CSIRO PUBLISHING

www.publish.csiro.au/journals/fpb Functional Plant Biology, 2006, 33, 931–940

Isoprene prevents the negative consequences of high temperaturestress in Platanus orientalis leaves

Violeta VelikovaA,D, Francesco LoretoB, Tsonko TsonevA, Federico BrilliB and Aglika EdrevaC

AInstitute of Plant Physiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.BCNR-Istituto di Biologia Agroambientale e Forestale, Monterotondo Scalo, Rome, Italy.

CInstitute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.DCorresponding author. Email: violet@obzor.bio21.bas.bg

Abstract. The phenomenon of enhanced plant thermotolerance by isoprene was studied in leaves of the same ageof 1- or 2-year-old Platanus orientalis plants. Our goals were to determine whether the isoprene emission dependson the age of the plant, and whether different emission rates can influence heat resistance in plants of differentage. Two-year-old plants emit greater amounts of isoprene and possess better capacity to cope with heat stress than1-year-old plants. After a high temperature treatment (38◦C for 4 h), photosynthetic activity, hydrogen peroxidecontent, lipid peroxidation and antiradical activity were preserved in isoprene emitting leaves of 1- and 2-year-oldplants. However, heat inhibited photosynthesis and PSII efficiency, caused accumulation of H2O2, and increased allindices of membrane damage and antioxidant capacity in leaves of plants of both ages in which isoprene was inhibitedby fosmidomycin. In isoprene-inhibited leaves fumigated with exogenous isoprene during the heat treatment, thenegative effects on photosynthetic capacity were reduced. These results further support the notion that isoprene playsan important role in protecting photosynthesis against damage at high temperature. It is suggested that isopreneis an important compound of the non-enzymatic defence of plants against thermal stress, possibly contributing toscavenging of reactive oxygen species (ROS) and membrane stabilising capacity, especially in developed plants.

Keywords: hydrogen peroxide, isoprene, lipid peroxidation, photosynthesis.

Introduction

Numerous volatile organic compounds (VOCs) are emittedby the leaves of many plant species, especially trees, into theatmosphere (Kesselmeier and Staudt 1999). Among biogenicVOCs, isoprene is the most abundant biogenic hydrocarbon.On a global scale the emission rate of isoprene is estimated tobe 500 Tg year−1 and represents ∼44% of all non-methanehydrocarbons emitted into the atmosphere (Guenther et al.1995). Typically in non-stressed conditions around 2% of theassimilated carbon is emitted as isoprene, which representsa non-trivial loss of carbon to the plant (Sharkey andYeh 2001).

The interest in isoprene has grown rapidly over thelast decade, not only because of its role in atmosphericchemistry, but also because of its putative involvementin protection of plants against oxidative stress caused bydifferent environmental factors (Loreto and Velikova 2001;Sharkey and Yeh 2001). Because isoprene production takes

Abbreviations used: DPPH., 2,2-diphenyl-1-picrylhydrazyl radical; FRAP, ferric reducing antioxidant power; ROS, reactive oxygen species;

TBARS, thiobarbituric acid reactive substances; VOCs, volatile organic compounds.

place in chloroplasts, it was suggested that a link may existbetween isoprene production and environmental stresses tothe photosynthetic apparatus (Sharkey and Singsaas 1995;Loreto et al. 2001; Loreto and Velikova 2001).

Isoprene emission is very sensitive to changes intemperature. The strong positive temperature response ofisoprene emission rate was reported in many studies (Loretoand Sharkey 1990; Sharkey and Loreto 1993; Sharkey et al.1996; Singsaas and Sharkey 1998, 2000). The hypothesisthat isoprene may exert a protective action against heat stresswas stated for the first time by Sharkey and Singsaas (1995).Recently the thermoprotection of photosynthetic capacityby isoprene has been further explored and demonstrated byother authors (Penuelas et al. 2005; Velikova and Loreto2005). The mechanism by which this protective action occursis unclear.

It is well established that unfavourable environmentalconditions may induce the development of oxidative stress

© CSIRO 2006 10.1071/FP06058 1445-4408/06/100931

932 Functional Plant Biology V. Velikova et al.

in plants. Oxidative stress is essentially a regulated process,the equilibrium between the oxidative and antioxidativecapacities determining the extent of the stress. Under normalconditions, plants rapidly metabolise ROS with help ofantioxidant enzymes and / or metabolites (Scandalios 1997;Edreva 2005). However, various environmental perturbationscan cause excess ROS production, overwhelming thesystem and necessitating additional defenses. Thegeneral plant response to environmental stresses is anenhanced generation of ROS such as superoxide, hydrogenperoxide and hydroxyl radicals (Able et al. 2003; Alscheret al. 1997). Unless efficiently metabolised, these ROSrapidly oxidise membrane lipids, proteins and othercellular constituents leading to their malfunction andultimately inducing cell death that is manifested by theappearance of necrotic lesions. Antioxidants are believedto play a very important role in the defence systemagainst ROS.

In plants, several enzymatic and non-enzymaticmechanisms with complementary and interdependentstrategies are involved in the antioxidant defence system(Polle and Rennenberg 1994; Camejo et al. 2006). Someantioxidant compounds are hydrophilic scavengers, suchas ascorbic acid and glutathione; others are lipophilicsubstances, such as carotenoids and tocopherols, which arefound in the membrane bilayer.

It has been proposed that isoprene is involved in plantprotection mainly by two molecular mechanisms. First,the presence of conjugated double bounds (delocalisedπ-electrons) in the isoprene molecule can mediate easyelectron and energy transfers, thus constituting a basis forROS-scavenging ability (Affek and Yakir 2002; Loreto andVelikova 2001; Velikova et al. 2004). Second, the shorthydrocarbon chain (C5H8) determining high hydrophobicity,may allow the allocation of isoprene within the lipidmembrane domain, thus preventing dissociation of themembrane bilayer (Loreto et al. 2001; Loreto and Velikova2001; Sharkey and Yeh 2001).

There is little evidence of the mechanism(s) and theinteractions involved in the phenomenon of enhancedplant thermotolerance by isoprene. The purpose of thepresent study was to test the possible thermotoleranceinduced by isoprene in leaves of Platanus orientalischaracterised by different isoprene emission levels, namelyleaves developing from 1- and 2-year-old plants, leaves inwhich isoprene biosynthesis was inhibited by fosmidomycin,a specific inhibitor of the deoxy-xylulose-phosphate pathwayin chloroplasts (Zeidler et al. 1998), and leaves that weresupplied exogenous isoprene after inhibiting endogenousisoprene synthesis with fosmidomycin. Thermotolerancewas tested with in vivo measurements of photosynthesis,stomatal conductance and photochemical efficiency. Theputative mechanisms by which isoprene was involved ininducing thermotolerance were tested by assessing the levels

of H2O2, an important ROS, and TBARS, a recognisedmarker of membrane damage. In this way we tried to assignto isoprene a ROS-scavenging and / or membrane-stabilisingfunction in thermotolerance. Moreover, we determined thetotal antioxidant and antiradical ability as dependent on thepresence or deficiency of isoprene in the leaves.

Materials and methodsPlant material

Platanus orientalis L., a common and widespread Balkan tree species,was used as a source of plant material. One- and 2-year-old plantswere used for the experiments performed. The two groups of plantswere grown in a greenhouse until the appearance of the first leaves.They were regularly watered to keep the pots at full water capacity,and were fertilised once a week with full-strength Hoagland solution.After appearance of the first leaves, the plants were transferredto a climatic chamber under controlled conditions: light intensity350 µmol m−2 s−1, day / night temperature 25 / 20◦C ± 2◦C, relativehumidity 65%, and a photoperiod of 12 h. The plants were grown underthese conditions for 1 month before starting the treatment. At this stageall plants had only developed the main branch. One-year-old plants were7–8 cm tall, and had four or five leaves, whereas 2-year-old plants were∼20 cm tall and had 5–7 leaves. The measurements were made on fullydeveloped leaves of the same age (70–75 days) in both 1- and 2-year-old plants. High temperature, isoprene inhibition and measurementsof isoprene emission rate, gas exchange and fluorescence parameterswere all performed on detached leaves, as described by Loreto andVelikova (2001) and also detailed in the following sections. Preliminarymeasurements have shown no impact of leaf cutting on physiologicalparameters (photosynthesis, stomatal conductance and photochemicalyield) and isoprene emission measured 1 h after the cutting.

High temperature treatment, isoprene inhibition, and fumigationof isoprene-inhibited leaves with exogenous isoprene

High temperature (38◦C) was applied to detached leaves fromboth groups of plants for 4 h. P. orientalis leaves were cut underwater and maintained in a glass vial with 15 mL of distilledwater for 1 h before starting the treatment. The experiments wereperformed with leaves emitting isoprene and leaves in which isopreneemission was preliminarily inhibited by fosmidomycin, a powerful andspecific inhibitor of the methyl-eritrythol-phosphate (MEP) pathwayof isoprenoid biosynthesis in chloroplasts (Zeidler et al. 1998).Fosmidomycin was added to the water of the vial and was taken upby the leaves through the transpiration stream. The fosmidomycinconcentration in the solution was 5 µM. In isoprene-inhibited leavesthe high temperature treatment started when isoprene emission wasinhibited to ∼10% of the original level and did not decrease further,i.e. 60 min after fosmidomycin feeding.

In a reconstitution experiment, isoprene inhibited leaves werefumigated with 3 µmol mol−1 of isoprene, a concentration yieldingan internal concentration of isoprene similar to the endogenousconcentration (Singsaas et al. 1997), throughout the high temperaturetreatment. Isoprene was added to the airstream entering the leaf cuvette,as described by Loreto and Velikova (2001).

Gas exchange and fluorescence measurements

Photosynthesis, stomatal conductance and transpiration were measuredwith a portable photosynthetic system (LI 6400, Li-Cor, Lincoln, NE).The measurements were performed on the intact leaves ata photosynthetically active radiation of 350 µmol m−2 s−1 after10–15 min to achieve steady-state conditions. A leaf portion (6 cm2)was clamped in a gas-exchange cuvette at a rate of 0.3 L min−1 air

Isoprene prevents the negative consequences of high temperature stress Functional Plant Biology 933

flow. Relative humidity within the chamber was controlled at 60–70%and supplied CO2 concentration was 380 µmol mol−1. Chlorophyllfluorescence was measured by mini-PAM (Heinz Walz GmbHEffeltrich, Germany) simultaneously to gas exchange measurements,and photosynthetic electron transport rate was calculated fromfluorescence measurements as detailed by Loreto et al. (1994). Thequantum yield of PSII (�PSII) was calculated according to Gentyet al. (1989): �PSII = (Fm

′ − Fs) / Fm′, where Fm

′ is the maximumfluorescence in the light and Fs is the steady-state fluorescence in thelight.

Isoprene measurements

Isoprene emission was measured on-line by diverting the air at the exitof the gas-exchange cuvette into a portable gas chromatograph (SyntechSpectras BTX Analyser GC 855; Syntech, Groningen, The Netherlands)as detailed elsewhere (Loreto and Velikova 2001). At the end of eachmeasurement the leaf disc was frozen in liquid nitrogen and then usedfor the biochemical assays.

Determination of H2 O2 content

Hydrogen peroxide levels were determined as described by Velikovaet al. (2000). Fresh leaf material (0.3 g) was homogenised in an ice bathwith 3 mL 0.1% (w / v) trichloroacetic acid (TCA). The homogenatewas centrifuged at 12 000 g for 30 min and 0.4 mL of the supernatantwas added to 0.4 mL 10 mM potassium phosphate buffer (pH 7.0) and0.8 mL 1 M KI. The absorbancy of supernatant was read at 360 nm. Thecoloured reaction product of H2O2 with KI develops within 25 min andis stable for at least 2 h. We have established that H2O2 does not degradeduring extraction in an ice bath, being stabilised by trichloroacetic acid(TCA). The stability of H2O2 at the above conditions was also shownby a recovery experiment with external standard. The content of H2O2

was given on a standard curve.

Lipid peroxidation

The level of lipid peroxidation was measured in terms of thiobarbituricacid reactive substances (TBARS) content according to Heath andPacker (1968). Leaf material (0.3 g) was homogenised in 3 mL 0.1%(w / v) TCA solution. The homogenate was centrifuged at 12 000 gfor 30 min and 0.5 mL of the supernatant was added to 1 mL 0.5%(w / v) TBA in 20% TCA. The mixture was heated at 95◦C for30 min, and then quickly cooled in an ice bath. Then the samples werecentrifuged at 10 000 g for 5 min, and the absorbance of supernatant wasrecorded at 532 nm. The value for non-specific absorption at 600 nm wassubtracted. A blank containing the components of the reaction mixtureexcept TBA was employed to eliminate the influence of interferingsubstances following the original procedure of Heath and Packer (1968).Anthocyanins, which have an absorption maximum at 536 nm and hencemay interfere with the measurement of TBARS, are not present in ourplant material. The TBARS content was calculated by its absorptioncoefficient of 155 mM−1 cm−1.

Evaluation of antioxidant activity

The antioxidant capacity was assessed by two different tests, the ferricreducing antioxidant power (FRAP) assay, and the antiradical activity(DPPH

.) assay. Preliminary tests on the reliability of these methods

concerning the stability of the reaction products and influence ofinterfering substances were carried out.

Sample preparation

Fresh leaf material (0.5 g) was homogenised in 5 mL 80% ethanol. Aftercentrifugation at 10 000 g for 30 min, the supernatant was immediatelyused for FRAP and DPPH

.assays.

FRAP assay

In the FRAP assay, antioxidants in the sample reduceFe(III) / tripyridyltriazine complex, present in excess, to the blueferrous form, with an increase in absorbance at 593 nm, this increasebeing the proportional to the total FRAP value of antioxidants in thesample. FRAP assays were performed according to Benzie and Strain(1999). The reagents included 300 mM acetate buffer (pH 3.6), 10 mM

tripyridyl-s-triazine (TPTZ) in 40 mM HCl and 20 mM FeCl3. FRAPreagent was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZsolution and 2.5 mL FeCl3. Diluted supernatant (50 µL) was added to1.5 mL of freshly prepared reagent. After 15 min the absorbance at593 nm was read. FeSO4 was used as a standard. Data were expressedas µM FRAP per g of fresh tissue.

DPPH.

radical scavenging assay

Radical scavenging activity of ethanol leaf extracts againststable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH

.) was determined

spectrophotometrically. When DPPH.

reacts with an antioxidantcompound, which can donate hydrogen, it is reduced. The changes incolour (from deep violet to light yellow) were measured at 515 nm.

Radical scavenging activity of extracts was determined after themethod of Brand-Williams et al. (1995). The solution of DPPH

.in methanol (6 × 10−5 M) was prepared daily. A volume of 1.95 mLof this solution was mixed with 0.05 mL extract. The decrease ofabsorption was measured at room temperature. Blank samples (withoutantioxidant) containing 0.05 mL ethanol and 1.95 mL DPPH

.solutions

were prepared and measured before every measurement of the sampleof the relevant extract. DPPH

.standard solution was freshly prepared

and kept in a flask protected against light with aluminium foil and storedat 4◦C between the measurements. The results are presented as a rate ofDPPH

.reduction.

Statistics

All data shown in the figures represent the means ± s.e. fromtwo independent experiments with at least three replications per plants ofeach treatment and age. Means were statistically separated by a Tukey’stest, and those significantly different at the 5% level are shown bydifferent letters.

Results

Isoprene emission

The temperature response of isoprene emission rate of leavesof 1- and 2-year-old plants is shown on Fig. 1. The leaveswere held at a temperature of 25◦C and 350 µmol m−2 s−1

light intensity (i.e. at growing conditions) until the isopreneemission reached a steady rate. At 25◦C, isoprene emissionwas slightly but not significantly higher in 2-year-oldplants than in 1-year-old plants. When the temperature wasincreased to 38◦C, isoprene emission increased by 175% andapproximately 4-fold over the initial rate in 1- and 2-year-oldplants, respectively.

Changes in photosynthetic activity

Photosynthesis measured at growing conditions (25◦C)was higher in 2-year-old plants than in 1-year-old plants.Isoprene inhibition alone was not related to changes inphotosynthesis in leaves in both groups of plants (Fig. 2A,B). The high temperature treatment resulted in a slight andnot statistically significant decrease of photosynthesis in

934 Functional Plant Biology V. Velikova et al.Is

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Fig. 1. Effect of high temperature treatment (38◦C for 4 h) on isopreneemission in leaves of 1- and 2-year-old Platanus orientalis plants.Measurements were made at 25◦C / 350 µmol m−2 s−1 PPFD (growingconditions) and 38◦C / 350 µmol m−2 s−1 PPFD (conditions of hightemperature treatment). Means ± s.e. (n = 6) are shown. Means wereseparated by Tukey’s test and different letters indicate means that arestatistically different with P>95%.

isoprene-emitting leaves of both 1- and 2-year-old plants.In isoprene-inhibited leaves the decrease of photosynthesisinduced by high temperature was complete in 1-year-oldplants, whereas in 2-year-old plants photosynthesis wasreduced by 56% with respect to the control rate (at 25◦C).In 1-year-old leaves in which endogenous isoprene wasinhibited by fosmidomycin, exogenous isoprene was suppliedthroughout the high temperature treatment to reconstitutethe internal pool of isoprene. In these leaves photosynthesisreduction was only 46% of control rate (Fig. 2A). The hightemperature treatment also caused a reduction of stomatalconductance (Fig. 2C, D). This reduction was, however,similar in isoprene-emitting and isoprene-inhibited leaves,and in isoprene-inhibited leaves supplied with exogenousisoprene, suggesting that changes in stomatal conductancedo not account for the negative effect of high temperature onphotosynthesis of isoprene-inhibited leaves.

The quantum efficiency at growing conditions (25◦C) washigher in 2-year-old plants in comparison with 1-year-oldplants. Isoprene inhibition did not change significantly theefficiency of PSII of both classes of plants age (Fig. 2E, F ).The high temperature treatment did not influence the�PSII ofisoprene emitting plants. On the contrary, �PSII of isoprene-inhibited leaves exposed to high temperature was reducedby 42 and 49% in 1- and 2-year-old plants, respectively, incomparison to the �PSII of leaves exposed to 25◦C. Theincomplete inhibition of �PSII in 1-year-old plants, in whichphotosynthesis was totally inhibited, was likely caused by alarge heat-induced photorespiration rate, or by the activationof alternative electron sinks.

H2O2 content

The H2O2 content was similar in 1- and 2-year-old plantsemitting isoprene, and was not influenced by the temperature

treatment in these plants (Fig. 3A, B). Isoprene inhibitiondid not significantly increase H2O2 level at 25◦C. However,the high temperature treatment caused significant H2O2accumulation in isoprene-inhibited leaves, whereas H2O2content in isoprene-emitting leaves was not significantlychanged. H2O2 level of isoprene inhibited leaves exposedto high temperature was enhanced by 28 and 24% in 1- and2-year-old plants, respectively, in comparison to the H2O2content of leaves exposed to 25◦C.

Lipid peroxidation

The level of TBARS was higher in 1-year-old plants thanin 2-year-old isoprene-emitting plants sampled at 25◦C(Fig. 3C, D). After inhibition of isoprene synthesis TBARScontent did not change in either 1- or 2-year-old plants. Hightemperature treatment of 1- and 2-year-old plants caused anincrease in TBARS level only in isoprene-inhibited leaves,which was more evident in 2-year-old plants.

Total antioxidant ability and radical scavenging activity

In leaves of both groups of plants isoprene inhibition causeda similar increase in total antioxidant ability (Fig. 4A, B).Heat treatment of isoprene-emitting plants caused a dramaticrise of antioxidant activity, reaching 386 and 465% in 1- and2-year-old plants, respectively, while in isoprene-inhibitedplants this response amounted to 252 and 266%, respectively,compared with the corresponding controls held at 25◦C.Irrespective of the treatment, the FRAP values in the leavesof 2-year-old plants were higher than in the leaves of 1-year-old plants. Moreover, the activation of antioxidant capacity ofthe leaves of 2-year-old plants following heat exposure washigher than in 1-year-old plants (Fig. 4A, B).

Isoprene inhibition and temperature treatment, aloneor in combination, did not increase the rate of DPPH

.reduction, indicating radical scavenging activity in leaves of1- and 2-year-old plants. However, this was not the case inisoprene-inhibited leaves of 2-year-old plants, where hightemperature exposure caused significant rise in antiradicalactivity (Fig. 4C, D).

Discussion

Platanus orientalis is widespread in the riparian areas ofthe Balkans, while it survives as an endangered species insome riparian areas of the west Mediterranean. P. orientaliswas first described as a strongly isoprene-emitting plantby Zhang et al. (2000). We show that the emission ofisoprene by this plant is ubiquitous and not restrictedto populations of China, expanding our knowledge ofisoprene emission by natural vegetation worldwide. Ourresults also support the observation that riparian speciesare widespread isoprene emitters and the consequentspeculation that isoprene emission trait may have evolvedto withstand stresses occurring at the water–soil interface,such as the association of heat and drought episodes(Loreto 2002).

Isoprene prevents the negative consequences of high temperature stress Functional Plant Biology 935

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Fig. 2. Photosynthesis (A, B), stomatal conductance (C, D) and photochemical quantumefficiency of PSII (E, F ) in isoprene-emitting and isoprene-inhibited leaves of 1- and 2-year-oldPlatanus orientalis plants in response to high temperature (38◦C for 4 h). Measurements weremade at 350 µmol m−2 s−1 PPFD and 380 µmol mol−1 CO2. The panels on the left represent dataof 1-year-old plants and the panels on the right represent data of 2-year-old plants. White barsrepresent data of control plants and grey bars represent data after high temperature treatment. Thecross-hatched bars represent photosynthesis in isoprene-inhibited leaves in which the isopreneleaf pool was reconstituted with fumigation of 3 µmol mol−1 exogenous isoprene (third pair ofbars in panels A and C ). Means ± s.e. (n = 6) are shown. Means were separated by Tukey’stest and different letters indicate means that are statistically different within the same panel,with P>95%.

936 Functional Plant Biology V. Velikova et al.

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Fig. 3. Hydrogen peroxide content (A, B) and TBARS level (C, D) in isoprene-emitting andisoprene-inhibited leaves of 1- and 2-year-old Platanus orientalis plants in response to hightemperature (38◦C for 4 h). Bar assignment, treatments, replications and statistical treatmentas in Fig. 2.

The high temperature treatment enhanced isopreneemission in both 1- and 2-year-old plants, the increasebeing particularly relevant in 2-year-old plants. This wasan expected response because the temperature-dependenceof isoprene emission and of isoprene synthase activity iswell documented (Loreto and Sharkey 1990; Monson et al.1992; Lehning et al. 1999). We found that the isopreneemission rate was higher in leaves developed from 2-year-old plants than in leaves developed from 1-year-old plants.There is considerable evidence that isoprene emission is alsostrongly dependent on leaf developmental state, and thatchanges in extractable isoprene synthase activity parallelisoprene emission rates during leaf development (Kuzmaand Fall 1993; Fischbach et al. 2002). However, here wereport that plant age, not leaf age, may also be an importantfactor in determining isoprene emission rates. Under eachset of growing conditions a positive relationship betweenthe isoprene emission capacity (Fig. 1) and photosynthesis(Fig. 2A, B) and quantum yield (Fig. 2E, F ) was also observed

in Platanus leaves. As photosynthesis provides reducedcarbon, ATP and NADPH for isoprenoid synthesis, thismay explain the plant-age-dependent rates of isoprenoidsynthesis / emission. It is, however, questionable whetherisoprenoid synthesis is ever limited by photosynthesis, asisoprenoid synthesis requires only a few percent of totalphotosynthetic turnover of reduced carbon, ATP and NADPH(Niinemets et al. 2002). Moreover, isoprene emission wasparticularly enhanced following the heat treatment in 2-year-old plants, which shows that the isoprene emission capacityof older plants is very large and may be sustained even whenphotosynthesis is reduced by environmental stresses, beingcontrolled by other physiological processes that are activatedunder environmental constraints (Lehning et al. 2001; Kuhnet al. 2004).

Isoprene inhibition did not cause changes inphotosynthesis under normal growing conditions(Fig. 2A, B), as we previously reported in our experimentswith other plant species (Loreto and Velikova 2001).

Isoprene prevents the negative consequences of high temperature stress Functional Plant Biology 937

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Fig. 4. Total antioxidant ability (A, B) and antiradical activity (C, D) in isoprene-emitting andisoprene-inhibited leaves of 1- and 2-year-old Platanus orientalis plants in response to hightemperature (38◦C for 4 h). Bar assignment, treatments, replications and statistical treatmentas in Fig. 2.

However, when plants were exposed to heat, isopreneinhibition resulted in a significant decrease of photosynthesis,in contrast to the preserved photosynthesis in isoprene-emitting plants. Hence, isoprene becomes a requisite tosustain photosynthetic performance when a stress constraint,such as heat, is imposed on plants. This is compatible withthe enhanced isoprene emission observed following heatexposure (Fig. 1). The reduced photosynthesis in isoprene-inhibited plants is not due to stomatal factors, as decreasedstomatal conductance after heat exposure is observedin both isoprene-emitting and isoprene-inhibited plants(Fig. 2C, D). Unaffected intracellular CO2 concentrations(data not shown) in isoprene-inhibited leaves support theassumption that non-stomatal (biochemical) factors areinvolved in the significant photosynthesis inhibition in theseleaves exposed to high temperature.

To further prove the thermoprotection ability ofisoprene, we performed a reconstitution experiment inwhich exogenous isoprene replaced the endogenouspool, previously inhibited by fosmidomycin. In this casephotosynthesis was less influenced by heat than in leavesin which isoprene was inhibited and not replaced. Thisconfirms that the inhibition of photosynthesis was notattributable to a non-specific action of fosmidomycinalso affecting carotenoids, which are formed through thesame biosynthetic pathway as isoprene, and which alsohave relevant antioxidant action. Rather, our results arein agreement with conclusion of Sharkey et al. (2001),as well as with our previous finding with other plantspecies (Velikova and Loreto 2005), that thermotoleranceof photosynthesis is a substantial benefit of isoprene andthat this protective action can be partly carried out also

938 Functional Plant Biology V. Velikova et al.

when the isoprenoid pathway is shut down and isoprene issupplied exogenously.

Throughout the chlorophyll fluorescence measurementsof plants grown at 25◦C no changes of PSII efficiency(Fig. 2E, F ) and electron transport (data not shown)following isoprene inhibition were established. However,we observed a considerable decrease in these parametersaccompanying an increased ROS formation in isoprene-inhibited leaves exposed to heat (Figs 2E, F, 3A, B). Thedecrease in �PSII indicates that there was a fall in thephotosynthetic energy conversion in PSII (Mohammed et al.1995). This indicates an increased amount of the reducedform of plastoquinone QA and a decreased effectiveness oftransfer through the electron transport chain. In isoprene-emitting leaves, in which photosynthetic capacity wasnot influenced by heat, photosynthetic electron transportis probably sufficient to prevent the build-up of excessenergy in PSII, and the powerful antioxidant defence systemefficiently protects the leaf photochemistry against enhancedgeneration of ROS. High temperature treatment significantlylimited photosynthesis and photochemical PSII efficiencyonly in isoprene-inhibited leaves. Under such conditions,the energy provided by light absorption greatly exceeds theenergy demand of photosynthesis, probably leading to anoverproduction of ROS (Elstner 1991; Foyer et al. 1994).

Most cell organelles, for instance chloroplasts,mitochondria, plasma membrane and peroxisomes, aresites where oxygen can be activated (Shigeoka et al. 2002).Hydrogen peroxide is especially toxic in the chloroplast,because it is known to be the powerful inhibitor of theCalvin cycle. Hydrogen peroxide, even at low concentration,inhibits the enzymes possessing exposed sulfhydryl groups,such as NADP+-dependent glyceraldehyde-3-phosphatedehydrogenase and fructose-1,6-biphosphatase, reducingphotosynthetic CO2 assimilation (Takeda et al. 1995).Our data are consistent with this finding, showing thatphotosynthesis restriction in isoprene-inhibited leavesexposed to high temperature (Fig. 2A, B) was associatedwith a significant increase of H2O2 content (Fig. 3A, B)measured in these leaves.

Free radicals cause degradation of membrane lipids.Therefore, oxidative stress was expected to produce anincreased level of lipid peroxidation in the plants subjectedto heat. Malondialdehyde is the main decomposition productof polyunsaturated fatty acids in biomembranes and itsenhancement seems reasonable, as it is known that thylakoidmembranes become leaky at moderately high temperatures(Pastenes and Horton 1996; Bukhov et al. 1999). Suchresults are consistent with those in the present investigation(Fig. 3C, D). A significant increase in the TBARS level(used as an indicator of the extent of membrane damage)was observed in isoprene-inhibited leaves exposed to hightemperature. The absence of change in TBARS levelin isoprene-emitting leaves under high-temperature stress

suggests that endogenous isoprene may sustain membraneintegrity. Similar results were obtained in Phragmitesaustralis exposed to heat (Velikova and Loreto 2005; Velikovaet al. 2005). The enhanced monoterpene emission and lackof changes in lipid peroxidation in oak plants at 45◦Cis considered as certain protection to the photosyntheticapparatus in these plants (Penuelas et al. 2005).

At growing temperature (25◦C), isoprene inhibitiontriggered an up-regulation of total antioxidant ability,suggesting the involvement of alternative protectivemechanisms in the absence of isoprene. High temperaturetreatment caused a significant increase in total antioxidantability of isoprene-emitting and isoprene-inhibited leaves(Fig. 4A, B). Noticeably, the high-temperature response ofisoprene-inhibited leaves was lower than in isoprene-emittingleaves. This may be accounted for by cross-protection, whicharises when one stress factor is followed by another. Inour case, the primary stress factor, isoprene inhibition, mayinduce some levels of defence that could attenuate the effectof the following stressor, high temperature.

The rise of antiradical activity following heat exposure(Fig. 4D) provides additional evidence that when isopreneemission was inhibited the plants triggered alternativeprotective mechanisms. Low-molecular-weight componentssuch as ascorbic acid and gluthathione, constituents ofthe antioxidant and antiradical pool of plants, can be up-regulated under stress conditions. The resulting membranedamage (Fig. 3C, D) suggests, however, that isoprenedeficiency cannot be compensated by the activation of theantioxidant and antiradical capacity, with this substantiatingthe basic protective function of isoprene in stress conditions.Moreover, the fact that heat exposure caused no changesin photosynthesis, PSII quantum efficiency, TBARS, H2O2and antiradical activity levels in isoprene-emitting plantsconfirms that isoprene alone is effective enough toprevent oxidative stress caused by heat, thus sustainingthermotolerance without damage to photosynthetic processesand membrane integrity.

In conclusion, it has been highlighted that isopreneemission depends on plant age and that this dependency isexacerbated by heat. Two-year-old plants emitted isoprenemore intensively, increased their antioxidant and antiradicalabilities after heat exposure more significantly, and weremore resistant to heat than 1-year-old plants. Our resultssupport the idea that isoprene protects against environmentalstresses, as it has been recognised in detailed studies withdifferent plants (Singsaas et al. 1997; Sharkey and Yeh 2001;Sharkey et al. 2001; Penuelas et al. 2005). The finding ofan isoprene-induced thermotolerance in Platanus orientalis,a widespread Balkan species, increases our knowledge onthe role of isoprene in stressful environment. Our resultsare consistent with previous studies on another isoprene-emitting plant, Phragmites australis (Velikova et al. 2004,2005; Velikova and Loreto 2005), suggesting the ROS-

Isoprene prevents the negative consequences of high temperature stress Functional Plant Biology 939

scavenging and membrane-stabilising properties of isopreneas a rationale of its protective functions in stress conditionscontributing to the non-enzymatic defence of plants.

Acknowledgments

This study was funded by a NATO Reintegration Grant(No 981279), by the European Science Foundation programVOCBAS, and by a bilateral project within the frameworkagreement between Italian National Research Council andBulgarian Academy of Sciences.

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Manuscript received 24 March 2006, accepted 7 June 2006

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