© 1999 Blackwell Science Ltd 1319

Plant, Cell and Environment (1999) 22, 1319–1335

tures of J and/or limited maximum fraction of electronsused for isoprene synthesis. The model provides good fitsto diurnal courses of field measurements of isoprene emis-sion, and is also able to describe the changes in isopreneemission under stress conditions, for example, the declinein isoprene emission in water-stressed leaves.

Key-words: atmospheric chemistry; emission modelling; iso-prene emission; photosynthetic electron transport; volatileorganic compounds.

INTRODUCTION

Volatile organic compounds (VOC) play an important rolein ozone forming reactions in the troposphere (Liu et al.1987; Chameides et al. 1988) as well as in determining accu-mulation rates of atmospheric greenhouse gases (Guentheret al. 1995).At the global scale, biogenic emission of volatileorganic compounds is estimated to be at least as large as,or larger than the anthropogenic emissions (Mueller 1992;Guenther et al. 1995). Yet the current estimates of biogenicemissions of VOC suffer from significant uncertainties,because of limited knowledge of physiological and envi-ronmental controls over VOC emission.

Isoprene (2-methyl-1,3-butadiene) is the major emittedhydrocarbon in many species, and its emission is closelycoupled to environmental conditions as well as to leaf physiological state. Current empirical algorithms employ anArrhenius temperature relation of enzyme activity and ahyperbolic relationship with light to calculate the rates ofisoprene emission in dependence upon leaf temperatureand incident quantum flux density (Guenther, Monson &Fall 1991; Guenther et al. 1993; Geron, Guenther & Pierce1994).Yet, within and between species, there is considerablevariation in light and temperature responses observed inlaboratory measurements of isoprene emission (Guentheret al. 1993; Harley, Guenther & Zimmerman 1996, 1997;Lerdau & Keller 1997; Sharkey et al. 1999), and the empir-ical parameterizations are valid for very limited situationsonly. Present models also do not simulate the emissionunder stress conditions in a satisfactory manner, e.g.

ABSTRACT

We present a physiological model of isoprene (2-methyl-1,3-butadiene) emission which considers the cost for isoprene synthesis, and the production of reductive equiv-alents in reactions of photosynthetic electron transport forLiquidambar styraciflua L. and for North American andEuropean deciduous temperate Quercus species. In themodel, we differentiate between leaf morphology (leaf drymass per area, MA, g m-2) altering the content of enzymesof isoprene synthesis pathway per unit leaf area, and bio-chemical potentials of average leaf cells determining theircapacity for isoprene emission. Isoprene emission rate perunit leaf area (mmol m-2 s-1) is calculated as the product ofMA, the fraction of total electron flow used for isoprenesynthesis (e, mol mol-1), the rate of photosynthetic electrontransport (J) per unit leaf dry mass (Jm, mmol g-1 s-1), andthe reciprocal of the electron cost of isoprene synthesis[mol isoprene (mol electrons-1)]. The initial estimate ofelectron cost of isoprene synthesis is calculated accordingto the 1-deoxy-D-xylulose-5-phosphate pathway recentlydiscovered in the chloroplasts, and is further modified toaccount for extra electron requirements because of pho-torespiration. The rate of photosynthetic electron transportis calculated by a process-based leaf photosynthesis model.A satisfactory fit to the light-dependence of isoprene emis-sion is obtained using the light response curve of J, and asingle value of e, that is dependent on the isoprene synthaseactivity in the leaves. Temperature dependence of isopreneemission is obtained by combining the temperatureresponse curves of photosynthetic electron transport, theshape of which is related to long-term temperature duringleaf growth and development, and the specific activity ofisoprene synthase, which is considered as essentially con-stant for all plants. The results of simulations demonstratethat the variety of temperature responses of isoprene emis-sion observed within and among the species in previousstudies may be explained by different optimum tempera-

Correspondence: Ü. Niinemets. Fax: 00372 7 383013; e-mail:ylo@zbi.ee

A model of isoprene emission based on energeticrequirements for isoprene synthesis and leafphotosynthetic properties for Liquidambar and Quercus

Ü. NIINEMETS,1 J. D. TENHUNEN,2 P. C. HARLEY3 & R. STEINBRECHER4

1Department of Ecophysiology, Institute of Ecology, Tallinn University of Educational Sciences, Kevade 2, EE-10137 Tallinn,Estonia, 2Department of Plant Ecology, University of Bayreuth, D-95440 Bayreuth, Germany, 3Atmospheric ChemistryDivision, National Center for Atmospheric Research, PO Box 3000, Boulder CO 80307, USA and 4Fraunhofer-Institut fürAtmosphärische Umweltforschung, Kreuzeckbahnstraße 19, D-82467 Garmisch-Partenkirchen, Germany

isoprene emission declines in water-stressed leaves (Tingey,Evans & Gumpertz 1981; Sharkey & Loreto 1993; Fang,Monson & Cowling 1996; Lerdau, Guenther & Monson1997; Steinbrecher et al. 1997), but the empirical emissionmodels lack a means for describing these shifts. For accu-rate prediction of isoprene emission rates (I), a reliablesimulation of stress periods is critical, because many impor-tant emitting plants function for long periods at suboptimalenvironmental conditions.

There is ample evidence of correlations between I andleaf carbon dioxide assimilation rates (A, Harley et al. 1994;Litvak et al. 1996) and of co-ordinated changes in isopreneemission and photosynthesis during stress periods (Sharkey& Loreto 1993; Fang et al. 1996). Since detailed informationis available on the mechanisms of environmental andphysiological controls (e.g. Farquhar & von Caemmerer1982), acclimation responses (e.g. Berry & Björkman 1980;Niinemets & Tenhunen 1997), and nutritional limitations(e.g. Evans 1989) of leaf-level photosynthesis rates, use of the correlation between I and A to calculate isopreneemission rates may reveal several important and excitingapproaches for examining the observed influences of plantnutrition (Harley et al. 1994), leaf acclimation (Monsonet al. 1992; Harley et al. 1994; Harley, Guenther & Zimmer-man 1996; Harley et al. 1997; Sharkey et al. 1996) and envi-ronmental stresses on I in modelling the emission rates overthe long-term. Yet, the responses of net assimilation and isoprene emission rates to environmental factors differ inseveral important respects. In general, light response curvesof I measured at current ambient air CO2 concentrationssaturate at higher quantum flux densities than those of A(Monson et al. 1992; Sharkey & Loreto 1993; Lerdau &Keller 1997), and the fraction of leaf current photosynthateproduction lost as isoprene increases with increasing leaftemperature (Monson & Fall 1989; Sharkey, Loreto & Delwiche 1991b; Sharkey & Loreto 1993; Sharkey et al.1996; Lerdau & Keller 1997). At the same time, changes inI are strongly correlated with changes in leaf ATP content(Loreto & Sharkey 1993), suggesting that the rate of photo-synthetic electron transport (J) rather than A may exertcontrol over I. Limitation of I by J may provide importantinsight into the non-correlations between A and I at highlight and in particular temperature regimes.

According to the model of leaf photosynthesis of Farquhar & von Caemmerer (1982), light-saturation of Aat current ambient CO2 concentrations results from limitedcapacity of ribulose-1,5-bisphosphate carboxylase/oxyge-nase (Rubisco), and J becomes rate-limiting at low light.Even though there is evidence that J is down-regulated tomatch the overall reaction capacity in Rubisco-limited con-ditions (Sharkey, Berry & Sage 1988), there is often anexcess capacity for photosynthetic electron transport inleaves carrying out photosynthesis at saturating light (Stitt1986; Bloom et al. 1989). Excess J may explain higher satu-rating light for I than for A, because isoprene emission mayproceed in high quantum flux densities at the expense ofthis extra capacity the leaves are unable to use for carbondioxide assimilation. Both the increases in photosynthetic

1320 Ü. Niinemets et al.

enzyme activity, photorespiration to photosynthesis ratio(Jordan & Ogren 1984) and mitochondrial respiration withincreasing temperature alter the temperature response ofA at current air CO2 concentrations. Proportionally greaterphotorespiration in combination with higher rate of mito-chondrial respiration will result in declining A to I ratioeven if J increases with temperature. Moreover, stomatalconductance (G) often declines at higher temperatures(Berry & Björkman 1980). A decline in stomatal conduc-tance restricting CO2 entry into leaf intercellular spacesmay result in dramatic increases in photorespiration at hightemperature, and accordingly, in decreases in A, but it doesnot necessarily alter I, which is independent of stomatalaperture (Sharkey 1991). A decrease in G affects J muchless than it affects A, because photorespiration is a relevantelectron sink in leaves with closed stomata (Cornic 1994;Kozaki & Takeba 1996). Collectively, the evidence suggeststhat J could be the critical leaf parameter to focus on formodelling changes in I.

Here we develop a physiological model of isoprene emis-sion, separating structural and biochemical factors control-ling I. The model is based on leaf photosynthetic electrontransport rate, leaf dry mass per area, electron requirementfor isoprene synthesis and the fraction of electrons allo-cated to the isoprene synthesis pathway. The latter variableis the result of competition for electrons between isoprenesynthesis and Calvin and photorespiratory cycles, and iscontrolled by leaf isoprene synthase activity (Kuzma & Fall1993; Silver & Fall 1995; Wildermuth & Fall 1996). Weconfirm that J may successfully be used to simulate fieldmeasurements of I in stressed and non-stressed leaves, anddemonstrate that separation of biochemical and morpho-logical determinants of I provides an useful means forlinking the model to calculations at the canopy scale.

METHODS

Biochemical model of isoprene emission based on the electron requirement for isoprene synthesis

According to recent investigations, 1-deoxy-D-xylulose-5-phosphate (DXP) pathway confined to chloroplasts(Appendix A) is the dominant route for foliar isoprene production (Lichtenthaler, Rohmer & Schwender 1997;Lichtenthaler et al. 1997). Although some isoprene emittedfrom the leaves may originate from cytosolic isopen-tenylpyrophosphate synthesized via the mevalonate-dependent pathway (Schwender et al. 1997; Appendix A),labelling experiments with stable carbon isotopes, indicat-ing that isoprene emission relies on a small pool of chloro-plastic carbon, suggest that the fraction of isopreneoriginating from cytoplasmic carbon is likely to be minor(Delwiche & Sharkey 1993). Assuming that all isopreneemitted from the leaves has been synthesized in chloro-plasts via the DXP pathway, the coenzyme requirement forthe synthesis of one mole isoprene is 14 mole NADPH and17 mole ATP (Appendix A), i.e. NADPH to ATP ratio is

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

0·82. This ratio is 0·67 for glyceraldehyde-3-phosphate syn-thesis from CO2 and H2O, which has a metabolic cost of 9mole ATP and 6 mole NADPH. Accordingly, isoprene pro-duction is more NADPH-limited than photosyntheticcarbon assimilation. Assuming that isoprene emission islimited by NADPH, and provided that 2 mole electrons areneeded to produce 1 mole NADPH, 1 mole isoprene costs28 mole electrons. For each molecule isoprene released, sixmolecules of CO2 must be assimilated in the Calvin cycle(Appendix A). Thus, NADPH requirement per assimilatedCO2 is 2·33 for isoprene production as compared with 2 forsugar synthesis. Using these synthesis costs, and the analogywith the requirement for electron transport for carbonassimilation, we calculate the rate of electron transport nec-essary to support the measured isoprene emission rate.Photosynthetic electron transport rate (JCO2+O2, mmolm-2 s-1) used to sustain the observed rates of net photo-synthesis (A, mmol m-2 s-1) may be computed from leaf gas-exchange measurements as (Brooks & Farquhar 1985):

(1)

where Rd is the rate of mitochondrial respiration continu-ing in the light (mmol m-2 s-1), G * is the CO2 compensationpoint in the absence of Rd (mmol mol-1, Laisk 1977), and Ci

is the intercellular CO2 concentration (mmol mol-1). Giventhat the NADPH requirement per mole CO2 assimilated is1·17 times higher for isoprene than for sugar synthesis (cf.above), the photosynthetic electron transport rate (JI,mmol m-2 s-1) required to sustain the observed rates of iso-prene emission (I, mmol m-2 s-1) is given by:

(2)

Because of the extra electrons needed to increase thecarbon reduction state from the level of sugars to isoprene,total electron transport rate per unit leaf area(JT = JCO2+O2 + Je, where Je is the extra electron transportrate needed to reduce the sugars to isoprene) is somewhatlarger than that calculated by Eqn 1. Thus, the fraction ofelectrons used for isoprene production is equal to:

(3)

e is the outcome of the competition for electrons betweenisoprene synthesis pathway, and other electron sinks in theleaves. Several chloroplastic biochemical pathways arecompetitive with respect to reducing equivalents, forexample, a significant part of photosynthetic electron flowis used for nitrate assimilation, and dependent on meas-urement conditions, enhanced nitrate reduction may resultin decreased carbon assimilation rates (Bloom et al. 1989;Holmes, Weger & Turpin 1989).

e =+

=+ ◊ + ◊( )[ ] -( ){ }

+

+

JJ J

JJ I C C

I

CO O e

I

CO O i i

2 2

2 2 6 0 67 1 33G G* *.

JI C

CI

i

i=

◊ + ◊( )-

6 4 67 9 33GG

**

.

JA R C

CCO O

d i

i2 2

4 8+ =

+( ) +( )-

GG

**

,

Isoprene emission in relation to photosynthetic electron transport 1321

Rearranging,

(4)

where Jm (mmol g-1 s-1) is the rate of total photosyntheticelectron transport per unit leaf dry mass and MA (g m-2) isleaf dry mass per unit area. When I is low (e.g. in conditionsof low temperature), Je is relatively small and may practi-cally be neglected. Substituting JmMA for JT allows us todistinguish between the morphological and biochemicaldeterminants of isoprene emission. In the current model,the light-dependence of isoprene emission (e.g. Tingey et al. 1979; Monson & Fall 1989; Harley, Guenther & Zimmerman 1996; Harley et al. 1997) results from theresponse of total electron flow (Jm) to incident quantumflux density. In the photosynthesis model used, J versus lightresponse is fitted by Smith’s equation (Harley & Tenhunen1991; Harley et al. 1992). Although Eqn 4 includes both Ci

and G *, precision of which may occasionally be low, espe-cially for field measurements under extreme environmen-tal conditions, the estimate of I obtained from net carbonassimilation rates is relatively insensitive to possible inac-curacies in Ci and G *, because these errors alter the calcu-lations of JT (Eqns 1 and 3) and I (Eqn 4) in oppositedirections.

In a derivation analogous to that above (Eqns 1–4), iso-prene emission rate may be expressed as dependent on theproduction of its immediate precursor 3-phosphoglycerate(PGA). However, although PGA production calculatedfrom leaf gas exchange gives the rate of PGA synthesis nec-essary to sustain the observed steady-state rates of netassimilation, it provides no information of chloroplasticconcentrations and pool sizes. Since in the data used formodel parameterization, PGA production was strongly cor-related with J (r2 = 0·99 for a linear regression analysis), theanalysis based on PGA described the same fraction of vari-ance of isoprene emission rates as that based on J. Giventhat the availability of PGA may be an important factorcurbing I in low CO2 (Loreto & Sharkey 1990), the modelmay further be improved by including a limitation imposedby chloroplastic PGA pool size.

Relationship between the temperaturedependence of isoprene synthase and e

The major assumption underlying Eqn 4 (Hypothesis 1) isthat a certain proportion of electrons, e, is going to isoprenesynthesis.To describe the variability in e between the leavesand possible variation within the leaf, we further assume(Hypothesis 2; see Kuzma & Fall 1993; Silver & Fall 1995;Wildermuth & Fall 1996) that the competitive strength ofisoprene synthesis pathway for electrons is proportional tothe total activity of isoprene synthase (ST, nmol m-2 s-1) inthe leaves. The development of leaf ability for isopreneemission is dependent on increases in leaf isoprene

I JC

C

J MC

C

=-( )

◊ + ◊( )

=-( )

◊ + ◊( )

e

e

Ti

i

m Ai

i

GG

GG

**

**

,

6 4 67 9 33

6 4 67 9 33

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

synthase activity (Kuzma & Fall 1993; Wildermuth & Fall1998), and seasonal variability in this enzymatic activity(Schnitzler, Lehning & Steinbrecher 1997; Lehning et al.1999) may explain the seasonal maximum in I in fullymature leaves (Monson et al. 1994) and in the forest atmos-phere in mid-season (Jobson et al. 1994).

An equation similar to those used to model temperaturedependencies of Rubisco activity and photosynthetic elec-tron transport in leaf photosynthesis models (Tenhunenet al. 1976; Harley et al. 1992; Niinemets & Tenhunen 1997)has been previously employed to fit the isoprene emissionversus temperature relationships (Guenther et al. 1991;Guenther et al. 1993; Geron et al. 1994). Given that an esti-mate of ST is given by the specific activity of isoprene syn-thase [Ss, mmol isoprene (g isoprene synthase)-1 s-1] timesleaf isoprene synthase content (mg m-2), we describe thetemperature response of Ss as:

(5)

where cs is a scaling constant, DHsa (J mol-1) is an activa-

tion energy, DHsd (J mol-1) is a deactivation energy, DSs

(J mol-1 K-1) is an entropy term, Tk (K) is leaf temperatureand R (8·314 J mol-1 K-1) is the gas constant. In the currentmodel parameterization, we use the temperature depen-dence of isoprene synthase in crude leaf extracts of Populustremuloides (data of Monson et al. 1992; Silver & Fall 1995;cf. Fig. 4a). Expressing Jm as:

(6)

where the definition of parameters is analogous to Eqn 5,an estimate of e [JI/(JmMA); Eqn 3] is given by:

(7)

where d is a scaling constant [(mmol electrons). (g isoprenesynthase) (mmol isoprene)-1 (g leaf dry mass)-1] that isdependent on total isoprene synthase content of the leavesas well as on the ratio of isoprene emitted to electrons usedin isoprene synthesis. The scaling constant, d, is truly con-stant only if intercellular CO2 and O2 concentrations areconstant, i.e., the electron cost of isoprene synthesis is con-stant (cf. Eqn 4). Thus, the way e responds to temperatureshould be dependent on both the temperature responses ofSs and of photosynthetic electron transport.

Data for model parameterization

Detailed measurements in Liquidambar styraciflua(Harley, Guenther & Zimmerman 1996) and Quercusspecies (Harley et al. 1997) were used to test the proposedscheme. The leaf photosynthesis model that forms the coreof the isoprene emission routine is described in detail in

e =

=+( )+( )

- -( )

- -( )

dSJ

dc H RT S T H RT

c H RT S T H RT

s

m

e e

e e

sas

kJ

k dJ

k

JaJ

ks

k ds

k

D D D

D D D

1

1,

Jc H RT

S T H RTme

e

JaJ

k

Jk d

Jk

=+

-

-( )D

D D1,

Sc H RT

S T H RTse

e

sas

k

sk d

sk

=+

-

-( )D

D D1,

1322 Ü. Niinemets et al.

Harley et al. (1992) and Niinemets & Tenhunen (1997).Temperature dependencies of all constants of Rubiscokinetics, that are necessary to calculate G * (Eqns 1, 2), werethose in Niinemets & Tenhunen (1997). Instantaneous ratesof photosynthetic electron transport were calculated frommeasurements of net assimilation rates as described inNiinemets et al. (1999b), whereas the leaf mitochondrialrespiration rate (Rd) for the data of Harley et al. (1997) wascalculated according to Harley & Baldocchi (1995). Anaverage Rd in L. styraciflua (Harley, Guenther & Zimmer-man 1996) was found as the intercept of the linear regres-sions between net assimilation versus quantum flux density(Q) using the data measured at Q < 150 mmol m-2 s-1 at aleaf temperature of 25 °C; the obtained estimate of Rd wasscaled to different leaf temperatures using an activationenergy (see Niinemets & Tenhunen 1997) of 55·0 kJ mol-1.For all measurements in L. styraciflua, leaf absorbtance nec-essary to calculate initial quantum yield of photosyntheticelectron transport was fixed at 0·85. For Quercus species,leaf absorbtance was calculated from leaf chlorophyll con-tents as described in Niinemets & Tenhunen (1997). Formeasurements missing chlorophyll estimations, a linearregression between leaf dry mass per area (MA) and chloro-phyll content per area (r2 = 0·34, P < 0·001) was used toderive the values of leaf chlorophyll content.

Daily time-courses of non-water-stressed (Harley et al.1997; Steinbrecher et al. 1997) and water-stressed (Steinbrecher et al. 1997) leaves in three Quercus specieswere used to examine the usefulness of the current modelin predicting isoprene emissions under field conditions. ForQuercus alba (Harley et al. 1997), the parameters of leafphotosynthesis model were obtained as described above.For Quercus pubescens and Quercus frainetto (Steinbrecheret al. 1997), all parameters of biochemical photosynthesismodel were derived using the mitochondrial respirationrates measured in the night and scaled to different leaf tem-peratures using an activation energy measured for Q. alba(Harley & Baldocchi 1995), and using a value of 0·85 forleaf absorptance.

RESULTS

Scaling of isoprene emission rate with leafphotosynthetic electron transport rate and leafdry mass per unit area

Equation 4 (Hypothesis 1) suggests that isoprene emissionrate (I, mmol m-2 s-1) should scale positively with the totalphotosynthetic electron transport rate per unit leaf area(JT) that is the product of leaf dry mass per unit area (MA)and the photosynthetic electron transport rate per unit leafdry mass (Jm). In all species examined, there was a strongpositive correlation between I and JT (Fig. 1a, Table 1), butMA and I were also correlated (Fig. 1c, Table 1). Given thestrong relationships between JT and MA (Fig. 1e), onereason for the positive scaling of I with JT may be the parallel accumulation of both isoprene synthesis pathwayenzymes and rate-limiting proteins of photosynthetic elec-

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

tron transport per unit leaf area with increasing MA.To testthis possibility, correlations between mass-based parame-ters were also examined. In these data, Jm increased signifi-cantly with increasing MA only in Quercus velutina, but notin the other species (Fig. 1f). Isoprene emission rate per unitleaf dry mass (Im) was strongly correlated with Jm (Fig. 1b,Table 1), but the correlation with MA was generally muchweaker (Fig. 1d, Table 1), indicating that the biochemicalcapacities of leaf cells for isoprene emission were moretightly coupled to leaf photosynthetic electron transportpotentials than to foliar morphology.

The slope of the I versus JT relationship was lower in L.styraciflua than in Quercus species (Fig. 1a, Table 1). Thiswas partly related to the lower measurement temperaturein L. styraciflua (25 °C) as compared with Quercus species(30 °C). Nevertheless, when the slopes were calculated for30 °C using the temperature dependence of the specificactivity of isoprene synthase (Fig. 4a) and that of photo-synthetic electron transport (Fig. 5a), they were still lowerin L. styraciflua than in Quercus species; for example, theslope of the I versus JT relationship calculated for zerointercept was 0·174 nmol mmol-1 at 25 °C (Table 1), and avalue of 0·257 nmol mmol-1 was calculated for 30 °C, butthe lowest slope value was 0·327 nmol mmol-1 for Quercusspecies (Table 1). Moreover, there were also significantinterspecific differences between the Quercus species in theslope values (Table 1), indicating that there is a broad vari-

Isoprene emission in relation to photosynthetic electron transport 1323

ability in the fraction of electrons allocated to isoprene production – a result that is consistent with previous obser-vations demonstrating large variability, zero to more than50%, in the percentage of assimilated carbon emitted as iso-prene (e.g. Monson & Fall 1989; Monson et al. 1991; Sharkey& Loreto 1993; Harley et al. 1994).

The intercepts of I versus JT and Im versus Jm relation-ships were generally different from zero (Table 1, Figs 1a& b), indicating that the fraction of electrons used for isoprene production increased with increasing rate of photosynthetic electron transport or/and rate of isopreneemission. When the fraction of electrons used for isopreneproduction was plotted against the rate of isoprene emis-sion, there was a single positive relationship across theentire set of values (Fig. 2), possibly because the competi-tive ability of isoprene synthesis pathway for electronsincreases with increasing the total activity of the enzymes,such as isoprene synthase, involved in isoprene synthesis(Hypothesis 2).

Light dependence of isoprene emission

There was a good correspondence (Fig. 3) between themeasured light-dependence of I, and isoprene emission ratecalculated from the light-response of leaf photosyntheticelectron transport using Eqn 4, and assuming that the frac-tion of electrons used for isoprene synthesis is independent

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

Figure 1. Correlation of isoprene emission rate (I) with (a, b) photosynthetic electron transport rate (J) and with (c, d) leaf dry massper area (MA), and the correlations between photosynthetic electron transport rate and MA (e, f) in leaves sampled from different lightenvironments along the canopy in Liquidambar styraciflua and Quercus species. Panels a, c, e present area-based variables, and panels b,d, f dry-mass-based variables. The measurements were conducted at an ambient CO2 concentration of 350 mmol mol-1 and either at a leaftemperature of 30 °C (Quercus ssp., Harley et al. 1997) or 25 °C (L. styraciflua, Harley, Guenther & Zimmerman 1996). Data are fitted bylinear regressions (Table 1). All linear regressions were significant at P < 0·001 on panel e, and the regression for Q. velutina at P < 0·05on panel f.

of quantum flux density (Q). However, there was evidenceof greater fractional investment of electrons in isopreneproduction at higher Q (Fig. 3). As a result of this, the lightresponse curves of isoprene emission tended to saturate atquantum flux densities beyond those saturating for electrontransport. Nevertheless, although slightly biased at higherlight, calculated and measured values of I were close acrossthe entire light range (Fig. 3), indicating that the lightresponse curve of photosynthetic electron transport pro-vides a satisfactory approximation for light effects on iso-prene emission.

Temperature response of isoprene emission

Comparison of temperature response curves of the specificactivity of isoprene synthase (Fig. 4a), and temperatureresponse curves of leaf isoprene emission rate (Figs 4b &c) revealed that I versus temperature relationship was wellparameterized by temperature dependence of isoprenesynthase up to leaf temperatures of about 35 °C (Fig. 4). Athigh leaf temperatures, there was a large within andbetween species variability in isoprene emission rate. Theoptimum temperature of in vivo isoprene emission rate(Figs 4b & c) was in all cases lower than that for the temperature dependence of isoprene synthase (Fig. 4a),

1324 Ü. Niinemets et al.

suggesting that other factors became limiting at high tem-perature.

Since we took the amount of electrons used for isoprenesynthesis to be proportional to isoprene synthase activity in modelling the temperature response curves (Eqn 7,Hypothesis 2), combination of Eqns 4 and 7, gives

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

Table 1. Dependencies of isoprene emission rate per unit leaf area (I, nmol m-2 s-1), and per unit leaf dry mass (Im, nmol g-1 s-1) onphotosynthetic electron transport rate per unit area (J, mmol m-2 s-1) and per unit dry mass (Jm, mmol g-1 s-1), and on leaf dry mass perarea (MA) in Liquidambar styraciflua (data from Harley, Guenther & Zimmerman 1996) and Quercus species (data from Harley et al.1997): linear regression equations

SpeciesIndependent

Intercept P Slope P r2Slope for

nvariable intercept = 0

A. Isoprene emission rate per unit leaf areaQ. alba J -1·53 0·6 0·398 0·001 0·76 0·380 33Q. prinus 12·1 0·02 0·206 0·001 0·52 0·327 18Q. coccinea -13·5 0·01 0·654 0·001 0·84 0·487 23Q. velutina -0·265 0·9 0·483 0·001 0·92 0·480 11L. styraciflua -2·38 0·01 0·205 0·001 0·92 0·174 32

Q. alba MA -13·9 0·001 0·611 0·001 0·85 0·44 35Q. prinus 9·05 0·3 0·233 0·02 0·28 0·323 19Q. coccinea -28·9 0·001 0·881 0·001 0·80 0·504 23Q. velutina -33·5 0·06 0·803 0·002 0·69 0·402 11L. styraciflua -3·40 0·01 0·243 0·001 0·85 0·193 32

B. Isoprene emission rate per unit leaf dry massQ. alba Jm 0·160 0·02 0·222 0·001 0·32 0·367 33Q. prinus 0·174 0·03 0·160 0·05 0·21 0·337 18Q. coccinea -0·131 0·3 0·597 0·001 0·44 0·467 23Q. velutina 0·0101 0·8 0·467 0·001 0·83 0·479 11L. styraciflua -0·142 0·005 0·284 0·001 0·69 0·155 32

Q. alba MA 0·198 0·001 0·00287 0·001 0·42 0·00541 35Q. prinus 0·397 0·001 -0·00069 0·4 0·04 0·0032 19Q. coccinea -0·0191 0·9 0·00665 0·001 0·51 0·00640 23Q. velutina -0·0232 0·8 0·00503 0·05 0·38 0·00476 11L. styraciflua 0·0817 0·001 0·00145 0·001 0·32 0·00265 32

The original data are plotted in Fig. 1. To allow a better virtual comparison among the slopes of the regressions, slopes were alsocalculated for a zero intercept.

Figure 2. The fraction of electrons used for isoprene synthesis(e) in relation to the rate of isoprene emission. Symbols,measurement conditions, and data sources as in Fig. 1. r2 = 0·69,P < 0·001 for a linear regression calculated over all data.

I = dSsMA(Ci – G *)/[6(4·67 Ci + 9·33G *)].This indicates thatthe assumption of proportionality between e and isoprenesynthase activity leads to independence of I on total photosynthetic electron flow. However, the collapse of photosynthetic electron transport at higher temperaturesinevitably halts the energy flow to all chloroplastic biochemical reactions, amongst them isoprene synthesis.Therefore, the shape of the temperature response curve ofI should not only depend on the temperature relationshipof isoprene synthase, but may also be influenced by the tem-perature stability of J. Liquidambar styraciflua has anoptimum temperature of 37·8 °C for photosynthetic elec-tron transport (Fig. 5a), but the optimum temperature forisoprene emission is 42·4 ± 0·3 °C (average ± SE calculatedfor five temperature response curves from the data ofHarley, Guenther & Zimmerman 1996), and a value of39·8 °C has also been measured (calculated from Guentheret al. 1993). In comparison with the optimum temperaturefor isoprene synthase of 44·8 °C (Fig. 4a), the in vivo esti-mates are low in this species, providing indirect evidencefor the influence of temperature behaviour of J on the temperature response of isoprene emission. Nevertheless,because I had a greater optimum temperature than J, thefraction of electrons in isoprene production, e, increasedwith increasing leaf temperature (Fig. 5b) as was also cal-

Isoprene emission in relation to photosynthetic electron transport 1325

culated by Eqn 7 taking account of both the temperatureresponses of isoprene synthase and photosynthetic electrontransport.

To explain the differences between the temperatureresponses of isoprene emission rate and isoprene synthaseactivity (Fig. 4) we postulate that there is an upper limit ofe (emax), above which the increases in isoprene synthaseactivity do not result in increases in the fractional alloca-tion of electrons to isoprene synthesis. If e < emax isopreneemission rate is controlled by isoprene synthase activity(Eqns 4 and 7, cf. above), and if e = emax by photosyntheticelectron transport (Eqn 4). Having fixed emax at 0·25 andcombining theoretical J response curves with differentoptimum temperature (Fig. 6a) with the temperaturedependence of isoprene synthase (Fig. 4a), a positive rela-tionship is evident between the optimum temperatures ofisoprene emission rate and J (Fig. 6b). This correlationresults from the circumstance that emax is reached at a lowertemperature in leaves having lower temperature optimumfor photosynthetic electron transport.

Alternatively, combining of the temperature responses ofisoprene synthase (Fig. 4a) and J (Fig. 5a), and varying themaximum e, a set of temperature response curves of I is alsogenerated (Fig. 6c). Thus, even if the temperature responsecurve of photosynthetic electron transport remains con-

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

Figure 3. Measured (Harley, Guenther & Zimmerman 1996) and modelled light-response curves of isoprene emission in L. styraciflua.The rate of isoprene emission was calculated from Eqn (4). e values depicted in the figure were held constant for all points of the curve,and were adjusted with a non-linear regression procedure giving the best coefficient of determination. The differences in the capacity forphotosynthetic electron transport (Jmax, calculated according to Niinemets et al. 1999b) provided one explanation for differing plateauvalues of isoprene emission rates.

stant, varying the maximum fraction of electrons going toisoprene synthesis may provide an alternative explanationfor different temperature responses of isoprene emission.

Isoprene emission in relation to intercellularCO2 and O2

Although Eqn 4 includes both intercellular CO2 concentra-tion (Ci) and G * (that at a common temperature increaseslinearly with intercellular concentration of O2), the way I isrelated to Ci and O2 is also dependent on the possibleresponses of total photosynthetic electron transport rateand e to Ci and O2. Provided I and photosynthetic electrontransport rate remain constant with changing Ci, the frac-

1326 Ü. Niinemets et al.

tion of electrons used for isoprene synthesis should increasewith decreasing intercellular CO2 concentration (Fig. 7a).This is because isoprene production costs more in mole elec-trons per mole carbon assimilated than sugar synthesis (cf.Eqns 1 and 2), and accordingly, is more strongly affected by electron allocation to photorespiration than to carbonassimilation. Simulations demonstrate that the fraction ofelectrons used for isoprene synthesis should be quasi-inde-pendent of intercellular CO2 concentration at low atmos-pheric O2 concentration (Fig. 7a). Ci response of e (Fig. 7b)calculated from measured values of isoprene emission in Q.rubra (data of Loreto & Sharkey 1990) was basically iden-tical to that depicted in Fig. 7a, except that e was not inde-pendent of Ci in low O2 in one experiment (Fig. 7b). In one

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

Figure 4. Temperature dependence of the catalytic activity of isoprene synthase in crude leaf extracts in Populus tremuloides (a, datafrom Monson et al. 1992), and the isoprene emission rates in P. tremuloides (b) and Quercus species (c). The scale for the temperatureresponse of the specific activity of isoprene synthase (Ss) was obtained using a value of Ss of 900 nmol (mg isoprene synthase)-1 min-1 at32 °C (Silver & Fall 1995).The scaling constant, c, depicted in panel (a) is for the specific activity of isoprene synthase. In in vivoestimations, incident quantum flux density (Q, mmol m-2 s-1) was 800–1000 (Tingey et al. 1979; Ohta 1986; Monson & Fall 1989; Sharkey,Loreto & Delwiche 1991b; Monson et al. 1992, 1994; Harley et al. 1997), 1200–1500 (Loreto & Sharkey 1990; Guenther et al. 1993) or 2000(Sharkey et al. 1996), and cuvette CO2 concentration was around 350 mmol mol-1 in all measurements, except for those of Loreto &Sharkey (1990) that were conducted both at 385 and 2170 mmol mol-1. The data were standardized to give an isoprene emission rate of1·0 at a leaf temperature of 30 °C. Equation 5 was used to fit the data by a non-linear regression routine. Except for the scaling constant,c, all regression parameters obtained for in vitro enzyme activity (a) were held constant in fitting the in vivo emission data in panels band c. The coefficients of determination (r2) were calculated over the whole set of values.

Figure 5. Temperature dependence of photosynthetic electron transport (a) and the fraction of electrons used for isoprene production(b) in L. styraciflua (data of Harley, Guenther & Zimmerman 1996). The rate of photosynthetic electron transport was calculated fromleaf net assimilation rate as described in Methods (see also Niinemets et al. 1999b). Data were fitted by Eqn 6 in panel (a) using a non-linear regression routine. The modelled curve in panel (b) was calculated according to Eqn 7 using the temperature dependence ofphotosynthetic electron transport found in panel (a) and temperature coefficients for in vitro isoprene synthase activity (Fig. 4a), andadjusting the scaling constant (d) to obtain the best fit to the data.

case, under low O2, the electron transport rate necessary tosustain observed isoprene emission rates (JI) was alsorelated to CO2 in the study of Loreto & Sharkey (1990). Inthis case, JI was low in both lowest and highest Ci, and quasi-constant over a broad range of CO2 concentrations (Fig. 7c).

Simulation of diurnal courses of isopreneemission measured in the field

It appeared that a single value of isoprene emission rate,which gives an initial estimate of total isoprene synthaseactivity in the leaves (cf. Fig. 2), is sufficient to predictdiurnal courses of isoprene emission from photosynthetic

Isoprene emission in relation to photosynthetic electron transport 1327

electron transport rate. A regression equation between Jm

and isoprene emission rate per unit leaf dry mass (Table 1,Fig. 1b) was used to calculate this initial estimate of iso-prene emission rate in Q. alba (Figs 8a–f), and isopreneemission rates measured at leaf temperatures around 30 °Cwere employed in Q. frainetto (Figs 8g–i) and Q. pubescens(Figs 8j–l). These ‘basal’ isoprene emission rates werefurther used to calculate the scaling constant, d, in Eqn 7.Isoprene emission rates calculated by Eqns 4 and 7 alongwith information regarding the temperature response ofisoprene synthase (Fig. 4a) were in a good accordance withthe measured estimates in both non-water-stressed (Figs 8a,d & g) and in water-stressed leaves (Fig. 8j).

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

Figure 7. Modelled influence of changing intercellular CO2 concentration on the fraction of electrons necessary to sustain a constantisoprene emission rate of 35 nmol m-2 s-1 at two different intercellular O2 concentrations (O) and at a constant photosynthetic electrontransport rate (J) of 100 mmol m-2 s-1 (a); the fraction of electrons in isoprene synthesis calculated from the measurements in Quercusrubra (Loreto & Sharkey 1990) at two different quantum flux densities (Q) and intercellular O2 concentrations (b); and the rate ofphotosynthetic electron transport necessary to sustain the observed rate of isoprene emission (c, symbols as in panel b). Leaf temperaturewas held at 25 °C in the model estimations (a), and was 30 °C in the measurements. The rate of photosynthetic electron transport wascalculated as described in Niinemets et al. (1999b), and G * as in Fig. 6.

Figure 6. Modelled effects of changing optimum temperature of photosynthetic electron transport (a, b) and maximum fraction ofelectrons in isoprene production (emax, c) on the temperature response curve of isoprene emission. To obtain the temperature responsecurves of J with varying optimum temperature (Topt) depicted in panel a, the activation energies of photosynthetic electron transport inFig. 5a were held constant and the entropy term (Eqn 6) was changed; the scaling constant, cJ, was adjusted such that J was equal at25 °C for all curves. Temperature responses of J and in vitro isoprene synthase activity (Fig. 4a) were combined to yield the isopreneemission versus temperature curves in panel b, whereas emax was fixed at 0·25, and the scaling constant d (Eqn 7) was the same for allcurves and was chosen to give an isoprene emission rate of 150 nmol m-2 s-1 at 25 °C. In panel c, the temperature response curve ofphotosynthetic electron transport was that for L. styraciflua (Fig. 5a), emax was varied as depicted in the figure, and d adjusted to give an Iof 150 nmol m-2 s-1 at 25 °C. In all model calculations, intercellular CO2 concentration was fixed at 250 mmol mol-1 and that of O2 at210 mmol mol-1. Temperature coefficients of the kinetic parameters of Rubisco that determine the CO2 compensation point ofphotosynthesis in the absence of mitochondrial respiration (G *, see Eqn 1) were those reported in Niinemets & Tenhunen (1997).

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Figure 8. Modelled and measured daily time-courses of isoprene emission in Q. alba (a–f, data from Harley et al. 1997; measurements of July 1992), Q. frainetto (g–i,Steinbrecher et al. 1997, measurements of May 26, 1994) and Q. pubescens (j–l, Steinbrecher et al. 1997, measurements of June 30, 1995 in water-stressed leaves). Isopreneemission rate was calculated from Eqn 7, whereas for all data, the shape of the temperature response curve of photosynthetic electron transport was that depicted in Fig. 5a. Inpanels a and d, the scaling constant, d, was adjusted to give isoprene emission rates of 40·8 nmol m-2 s-1 (panel a) and 20·1 nmol m-2 s-1 at 30 °C (panel d). The latter valueswere calculated from the measured rates of photosynthetic electron transport at leaf temperatures around 30 °C (± 0·5 °C) according to the regression equations in Table 1(Fig. 1b). In panels g and j, d was adjusted to give an emission rate compatible with the measurements at 30 °C. Photosynthetic electron transport rates were calculatedaccording to Niinemets et al. (1999b), and G * at different leaf temperatures as in Fig. 6.

DISCUSSION

Relationships between isoprene emission, leafstructure and photosynthetic electron transport

Leaf dry mass per unit area (MA) is an important structuralparameter linking area- and mass-based physiologicalparameters: area basis is the product of mass basis and MA.Over the long-term, MA often responds more readily toenvironmental factors than the biochemical potentials ofindividual leaf cells, characterized by the process rate perunit leaf dry mass (Niinemets & Tenhunen 1997). Increasesin MA with increasing growth irradiance in the canopy (e.g.Ellsworth & Reich 1993; Niinemets 1997; Niinemets & Kull1998) belongs to the most important acclimation responsesof tree foliage to long-term light receipt, because this resultsin accumulation of foliar photosynthetic compounds perunit leaf area, and accordingly, greater photosynthesis ratesper unit area at greater light (Niinemets & Tenhunen 1997;Niinemets, Kull & Tenhunen 1998; Niinemets et al. 1998,1999a; Fig. 1e). In a like manner, isoprene emission ratesper unit leaf area consistently increase with increasing MA

(Harley et al. 1994, 1996; Harley, Guenther & Zimmerman1996; Harley et al. 1997; Litvak et al. 1996) as was also seenin reviewing the published data (Fig. 1c, Table 1). There wasalso a large variability in isoprene emission rates per unitleaf dry mass (Im), but the positive relationships were weakwith MA in most species (Fig. 1d, Table 1) and strong withthe rate of photosynthetic electron transport per unit leafdry mass (Jm, Fig. 1b, Table 1). This indicates that the posi-tive correlation between isoprene and photosynthetic elec-tron transport rates per unit leaf area is not attributable tothe covariation of both variables with MA (Fig. 1a,b), butthat leaf biochemical potentials for isoprene emission trulyscale with Jm.

Long-term light and temperature environmenteffects on isoprene emission

Since the variability in MA in the data (Fig. 1), which werecollected over canopy light gradients, was the result of theexposure of leaves to different long-term average quantumflux densities, the weak positive relationships between Im

and MA are compatible with positive scaling of Im withlong-term irradiance. Given that potential excess quantumflux densities increase with average integrated light in thecanopy (Niinemets et al. 1998), disproportionate increasesin isoprene emission rate with MA (Figs 1c & d) may berelated to avoiding excess light damage of the leaves. Com-patible with the results of the current analysis, studies havedemonstrated that the fractional allocation of assimilatedcarbon to isoprene increases with increasing long-term irra-diance (Sharkey, Loreto & Delwiche 1991b; Harley et al.1994; Harley, Guenther & Zimmerman 1996; Litvak et al.1996; Sharkey et al. 1996). Furthermore, there are importantinteractions between the environmental factors – a positiverelationship between irradiance and air temperature, and anegative relationship between irradiance and humidity –across the canopy (Chiariello 1984; Shuttleworth et al.

Isoprene emission in relation to photosynthetic electron transport 1329

1985). Recent evidence suggests that the emission of iso-prene (Sharkey & Singsaas 1995; Singsaas et al. 1997) andmonoterpenes (Loreto et al. 1998) may protect leaves from heat-stress damage, and that isoprene emission mayrespond fast enough to temperature ramps during brightsunflecks to protect the leaves from heat injury (Singsaas& Sharkey 1998). Thus, greater capacity for isoprene emis-sion of the leaves grown in higher light may have developedto cope with the high leaf temperatures that are more frequent in the upper canopy. This hypothesis is also con-sistent with the observations indicating relatively short-term acclimation of isoprene emission rates at standardconditions to both leaf average temperature and light environment in Quercus species (Sharkey et al. 1999). Incontrast, basal emission rates were similar in warm- andcool-temperature-grown specimens of Mucuna pruriens(Monson et al. 1992). Nevertheless, the optimum tempera-ture of I was greater for warm temperature grown leavesin this study (Monson et al. 1992), likely because of changesin temperature response curve of photosynthetic electrontransport (cf. below).

The competition for electrons in chloroplasts:light-dependence of isoprene synthesis

Although the phenomenon of electron competition is well know (cf. Material and Methods), little information isavailable of the regulatory mechanisms controlling the partitioning of electron flow between various biochemicalprocesses, for example, to nitrate and carbon assimilation.Data concerning electron allocation to isoprene synthesisare even more scarce. Since the fraction of electrons in iso-prene synthesis, e, was related to isoprene emission rate(Fig. 2), the total activity of enzymes of isoprene synthesispathway may be one determinant of the maximum fractionof electrons allocated to isoprene synthesis. It is possiblethat total activity of isoprene synthase, which catalyses theformation of isoprene from dimethylallyl pyrophosphate(Silver & Fall 1995; Wildermuth & Fall 1996, 1998), deter-mines the leaf capacity for isoprene emission.

Light dependence of J was generally a good predictor oflight effects on isoprene emission for a fixed fraction ofelectrons in isoprene synthesis (Fig. 3). Yet, e tended toincrease at highest instantaneous quantum flux densities(Fig. 3). In a like manner, nitrate photo-assimilation rela-tive to carbon assimilation consumed a greater fraction ofelectrons at higher incident irradiances (Bloom et al. 1989).Thus, either (1) isoprene synthesis and nitrate reduction arestronger competitors for electrons in higher quantum fluxdensities, or (2) there is a capacity of chloroplastic electrontransport that leaves are unable to utilize for carbon assim-ilation because of Rubisco limitations (cf. Introduction),and that may be used for other energy-consuming processessuch as isoprene synthesis and nitrate assimilation. Detaileddata regarding the regulation of isoprene synthase activityas well as of fine-tuning of photosynthetic electron trans-port are needed to provide conclusive support towardshypotheses (1) or (2).

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

Temperature dependence of isoprene synthesis

The data reviewed by us demonstrate that the initial partof the temperature response curve of I is very similar fordifferent species (Figs 4b & c), and fits well the tempera-ture response curve of in vitro isoprene synthase activity(Fig. 4a). Thus, in addition to greater photosynthetic elec-tron transport rates in higher temperature, the temperaturedependence of isoprene synthase plays an important partin the changes in I with leaf temperature. From our analy-sis (Fig. 5), and from other studies (Monson et al. 1992;Sharkey et al. 1996) it is evident that the fraction of elec-trons invested in isoprene synthesis increases, reflectingincreasing competitive ability of isoprene synthesispathway, with increasing temperature.

However, the temperature optima (Topt) of in vivo iso-prene synthesis differ widely between various species andwithin single species (Guenther et al. 1993; Simpson et al.1995; Fig. 4) and are generally lower than those observedin in vitro measurements. Optimum temperatures for iso-prene synthase as high as 44·8 °C for P. tremuloides (Fig. 4a)and 48 °C for Q. robur (Lehning et al. 1999) have been mea-sured, but the temperature optima of in vivo isoprene emis-sion rates rarely exceed 40 °C (Figs 4b & c). According tomodel calculations, differences in the temperature responsecurve of photosynthetic electron transport (Figs 6a & b)and/or the maximum possible fraction of electrons divertedtowards isoprene synthesis (Fig. 6c) may alter the optimumtemperature for isoprene emission. It is unclear what deter-mines the upper limit for e, but it is reasonable to assumethat there is one, because both excess electron or carbonusage in isoprene synthesis may potentially limit ribulose-1,5-bisphosphate (RUBP) regeneration; excess carbondrawn for isoprene formation may deplete the Calvin cycleand lead to depression of isoprene synthesis because ofcarbon limitations. Increases in optimum temperature of Iwith increasing growth temperature have been demon-strated in Mucuna pruriens (Monson et al. 1992).That study,where the positive effect of growth temperature on thetemperature optimum of I was paralleled by increased pho-tosynthesis at higher temperatures (Monson et al. 1992),contributes toward the mechanism depicted in Figs 6a & b.

CO2 effects on isoprene emission: implicationsfor modelling water-stress influences

Net assimilation versus intercellular CO2 response curvesprovide fundamental information of the limitations of photosynthetic processes. Thus, CO2 responses of isopreneemission and net assimilation measured concurrently mayprovide a means to differentiate between the controlsexerted over isoprene emission by the activity of isoprenesynthase and leaf photosynthesis parameters.

However, the reports of the influences of CO2 on iso-prene emission are contrasting, and demonstrate that therelationship between J and I is complex. There is evidencethat I declines at lower intercellular CO2 concentrations(Ci), and is constant over a wide range of Ci up to 600–

1330 Ü. Niinemets et al.

800 mmol mol-1 (Loreto & Sharkey 1990; Guenther,Monson & Fall 1991; Sharkey, Loreto & Delwiche 1991b;but see Monson & Fall 1989). As calculations (Fig. 7a) aswell as measurements (Fig. 7b) demonstrate, the constancyof I over widely varying Ci partly results from changes inthe fraction of electrons allocated to isoprene synthesis.Higher competitive ability for electrons of isoprene syn-thesis pathway than RUBP regeneration at low CO2 con-centration (Fig. 7b), may be related to greater ‘excess’photosynthetic electron transport rate at low Ci. Given thatthe specificity of Rubisco is considerably higher for CO2

than for O2 (Jordan & Ogren 1984; Brooks & Farquhar1985), RUBP regeneration cannot be maintained at thesame rates at low than at high CO2 concentrations.

Provided there is an upper limit of e, above which iso-prene synthesis would be limited by carbon availabilitybecause of the depletion of the Calvin cycle (cf. above), theoccasional declines in I at low Ci (Loreto & Sharkey 1990;Fig. 7c) may be related to reduced electron or carbon avail-abilities in low CO2 as well as greater electron cost of iso-prene synthesis at lower Ci (Fig. 7a). This may also explainthe decrease in I with increasing atmospheric O2 concen-trations in Populus (Sanadze & Tarhnishvili 1986). Inseveral experiments with P. tremuloides (Monson & Fall1989) and Arundo donax (Hewitt, Monson & Fall 1990) asimilar stimulatory effect of low O2 on isoprene emission(see also Fig. 7a) has been observed.

Although isoprene is emitted via stomata, isoprene emis-sion does not directly depend on stomatal conductance (G;Monson & Fall 1989; Sharkey 1991; Fall & Monson 1992).Because the atmosphere is an infinite sink for isoprene, thisindependence has been explained by the compensatoryincreases in isoprene vapour pressure difference betweenthe leaf and air in response to declining conductance(Sharkey 1991), possible only when a mild build-up of theisoprene concentration within the leaf does not inhibit itssynthesis rate. Nevertheless, I and G may be positively cor-related (Monson & Fall 1989), and I and stomatal conduc-tance may decline in a parallel manner in conditions ofdeveloping water stress (Tingey, Evans & Gumpertz 1981).We hypothesize that the correlations between isopreneemission and G result from low CO2 concentration effectson I (Fig. 7a) as well as on leaf photosynthetic electrontransport. Similarly, nitrate photo-assimilation was low inconditions of low Ci, which resulted from moderate leafwater stress (Kaiser & Förster 1989).

Isoprene emission rate may decrease in high CO2 con-centrations (Monson & Fall 1989; Loreto & Sharkey 1990).This is not well parameterized with the current model, pos-sibly because the lack of feedback limitations on photo-synthetic electron transport (Sharkey 1990) in the model.Loreto & Sharkey (1990) suggested that high CO2 declinein isoprene emission is related to the feedback-limitation-caused decline in leaf ATP content. Nevertheless, becausethe decrease in isoprene emission rates occurs at muchhigher CO2 concentrations than current ambient CO2, itshould not be of too much concern for modelling currentisoprene emissions.

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335

Usefulness of the proposed schema:simulation of daily time-courses

The analysis of daily time-courses of isoprene emissiondemonstrates that the algorithm based on leaf photosyn-thetic electron transport gives good fits to the data (Fig. 8)and describes as much variability in I as the empiricalmodels (cf. Harley et al. 1997; Steinbrecher et al. 1997). Themodel adequately described instantaneous responses of Ito temperature and light as well as to long-term variabilityin irradiance (Figs 8a & d). Yet, the maximum rate of I inhigh light was somewhat underestimated by the model insun leaves of Q. alba (Fig. 8a), possibly because of thegreater competitive ability of isoprene synthesis for elec-trons in high light (Fig. 3), or because the actual electrontransport rate was greater than that calculated from leafcarbon assimilation parameters (cf. above). The model alsoprovided good fits to the data measured under conditionsof water stress as characterized by the occurrence of mid-day stomatal closure and depression of net photosynthesis(Figs 8j–l), suggesting that the model is applicable over awide range of environmental conditions.

CONCLUSIONS

The results of the current study provide encouraging evidence that leaf photosynthetic electron transport maysuccessfully be employed for simulating foliar isopreneemission. Although the light- and temperature functionsused were similar to those applied previously (Guenther,Monson & Fall 1991; Guenther et al. 1993), we were able to achieve the same descriptive power with one leaf-dependent coefficient characterizing the leaf capacity forisoprene emission (scaling constant d in Eqn 7) as theformer models containing six empirical leaf-dependentparameters. Nevertheless, previous studies and our analysisindicate that further information of fine-regulation of iso-prene synthase activity in chloroplasts as well as of devel-opmental (Grinspoon, Bowman & Fall 1991; Kuzma & Fall1993; Harley et al. 1994; Monson et al. 1994; Lehning et al.1999) and environmental (Lehning et al. 1999; Sharkey et al.1999) factors influencing the isoprene synthase content ofleaves is necessary to more efficiently describe the com-petitive ability of isoprene synthesis pathway for electrons.Separation of morphological (MA, Figs 1c & e) and bio-chemical (J, fraction of electrons in isoprene synthesis,Figs 1b & 2) controls over isoprene emission gives animportant framework to scale isoprene emission rates upfrom leaf to a canopy level. As studies demonstrate, I scalespositively with MA along the canopy (Harley, Guenther &Zimmerman 1996; Harley et al. 1997), and MA may give arough approximation of leaf isoprene emission capacity.However, MA varies by a factor of five, but I by a factor of20 across the canopy (Fig. 1c), indicating that accounting foracclimation responses in leaf isoprene emission potentialsas related to changes in J and leaf isoprene synthase activ-ities will provide additional improvement of the estimatesof canopy level isoprene fluxes.

Isoprene emission in relation to photosynthetic electron transport 1331

ACKNOWLEDGMENTS

We thank Professors Graham D. Farquhar (ResearchSchool of Biological Sciences, Australian National Univer-sity, Canberra, Australia) and Russell K. Monson (Univer-sity of Colorado, Boulder, CO, USA) for the constructivecomments on the manuscript. The study was supported byEstonian Science Foundation (Grant 3235), by GermanFederal Minister of Research and Technology (BMFT,grants BEO 51–0339476 A, EST 001–98), and by EuropeanUnion (VOCAMOD, Contract Nr. ENV4-CT97–0424).TheNational Center for Atmospheric Research is sponsored bythe National Science Foundation.

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Energetic and carbon requirements for isoprene production13C labelling experiments indicate that isoprene emitted byplant leaves originates from a small pool of photosyntheticintermediates and is labelled as fast as the immediateproduct of ribulosebisphosphate carboxylation, 3-PGA(Delwiche & Sharkey 1993). This, in combination with thefinding that the enzyme, isoprene synthase, responsible forisoprene synthesis is located in the chloroplasts (Silver &Fall 1995; Wildermuth & Fall 1998) provides conclusive evi-dence that isoprene is synthesized in the chloroplasts. Eventhough the synthesis of chloroplastic isoprenoids is not sen-sitive to the inhibitors of hydroxymethylglutaryl-CoA(HMG-CoA) reductase (Schulze-Siebert & Schultz 1987),which catalyses the synthesis of mevalonic acid from HMG-CoA, there was a wide consensus that isoprenoid synthesisin chloroplasts proceeds via mevalonate-dependentpathway until recently (Gershenzon & Croteau 1993).Latest studies demonstrate that the immediate precursor of isoprenoids in chloroplasts is 1-deoxy-D-xylulose-5-phosphate (DXP), while mevalonate-dependent pathway isengaged in the cytosol (Lichtenthaler, Rohmer &

Schwender 1997; Lichtenthaler 1998). For comparative pur-poses, we calculate here the carbon and coenzyme require-ments for both the mevalonate pathway, which has beenused to calculate the energetic requirements for andpropose possible controlling steps of isoprene synthesis inthe majority of studies (Sharkey, Loreto & Delwiche 1991a;Steinbrecher 1997), and for DXP pathway. According to arecent study in Populus nigra, where some isoprene emittedfrom the leaves was derived from the mevalonate fed to theleaves (Schwender et al. 1997), both pathways may con-tribute to isoprene emission, possibly because cytosolicisopentenylpyrophosphate (IPP) may be transported to thechloroplasts.

(a) Mevalonate-dependent pathway

Gershenzon & Croteau (1993) give a detailed review of themevalonate-dependent pathway for isoprenoid biosynthe-sis. According to this route, three molecules of acetyl-CoAare combined in several enzymatic steps to yield one mol-ecule of mevalonic acid, that is further converted to isopen-tenylpyrophosphate (IPP). All energy requiring steps ofthis pathway are well-known (Fig. A1). Acetyl-CoA for

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influence of light and temperature on isoprene emission ratesfrom live oaks. Physiologia Plantarum 47, 112–118.

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Received 23 October 1998; received in revised form 6 April 1999;accepted for publication 5 June 1999

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Figure A1. Comparison of the energetic requirements of isoprene synthesis by mevalonate-dependent (Gershenzon & Croteau 1993)and mevalonate-independent (Lichtenthaler, Rohmer & Schwender 1997; Lichtenthaler 1998) pathways for isoprene biosynthesis. Sincethe intermediate compounds and participating enzymes leading from 1-deoxy-D-xylulose-5-phosphate to isopentenylpyrophosphate arecurrently not known, the given coenzyme requirements should be interpreted as minimum estimates.

APPENDIX A

mevalonate production in the chloroplasts may either beformed by acetyl-CoA synthetase from the acetate diffusedfrom the mitochondria (Murphy & Stumpf 1981), or fromchloroplastic pyruvate by pyruvate dehydrogenase complex(Givan 1983). Since all enzymes required for pyruvate syn-thesis are present in chloroplasts (see Sharkey, Loreto &Delwiche 1991a for a review), we first assume that chloro-plastic pyruvate is the source of acetyl-CoA. Taking 3-phosphoglycerate (PGA) formed in the Calvin cycle as theprecursor of pyruvate, production of 3 mole pyruvate fromCO2 and H2O costs 21 mole ATP and 15 mole NADPH(Fig. A1). Three mole of NADPH are recovered in the for-mation of acetyl-CoA, and 2 mole used for reduction ofhydroxymethylglutaryl-CoA to form mevalonic acid. Thelatter is converted to IPP at the expense of 3 mole ATP(Fig. A1). The IPP conversion to dimethylallyl pyrophos-phate (DMAPP) by IPP-isomerase and formation of iso-prene from DMAPP by isoprene synthase do not requireextra metabolic energy. Thus, the total cost of 1 mole iso-prene via mevalonate-dependent pathway is 24 mole ATPand 14 mole NADPH. In addition, 4 mole CO2 are also lostduring the synthesis (Fig. A1). Thus, in terms of carbonequivalents, 1 mole isoprene costs 3 mole PGA. If isopreneoriginates from mitochondrial acetyl-CoA, additional ATPrequirements are either 2 mole ATP per each mole acetyl-

Isoprene emission in relation to photosynthetic electron transport 1335

CoA in the acetyl-CoA hydrolase/synthetase reactions, or1 mole ATP if acetyl-CoA is transported to chloroplasts viacitrate transport system.

(b) 1-Deoxy-D-xylulose-5-phosphate (DXP) pathway

1-Deoxy-D-xylulose-5-phosphate synthesis from glycer-aldehyde-3-phosphate (GAP) and pyruvate by a thiamin-dependent DXP synthase (Sprenger et al. 1997) is the key event in the second isoprenoid synthesis pathway(Lichtenthaler, Rohmer & Schwender 1997; Lichtenthaler1998; Putra et al. 1998). However, further steps and inter-mediates leading to IPP formation are still uncertain.2-Methyl-D-erythritol-4-phosphate (MEP) has been sug-gested as the compound directly following DXP (Lichten-thaler 1998). Synthesis of MEP from DXP should involveone reduction step, and further at least two reductions andthree dehydrations as well as one phosphorylation arerequired to finally yield IPP. Thus, at least 3 mole NADPHand 1 mole ATP are necessary to form IPP from DXP.Given the coenzyme requirements for GAP and pyruvatesynthesis, formation of 1 mole isoprene from CO2 and H2Ocosts 17 mole ATP and 14 mole NADPH (Fig. 1). Since only1 mole CO2 is lost (Fig. 1), 2 mole PGA are necessary toform 1 mole isoprene.

© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 1319–1335