericEnvironment 40, 4649–4662.

Oguchi, R., Terashima, I., Chow, W.S., 2009. The involvement of dual mechanismsof photoinactivation of Photosystem II in Capsicum annuum L. plants. Plant andCell Physiology 50, 1815–1825.

Olesen, T., 1992. Daylight spectra (400–740 nm) beneath sunny, blue skies in Tasma-nia, and the effect of a forest canopy. Australian Journal of Ecology 17, 451–461.

Rasulov, B., Hüve, K., Välbe, M., Laisk, A., Niinemets, Ü., 2009. Evidence that light,carbon dioxide and oxygen dependencies of leaf isoprene emission are drivenby energy status in hybrid aspen. Plant Physiology 151, 448–460.

Rodeghiero, M., Niinemets, Ü., Cescatti, A., 2007. Major diffusion leaks of clamp-onleaf cuvettes still unaccounted: how erroneous are the estimates of Farquharet al. model parameters? Plant, Cell and Environment 30, 1006–1022.

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.

Rosenstiel, T.N., Potosnak, M.J., Griffin, K.L., Fall, R., Monson, R.K., 2003. Increased CO2

uncouples growth from isoprene emission in an agriforest ecosystem. Nature421, 256–259.

Sanadze, G.A., 1969. Light-dependent excretion of molecular isoprene. Progress inPhotosynthesis Research 2, 701–706.

Sarala, M., Taulavuori, E., Karhu, J., Savonen, E.-M., Laine, K., Kubin, E., Taulavuori, K.,2009. Improved elongation of Scots pine seedlings under blue light depletion isnot dependent on resource acquisition. Functional Plant Biology 36, 742–751.

Sharkey, T.D., Raschke, K., 1981. Effect of light quality on stomatal opening in leavesof Xanthium strumarium L. Plant Physiology 68, 1170–1174.

Sharkey, T.D., Yeh, S.S., 2001. Isoprene emission from plants. Annual Review of PlantPhysiology and Plant Molecular Biology 52, 407–436.

Shi, Z., Liu, S., Liu, X., Centritto, M., 2006. Altitudinal variation in photosyntheticcapacity, diffusional conductance, and ı13C of butterfly bush (Buddleja davidiiFranch.) plants growing at high elevations. Physiologia Plantarum 128, 722–731.

Staudt, M., Bertin, N., 1998. Light and temperature dependence of the emission ofcyclic and acyclic monoterpenes from holm oak (Quercus ilex L.) leaves. PlantCell and Environment 21, 385–395.

Staudt, M., Bertin, N., Hansen, U., Seufert, G., Ciccioli, P., Foster, P., Frenzel, B., Fugit,J.L., 1997. Seasonal and diurnal patterns of monoterpene emissions from Pinuspinea (L.) under field conditions. Atmospheric Environment 31, 145–156.

Sun, P., Grignetti, A., Liu, S., Casacchia, R., Salvatori, R., Pietrini, F., Loreto, F., Cen-tritto, M., 2008. Associated changes in physiological parameters and spectralreflectance indices in olive (Olea europaea L.) leaves in response to differentlevels of water stress. International Journal of Remote Sensing 29, 1725–1743.

Terashima, I., Hanba, Y.T., Tholen, D., Niinemets, Ü., 2011. Leaf functional anatomyin relation to photosynthesis. Plant Physiology 155, 108–116.

Tholen, D., Boom, C., Noguchi, K., Ueda, S., Katase, T., Terashima, I., 2008. The chloro-plast avoidance response decreases internal conductance to CO2 diffusion inArabidopsis thaliana leaves. Plant Cell and Environment 31, 1688–1700.

Wada, M., Kagawa, T., Sato, Y., 2003. Chloroplast movement. Annual Review of PlantBiology 54, 455–468.