Competition between isoprene emission and pigmentsynthesis during leaf development in aspen

BAHTIJOR RASULOV1, IRINA BICHELE1, AGU LAISK1 & ÜLO NIINEMETS2

1Institute of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51010, Estonia and 2Institute of Agricultural andEnvironmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia

ABSTRACT

In growing leaves, lack of isoprene synthase (IspS) is consid-ered responsible for delayed isoprene emission, butcompetition for dimethylallyl diphosphate (DMADP), thesubstrate for both isoprene synthesis and prenyltransferasereactions in photosynthetic pigment and phytohormone syn-thesis, can also play a role. We used a kinetic approach basedon post-illumination isoprene decay and modelling DMADPconsumption to estimate in vivo kinetic characteristics ofIspS and prenyltransferase reactions, and to determine theshare of DMADP use by different processes through leafdevelopment in Populus tremula. Pigment synthesis rate wasalso estimated from pigment accumulation data and distribu-tion of DMADP use from isoprene emission changes dueto alendronate, a selective inhibitor of prenyltransferases.Development of photosynthetic activity and pigment synthe-sis occurred with the greatest rate in 1- to 5-day-old leaveswhen isoprene emission was absent. Isoprene emission com-menced on days 5 and 6 and increased simultaneously withslowing down of pigment synthesis. In vivo Michaelis–Menten constant (Km) values obtained were 265 nmol m−2

(20 μM) for DMADP-consuming prenyltransferase reactionsand 2560 nmol m−2 (190 μM) for IspS. Thus, despite deceler-ating pigment synthesis reactions in maturing leaves, iso-prene emission in young leaves was limited by both IspSactivity and competition for DMADP by prenyltransferasereactions.

Key-words: competition within isoprenoid synthesispathway; geranyl diphosphate synthase; isoprenoid synthesis;photosynthesis development; prenyltransferase reactions;terpenoid synthesis.

INTRODUCTION

Isoprene (2-methyl-1,3-butadiene) is a volatile leaf hydrocar-bon importantly modulating the balance of photochemicallyinduced oxidative processes in troposphere, particularly con-tributing to ozone formation in atmospheres enriched withnitrogen oxides (e.g. Atkinson & Arey 2003; Sanderson et al.2003; Pike & Young 2009). Isoprene is produced throughthe chloroplastic 2-C-methylerythritol 5-phosphate (MEP)isoprenoid synthesis pathway, and there is major interest inunderstanding the regulation of the isoprene synthesis pathway

to predict isoprene emissions from biosphere (Sharkey,Wiberley & Donohue 2008; Sharkey 2009; Niinemets et al.2010b; Monson et al. 2012; Grote, Monson & Niinemets 2013;Guenther 2013; Niinemets & Monson 2013; Li & Sharkey2013b). Not all plant species emit isoprene, and in the emitters,the rate of isoprene emission can vary orders of magnitudeduring foliage lifespan (Grinspoon, Bowman & Fall 1991;Monson et al. 1992; Kuzma & Fall 1993; Sharkey et al. 1996).

Leaf development comprises an important part of leaflifetime (Miyazawa, Satomi & Terashima 1998; Miyazawa,Makino & Terashima 2003; Niinemets, García-Plazaola &Tosens 2012), but the factors controlling the induction ofisoprene emission and variation during leaf ontogeny arestill not fully understood and empirical approaches areused in models predicting isoprene emission during leafdevelopment (Niinemets et al. 2010a; Monson et al. 2012;Grote et al. 2013). Development of leaf photosyntheticactivity is a highly orchestrated process characterized bycoordinated accumulation of photosynthetic pigments, pro-teins and membrane lipids in developing chloroplasts regu-lated by phytohormones, especially cytokinins (Skoog &Armstrong 1970; Fletcher & McCullagh 1971; Shesták 1985;Schmülling, Schafer & Romanov 1997; Nakano et al. 2001)and gibberellins (Nelissen et al. 2012). As carotenoids andgibberellins, the phytyl residue of chlorophylls, plastoquinone(Lichtenthaler 2007, 2009) and the 4-hydroxy-3-methyl-2-butenyl-diphosphate side chain of cytokinins (Kakimoto2003; Hwang & Sakakibara 2006) are formed by the MEPpathway, the MEP pathway is central in leaf development.

Despite the important role of the MEP pathway in photo-synthetic machinery development, delays between the devel-opment of photosynthetic activity and isoprene emissionhave been observed in previous studies (Grinspoon et al.1991; Sharkey & Loreto 1993; Harley et al. 1994; Monsonet al. 1994; Wiberley et al. 2005; Niinemets et al. 2010a;Guidolotti, Calfapietra & Loreto 2011). The delay in iso-prene emission has been associated with the time lagbetween the development of photosynthetic activity andexpression of isoprene synthase (IspS) (Schnitzler et al. 1996;Fall & Wildermuth 1998; Wiberley et al. 2005, 2009; Sharkeyet al. 2008; Cinege et al. 2009; Vickers et al. 2010), suggestingthat the first step towards induction of isoprene emissionis the activation of the promoter of IspS (Sharkey et al.2008; Cinege et al. 2009; Wiberley et al. 2009). Increases inIspS activity are strongly correlated with time-dependentincreases in isoprene emission in developing leaves (Kuzma& Fall 1993; Monson et al. 1994; Schnitzler et al. 1996;

Correspondence: Ü. Niinemets. Fax: +372 731 3738; e-mail:ylo.niinemets@emu.ee

Plant, Cell and Environment (2013) doi: 10.1111/pce.12190

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© 2013 John Wiley & Sons Ltd 1

Lehning et al. 1999, 2001; Vickers et al. 2010), further empha-sizing that the induction of IspS protein synthesis is the pointof no return for the onset of isoprene emission in developingleaves.

Although IspS is needed for the onset of isoprene, it ischaracterized by a very high Michaelis–Menten constant(Km) for its immediate substrate, dimethylallyl diphosphate(DMADP) with estimates varying between 0.3 and 9.0 mm(for recent reviews, see Li & Sharkey 2013b; Rajabi Memari,Pazouki & Niinemets 2013; Weise et al. 2013). Especiallyhigh estimates corresponding to in vitro studies are likelyoverestimates (Rasulov et al. 2009a,b; Weise et al. 2013), butthe available data collectively do demonstrate that highDMADP concentrations are needed for high isoprene emis-sion rates. Given that isoprene emission relies on carbon,ATP and NADPH provided by photosynthesis (Sanadze &Dzhaiani 1972; Loreto & Sharkey 1990; Sharkey, Loreto &Delwiche 1991; Rasulov et al. 2009b; Li & Sharkey 2013b),low isoprene emission rates in young leaves can reflectlimited supply of carbon and energetic and reductivecofactors in young leaves with low photosynthetic activity,reducing the key substrate, DMADP, availability for isopreneformation. However, synthesis of the components of thephotosynthetic machinery (carotenoids, chlorophylls, plasto-quinone), and gibberellins and cytokinins during the initialperiod of leaf development may also reduce the availabilityof DMADP for isoprene synthesis (Owen & Peñuelas 2005).Given the first reaction downstream of DMADP, synthesis ofgeranyl diphosphate (GDP) by GDP synthase has a muchlower Km value for DMADP (Orlova et al. 2009; Wang &Dixon 2009; Chang et al. 2010) than IspS (Schnitzler et al.2005; Rasulov et al. 2009a,b; Köksal et al. 2010); competitionfor DMADP may constrain isoprene emission rate in devel-oping leaves. This trade-off between the synthesis of ‘essen-tial’ isoprenoids with well-known structural and protectivefunctions, and ‘non-essential’ isoprenoids such as isoprenewith still partly unknown functions has been postulated(Rosenstiel et al. 2004; Owen & Peñuelas 2005), but to ourknowledge, has not been experimentally confirmed.

We investigated the kinetics of isoprene emission and pho-tosynthetic machinery development from leaf unfolding tomaturation in aspen (Populus tremula) using a recentlydeveloped method to estimate chloroplastic DMADP poolresponsible for isoprene emission and IspS kinetics (Rasulovet al. 2009a,b; Li, Ratliff & Sharkey 2011). The non-destructive method based on integration of post-illuminationisoprene release provides estimates of DMADP pool sizethat are well correlated with estimates by alternative destruc-tive methods (Rasulov et al. 2009a; Weise et al. 2013). Wehypothesized that isoprene synthesis during leaf develop-ment is simultaneously limited by IspS activity (proteincontent) and competition by other DMADP-consumingreactions. Model- and inhibitor-based methods were devel-oped to determine simultaneously the chloroplasticDMADP pools used for isoprene and for other competingreactions, and to determine the extent to which synthesis of‘essential’ isoprenoids limits isoprene emission during leafdevelopment. The results demonstrate that the control of

isoprene synthesis in developing leaves is shared betweenIspS and DMADP pool size, and overall support the hypoth-esis of a trade-off between ‘essential’ and ‘non-essential’isoprenoid synthesis.

MATERIAL AND METHODS

Plant material

Aspen (P. tremula L.) trees (age 6–7 years, height 7–8 m)growing in the field in the vicinity of Tartu, Estonia (58.39°N,26.70°E, elevation 41 m), were used in these experiments(Sun, Copolovici & Niinemets 2012a for the site description).During bud-burst (May 18) and rapid leaf expansion in May,average air temperature was 13.8 °C (average daytime airtemperature of 16.6 °C), whereas average air temperatureduring foliage maturation in June was 18.8 °C (averagedaytime air temperature of 21.3 °C) according to the weatherstation of the Laboratory of Environmental Physics, Univer-sity of Tartu (http://meteo.physic.ut.ee, 58.37°N, 26.73°E,elevation 76 m; inset in Fig. 1a).

For experiments, leaves were taken from young fully-exposed long shoots where new leaves developed continu-ously. Leaf age and rate of leaf formation were determinedby marking the leaves and frequently monitoring new leafformation. Gas exchange measurements were conductedwith detached leaves sampled in the morning to assure fullhydration of leaves.The leaf petiole was cut under water, andthe leaf with petiole in water was immediately transportedto the laboratory where it was stabilized in dim light of50 μmol m−2 s−1 and room temperature of 22–25 °C until themeasurements, 1–2 h after sampling.

Gas exchange system for CO2, water vapour andisoprene measurements

A fast-response gas exchange system with a circular clip-on-type thermostated leaf chamber (8.04 cm2 area and 0.3 cmheight) was used (Laisk et al. 2002). Given the chambervolume and gas flow rate maintained at 0.5 mmol s−1, thesystem full response time is ca. 1 s, belonging to the fastestcurrently available gas exchange systems (see Niinemets2012 for a review). The upper surface of the leaf was gluedby a starch paste to the glass window of the leaf chamberwater jacket. This strongly enhanced the heat exchange suchthat leaf temperature was maintained within 0.5 °C of thethermostated water jacket.

The system has two identical gas lines (channels) thatallow for rapid alternate measurements of reference andsample lines. While the leaf was in the gas stream of channel1, gas analysers connected to channel 2 recorded the refer-ence (zero) line. The channels could be switched in less thanin 1 s, and as soon as the leaf chamber was connectedto channel 2, the analysers immediately measured watervapour, CO2 and isoprene mole fractions at the gas concen-trations pre-adjusted in channel 2. This set-up is especiallyuseful for fast measurement of transient responses, avoidingdelays in the detection of leaf responses because of stabiliza-tion of gas concentrations.

2 B. Rasulov et al.

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

A Schott KL 1500 halogen light source with a heat-reflecting filter (Optical Coating Laboratory, Santa Rosa,CA, USA) was used for actinic light. Measurement air wasmixed from pure N2, O2 and CO2 by computer-controlledmanostatic mixers (Laisk & Oja 1998). The air was humidi-fied to maintain a water vapour pressure deficit of 1.7 kPa. ALI-6251 (Li-Cor, Inc., Lincoln, NE, USA) infrared gas ana-lyser was used to measure chamber CO2 concentration,whereas water vapour concentration was gauged by amicropsychrometer integrated in the gas exchange system(Laisk & Oja 1998). Isoprene concentration (m/z = 69) wasmeasured by a proton transfer reaction mass spectrometer

(PTR-MS; high-sensitivity version; Ionicon Analytik GmbH,Innsbruck, Austria), with a response time of approximately0.1 s and a limit of detection of ca. 10 pmol mol−1 for isoprene(Hansel et al. 1995; Lindinger, Hansel & Jordan 1998). Inaddition, masses corresponding to monoterpenes (m/z = 137)and lipoxygenase (LOX) pathway volatiles, main fragmentsof hexenals (m/z = 81) and other C6 volatiles (m/z = 83, sumof hexenols, hexanal and hexenyl acetates) were routinelyrecorded following Graus et al. (2004). PTR-MS was cali-brated with a standard gas containing all key volatiles(Ionimed GmbH, Innsbruck, Austria).

Chlorophyll fluorescence estimations

Chlorophyll fluorescence characteristics were measuredtogether with foliage gas exchange characteristics by a PAM101 fluorimeter (Walz GmbH, Effeltrich, Germany) operatedat 1.6 kHz for darkened leaves and at 100 kHz for illumi-nated leaves and during saturation pulses. Two Schott KL1500 light sources were employed for chlorophyll fluores-cence measurements. One light source was for 2 s saturatedpulses of white light of 14 000 μmol m−2 s−1 to estimate eitherthe dark-adapted (Fm) or light-adapted (Fm′) maximumfluorescence yields of photosystem II (PSII). The otherlight source with a 720 nm narrow-pass interference filter(Andover Corp, Salem, NH, USA) was for far-red light(50 μmol m−2 s−1) needed to obtain the true dark-adaptedminimum fluorescence yield (F0).

Measurement protocol

After leaf enclosure in the chamber, standard experimentalconditions were established. These included gas concentra-tions of 21% O2 and 360 μmol mol−1 CO2, relative humidityof 60% and leaf temperature of 30 °C in darkness. When leafgas exchange rates had stabilized, but at least 20 min afterleaf enclosure, dark respiration rate (Rd) and dark-adaptedminimum (F0) and maximum (Fm) fluorescence yieldswere measured. After these measurements, actinic light of650 μmol m−2 s−1 was switched on, and the leaf was stabilizedagain until stomata opened and gas exchange rates reached asteady state, typically in 20–30 min. Steady-state rates of netassimilation, transpiration and isoprene emission, and light-adapted steady-state (F) and maximum (Fm′) fluorescenceyields were recorded, and the leaf was subjected to a light–dark transient to measure DMADP pool size.

To determine the initial quantum yield of net assimilation,net assimilation rate was measured in separate leaves at threelight intensities between 15 and 55 μmol m−2 s−1 where Kokeffect is absent (Sharp, Matthews & Boyer 1984). After com-pletion of the gas exchange and chlorophyll fluorescencemeasurements, leaf absorptance (ζ ) was measured by anUlbricht-type integrated sphere.

All gas exchange rates were calculated according to vonCaemmerer & Farquhar (1981) and Niinemets et al. (2011).The dark-adapted quantum yield of PSII was computed as(Fm – F0)/Fm and the light-adapted quantum yield (ΦPSII) as(Fm′ – F)/Fm′. The rate of photosynthetic electron transport

0

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2

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LMA

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300

400

500

600

0 5 10 15 20 25 30Leaf age (d)

)mlo

m(tnetnoc)b

+a

b+a

(llyhporolhC

–2

–2–2

0

50

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250

300

Car

Leafarea

)mlo

m(tnetnoc

dionetoraC

(b)

(a)

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º

MeanMax

Min

µ

µ

Figure 1. Age-dependent changes in (a) the size of main stemleaves, leaf dry mass per unit area and (b) contents of totalchlorophyll (Chl a + b) and total carotenoids (Car) in aspen(Populus tremula L.). In (b), the data were fitted by exponentialrelationships by minimizing the sum of the squares betweenpredicted and measured values (Eqn 8; r2 = 0.994 for chlorophylland r2 = 0.986 for carotenoids, P < 0.001 for both). The fittingcoefficients (Eqn 8) obtained were for chlorophyll (a + b):a1 = 601 μmol m−2, a2 = 0.443 d and a3 = 0.125 d−1; for carotenoids:a1 = 185 μmol m−2, a2 = 0.775 d and a3 = 0.130 d−1. Data aremeans ± SE of at least four replicate experiments. Day 0corresponds to the day of bud-burst (May 18). Data in the insetdemonstrate variation in daytime minimum, maximum and meantemperature during the study period (derived from the data of theweather station of the Laboratory of Environmental Physics,University of Tartu, http://meteo.physic.ut.ee).

Competition between ‘essential’ and ‘non-essential’ isoprenoids 3

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

from chlorophyll fluorescence was further calculated as0.5QζΦPSII (Genty, Briantais & Baker 1989; Schreiber, Bilger& Neubauer 1994). The maximum quantum yield of photo-synthesis for an absorbed light was found as the slope of thelinear regression of net assimilation versus ζQ over low Qvalues of 15–55 μmol m−2 s−1.

Determination of the apparent DMADP pool size

The size of the immediate precursor pool responsible for iso-prene emission in steady state was estimated by the method ofRasulov et al. (2009a, 2010). This method is based on integrat-ing the fast, 200–400 s after darkening, dark decay of isoprenerelease, providing the amount of substrate available for iso-prene emission (Rasulov et al. 2009a, 2010; Li et al. 2011; Weiseet al. 2013). Typically, application of this method requires cor-rection for chamber responsiveness (Rasulov et al. 2009a; Liet al. 2011; Sun et al. 2012b), but this was not required with theultra-fast system used here (Rasulov et al. 2010, 2011). Aschloroplastic DMADP/isopentenyl diphosphate (IDP) ratiosare high in mature chloroplasts (Ramos-Valdivia, Heijden &Verpoorte 1997), the integral of the fast isoprene release hasbeen generally assumed to mainly consist of the primary iso-prene precursor DMADP, with minor contribution of IDPconverted to DMADP by IDP isomerase (IDI) (Rasulov et al.2009a; Li et al. 2011). However, recently, a relatively low ratioof whole-leaf DMADP to IDP of ca. 2 was reported for kudzuleaves (Zhou et al. 2013). Possible dark conversion of IDP toDMADP and use by IspS would result in moderate overesti-mation of DMADP pool size prior to the dark transient.Nevertheless, only a slight overestimation of the chloroplasticDMADP pool by the post-illumination method comparedwith a mass spectrometric method has been reported forpoplar leaves (Weise et al. 2013).

There is a secondary dark rise of isoprene emissionbetween 400 and 1200 s that has been attributed to MEPpathway metabolites upstream of DMADP and IDP that areslowly converted to DMADP in darkness (Li et al. 2011;Rasulov et al. 2011; Li & Sharkey 2013a). This secondary risewas not investigated in the current study.

Apart from the assumption that DMADP/IDP ratio is highin chloroplasts, implicit in this method is also that isoprenesynthesis is the only process consuming DMADP. This latterassumption is generally satisfied in mature leaves whereother processes competing for DMADP are operating onlyat a low level, but may not be necessarily valid for youngleaves. Therefore, we denote the pool of DMADP deter-mined here as an apparent DMADP pool.

Determination of IspS rate constant andapparent Km and Vm values

The time courses of post-illumination isoprene emission werefurther employed to determine the isoprene emission rateversus DMADP pool size relationships and derive the kineticcharacteristics of IspS as in Rasulov et al. (2010, 2011). Thiswas carried out by integrating the dark decay curves of iso-prene emission rate at different times after leaf darkening,

resulting in paired values of isoprene emission rate versusremaining DMADP pool size.

The initial slope of isoprene emission rate versus DMADPpool size (s−1) provides the first-order rate constant of IspS(ka). The Michaelis–Menten constant for IspS (Km,iso) andthe capacity (Vm,iso) were derived from Hanes–Woolf plot(DMADP pool size per emission rate versus apparentDMADP pool size). The slope of this plot is 1/Vm,iso, and theintercept is Km,iso/Vm,iso. As with the DMADP pool size,when isoprene is not the only process consuming DMADP,the derived values of Km,iso and Vm,iso are apparent values(Km,iso,app and Vm,iso,app). We note that dark conversion of IDPto DMADP would only moderately affect the estimates ofKm,iso,app and Vm,iso,app by this method. For example, for a rela-tively low DMADP to IDP ratio of 2 (Zhou et al. 2013), andDMADP pool size of 1500 nmol m−2 and assuming an IDPdark conversion rate constant of 0.002 s−1 (chloroplastic IDPpool reaches to half of the initial value in ca. 350 s), Km,iso,app isonly overestimated by ca. 20% with the effect becoming pro-gressively less with decreasing the conversion rate.

To convert Km,iso to molar units, a leaf water content of0.84 g g−1 fresh mass for young and 0.79 g g−1 for matureleaves was used, and the fraction of chloroplast water of totalleaf water was taken as 0.15 (Wolfertz et al. 2004).The resultsof these calculations are demonstrated in Supporting Infor-mation Figs S1.1 and S1.2.

Derivation of true DMADP pool size, and Km andVm values for IspS and GDP synthase in vivo

When multiple processes compete for chloroplastic DMADP,the change in DMADP pool size (SDMADP, nmol m−2) is drivenby all of these processes. Provided isoprene (rate vIso,nmol m−2 s−1) and pigment synthesis (vPig, sum of the rates ofchlorophyll and carotenoid synthesis, vChl + vCar, nmol m−2 s−1)are the dominant DMADP-consuming processes (with someadditional share for isoprenoid-containing phytohormone andplastoquinone synthesis), the dynamics of SDMADP is given as

dSdt

v v S v SDMADPDMADP Iso DMADP Pig DMADP= − ( ) − ( ), (1)

where vDMADP is the rate of DMADP synthesis. In a steadystate, vDMADP = vIso + vPig. When light is switched off, vDMADP

becomes zero, at least for a time period of 200–400 s followedby switching off the light, until NADPH becomes availableagain and phosphorylated intermediates of MEP pathway areconverted to DMADP (Li et al. 2011; Rasulov et al. 2011; Li &Sharkey 2013a). Thus, the change in DMADP pool corre-sponding to a light–dark transient is given as

dSdt

v S v SDMADPIso DMADP Pig DMADP= − ( ) − ( ). (2)

Assuming Michaelis–Menten kinetics, we write Eqn 2 as

dSdt

V S tK S t

V S tDMADP m iso DMADP

m iso DMADP

m pig DMADP= −( )

+ ( )−

( ),

,

,

KK S tm pig DMADP,

,+ ( ) (3)

4 B. Rasulov et al.

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

where Vm,iso is the capacity of IspS reaction and Vm,pig that forpigment synthesis, and Km,iso is the Michaelis–Menten con-stant for IspS and Km,pig that for pigment synthesis. As GDPsynthase is generally considered as the main enzyme consum-ing DMADP in the pathway leading to pigment synthesis (Li& Sharkey 2013b; Rajabi Memari et al. 2013), Vm,pig and Km,pig

most likely reflect the kinetics of GDP synthase. However, inaddition to GDP synthase, some forms of geranylgeranyldiphosphate (GGDP) synthase (Tholl et al. 2004; Orlovaet al. 2009; Wang & Dixon 2009) may also use DMADP as adonor. As in such a case, GDP is formed as an intermediate;Vm,pig and Km,pig correspond to the GDP synthase activity ofGGDP. According to literature data, GDP synthase (andDMADP-consuming GGDP synthases) has a very high affin-ity towards DMADP (Orlova et al. 2009; Wang & Dixon2009; Chang et al. 2010), whereas IspS has a low affinity(Schnitzler et al. 2005; Rasulov et al. 2009a,b; Köksal et al.2010). However, as GDP synthase activity can be feedbackinhibited by GDP (e.g. Tholl et al. 2004), reactions down-stream of GDP synthesis can alter the Vm,pig and Km,pig values,and thus, they characterize the effective kinetics of DMADPconsumption by processes other than isoprene synthesis.

Integrating Eqn 3 from the start of darkening (t0) when theDMADP pool has a maximum value, SDMADP,0, to a given timeof the light–dark transient, t1, provides the size of theDMADP pool size corresponding to the rate of the sum ofDMADP-consuming reactions at time t1:

S tV S t

K S tV S t

DMADPm iso DMADP

m iso DMADP

m pig DMADP1( ) =

( )+ ( )

+( ),

,

,

KK S tdt

t

t

m pig DMADP,

.+ ( )

⎡⎣⎢

⎤⎦⎥∫

1

0

(4)

The first part of the integral is equal to the apparentDMADP pool size, SDMADP,app, measured by integrating thedark release of isoprene emission:

S tV S t

K S tdt

V

t

t

DMADP appm iso DMADP

m iso DMADP

m i

,,

,

,

1

1

0

( ) =( )

+ ( )

=

∫sso app DMADP app

m iso app DMADP app

, ,

, , ,

,S t

K S tdt

t

t ( )+ ( )∫

1

0

(5)

where Vm,iso,app is the apparent maximum IspS activity andKm,iso,app is the apparent Km value of IspS found by fitting theisoprene emission rate versus SDMADP,app responses. The keyimplication of Eqns 4 and 5 is that if Km values for differentDMADP-consuming processes are different, Km,iso,app valuesderived according to Eqn 5 will depend both on Km,iso andKm,pig. The value of Km,iso,app will approximate Km,iso the morethe greater is the pathway flux towards isoprene synthesis, thatis, the larger is Vm,iso relative to Vm,pig. Thus, variations in Vm,iso

relative to Vm,pig are expected to result in modified shapes ofthe responses predicted by Eqn 5 (Fig. 4 for sample responsespredicted by Eqns 4 and 5). Such modifications in curve shapecan be used to estimate the true value of Km,iso and Km,pig andthe true pool size of DMADP, and partition DMADP con-sumption between isoprene and pigment synthesis.

We used an iterative least-squares technique to estimate allthe parameters by fitting Eqns 4 and 5 to SDMADP,app(t) versus

isoprene emission relationships of all leaf ages pooled. Con-stant values of Km,iso and Km,pig were fitted to all leaves ofdifferent age, whereas age-specific values of Vm,iso, Vm,pig andSDMADP,app were used in fitting. The model parameters werefound by simultaneously minimizing the weighted sum ofsquares of [SDMADP,app(measured) − SDMADP,app(predicted)]2

and [vIso(measured) − vIso(predicted)]2. Excellent fits betweenmeasured and predicted SDMADP,app (r2 = 0.994 for all leavespooled) and vIso (r2 = 0.981) values were observed using con-stant Km,iso and Km,pig values for all leaves of different ages,suggesting that the model reliably described the data (Sup-porting Information Fig. S2.1).

Alendronate inhibitor experiments

Alendronate (Fosamax; Merck Sharp & Dohme Corp.,Whitehouse Station, NJ, USA), a highly specific inhibitor ofGDP (Lange, Ketchum & Croteau 2001; Burke, Klettke &Croteau 2004) and farnesyl diphosphate synthases(Bergstrom et al. 2000; Burke et al. 2004), and less specificinhibitor of GGDP synthase (Szabo et al. 2002) was used toinhibit isoprenoid synthesis pathway downstream ofDMADP. The inhibitor was applied by feeding the 10 mmaqueous solution through the cut petiole of leaves enclosedin the gas exchange system under standard experimental con-ditions. Feeding times of 20, 40 and 60 min were employed,and isoprene emission rates and DMADP pool sizes wereestimated as before.

Provided alendronate does not affect the rates of DMADPand isoprene synthesis, Eqn 1 simplifies to

dSdt

v v SDMADPDMADP Iso DMADP= − ( ). (6)

As pigment synthesis is inhibited, DMADP pool is expectedto increase until a new equilibrium is reached. Thus, monitor-ing time-dependent changes in apparent maximum DMADPpool size (SDMADP,app,0) and steady-state isoprene emission ratein response to alendronate treatment can be used to estimatethe rates of DMADP synthesis from the rate of DMADP poolaccumulation in alendronate-inhibited leaves. Having deter-mined vDMADP, the rate of pigment synthesis can be estimatedas the difference between vDMADP and vIso in control leaveswithout alendronate treatment. We fitted SDMADP,app,0 andsteady-state isoprene emission rate versus time of alendro-nate treatment relationships with non-linear regressions, andestimated vDMADP from the rate of DMADP pool size accumu-lation, allowing us to get an alternative estimate of vPig.

Another implication of alendronate inhibition is that uponleaf darkening, Eqn 2 simplifies to

dSdt

v SDMADPIso DMADP= − ( ). (7)

Thus, true Km and Vm values of IspS can be determined inalendronate-inhibited leaves, provided pigment synthesishas been fully inhibited and DMADP pool has reached asteady state before the start of the dark transient. As with

Competition between ‘essential’ and ‘non-essential’ isoprenoids 5

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

non-treated leaves, Km and Vm values were determined fromHanes–Woolf plots at different times after alendronatetreatment.

Foliage structural analyses and determination ofpigment contents

Immediately after gas exchange measurements, discs of2–4 cm2 were taken for fresh mass and dry mass estimationand for pigment extraction.The pigment samples were frozenin liquid nitrogen and stored at −80 °C. The fresh mass of therest of the discs was determined immediately, and dry masswas determined after oven-drying at 70 °C for 48 h.

Pigment samples were ground in liquid N2 and extractedwith 80% aqueous acetone. As we were interested in totalpigment pool sizes, chlorophylls and carotenoids were meas-ured according to the method of Lichtenthaler & Buschmann(2001) using a Shimadzu UV-Vis UV2550PC spectrophoto-meter (Shimadzu, Kyoto, Japan).

Modelling the rate of Chl and Car synthesis fromchanges in pigment contents

We model the time-dependent (t) changes in leaf chlorophylland carotenoid contents in greening leaves (Fig. 1b) by anasymptotic relationship often used in chemical kinetics todescribe the first-order increase of reaction product:

S a e a t ai i

i, i= −( )− +( ),

, ,1 1 3 2 (8)

where Si is the content of given leaf pigment pool (μmol m−2),and the pool-specific coefficients are defined as follows: ai,1 isthe asymptotic value of the pigment pool size (the pigmentcontent in mature leaves), ai,2 (d) is the time offset that cor-responds to the time at the start of leaf greening, and ai,3 is thetime constant (d−1). This equation was fitted to the data byiteratively minimizing the sum of squares between measuredand predicted values.

The rates of Chl and Car synthesis were found by differ-entiating Eqn 8 with respect to time:

dS dt a a e a t ai i i

i i= − +( ), ,

, , .1 33 2 (9)

We further assumed that synthesis of both pigments occursduring daytime hours with 12 h light period. Given thatcarotenoids contain 40 carbon atoms and the phytyl residueof chlorophyll 20 carbon atoms, and given the correspondingmolecular masses of carotenoids and chlorophylls, the rateswere converted to nmol C m−2 s−1 (vChl for the rate of phytylresidue synthesis in chlorophylls and vCar for the rate ofcarotenoid synthesis). The total rate of isoprenoids goinginto pigment synthesis (nmol C m−2 s−1) was calculated asvChl + vCar.

Data analyses

For all leaf ages, 4–15 replicate measurements were con-ducted and mean ± SE were calculated. Whenever pertinent,

data among different leaf ages and alendronate treatmentswere compared by analysis of variance and considered dif-ferent at P < 0.05. Correlations between pigment synthesisrate and isoprene emission rate through leaf ontogeny wereanalysed by non-linear regressions. As leaf temperatureduring isoprene emission measurements differed from theambient temperature at leaf growth location, this analysiswas conducted both with the actual isoprene emission ratesand after scaling the emission rates to daytime average tem-perature during the experimental period (19.2 °C) using thetemperature response of isoprene emission from Rasulovet al. (2010).

RESULTS

Ontogenetic modifications in leaf size andpigment content

Leaves on long shoots developed continuously with a timeinterval of 2–3 d between successive leaves. Full leaf expan-sion of about 50 cm2 was reached in 12–14 d after bud-burst(Fig. 1a), whereas leaf dry mass per unit area stabilized a fewdays later, between 15 and 20 d after bud-burst.

Foliage pigment contents accumulated with the maximumrate in the youngest leaves (Fig. 1b) and approachedmaximum values of ca. 570 μmol m−2 for chlorophylls and180 μmol m−2 for carotenoids at days 20–22. The asymptoticmodel (Material and Methods, section Modelling the rate ofChl and Car synthesis from changes in pigment contents;Eqn 8) provided excellent fits to the data (Fig. 1b).

Time-dependent changes in photosyntheticcharacteristics

Net CO2 assimilation rate (A) became positive at day 2 andcontinued to increase until day 20 (Fig. 2a). Stomatal con-ductance increased in parallel with A, except in older leaveswhere stomatal conductance decreased somewhat, resultingin reductions in net assimilation rate as well (Fig. 2a). Darkrespiration rate (Rd) was high in juvenile leaves, up to8 μmol m−2 s−1, and exponentially decreased to a stable valueof ca. 3 μmol m−2 s−1 in mature leaves (Fig. 2a).

The maximum quantum yield of photosynthesis for anabsorbed light, estimated either as the initial slope of the lightresponse curve of net CO2 assimilation or as the dark-adapted quantum yield of PSII from chlorophyll fluores-cence, was constant through leaf ontogeny (Fig. 2b).However, quantum yield for an incident light increased withincreasing leaf age (data not shown), reflecting increases inleaf absorptance due to chlorophyll accumulation (Fig. 2c).

The light-adapted quantum yield of PSII (Fig. 2b) and pho-tosynthetic electron transport rate increased simultaneouslywith net assimilation rate (Fig. 2c). The increase in electrontransport rate was less rapid than in A, and no reduction wasobserved in older leaves with reduced stomatal conductance(cf. Fig. 2a,c). These discrepancies reflect the reductions of Aby Rd in young leaves and by stomatal conductance in olderleaves.

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© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

Isoprene emission in relation to leaf age

Isoprene emission was activated with a delay of about 4–5 dafter net assimilation rate became positive (Fig. 3a), but itincreased fast and approached the maximum rate almostsimultaneously with photosynthesis between days 18 and20 (cf. Figs 2a & 3a). Under steady-state conditions, onlyisoprene emission was observed without any significantmonoterpene emissions neither in young nor mature leaves(Supporting Information Fig. S3.1).

Integration of the first peak of the post-illumination iso-prene release after turning off the light was employed to

estimate the pool of the substrate DMADP that was con-verted to isoprene in the dark subsequent to illumination(SDMADP,app, Supporting Information Fig. S1.1). After day 4following the onset of isoprene emission,this pool consistentlyincreased, approaching a maximum of about 2000 nmol m−2

at day 20 simultaneously with the maximum of isopreneemission rate (Fig. 3a).

From dark decay data of isoprene emission, isoprene emis-sion rates (vIso) in relation to remaining DMADP pool werederived (Supporting Information Fig. S1.1), and the apparentcharacteristics of IspS were estimated. The initial slope ofisoprene emission versus remaining DMADP pool size(apparent rate constant of IspS) increased sharply withincreasing leaf age, reaching a maximum on day 15, a fewdays before maximum isoprene emission rate was observedon day 20 (Fig. 3a). The increase was also initially fasterthan changes in either isoprene emission rate or SDMADP,app

(Fig. 3a).The initial slope was positively correlated with bothisoprene emission rate and SDMADP,app through leaf develop-ment, but the correlation was curvilinear and the initial slopebecame almost invariable at SDMADP,app values greater than ca.1000 nmol m−2 (Fig. 3b).

The shapes of isoprene emission rate versus SDMADP,app rela-tionships were different among young and mature leaves. In

(a)

(b)

(c)

Figure 2. Dynamics of net CO2 uptake, dark respiration, stomatalconductance to water vapour (a), quantum yields (QY) ofphotosystem II (PSII) in dark- and light-adapted leaves andQY of CO2 uptake for an absorbed light (b), and the rate ofphotosynthetic electron transport and leaf absorptance (c) in aspenleaves. Data presentation as in Fig. 1.

(a)

(b)

Figure 3. Age-dependent changes in isoprene emission rate,apparent dimethylallyl diphosphate (DMADP) pool size andthe apparent rate constant of isoprene synthase (IspS) (a), andthe apparent rate constant of IspS in relation to apparentDMADP pool size (b) in different-aged aspen leaves. SupportingInformation Fig. S1.1 demonstrates determinations of the apparentDMADP pool size and the rate constant of IspS (SupportingInformation S1). Data are means ± SE of at least four replicatemeasurements.

Competition between ‘essential’ and ‘non-essential’ isoprenoids 7

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

young leaves,saturation of isoprene emission rate was observedat low values of SDMADP,app of about 400–500 nmol m−2, but nosaturation even at maximum observed values of ca. 1500–2000 nmol m−2 was observed for mature leaves (SupportingInformation Fig. S1.1b; Fig. 3a for maximum SDMADP,app values).Thus, both the apparent Michaelis–Menten constant (Km,iso,app)and maximum rate (Vm,iso,app) increased with increasing leaf age(Supporting Information Fig. S1.2).Differences in the isopreneemission versus SDMADP,app response shapes were reflected inlower Km,iso,app values of ca. 200–300 nmol m−2 (17–26 μm inchloroplast water) in young compared with 2000–2800 nmol m−2 in mature leaves (135–160 μm,Supporting Infor-mation Fig. S1.2).

True kinetic characteristics of IspS: the role ofpigment synthesis

Model analyses (Eqns 1–5) demonstrated that when multipleprocesses with differing Km values compete for the samechloroplastic DMADP pool size, the bias in Km,iso,app can belarge, especially if the second process proceeds with a ratehigher than or comparable with the rate of isoprene emission(Fig. 4). Under such circumstances, integration of post-illumination isoprene emission results in an apparentDMADP pool that seemingly depletes much faster than thetotal chloroplastic DMADP pool (Fig. 4). Given the presenceof active pigment synthesis in young leaves (Fig. 1b), and lowIspS activity (Fig. 3b) and that pigment synthesis at the levelof GDP synthase and isoprene emission compete for thesame chloroplastic DMADP pool, competition for DMADPcan explain the low Km,iso,app in young leaves.

The data of vIso versus SDMADP,app were iteratively fittedby Eqns 4 and 5 by adjusting globally constant values ofMichaelis–Menten parameters for isoprene (Km,iso) andpigments (Km,pig, mainly reflecting GDP synthase) simulta-neously to all leaves, and maximum activities of IspS (Vm,iso)and pigment synthesis (Vm,pig) and initial DMADP pool size(SDMADP,0) individually to leaves of different age. The fittedmodel with a constant Km,iso of 2560 nmol m−2 (188 μm inchloroplast water, converted by using an average watercontent for young and mature leaves) and Km,pig of265 nmol m−2 (20 μm) provided excellent fits to the data(r2 > 0.99; Supporting Information Fig. S2.1).

The rate of pigment synthesis was predicted to be minor inmature leaves (Vm,pig = 0.62 nmol m−2 s−1), with the overallcontribution of pigment synthesis being less than 10% of totalDMADP consumption (Fig. 5a). In contrast, in young leaves,Vm,pig was predicted to be much higher (5.8 nmol m−2 s−1), andthe bulk of DMADP, ca. 80%, was used for pigment synthesis(Fig. 5c). Overall, the effect of pigment synthesis on darkdecay patterns of DMADP pool increased with increasing therate of pigment synthesis (Fig. 5). The predicted DMADPpool sizes in steady state (SDMADP,0) were similar in leaves ofdifferent age as soon as isoprene emission started. However,the predicted decay of DMADP pool size after leaf darkeningwas slower in young than in mature leaves, reflecting thecircumstance that the achieved maximum rate of DMADPconsumption (vIso + vPig) was much larger in mature leaves

(24.7 nmol m−2 s−1) than in youngest leaves (6.8 nmol m−2 s−1)(Fig. 5) because of high isoprene emission rate in matureleaves.

Effects of alendronate on isoprene emission inleaves of different age

Application of the inhibitor alendronate that blocks the syn-thesis of isoprenoids downstream of DMADP and IDPresulted in enhanced isoprene emission rate, by approxi-mately one- to eightfold, in young leaves already after 20 minof application (Fig. 6a). The enhancement of isoprene emis-sion was associated with a major increase of DMADP poolsize available for isoprene emission, threefold increase after20 min and seven- to eightfold after 60 min of application

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Figure 4. Simulation of isoprene emission rate (vIso) versus true(SDAMAP, solid line) and apparent dimethylallyl diphosphate(DMADP) pool size (SDMADP,app, dashed line) relationships duringlight–dark transients (Supporting Information Fig. S1.1a for samplerelationships) for a mature leaf (a) with a high capacity forisoprene emission and a low capacity for pigment synthesis, and fora young leaf (b) with a low capacity for isoprene emission and ahigh capacity for pigment synthesis. After switching off the light,isoprene emission rate and DMADP pool size start to decreaseuntil the pre-existing pool of DMADP is exhausted. Whenisoprene emission rate is the only process consuming DMADP,integrating isoprene emission to a certain moment of time providesDMADP pool size that is remaining at that moment of time.However, when other processes compete for chloroplasticDMADP, this integration results in an apparent DMADP poolsize (SDMADP,app). In both panels, the relationships of vIso versusSDAMAP were derived by Eqns 3 and 4 using constant Km values forisoprene and pigments based on simultaneous fitting of all data.SDMADP,app was thereafter derived by integrating the values ofisoprene emission (Eqn 5), and the apparent Km,iso values derivedfrom vIso versus SDMADP,app relationships (Hanes–Woolf plots). Thepie diagrams show relative contributions of isoprene emission andpigment synthesis to overall consumption of DMADP pool sizeaccumulated before the light–dark transient.

8 B. Rasulov et al.

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

(Fig. 6a). Increased time of alendronate application moder-ately affected isoprene emission by further 16% for 1 h treat-ment, but the pool size of DMADP available for isopreneemission increased almost twofold in 1 h treatment relativeto 20 min treatment (Fig. 6a). Assuming that the enhance-ment of pool size of DMADP results from inhibition ofpigment synthesis, the average rate of pigment synthesis innon-inhibited leaves estimated from the change in the appar-ent DMADP pool size (Eqns 6 and 7) was predicted to be4.6 nmol m−2 s−1.

Differently from young leaves, isoprene emission rate inolder leaves was affected to a minor extent by 20 min treat-ment with alendronate, but longer-term application of

alendronate even appeared somewhat inhibitory to isopreneemission (Fig. 6b). The pool size of DMADP was increasedby ca. 20% by 20 min application of alendronate, and withincreasing the time of treatment up to 60 min, DMADP poolsize further increased by 10% relative to 20 min treatment(Fig. 6b). From the initial 20 min increase of DMADP poolsize, the average rate of pigment synthesis (Eqns 6 and 7) wasestimated to be 1.3 nmol m−2 s−1.

The effects of alendronate treatment on the initial slope ofisoprene emission versus DMADP pool size (apparent rateconstant of IspS) were moderate, ca. 35% reduction for youngleaves and 15% reduction for mature leaves (Fig. 6a,b).The apparent Vm,iso increased in young leaves after 20 minalendronate treatment by 1.6-fold and by additional 30% byextending the treatment to 60 min (Fig. 7a), but no effect ofalendronate on Vm,iso could be statistically confirmed inmature leaves. In addition, a major increase in Km,iso,app by3.3-fold for the longest treatment was observed for youngleaves (Fig. 7a). In mature leaves, the enhancement of Km,iso,app

was moderate, at most 1.2-fold for the longest alendronatetreatment (Fig. 7b).These continued alendronate treatments,however,appeared to constitute a moderate stress to the plantas suggested by somewhat enhanced emissions of typical dark-induced LOX pathway volatiles (Graus et al. 2004) (Support-ing Information Fig. S3.1).

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Figure 5. Comparison of the decay of dimethylallyl diphosphate(DMADP) pool size (SDMADP for true pool, and SDMADP,app for theapparent pool) and the contribution of isoprene emission rate tooverall rate of DMADP consumption (fIso) following light–darktransients in hybrid aspen leaves of different age (SupportingInformation Fig. S1.1a for corresponding measurements). fIso isdefined as the ratio of isoprene emission, vIso, to total rate ofDMADP consumption (sum of vIso and pigment synthesis, vPig). Forbetter visual comparison of patterns, DMADP pool sizes werenormalized to DMADP pool size at the steady state before thetransient (SDMADP,0). An iterative least-squares approach wasemployed to fit simultaneously isoprene emission rate andDMADP pool size by Eqns 4 and 5 and estimate theMichaelis–Menten constants (Km) for isoprene synthase (IspS)(Km,iso obtained was 2560 nmol m−2) and pigment synthesis(Km,pig = 265 nmol m−2) for all leaves simultaneously, as well as tofit the capacities for IspS (Vm,iso) and pigment synthesis (Vm,pig)reactions and SDMADP,0 for leaves of different age (SupportingInformation Fig. S2.1 for the overall fit of predicted and measuredvalues). The pie graphs indicate relative contributions of isopreneemission and pigment synthesis to consumption of DMADP poolsize accumulated before the light–dark transient.

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Figure 6. Isoprene emission rate, dimethylallyl diphosphate poolsize and the rate constant of isoprene synthase of young 6-day-old(a) and mature 20-day-old leaves (b) of aspen treated with 10 mmsolution of alendronate for 0 (control) to 60 min. Alendronatespecifically inhibits geranyl diphosphate (Lange et al. 2001; Burkeet al. 2004) and farnesyl diphosphate synthase (Bergstrom et al.2000; Burke et al. 2004) activities. Data are means + SE of at leastfour replicate measurements.

Competition between ‘essential’ and ‘non-essential’ isoprenoids 9

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

Interrelationships between isoprene emissionand pigment synthesis

The rate of pigment synthesis predicted on the basis ofpigment accumulation (Eqn 9) assuming a constant pigmentaccumulation rate over 12 h light period yielded pigmentsynthesis rates between 2.2 nmol m−2 s−1 (mol pigments) forthe youngest leaves and ca. 0.2 nmol m−2 s−1 for matureleaves. Considering that a C20 moiety production requires1 mol DMADP, and a C40 moiety requires 2 mol DMADPand the rest comes from IDP, these rates correspond to2.7 nmol m−2 s−1 and ca. 0.3 nmol m−2 s−1 in DMADP equiva-lents. In carbon equivalents, these rates correspond to54 nmol C m−2 s−1 in youngest leaves and 2 nmol C m−2 s−1 inmature leaves. Isoprene emission started when the rate ofpigment synthesis began to slow down after 4–5 d of leafgrowth (Fig. 8a). The overall isoprenoid pathway fluxes to

isoprene and to pigment synthesis were negatively correlated(Fig. 8b), but there was a positive x-intercept, indicatingthat isoprene did not evolve until the daily average rate ofpigment synthesis decreased to ca. 40 nmol C m−2 s−1. Furtherdecrease in pigment synthesis was accompanied by propor-tional increases in isoprene emission. When corrected forthe average ambient temperature (Fig. 1a), the maximumpigment and isoprene emission rates were almost numeri-cally equivalent (inset in Fig. 8b).

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Figure 7. Effects of alendronate on the apparent characteristicsof isoprene synthase (IspS), Vm,iso,app and Km,iso,app, in young 6- to7-day-old (a) and mature 20-day-old leaves (b) of aspen treatedfor various times with alendronate. The IspS characteristics werederived from Hanes–Woolf plots as in Supporting InformationFig. S1.2. If isoprene synthesis was the only process consumingdimethylallyl diphosphate as is expected in alendronate-inhibitedleaves, the derived values reflected the true Km and Vm of IspS.Data presentation and the number of replicates as in Fig. 6.

(a)

(b)

Figure 8. Dynamic changes in the rates of isoprene emission andsynthesis of photosynthetic pigments (Chl + Car) (a) and thecorrelation between isoprene emission and pigment synthesis rate(b) through the development of aspen leaves. Both the rates ofisoprene emission and pigment synthesis are expressed in nmolC m−2 s−1 (isoprene contains 5, phytyl residue of chlorophylls 20and carotenoids 40 carbon atoms). The rates of pigment synthesisare calculated from the rate of pigment accumulation by Eqn 9assuming a constant pigment synthesis rate for 12 h light period.The rates of isoprene emission were measured at a light intensityof 650 μmol m−2 s−1 and leaf temperature of 30 °C (data in themain panels). For quantitative comparison, isoprene emissionswere also scaled to an average daytime temperature of 19.2 °C(inset in Fig. 1a for variations in temperature during theexperiment) using the temperature response from Rasulov et al.[2010; inset in (b)]. Data in (b) were fitted by a non-linearregression in the form y = a ln(x) + b.

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DISCUSSION

Photosynthetic activity is present sinceleaf unfolding

Our results on the coordinated development of photosyn-thetic machinery during leaf ontogeny are in broad agree-ment with previous findings (for reviews, see Shesták 1985;Suzuki et al. 1987; Niinemets et al. 2012). Photosynthetic pig-ments and photosynthetic electron transport were detectableimmediately after leaf unfolding, whereas positive net assimi-lation rate was observed a few days after leaf unfolding(Figs 1 & 2). As observed in previous studies (Osborne &Garrett 1983; Long, Postl & Bolhár-Nordenkampf 1993;Harley et al. 1994; Singsaas, Ort & DeLucia 2001; Yoo et al.2003; Eichelmann et al. 2004), the maximum quantum yieldfor an absorbed light was constant from leaf unfolding to fullexpansion and photosynthetic maturation (Fig. 2b,c), indicat-ing that photosynthetically active chloroplasts with fullycoupled light and dark reactions of photosynthesis werepresent in juvenile leaves (Cai, Slot & Fan 2005). Furtherchanges in photosynthetic activity are mainly due to expan-sive developments, such as increased size (Hernandez-Gil &Schaedle 1973) and number of chloroplasts, cells and celllayers per unit leaf area (Dickmann & Gordon 1975;Kennedy & Johnson 1981; Yoo et al. 2003; Lichtenthaler &Babani 2004). Increased photosynthetic activity of mesophyllis accompanied by enhanced density of stomata, resulting ingreater stomatal conductance (Stitt 1991; Eichelmann et al.2004; Miyazawa, Livingston & Turpin 2006; Mott, Sibbernsen& Shope 2008; Mott 2009).

The rates of development of the traits characterizing leafphotosynthetic function were the highest during the first 8 dafter leaf unfolding when the photosynthetic developmentmatched the rate of leaf expansion (Figs 1 & 2).After full leafexpansion, the pigments still accumulated and photosyn-thetic activity increased, but with much weaker rate (Figs 1 &2). Thus, aspen leaves exhibited the phenomenon of ‘delayedleaf greening’ (Miyazawa & Terashima 2001; Miyazawa et al.2003; Niinemets et al. 2012), although the delay in greeningwas relatively weak.

Onset of isoprene emission duringleaf development

Although non-zero photosynthetic electron transport ratewas detected already on day 1 (Fig. 2c) and positive net CO2

uptake on day 2 (Fig. 2a), isoprene emission rate becameabove the limit of detection on day 6, further increasingrapidly to a maximum value on day 20 (Fig. 3a). Thus, ourdata are in agreement with the past evidence of a time lagbetween the onset of photosynthesis and isoprene emission(see Introduction section).

There is evidence that delayed isoprene emission can beexplained by deferred induction of isoprene synthase activity.Numerous reports have suggested that in developing leaves,IspS protein plays a key role in controlling the rate of iso-prene synthesis (Silver & Fall 1991; Monson et al. 1992;Kuzma & Fall 1993; Lehning et al. 2001; Wiberley et al. 2005;

Vickers et al. 2010, 2011) and that the delay in isoprene emis-sion is regulated by the transcription of the IspS gene(Mayrhofer et al. 2005; Wiberley et al. 2005).The level of IspSmRNA increases somewhat in advance of IspS protein level,and a certain expression activity maybe already presentbefore the onset of detectable isoprene emission, but IspSactivity approaches a maximum simultaneously with leafmaturation (Sharkey et al. 2008; Cinege et al. 2009). Paralleltrends in isoprene emission and IspS protein level have beenobserved in several studies (Mayrhofer et al. 2005; Wiberleyet al. 2005, 2009; Vickers et al. 2010). In fact, the share ofdevelopmental and environmental control on the onset ofisoprene emission is also likely determined at the levelof IspS. Induction of isoprene emission in developing leavesoccurs earlier at higher ambient temperatures (Monson et al.1994) that enhance the rate of leaf development and pigmentaccumulation (Niinemets et al. 2012) and IspS expression(Cinege et al. 2009; Li & Sharkey 2013b; Rosenkranz &Schnitzler 2013).

The importance of expression of IspS is underscored by ourdata demonstrating increases in the apparent rate constant ofisoprene emission (Fig. 3) and apparent Vm (Supporting Infor-mation Fig. S1.2) with increasing leaf age. Although both ofthese characteristics were likely somewhat underestimateddue to competition by other DMADP-consuming reactions inyoung leaves (Fig. 4, see below), both of them were alsosignificantly less in young than in old leaves according toglobal fitting of data (Fig. 5) and according to alendronateinhibition experiments (Figs 6 & 7), demonstrating that IspSdoes exert an important control on developmental changes inisoprene emission rate during leaf development.

Low isoprene emission in young leaves ispartly driven by competition by otherDMADP-consuming processes

When the amount of catalytically active enzyme is increasing,its Vm is expected to increase, but its Michaelis–Menten con-stant (Km) should remain constant as long as the catalyticmechanism is not modified. Thus, the finding of age-dependent increase of isoprene apparent Km (Km,iso,app)derived from post-illumination isoprene release data (Sup-porting Information Figs S1.1 & S1.2) is initially puzzling.However, the method applied assumes that during thepost-illumination decay, chloroplastic DMADP pool sizedecreases only because of isoprene emission, and no otherreactions besides IspS compete for DMADP. In fully matureleaves, where the build-up of the photosynthetic machineryhas been completed, this assumption is very likely to be trueeven if a certain part of DMADP is used for maintenance ofessential cellular functions such as synthesis of abscisic acidand for pigment turnover (Barta & Loreto 2006).

In contrast, in young leaves, synthesis of pigments,plastoquinone and isoprenoid-based plant hormones suchas gibberellins and cytokinins can potentially importantlycompete for chloroplastic DMADP, especially at the level ofGDP synthase that forms GDP from DMADP and IDP.According to in vitro analyses, GDP synthase has a much

Competition between ‘essential’ and ‘non-essential’ isoprenoids 11

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higher affinity for DMADP (Km,GDP) than IspS with valuesbetween 8 and 27 μm (Tholl, Croteau & Gershenzon 2001;Orlova et al. 2009; Wang & Dixon 2009) reported in the lit-erature. Accordingly, consumption of DMADP by GDP for-mation is expected to importantly alter the isoprene emissionversus apparent DMADP pool size relationships with themagnitude depending on the rate of IspS and otherDMADP-consuming reactions (Fig. 4). Analogously, Km

values for other prenyltransferases and terpenoid synthasesdownstream GDP are low, typically between 1 and 20 μm(Fraser, Schuch & Bramley 2000; Tholl et al. 2001, 2004). Forexample, phytoene synthase that is considered as the rate-limiting enzyme in carotenoid synthesis has a Km for GGDPof 5 μm (Fraser et al. 2000), indicating that downstream reac-tions unlikely limit consumption of DMADP.

We used this vast difference in Km values to derive globalvalues of Km for isoprene and other DMADP-consumingreactions (assumed to be mainly GDP formation for pigmentsynthesis, Km,pig) and to determine the Vm values for isoprene(Vm,iso) and pigment synthesis (Vm,pig) and steady-stateDMADP pool sizes for individual leaves (Eqns 4 and 5;Figs 4 & 5). The in vivo effective Km for prenyltransferasesderived in our study by global fitting of the model ofchloroplastic DMADP use after switching off the light(Eqns 4 and 5) was 265 nmol m−2 (20 μm), agreeing with lit-erature observations on GDP synthase affinity towardsDMADP in vitro (Tholl et al. 2001, 2004; Orlova et al. 2009;Chang et al. 2010).

Excellent fits to the data were obtained by globally fittingconstant Km,iso and Km,pig values to all leaves (SupportingInformation Fig. S2.1), suggesting that the observed age-dependent changes in Km,iso,app (Supporting InformationFig. S1.2) reflect consumption of DMADP by other ongoingprocesses, particularly pigment synthesis. This model analysisfurther indicated that in mature leaves, the competition frompigment synthesis was minor, and thus, the kinetic method forchloroplastic DMADP pool size estimation provided a real-istic estimate of chloroplastic DMADP pool size (Fig. 5a). Infully mature isoprene-emitting leaves, it has been suggestedthat the metabolic flux to carotenoids makes up only 1–2% ofthe MEP pathway flux to isoprene (Sharkey, Chen & Yeh2001). Our study suggested that the flux to alternativeDMADP-consuming reactions may be somewhat larger5–10% (Fig. 5a), but nevertheless demonstrating that iso-prene emission is the dominant sink of the MEP pathway.Differently from mature leaves, in young leaves just startingisoprene emission, DMADP used for isoprene emission con-stituted a small part of overall DMADP consumption, and inaddition to low IspS activity, isoprene emission rate was sig-nificantly constrained by competition from pigment synthesis(Fig. 5c).

This model-based analysis was confirmed by experi-ments with alendronate inhibitor that specifically inhibitsisoprenoid synthesis reactions downstream of DMADP(Bergstrom et al. 2000; Lange et al. 2001; Burke et al. 2004).Significantly higher isoprene emission rate was obtained inyoung alendronate-inhibited leaves (Fig. 6a), whereas theeffect was minor for mature leaves (Fig. 6b). In addition,

apparent Km,iso value was increased in alendronate-inhibitedyoung leaves, whereas no effect was observed for matureleaves (Fig. 7). Although possible accumulation of DMADPand IDP in alendronate-feeding experiments can result infeedback inhibition of deoxyxylulose 5-phosphate synthase(DXS), the first enzyme in the pathway, the inhibition con-stants (Ki) for both DMADP and IDP are relatively high(Banerjee et al. 2013), and our data did not suggest changes inthe degree of DXS control on pathway flux in response toalendronate feeding (Figs 6 & 7). We conclude that thealendronate inhibition experiments are in broad agreementwith the control of isoprene emission by competition fromother DMADP-consuming reactions that have greater affin-ity for DMADP.

In our study, no evidence of emission of other volatileisoprenoids such as monoterpenes was observed (SupportingInformation Fig. S3.1). Monoterpene emissions have occa-sionally been observed from aspen (Hakola, Rinne & Laurila1998) and young leaves of hybrid poplar (Brilli et al. 2009)and tropical evergreens (Kuhn et al. 2004), and such episodicemissions are not currently understood. Nevertheless,monoterpene emissions are typically elicited by bioticstresses (Blande et al. 2007; Toome et al. 2010; Copoloviciet al. 2011), suggesting that these emissions in past studiesmay have reflected biotic stress.

The rate of pigment synthesis in growing leaves

There is surprisingly little quantitative information on therate of pigment synthesis in growing leaves and on pigmentturnover in mature leaves. So far, 14C (Bhaya & Castelfranco1985; Schulze-Siebert & Schulze 1987; Goericke &Welschmeyer 1993; Beisel et al. 2010; Beisel, Schurr &Matsubara 2011), or 15N (Vavilin, Brune & Vermaas 2005;Vavilin & Vermaas 2007) pulse–chase labelling methods havebeen employed to estimate the rate of pigment synthesis.However, these methods typically have been conducted inchloroplastic or cellular systems (Bhaya & Castelfranco1985; Schulze-Siebert & Schulze 1987; Vavilin et al. 2005;Vavilin & Vermaas 2007) with a few exceptions (Beisel et al.2010, 2011), and yield relative information that is hard toquantitatively relate to foliage absolute pigment contents inintact leaves. We used three methods to estimate the rate ofphotosynthetic pigment synthesis during leaf development.Estimation of pigment synthesis rate from the rate ofpigment accumulation (Figs 1b & 8) yielded estimatesbetween 0.3 nmol m−2 s−1 (DMADP equivalents) in matureleaves and 2.7 nmol m−2 s−1 in young leaves. However,the rates of alternative DMADP-consuming reactions esti-mated from fitting of post-illumination isoprene decay dataand from alendronate inhibition experiments were 0.6–1.3 nmol m−2 s−1 for mature leaves and 4.6–5.8 nmol m−2 s−1

for young leaves.Somewhat lower estimates from pigment accumulation

data can be explained by several factors. Firstly, the valuesderived from the kinetic analysis of isoprene emission dataprovide the rate of whole set of alternative DMADP-consuming reactions, including synthesis of plastoquinone

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pool and isoprenoid-based phytohormones. In addition, thepigment accumulation-based method does not consider turn-over of pigments that is possibly low in developing leaves butcould still contribute to a certain extent to DMADP con-sumption (Beisel et al. 2010, 2011). Nevertheless, there is evi-dence that phytol can be reused in chlorophyll turnover(Vavilin & Vermaas 2007), implying no DMADP consump-tion. Secondly, the pigment accumulation-based methodassumes a constant rate of pigment synthesis over 12 h lightperiod. However, DMADP pool size varies during the daybecause of variations in light and temperature (Rosenstielet al. 2002; Rasulov et al. 2009b, 2010; Li et al. 2011), alsolikely altering the rate of pigment synthesis. In fact, scalingthe pigment synthesis rate estimated during the gasexchange measurements at 30 °C to the average ambienttemperature results in very similar pigment synthesis ratesin the field and in the laboratory estimations (data notshown, see Fig. 8b inset for temperature correction forisoprene). This evidence suggests that the average dailypigment synthesis rate possibly underestimated themaximum pigment synthesis rate. Finally, we assumed that1 mol DMADP is used for synthesis of 1 mol pigmentduring formation of GDP, whereas the rest of the pigmentisoprenoid residues comes from IDP. However, chloroplasticDMADP is in equilibrium with IDP, with the equilibriumdepending on the rate of DMADP and IDP consumptionand IDI activity (Brüggemann & Schnitzler 2002a). Thus,because of partial conversion of DMADP to IDP, activepigment synthesis may draw down DMADP pool fasterthan in the case of lower pigment synthesis rate. This wouldsuggest that isoprene emission-based methods overestimatethe rate of synthesis of compounds downstream of GDP.Despite these discrepancies between the different methods,all methods provided a similar magnitude of alternativeDMADP-consuming reactions, collectively providing con-clusive evidence of high rate of alternative DMADP con-sumption in developing leaves.

Balance between ‘essential’ and ‘non-essential’isoprenoid synthesis during leaf development

Development of leaf photosynthetic activity in aspen con-sisted of two different phases: a rapid increase from day 0 today 6, followed by a slower phase from day 6 to day 20 (Figs 2& 3).The induction of IspS occurred during the second phasewhen photosynthesis was slowly approaching the maximum.Thus, isoprene emission was induced when the rates of pho-tosynthetic pigments and hormones such as cytokinins (Shaniet al. 2010) synthesized via chloroplastic isoprenoid pathwaywere decelerating. As the long-chain length isoprenoids suchas the phytyl residue of chlorophyll and carotenoids mainlyrely on IDP, during most active pigment synthesis, DMADPis expected to be just a smaller branch of the pathway,whereas dominant carbon stream goes through IDP, bothsynthesized by the hydroxymethylbutenyl diphosphatereductase (HDR). The ratio of these two parallel products ofthe HDR reaction is strongly shifted towards IDP (Tritschet al. 2010), but both products, IDP and DMADP, are

mutually bound through the IDI reaction (Berthelot et al.2012) and a significant part of DMADP needed forisoprenoid synthesis is formed from IDP (Tritsch et al. 2010;Berthelot et al. 2012). We suggest that as long as IDP israpidly consumed (e.g. in developing leaves), DMADP doesnot accumulate significantly because the pathway is shiftedtowards IDP synthesis. As soon as the consumption of IDPand DMADP for production of higher order isoprenoidsslows down during maturation of chloroplasts in maturingleaves, IDP accumulates, inducing accumulation of the paral-lel product DMADP. It has also been demonstrated that theequilibrium of IDI reaction is shifted towards DMADP(Page et al. 2004; Berthelot et al. 2012; Zhou et al. 2013),implying that as IDP consumption slows down, DMADPgradually becomes the dominant product of the MEPpathway. Implicit in this reasoning is that IDI activity is largeenough for adequate conversion rates such that IDI simplyoperates to meet the downstream metabolic pulls. In fact, IDIactivity does vary and in oak (Quercus robur), it was corre-lated with IspS activity (Brüggemann & Schnitzler 2002b),suggesting that changes in IDI activity can constitute an addi-tional control point affecting DMADP pool size.

Clearly, a complementary character of isoprene emissionand pigment synthesis is evident in our data. Quantificationof carbon streams going into isoprene emission and pigmentsynthesis resulted in comparable estimates, especially ifisoprene emission rate was scaled to ambient temperature(Fig. 8). This evidence collectively suggests a balancebetween the synthesis of ‘essential’ and ‘non-essential’isoprenoids. As a key MEP pathway sink became weakerwith leaf maturation, it was replaced by isoprene emission.

However, there is also evidence that the enzymatic capac-ity of the whole MEP pathway can simultaneously increasewith increasing the activity of IspS. Activation of the tran-scription of MEP pathway genes DXR and DXS (Mayrhoferet al. 2005; Wiberley et al. 2009), accompanied by increasingcatalytic activities of both enzymes (Ghirardo et al. 2010),paralleled the synthesis of IspS protein (Mayrhofer et al.2005; Wiberley et al. 2009; Ghirardo et al. 2010). Such syn-chronous expression of IspS, DXR and DXS genes, andcorresponding enzymatic activities suggests that isopreneemission is cooperatively regulated not only by processesdownstream of HDR but also by enzymes upstream of HDR(Wiberley et al. 2009). Given the high Km value of IspS, highDMADP pool sizes are needed to maintain high isopreneemission rates.

An order of magnitude lower Km value for prenyl-transferases (in particular GDP synthase) than for IspS hasoverall important consequences for leaves supporting highDMADP pool, for example, in mature leaves with high iso-prene emission capacity. In such leaves, synthesis of ‘essen-tial’ isoprenoids can be elicited with a significant rate as soonas these isoprenoids are needed such as during repair pro-cesses in leaves recovering from photoinhibition and duringphotosynthetic apparatus acclimation to modifications inenvironmental conditions (e.g. Niinemets & Anten 2009).Although the permanent running of the MEP pathwaytowards isoprene in the constitutively emitting species may

Competition between ‘essential’ and ‘non-essential’ isoprenoids 13

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment

seem futile in the first glance, it can play a major role in fastplant responses to stress conditions requiring turning on thesynthetic reactions, and allowing for rapid switch between thesynthesis of ‘non-essential’ and ‘essential’ isoprenoids.

CONCLUSIONS

In this work, a kinetic gas exchange approach combined withmodelling was applied to gain insight into processes determin-ing the induction and regulation of isoprene emission duringfoliage development.This approach is unique in that it resolvesthe chloroplastic DMADP pool that is difficult to estimate byother methods due to a large cytosolic DMADP pool (Loretoet al. 2004; Falbel & Sharkey 2005; Rasulov et al. 2009a; Weiseet al. 2013). In addition, methods based on 13C-labelling can beproblematic due to inclusion of ‘older’ not readily labelledcarbon in isoprene that can be of cytosolic origin and enterthe MEP pathway at the level of pyruvate or originate fromchloroplastic starch (e.g. Trowbridge et al. 2012).

Induction of IspS activity is a crucial event for the onset ofisoprene emission, and this was not observed before pigment-synthesizing reactions consuming the bulk of MEP pathwaymetabolites started to slow down, and substrate became avail-able for isoprene synthesis. Thus, the presence of a certainthreshold chloroplastic DMADP pool may be the key factorleading to accumulation of functionally active IspS in chloro-plasts. Despite decreasing rates of pigment synthesis, isopreneemission in young leaves was still strongly curbed by compe-tition by other DMADP-consuming reactions, reflectinggreater affinity of the prenyltransferases consuming DMADP(mainly GDP synthase). Thus, isoprene emission in growingleaves was co-limited by DMADP and IspS activity, and thebalance of MEP pathway was almost stoichiometricallyshifted towards isoprene during leaf maturation as IspS activ-ity increased and rate of synthesis of isoprenoid componentsof photosynthetic machinery slowed down. The competitionfor DMADP by IspS and prenyltransferases observed in ourstudy supports the hypothesis of a role of isoprene emission, inaddition to its antioxidant and membrane-stabilizing role (forrecent reviews,see Fineschi et al. 2013;Possell & Loreto 2013),in maintaining MEP pathway active, thereby allowing forrapid synthesis of ‘essential isoprenoids’ when they areneeded, for example, during repair processes elicited afterstress events (e.g. for rapid response of carotenoids and toco-pherols to high irradiance stress in aspen; Niinemets et al.2003; García-Plazaola et al. 2004).

ACKNOWLEDGMENTS

We thank three reviewers and Tom Sharkey for insightfulcomments on the manuscript. This work was supported bygrants from the Estonian Ministry of Science and Education(institutional grant IUT-8-3 and grant SF0180045s08), theEstonian Science Foundation (grant 9253), the EuropeanCommission through European Regional Fund (the Centerof Excellence in Environmental Adaptation) and the Euro-pean Research Council (advanced grant 322603, SIP-VOL+).

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Received 27 May 2013; received in revised form 17 August 2013;accepted for publication 20 August 2013

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article at the publisher’s web-site:

Figure S1.1. Illustration of (a) post-illumination isopreneemission transients in 6-, 8- and 20-day-old (b) and isopreneemission rate in relation to the apparent DMADP pool sizein 6-, 12- and 30-day-old leaves of aspen (Populus tremula).The leaf measured was maintained under chosen environ-mental conditions until steady-state net assimilation andisoprene emission rates were achieved.A reference measure-ment was then conducted by replacing the leaf chamber withan equivalent empty reference at time t = 40 s since the startof recording. The leaf chamber was returned to the measure-ment channel at time t1 = 100 s, and light was turned offimmediately. The following recordings demonstrate isoprenerelease in the dark. The integral of isoprene emission in thedark reflects the whole pool of DMADP that was convertedto isoprene. Apparent kinetic curve of IspS was obtained byrelating the rate at any moment of the post-illuminationprocess to the remaining area (pool of DMADP remainingunused) at that moment. The inset in (a) demonstrates esti-mations of the apparent rate constant of isoprene synthase(ka) from the initial part of this curve.Figure S1.2. Illustration of Hanes–Woolf plots of the rela-tionships of isoprene emission rate and apparent DMADPpool size in aspen leaves of different age (a) and derivedcharacteristics of isoprene synthase, the maximum isoprenesynthase activity (Vm) and Michaelis–Menten constant (Km)(b). The inverse of the slope of Hanes–Woolf plot providesVcmax, while the intercept is Km/Vm. Representative relation-ships of isoprene emission versus apparent DMADP poolsize are demonstrated in Supporting Information Fig. S1.1.The data in (b) were replicated at least four times with dif-ferent leaves. Error bars show ± SE.Figure S2.1. Correlations between predicted and measuredapparent pools of DMADP (SDMADP,app) (a) and isopreneemission rate (b) during light–dark transients in aspen(Populus tremula) leaves of different age (6–20 days old)together with 1:1 lines.The apparent pool of DMADP at timet was obtained by integrating the isoprene emission rate afterleaf darkening (time t0) to time t. When multiple processes

Competition between ‘essential’ and ‘non-essential’ isoprenoids 17

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consume DMADP, this pool is significantly less than the trueDMADP pool. Especially in young leaves, chlorophyll andcarotenoid synthesis strongly competes for chloroplasticDMADP at the level of geranyl diphosphate synthesis. Pro-vided the competing processes have different Michaelis–Menten constants, data of isoprene emission versus SDMADP,app

size can be employed to estimate the rate of the competingprocesses and true Michaelis–Menten constants for isoprenesynthase and the competing reactions. An iterative least-squares fitting approach was used to fit simultaneously iso-prene emission rate and DMADP pool size relationships byEqns 4 and 5 and estimate the Michaelis–Menten constants(Km) for isoprene synthase and pigment synthesis as well asthe capacities for isoprene synthase and pigment synthesisreactions, and derive the true initial DMADP pool sizes. Forbetter visual assessment, only the data corresponding tosample transients in Fig. 4a are demonstrated.Figure S3.1. Illustration of the kinetics of volatiles releasedduring light–dark transients from mature (a, b) and young (c,d) control (a, c) and alendronate-treated aspen (Populustremula) leaves. The transients demonstrate periods ofmeasurement of reference lines (Ref) and foliage (Mea).Alendronate is a specific inhibitor of prenyltransferases, inparticular,geranyl diphosphate (Lange et al. 2001;Burke et al.

2004) and farnesyl diphosphate synthase (Bergstrom et al.2000;Burke et al. 2004).The leaf was maintained at a quantumflux density of 650 μmol m−2 s−1, leaf temperature of 30 °C andambient CO2 concentration of 360 μmol mol−1 until a steady-state isoprene emission rate was observed. At t = 550 s, lightwas switched off, the leaf was simultaneously returned to thereference channel and dark release of volatiles was measured.The measurements were conducted with a high-sensitivityproton transfer reaction mass spectrometer (PTR-MS) and inaddition to protonated isoprene (m/z = 69), also masses cor-responding to monoterpenes (m/z = 137) and lipoxygenasepathway volatiles (LOX), main fragments of hexenals(m/z = 81) and other C6 volatiles (m/z = 83, sum of hexenols,hexanal and hexenyl acetates) were measured followingGraus et al. (2004). Dark release of these lipoxygenasevolatiles has been studied in detail in grey poplar (P. xcanescens) and suggested to reflect disturbances generated bylight–dark transients with corresponding release of free poly-unsaturated fatty acids and their oxidation by lipoxygenases(Graus et al. 2004).The kinetics and magnitude of the releaseof LOX volatiles from the control leaves in our study broadlycorresponds to the results of Graus et al. (2004), but the LOXemissions in alendronate-inhibited leaves were larger andcontinued for a longer period.

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