A unified mechanism of action for volatile isoprenoidsin plant abiotic stressClaudia E Vickers, Jonathan Gershenzon, Manuel T Lerdau & Francesco Loreto

The sessile nature of plants has resulted in the evolution of anextraordinarily diverse suite of protective mechanisms againstbiotic and abiotic stresses. Though volatile isoprenoids areknown to be involved in many types of biotic interactions, theyalso play important but relatively unappreciated roles in abioticstress responses. We review those roles, discuss the proposedmechanistic explanations and examine the evolutionarysignificance of volatile isoprenoid emission. We note that abioticstress responses generically involve production of reactiveoxygen species in plant cells, and volatile isoprenoids mitigatethe effects of oxidative stress by mediating the oxidative statusof the plant. On the basis of these observations, we propose a‘single biochemical mechanism for multiple physiologicalstressors’ model, whereby the protective effect against abioticstress is exerted through direct or indirect improvement inresistance to damage by reactive oxygen species.

Owing to their sedentary lifestyle, plants must be capable of copingwith a variety of changes in light intensity, temperature, moistureand other abiotic factors in their environments (Fig. 1). Whenthese factors shift out of a certain range, plants are subjected tostress; this can lead to decreased growth rate, reduced reproductionand even death. Beyond the single-plant level, changes in environ-mental stress can also select for short- and long-term shifts inecological traits, potentially affecting biological diversity, ecosystemfunctioning and carbon sequestration. Abiotic stress also resultsin significant losses in crop yields, the magnitude of whichfluctuates from year to year. Sources of abiotic stress includeextremes of temperature (including freezing), high light intensity,drought, air pollutants, salinity and mechanical damage. Thesestresses are rarely experienced singularly: they often occur incombination. The simultaneous occurrences of rapidly rising tem-peratures, drought and pollutants are among the most strikingphenomena associated with global change, and they threaten plantsthat have not adapted and are not able to rapidly acclimate tothese factors1.

Few responses of plants are stress-specific. Most often, stresses elicitgeneric responses—in particular, production of excess reactive oxygenspecies (ROS) such as singlet oxygen (1O2), superoxide (O2

��),hydrogen peroxide (H2O2) and hydroxyl radicals (�OH). ROS areimportant signaling molecules and also serve to initiate defenseresponses2. The cellular balance of ROS is normally kept under tightcontrol3; however, when this control is lost, damage occurs. ROS causedirect damage to plant cells through oxidation of biological compo-nents (nucleic acids, proteins and lipids) and can instigate chainreactions resulting in accumulation of more ROS and initiation ofprogrammed cell death2. Plants have a complex response network oflipid-phase and aqueous-phase antioxidant compounds and enzymesthat defend against conditions of excess ROS. Direct reactions toquench and remove ROS occur (for example, Fig. 2), as well asindirect responses including hormone-mediated signaling to upregu-late primary defense genes and activate secondary defense genes(reviewed previously2–4). When the antioxidant defense network isoverloaded, oxidative stress results. Measuring abiotic stress responsesand attributing mechanistic behavior can be problematic because ofthe complexity of the ROS response network, which makes itdifficult to distinguish cause-and-effect relationships. The network islinked between lipid and aqueous phases, so understanding physicalcompartmentalization is fraught. Many ROS are short-lived, sodirect measurements are also difficult; further, many moleculesinvolved in signaling cascades have not been identified and thuscannot be measured.

Although most research on plant antioxidants has focused onnonvolatile compounds, certain volatiles belonging to the isoprenoidfamily have also been implicated in protection against oxidative andother abiotic stresses. The isoprenoids are a very large and extremelydiverse group of organic compounds. Isoprenoid carbon skeletons arecomposed of five-carbon building blocks that may be assembled in avariety of formations and contain many different modifications. Inplants, two separate metabolic pathways are responsible for theproduction of the C5 building block of isoprenoids: the cytosolicmevalonic acid (MVA) pathway, and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway5–7 (Fig. 3). The two pathwayscan be linked through exchange of metabolic precursors across theplastid membrane8–11. It is generally assumed that later steps in theformation of hemiterpenes (C5), monoterpenes (C10), diterpenes (C20)and tetraterpenes (C40) are present in the plastids, whereas theformation of sesquiterpenes (C15) and triterpenes (C30) takesplace in the cytosol. Volatile isoprenoids are generally lipophilic, low-molecular-weight compounds with masses under 300 (see Fig. 4).Only hemiterpenes (isoprene and methylbutenol), monoterpenes,Published online 17 April 2009; doi:10.1038/nchembio.158

Claudia E. Vickers is at the University of Queensland, Australian Institute forBioengineering and Nanotechnology, St. Lucia, Australia. Jonathan Gershenzonis at the Max Planck Institute for Chemical Ecology, Jena, Germany. Manuel T.Lerdau is in the Environmental Sciences and Biology Departments, Universityof Virginia, Charlottesville, Virginia, USA. Francesco Loreto is at ConsiglioNazionale delle Ricerche, Istituto di Biologia Agroambientale e Forestale,Monterotondo Scalo (Roma), Italy. e-mail: c.vickers@uq.edu.au.

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sesquiterpenes and some diterpenes have sufficient vapor pressure tovolatilize at ordinary biological temperatures12. Volatility bestowsparticular properties on these compounds—for example, the abilityto carry chemical messages away from the sites of synthesis in the plant.

Production of volatile isoprenoids represents a substantial invest-ment for the plant in terms of carbon (loss of which in the form ofvolatiles is irretrievable) and energy. Constitutive emissions in isopreneemitters and strong monoterpene emitters are generally in the rangeof 1–100 nmol m�2 s�1 (http://www.es.lancs.ac.uk/cnhgroup/download.html). This amount is equivalent to 1–2% of photosyntheticcarbon fixation13. Even when the carbon budget becomes negativeunder stress conditions and photosynthesis is severely or completelyinhibited, isoprenoid emission is often sustained14–16. This large cost,especially under stressful conditions, suggests the strong possibility thatisoprenoid emission also confers benefits to the plant. Though the roleof some volatiles in biotic interactions is well established and hasbeen well reviewed12,17,18, there are many volatiles for which thebenefit remains obscure. Higher order (nonvolatile) isoprenoids havea variety of roles in plant cells, including in abiotic stress defense. Themechanisms by which they act are diverse; some are hormonal signalsthat can carry messages throughout the plant and elicit systemicresponses (for example, abscisic acid), whereas others act directly asantioxidants (for example, carotenoids and tocopherols; see Fig. 4).Recent research, which we will highlight here, has revealed that certainvolatile isoprenoids also play an important role in abiotic stressresponses. Though the bulk of this research has focused historicallyon isoprene (2-methyl-1,3-butadiene), there is increasing evidence thatmany (and perhaps all) volatile isoprenoids are involved in abioticstress responses.

Changes in volatile emission patterns under stress conditions origi-nally supplied circumstantial evidence that volatiles are linkedwith stress responses. Emissions often increase under abiotic stress

conditions, particularly under heat andlight stress13,15,19,20. Stressed plants can main-tain high isoprenoid emissions, and theemission can be transiently enhanced inplants recovering from stresses, particularlyin drought-stressed plants14,21,22. Applicationof jasmonates, which trigger defense responsepathways in both biotic and abiotic stresses,stimulates production of volatile isopre-noids23,24. Stored carbon can even be mobi-lized to maintain volatile production understress conditions14,25,26. Subsequent experi-ments have demonstrated that volatile isopre-noids play a protective role under thermal,radiative, oxidative, drought and salt stress.Here we will examine the evidence showingthat volatile isoprenoids confer protectionagainst abiotic stress, and we will argue thatthe common mechanism driving abioticstress protection is an antioxidant effect ofthese compounds.

It should be noted that other functionalpossibilities, including balancing of sub-cellular supplies of phosphoenolpyruvate27

and dissipation of excess carbon28 andenergy29 from photosynthesis, have beenput forward to explain isoprene emission;however, there are stoichiometric and bio-chemical arguments against these proposed

functions30, and they have not been extended to volatile isoprenoids ingeneral. Isoprene emissions have also been shown to affect bioticinteractions31,32. The roles of volatile isoprenoids may be multifaceted,and different functional hypotheses are not necessarily mutuallyexclusive. Here we will focus on the behavior and mechanism ofaction of volatile isoprenoids in abiotic stress conditions.

Volatile isoprenoids protect against abiotic stressExperimental evidence has shown that volatile isoprenoids confer aprotective effect to photosynthesis under thermal and oxidativestress conditions. This evidence comes from three types of experi-ment: (i) the use of fosmidomycin, an antibiotic/herbicide thatselectively inhibits the activity of 1-deoxy-D-xylulose 5-phosphatereductoisomerase (DXR), thereby inhibiting the MEP pathway33

(see Fig. 3), (ii) fumigation of non-emitting species with exogenousgaseous isoprenoids, also followed by reconstitution experiments inleaves in which endogenous emission was previously inhibitedchemically, and (iii) the use of transgenic plants in which iso-prenoid synthesis has been either engineered by insertion of theappropriate terpene synthase genes, or repressed by silencing thesame genes.

Thermal (high temperature) stress. The role of volatiles in protec-tion against thermal stress has been relatively well studied. Sharkeyand co-workers have been investigating the isoprene effect for overa decade13,30,34. Under sun-flecking conditions, leaf temperaturecan fluctuate dramatically, with variations of up to 20 1C occurringin very short time periods; in this scenario, co-incident lightand heat stress are experienced in a punctuated fashion. Recoveryfrom temperature-induced decreases in photosynthetic efficiencyis poorer in isoprene-emitting plants that are treated withfosmidomycin, and fumigation with isoprene can partially restore

High light causes production of excess excitation energy in the photosynthetic reaction centers, resulting in direct accumulation of a variety of reactive oxygen species.

High temperature stress denatures proteins and causes lipid peroxidation.

Water deficit, or drought, interferes with metabolism. ROS produced under drought conditions trigger signaling pathways that generate defense responses.

Soil salinity is usually caused by excess salts of chloride and sulfate. Salinity results in ion cytotoxicity and osmotic stress, and decreases uptake of nutrients. Resulting metabolic imbalances lead to oxidative stress.

Air pollution with oxidizing species (including ozone and sulfuric acid) causes direct oxidative damage to tissues. Local and systemic signaling responses also occur.

Mechanical damage—both biotic(e.g., from insect feeding) and abiotic (e.g., from wind damage)—triggers expression of defense-related genes.

Cold stress interferes with metabolic processes (particularly enzyme activity) and alters membrane properties. Frosting can severely damage tissues when ice forms. Extracellular ice formation alsocauses intracellular water deficit.

O3

Na+

Cl–K+

SO2

Ca2+Mg2+

Figure 1 Plants are exposed to a variety of abiotic stresses. Complex response and protective systems

are triggered under these stress conditions (reviewed previously86–90). All of these abiotic stresses

result in production of reactive oxygen species (ROS). Excess light and heat, as well as exposure to

oxidizing air pollutants, cause direct accumulation of ROS (see Fig. 2). High temperatures are often

coincident with high light stress. Drought results in osmotic stress and intracellular water deficit; soilsalinity and cold stress (particularly frosting) also result in water deficit, and the molecular responses to

these three stresses are similar (though not identical). When stresses are combined, responses are often

amplified; for example, high light/low temperature stress and high light/low water stress can result in

very high production of ROS. ROS are particularly important for initiating signal cascades that trigger

defense gene transcription and adaptive responses. Phytohormones are also important in these

responses and are involved in signaling pathways. ABA is particularly important in water deficit

responses, and ethylene, salicylic acid and jasmonic acid are often involved in wound responses.

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recovery in fosmidomycin-poisoned leaves. These results wereconfirmed in an independent laboratory35–37. However, giventhat fosmidomycin inhibition of the MEP pathway acts by inhibitionof one of the early pathway enzymes, compounds besides isoprenemight also be affected (for example, fosmidomycin can affect abscisicacid biosynthesis38). The inhibition studies were followed up usingtransgenic Arabidopsis thaliana plants bearing heterologously expressedisoprene synthase genes39,40. Prolonged heat stress experimentsconfirmed that isoprene emission could confer a thermotolerantgrowth phenotype relative to non-emitting plants, but protection ofphotosynthesis could not be tested in this system because controlplants did not show inhibition of photosynthesis after heat stressepisodes39. However, these transgenic plants emitted only low levelsof isoprene relative to a naturally emitting plant. A second approachwas the use of transgenic poplar plants with silenced isoprenesynthase expression41. These plants showed little or no isopreneemission, and demonstrated greater sensitivity of photosynthesis torepeated heat/light stress events. However, interpretation of theseexperiments is complex because stress sensitivity often occurs inprimary-generation transgenic plants due to collateral damageduring transformation and tissue culture processes, and can becarried through several transgenic generations42. Consequently, analysis

of stress responses in transgenic plants in species with long gene-ration times is fraught with difficulties.

To address these issues, transgenic tobacco plants heterologouslyexpressing isoprene synthase have been engineered43. These linesproduced high levels of isoprene, and azygous plants were generatedfor use as controls. When fourth-generation plants were placed undera repeated heat/light stress regime, photosynthesis recovered better inhomozygous emitting plants compared to azygous non-emittingcontrols. The effect was only distinguishable by longitudinal analysisand was not as strong as that observed in fosmidomycin-poisonedleaves, which suggests that (i) inhibition of other MEP products mightplay a role in the phenotype observed using fosmidomycin treatments,and/or (ii) species-dependent variation exists. However, these resultsdemonstrated that endogenously emitted isoprene plays a role inthermotolerance of photosynthesis. The subtlety of the effect mightexplain why it was not observed in the low-emitting transgenicA. thaliana plants.

Protection of photosynthesis against high temperature stress is alsoobserved in monoterpene-emitting plants, when emission of mono-terpenes inhibited by fosmidomycin is restored with exogenousmonoterpene fumigation19. Monoterpene fumigation can alsoconfer thermotolerance on low-emitting species that are not treated

Thylakoid lumen

APXPSII PSI

SOD APX

APX

PL-OH PHGPX PRX

NTR

PSIIPL

3

4

T(OOH)

1O2

1O2

H2O2

H2O2

H2O2

H2O2

H2O2

PL-OOH PL-OO.

O2.–

.OH

Chloroplast stroma

O2H2O

H2O

H2O

H2O

1 22 H+

NAD(P)H

NAD(P)HGSSG

GSH

GSH GR

NTR

PRX

TRX-SH

TRX-SS

GPX

GR

MDAR

MDA

DHAR GSSG

Asc

Asc

MDA DHA

O2.–

αT(OH)

1O2 αC(O.) αT(OH)

H2O2 H2O

TRX-SS

NAD(P)H NAD(P)+

NAD(P)+

NAD(P)+

TRX-SH

Figure 2 The chloroplastic antioxidant/enzyme defense network reactions in response to increased light and temperature. When absorbed energy is in excess

of that used in photosynthesis, various different ROS are formed at the photosystems in the thylakoid membranes of the chloroplast, and a complex

scavenging system activates2,70,91. At photosystem II (PSII), O2��, H2O2, singlet oxygen (1O2) and hydroxyl radicals (�OH) are produced from molecular

oxygen (1). Superoxide ions are produced during the Mehler reaction by Fd-NADPH oxidase at photosystem I (PSI) (1O2 may also be produced at PSI) (2).

O2�� is dismutated by superoxide dismutase (SOD) to hydrogen peroxide (H2O2). 1O2 is highly reactive and has an extremely short half-life, reacting very

quickly with molecules close to the site of synthesis (proteins, pigments and lipids), and recent research in fact suggests that 1O2 is the major cause of

photo-oxidative damage under high light stress71. It is quenched by b-carotene in the PSII reaction center and a-tocopherol (aT(OH)) in the thylakoid

membranes; excess 1O2 reacts with D1 protein, resulting in degradation of D1 and loss of PSII activity (photoinhibition). Unquenched 1O2 also causes lipid

peroxidation (3) and can trigger defense gene expression via signal transduction (1O2 reacts with free radical nitric oxide (�NO) to produce peroxynitrite

(ONO2�), which acts as a signaling molecule). 1O2 and �OH can be scavenged by ascorbate (Asc), tocopherols and glutathione (GSH). H2O2 is produced

during a variety of different reactions under stress conditions, often from detoxification of other, more dangerous ROS. It is scavenged by three different

antioxidant/enzyme reactions: the Asc, GSH and peroxiredoxin (PRX) cycles. These cycles are interlinked through shared metabolites and reducing

equivalents. Increased temperature, which is often co-incident with high light stress, also causes lipid peroxidation and results in phospholipid peroxy

radicals (PL-OO�) (4). PL-OO� is converted to phospholipid hydroperoxide (PL-OOH) by oxidation of a-T(OH); PL-OOH is reduced to phospholipid alcohol

(PL-OH) by the action of phospholipid hydroperoxide–dependent glutathione peroxidase (PHGPX). Enzymes controlling regeneration of metabolites can be

found in the aqueous environment (stroma) or bound to the thylakoid lipid membrane. Lipid- and aqueous-phase antioxidant networks are linked through

Asc-mediated regeneration of a-T(OH) and activity of PHGPX. Not all chloroplast redox reactions are shown here. Similar antioxidant reaction networks can

be found in the cytosol and in other organelles; cross-talk between these compartments also occurs. Additional abbreviations: aC(O�), a-chromanoxyl radical;

APX, ascorbate peroxidase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG,

glutathione disulfide; GPX, glutathione peroxidase; MDA, monodehydroascorbate; MDAR, monodehydroascorbate free radical reductase; NTR, NADPH-

thioredoxin reductase; T(OOH), hydroperoxytocopherone; TRX, thioredoxin; TRX-SH, reduced thioredoxin; TRX-SS, thioredoxin disulphide.

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with fosmidomycin44. Transgenics that over- or underexpresshigher order volatile isoprenoids have not yet been studied forresistance to abiotic stress.

Oxidative stress. Several studies show that isoprene protects leaftissues against oxidative stress. Leaves show lower accumulation ofROS, less cellular damage and less damage to photosynthetic processesin response to ozone (O3) fumigation when isoprene is also applied45.Similarly, fosmidomycin-poisoned leaves show more photosyntheticdamage from ozone fumigation than leaves that are producingisoprene46. Under photo-oxidative stress, singlet oxygen is producedat the photosynthetic membranes, and photosynthetic assimilation

is inhibited47. Isoprene fumigation of non-emitting leaves resultsin protection of photosynthetic processes when singlet oxygen isproduced by addition of the photosensitizer Rose Bengal48; fosmido-mycin-based inhibition studies support this49. Experiments usingtransgenic tobacco plants heterologously expressing isoprene synthase(described above) confirmed that isoprene-emitting plants showmuch greater resistance to ozone-induced oxidative stress comparedto azygous control plants43. This showed that the protective effect wasassociated with endogenous isoprene production.

As is the case for thermal stress, much less evidence has beencollected from monoterpene-emitting plants than from isopreneemitters. However, it has been demonstrated clearly that the

Figure 3 Isoprenoid biosynthetic pathways

(simplified). Two linked isoprenoid biosynthetic

pathways are found in plant cells: the cytosolic

MVA pathway and the chloroplastic MEP

pathway. The universal five-carbon building

blocks produced by these pathways are isopentyl

pyrophosphate (IPP) and its isomer dimethylallyl

diphosphate (DMADP); conversion between

isoforms is catalyzed by isopentyl diphosphate

isomerase (IDI). The cytosolic MVA pathway

produces sesquiterpenes, triterpenes,

homoterpenes and precursors for sterols and

ubiquinone via farnesyl diphosphate (FPP);

the chloroplastic MEP pathway produces the

hemiterpene isoprene, monoterpenes (via geranyldiphosphate, GPP), diterpenes, tetraterpenes and

higher order isoprenoids (via geranylgeranyl

diphosphate, GGPP). Fosmidomycin inhibits

1-deoxy-D-xylulose 5-phosphate reductoisomerase

(DXR), thereby inhibiting formation of IPP and

DMADP from the MEP pathway. Members of the

isoprenoid group have an extraordinarily wide

range of roles in the plant; these include well-

characterized physiological functions in primary

metabolism such as growth regulation (for

example, hormones), photosynthetic components (phytol side chains of chlorophylls, prenyl side chains of plastoquinones) and structural (for example, sterol

membrane components) roles, and also secondary functions such as defense (for example, some phytoalexins) and antioxidants (for example, tocopherols

and carotenoids). In addition to this, isoprenoids fulfill a variety of roles in secondary metabolism; these roles are also very diverse, and many remain to be

fully characterized. ABA, abscisic acid; DXP,1-deoxy-D-xylulose 5-phosphate; GAs, gibberellins; GA3P, glyceraldehyde 3-phosphate; HMG-CoA, 3-hydroxy-3-

methylglutaryl-CoA; HMGR, HMG-CoA reductase; PEP, phosphoenolpyruvate; PPi, pyrophosphate; PQ, plastoquinone.

MVA

GA3P

PEP PEP Pyruvate

DXP

MEP

IPPIDI

IPP

HMGR

MVA

DMAPP

Squalene

SterolsPolyprenols

Sesquiterpenes (C15)Triterpenes (C30)

Homoterpenes (C11, C30)

FPP (C15)

DMAPP(C5)

GPP (C10) Monoterpenes (C10)

PQ-9 (C45)(prenyl chain)

+5 IPP

Tetraterpenes (C40)

CarotenoidsAbscisic acid

Phytyl chains Chlorophylls TocopherolsPhylloquinonesPhytoalexinsGibberellinsTaxol

2xDiterpenes (C20)GGPP (C20)

Chloroplast

Isoprene (C5)

IPP

2x

2 x IPP

2x

DXRPyruvate

Acetyl-CoA

HMG-CoA

GA3P

MEP

Tocopherols Hemiterpenes

Isoprene

Sesquiterpenes

Limonene Sabinene

Myrcene

Monoterpenes

Tocotrienols

Carotenoids

α-Carotene

α-Pinene

α-Humulene

β-Pinene

(E )-β-Ocimene

(E )-β-Farnesene

(E )-β-Caryophyllene δ-Cadinene

(E ,E )-α-Farnesene

(Z )-β-Ocimene

β-Carotene

Violoxanthin

O

O

HO

OH

R2

R3

R1R2

R3

R1

O

O

HO

HO

Figure 4 Chemical structures of isoprenoids

with antioxidant properties. Many higher order

nonvolatile isoprenoids have been shown to have

antioxidant functions (left panel). Tocopherols

and tocotrienols are lipid-phase antioxidants.

They scavenge lipid peroxy radicals and react

with and physically quench singlet oxygen;

R groups are methyl (-CH3) or hydrogen (-H).Carotenoids are photosynthetic pigments that

provide photoprotection through antioxidant

activity in addition to absorbing light energy

for photosynthesis. There are two classes of

carotenoids: unoxygenated (carotenes) and

oxygenated (xanthophylls). Carotenes quench

triplet chlorophyll, and xanthophylls such as

violoxantin participate in the xanthophyll cycle,

which is responsible for quenching singlet

chlorophyll. The volatile isoprenoids shown

in the right panel have either been shown to

have antioxidant properties (for example,

isoprene) or have chemical properties

conducive to antioxidant activities.

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photosynthesis of monoterpene-emitting plants becomes moresensitive to ozone if the emission is inhibited by fosmidomycin, andthat, in contrast, photosynthesis becomes less sensitive to ozone innon-emitting plants that are exogenously fumigated with volatileisoprenoids50. Like isoprene and monoterpenes, many volatile plantsesquiterpenes combine rapidly with ROS51, and their emission isstimulated by high light and temperature conditions52; thus, thesecompounds might also be involved in resistance to abiotic stress.Unfortunately, sesquiterpenes have not been well studied owing todifficulties in accurately quantifying emission rates as a consequenceof their high reactivity and sensitivity to disturbance53.

Mode of actionThe mechanisms by which isoprenoids exert their protective effect areas yet undetermined, though two primary mechanistic hypotheseshave been put forward in the context of thermal54 and oxidative46

stress tolerance. Mechanistic separation in this way assumes a complex‘multiple mechanisms for multiple stressors’ model. However, as allenvironmental stress responses are characterized by the release ofdangerous oxidative species, it is parsimonious to argue that theabiotic stress tolerance enhancement conferred by these compoundscan be grouped under a common rubric of oxidant protection. Herewe propose a ‘single biochemical mechanism for multiple physio-logical stressors’ model (Fig. 5) that attempts to unify the diverseempirical studies of volatile isoprenoid compounds and their role inabiotic stress tolerance.

Membrane stabilization. Membrane stabilization as a mechanisticexplanation for isoprene action was first proposed by Sharkey and co-workers34. Owing to its lipophilic properties and the site of synthesis(the chloroplast), isoprene is likely to partition into lipid phases ofthylakoid membranes. When heat stress occurs, membranes becomemore fluid, and photosynthetic processes (which are membrane-associated) exhibit a decrease in efficiency. It was therefore proposedthat the mechanism of the protective effect is through physicalstabilization of hydrophobic interactions (lipid-lipid, lipid-protein

and/or protein-protein) under increasing temperatures. According totheoretical modeling of molecular dynamics, isoprene does indeedpartition into the center of phospholipid membranes54. This enhancesmembrane order without a significant change in the dynamic proper-ties of the membrane. Decreased rates of electron transport are alsoobserved in photosynthetic membranes after inhibition by fosmido-mycin, which also suggests that the presence of isoprene facilitatesphotosynthetic processes under heat stress36. Given that other volatileisoprenoids tend to also be hydrophobic, this mechanism mightbe generic. However, direct testing of this mechanistic theory in anin vivo system is difficult.

Despite this indirect empirical support, there are certain physico-chemical problems with membrane stabilization as a mechanism forthe isoprene effect. Given that it is a hydrocarbon, isoprene is highlyhydrophobic; it is virtually insoluble in pure water, and the Henry’sLaw constant even in seawater has been estimated at KH B 3.1(ref. 55). As the site of isoprene production is the chloroplast, it seemsreasonable to assume that, even at low emission rates, lipid mem-branes (particularly chloroplast membranes) must be saturated withisoprene by the time isoprene is measured at the leaf surface. At anygiven temperature, once membranes are saturated, increasing theproduction of isoprene must increase the rate that isoprene travelsthrough the membrane rather than the concentration of isoprene inthe membrane. If membranes are always isoprene-saturated in emit-ting species, it follows that fumigation by isoprene should not conferfurther protection—a result that has been observed56. Interestingly,exogenously supplied isoprene also does not supply protection toisolated membranes under thermal stress57; such membranes presum-ably have no endogenous isoprene. On the other hand, fumigationwith isoprene can partially complement the effects of fosmidomycin-induced inhibition of the MEP pathway on photosynthesis under hightemperature30,35. These results are somewhat contradictory and canonly be reconciled if we assume that isolated membranes are notphysiologically the same as intact plant membranes and do notrespond the same way to the presence of isoprene. Partial comple-mentation also suggests that other products from the MEP pathway

Figure 5 The ‘single biochemical mechanism for multiple physiological

stressors’ model. The model shows how oxidative damage resulting from

environmental stress occurs (in gray), and how volatile isoprenoids (VIPs)

may exert protective effects through antioxidant activity (in black). Solid

lines represent direct reactions, and broken lines represent indirect

reactions. Environmental stress (high light, temperature, ozone exposure)

causes oxidative stress (Ox), which results in production of ROS (for

example, hydrogen peroxide, singlet oxygen and superoxide) and reactive

nitrogen species (RNS; for example, nitric oxide, peroxynitrite). These

compounds initiate cell signaling directly and also through interactions with

the hormonal response network, as well as causing further direct oxidative

damage. Different stresses trigger different response pathways. For example,

ozone exposure also triggers a response that overlaps with biotic stress

responses though the plant hormone network; salicylic acid (SA), jasmonic

acid (JA) and ethylene (ET) trigger signal cascades that initiate programmedcell death (PCD), resulting in accelerated senescence via an inappropriate

hypersensitive response (HR). VIPs may act at several different levels to

arrest oxidative stress response processes. (1) Because it is lipophilic, VIP

may physically stabilize hydrophobic interactions in membranes, minimizing

lipid peroxidation and reducing oxidative stress and downstream buildup of

ROS/RNS. (2) VIP may react with ROS/RNS to produce reactive electrophile

species (RES) such as methacrolein and methylvinylketone (products of

isoprene/ozone reaction), which are known to induce antioxidant and other defenses. If the stressor is itself an ROS (for example, ozone), VIP may react

directly with the stressor. (3) Direct antioxidant behavior (scavenging ROS/RNS) also prevents accumulation to damaging levels, thus preventing further

oxidative damage. As a consequence, ROS/RNS-activated signal cascades and PCD pathways that normally result in tissue necrosis are prevented. Figure

prepared with assistance from P. Mullineaux (Essex University).

Environmentalstress

1

2

33

3

2Ox

ROSRNS

Hormones(SA, JA, ET)

Signaltransduction

PCD-associatedgene expression

HRAcceleratedsenescence

Tissuenecrosis

Oxidativedamage

Defense-associated

geneexpression

RES

+ VIP

+ VIP

+ VIP

+ VIP

+ VIP

+ VIP

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(apart from isoprene) contribute to protection of photosynthesisunder thermal stress. Finally, if the isoprene effect were purely aphysical stabilization of membranes, it should be relatively species-independent. However, a species-dependent effect is observed: inspecies that do not normally emit isoprene, fumigation with isoprenecan confer protection in some cases but not in others30,40,56,58. Thisvariability may arise from differences in membrane properties amongspecies, but such differences have not been identified.

The direct antioxidant hypothesis. An alternative mechanistichypothesis is that isoprene behaves directly as an antioxidant, scaven-ging ROS by reactions through the conjugated double bond sys-tem45,46,48,49. In the atmosphere, highly reduced isoprenoids react withreactive oxygen and nitrogen species, including ozone, singlet oxygen,hydroxyl radicals and nitrous oxides (NOx)59–61. However, liquid-phase chemistry may be different from gas-phase chemistry. In leaves,these reactions potentially occur either in the aqueous environmentwithin the cell (probably at membrane surfaces) or in the humidenvironment in intercellular spaces and at the boundary layer of theleaf lamina. Lipid-phase reactions may also occur.

Under stress conditions, a variety of ROS are produced in plantcells. These ROS cause oxidative damage. The plant responds to thepresence of excess ROS through an antioxidant defense system thatconsists of antioxidant compounds that are either lipophilic (forexample, tocopherols and carotenoids) or hydrophilic (for example,ascorbate and glutathione) and enzymes that either mediate regenera-tion of antioxidants (for example, monodehydroascorbate reductase,dehydroascorbate reductase and glutathione reductase) or dismutatetoxic ROS to water or less reactive compounds (for example, super-oxide dismutase, catalase and peroxidase)2 (example shown in Fig. 2for chloroplast ROS response). Lipid- and aqueous-phase oxidativestates are closely linked through action of antioxidant enzymes(Fig. 2). Hydrogen peroxide is produced as a byproduct of detoxifica-tion of the more dangerous ROS and is involved in initiating stresssignaling networks. ROS are also constitutively present in plant cellsunder nonstress conditions and are involved in a number of cellularresponses. For example, production of ROS at the photosyntheticmembranes is integral to photosynthetic processes, and ROS signalingis involved in regulation of those processes2,62,63. The regulationof ROS is normally tightly controlled, but under stress conditionsthe cellular balance of ROS is often perturbed2,3. The cellular networkof antioxidants and antioxidant enzymes is then required to scavengethe excess ROS and prevent cytotoxic effects. It is well knownthat nonvolatile isoprenoids such as tocopherols, zeaxanthin andcarnosic acid can scavenge ROS directly by reactions through hydroxylradicals64. Volatile isoprenoids are typically olefins with reactiveconjugated and/or terminal double bonds. We propose that volatileisoprenoids also form part of the non-enzymatic oxidative defensesystem. This hypothesis rests on the following evidence.

Isoprene affects the oxidative status of plants under stress. Inhibi-tion of isoprene emission by fosmidomycin results in greater accu-mulation of H2O2, increased lipid peroxidation levels and increases inantioxidant enzyme activities when plants are placed under thermalstress35–37,46. Fumigation of fosmidomycin-inhibited leaves with exo-genous isoprene partially restores H2O2 and lipid peroxidation levelsto those found in non-inhibited leaves. These results were confirmedin a transgenic tobacco system using plants azygous and heterozygousfor isoprene synthase43. Further, pools of reduced antioxidant (ascor-bate) were higher in transgenic isoprene-emitting plants relative tonon-emitting plants, which suggests that the emitting plants had a

reduced requirement for antioxidant capacity compared to non-emitting plants. These findings all indicate that isoprene plays somerole in reducing the oxidizing load under stress conditions.

Direct reactions can occur between volatile isoprenoids and oxidiz-ing species. It has been suggested that volatile isoprenoids, especiallyisoprene, can react directly with ozone, either in planta or at the leafsurface, thus decreasing ozone levels and potentially mitigating oxi-dative damage caused to the leaf45,46. In highly oxidizing, humidatmospheric environments, isoprene does react with ozone65; similarconditions might occur at the leaf boundary layer and in the inter-cellular spaces of the mesophyll tissue when ozone is present. How-ever, only monoterpenes have been experimentally demonstrated toscavenge a significant amount of ozone in the boundary layer66. Thereaction between ozone and isoprene is relatively slow, and directremoval of ozone in this way is insufficient to result in the observedprotection of fumigated tissues66. Furthermore, ozonolysis of isoprenein humid environments results in production of hydrogen peroxide65;this is inconsistent with the lowered hydrogen peroxide levels observedin tissue extracts from ozone-fumigated leaves, which emit isoprene46.Methacrolein (MACR) and methylvinylketone (MVK) are the firstproducts of isoprene ozonolysis65; reactive electrophile species (RES)such as these are known to activate expression of defense genes67.Fares et al. observed a surprisingly low production of MACR andMVK from isoprene-emitting leaves that were fumigated withozone66; this implies that secondary reactions either with ozone orwith other metabolites inside the mesophyll remove these RES.

When ozone enters the leaf, it is degraded to other ROS: superoxide,singlet oxygen, hydroxyl radicals and hydrogen peroxide. Scavengingof some or all of these ROS by volatile isoprenoids might help explainthe protective effect observed. In aqueous solution, isoprene reactswith hydroxyl radicals to produce 2-methyltetrols68, and it has beensuggested that isoprene might act as a hydroxyl radical scavenger toprotect from oxidative damage69. The presence of conjugated doublebounds (delocalized p-electrons) in the isoprene molecule may serveto mediate electron and energy transfers, conferring a ROS-scavengingability to the molecule46,48,49. There is strong evidence that isoprenescavenges singlet oxygen. Singlet oxygen is produced (in addition toother ROS) at the thylakoid membranes when absorbed energy is inexcess of that used in photosynthesis70 (see Fig. 2). This may occurbecause excess light is present (at high light intensities) and/or becauseuse of excitation energy is retarded (for example, under various abioticstress conditions). Recent research suggests that singlet oxygen is themajor cause of photo-oxidative damage under high light stress71.When production of singlet oxygen at the photosynthetic membranesis exacerbated under light stress using chemical treatments, youngnon-emitting leaves of isoprene-emitting species that are fumigatedwith isoprene show higher net assimilation rates than non-fumigatedleaves48. Similar results are observed in mature leaves when isopreneemission is inhibited by application of fosmidomycin49. In isoprene-inhibited leaves, H2O2 and malonyldialdehyde (MDA) levels wereincreased compared to uninhibited leaves. MDA is an indicator oflipid peroxidation and is produced at the thylakoid membranes underoxidative stress (see Fig. 2).

Changes in signaling responses may also contribute to protectiveeffects. Recent studies show that isoprene may also have indirecteffects on oxidative state. Ozone damage is typified by accumulationof hydrogen peroxide followed by biochemical and transcriptionalresponses similar to those observed during the hypersensitive responsein an incompatible plant-pathogen interaction72. Nitric oxide (NO) is

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involved in the signal cascade that initiates this cellular response; inaddition, NO is involved in a number of physiological processes, manyof which are stress-related73,74. NO may also directly scavenge ROS,attenuating the effects of photo-oxidative stress75. Isoprene-emittingleaves produce less NO compared to fosmidomycin-treated leaveswhen fumigated with ozone, which leads to the suggestion thatisoprene can also quench NO58. Isoprene can react with oxygenatednitrogen species in the troposphere, but one reaction product is ozone.If this happened also in planta, it would obviously result in furtheroxidative damage. It therefore seems unlikely that this reaction isoccurring in plant cells. The observed decrease in relative NO levelscould also be explained if there was a decreased requirement for NO inthe presence of isoprene. NO and isoprene may indeed cooperate inprotecting leaves against oxidative stresses when they are present atphysiological levels76. Although the mechanism for this effect isunknown, a reduction in the amount of NO might attenuate theinduction of stress-induced hypersensitive response, thus avoiding thesignal cascade that results in accumulation of H2O2 and subsequentfoliar necrosis. This is supported by the decreased levels of H2O2 andlipid peroxidation markers observed in isoprene-emitting leavescompared to non-emitting leaves after ozone fumigation35. If isopreneinteracts with NO, then a number of other processes that are initiatedby the hypersensitive responses are also likely to be affected, princi-pally the MAPK cascade and the elicitation of jasmonate and salicylatestress signaling pathways. This has yet to be elucidated, and maybecome a major field of study in stress physiology.

Evolution of the stress tolerance functionEnzymatic production and emission of volatile isoprenoids occurs inmany plants, from bryophytes to highly derived angiosperms. TheDOX-MEP pathway appears to be primitive to green plants, and theterpene synthase enzyme that converts the substrate dimethylallyldiphosphate (DMADP) to isoprene has evolved multiple times inmosses, ferns, gymnosperms and angiosperms77,78. Isoprene biosynth-esis appears not to have evolved (or to have been lost) in severalgroups defined taxonomically or physiologically, most notably in twoancient divisions—hornworts (anthocerotophytes) and liverworts(marchantiophytes)—and in two physiologically defined groupswhose members are much more recently evolved—C4 and crassula-tion acid metabolism (CAM) plants. In the oaks (Quercus spp.), whereisoprene emission appears to be a primitive trait, the subgenus thathas lost the trait, the European live oaks, has replaced enzymaticallycontrolled light-dependent isoprene emission with enzymatically con-trolled light-dependent monoterpene emission79. This offers thestrongest phylogenetic evidence of an adaptive significance for volatilediene production and emission by plants, as monoterpenes appear tobe more effective in scavenging antioxidants in the gas phase thanisoprene66 and, because of their lower volatility, form larger pools inmembranes and intercellular spaces80,81. In other groups of non-isoprene-emitting taxa, monoterpenes or sesquiterpenes may playthe same role as isoprene in protection against abiotic stress. However,much more information about the phylogenetic occurrence of foliarmonoterpene and sesquiterpene emission in the plant kingdom isneeded to support or reject this conjecture.

Analysis of online emission databases (http://www.es.lancs.ac.uk/cnhgroup/download.html) reveals very few patterns between isopreneemission and particular growth forms, ecologies, habitats or phylo-genies. However, some strong correlations have been noted. Nearly allisoprene emitters are woody; within taxa that contain both woody andherbaceous groups (for example, the grasses), isoprene emission is farmore common in woody groups (for example, bamboos and reeds).

Based on the phylogenetic distribution of isoprene emission amongnonvascular plants, one may speculate that isoprene emission firstdeveloped when plants abandoned the aquatic environment toconquer the land82. Terrestrial plants may have developed isopreneas a quick and primitive method to cope with rapid, short-termchanges of temperatures that occur on land but that do not occur inthe more thermally buffered aquatic environment. In support of this,desert ecosystems tend to be lacking in isoprene-emitting taxa; thiscan be considered as evidence in favor of a role for isoprene in planttolerance against short-term heat stress (a rare event in desertsystems). Exposure to the oxygen-rich terrestrial atmosphere mayalso have selected for isoprene biosynthesis as an antioxidant mechan-ism, though this selective pressure does not account for the phyloge-netic distribution of the trait or for the sensitivity of isoprene synthesisto light and temperature83.

There is no clear evidence that isoprenoid-emitting species copebetter in the long term than non-emitting species in the presence ofenvironmental stresses. However, it should be noted that a widevariety of complex and efficient mechanisms for protection againstabiotic stresses can be found in plants. In species where isoprenoidemission has not evolved or has been lost, these mechanisms may bean alternative response. Nonetheless, the potential exists for changes inpopulation structure to occur under global warming conditions, giventhat (i) emissions specifically increase with increasing temperature,and (ii) oxidative stress (in particular the occurrence of increasedozone pollution) is increasing in parallel83. On the other hand, risingCO2, which drives the temperature increase globally, is expected toenhance photosynthesis, thus reducing the oxidative stress in plants.This might negatively feed back on isoprenoid emission. An inde-pendent, negative feedback of rising CO2 on isoprene emission hasbeen observed84 and may be explained by the insufficient supply ofphosphoenolpyruvate (PEP) to isoprene, as PEP is increasinglydiverted to oxaloacetate production for anabolic support of mito-chondrial respiration under rising CO2 (ref. 85).

Summary and conclusionsIt is now clear that volatile isoprenoids play an important role inprotection against a variety of abiotic stresses, including high light,temperature, drought and oxidizing conditions of the atmosphere.These stresses all result in oxidative stress, and the presence ofisoprenoids improves the ability of plants to deal with internaloxidative changes regardless of the nature of the external (physio-logical) stressor. Our ‘single biochemical mechanism for multiplephysiological stressors’ model provides a unified mechanistic explana-tion for the protection provided by volatile isoprenoids under diversestress events. Carbon is redirected to volatile production under stressconditions, and the presence of these compounds results in protection,thus justifying the metabolic expense of production. The importanceof this defense mechanism is further evidenced by the breadth of taxathat emit volatiles and the apparent repeated evolution of this trait.

The mechanism of volatile isoprenoid action is difficult to testdirectly in planta because current techniques do not allow discrimina-tion between cause and effect. Activity may be mediated through(i) direct reactions of isoprenoids with oxidizing species, (ii) indirectalteration of ROS signaling, and/or (iii) membrane stabilization.Stabilization of lipid membranes also presumably decreases lipidperoxidation, thus directly impacting the oxidative state of the cell;this mechanism might explain a generic oxidative protection that isnot necessarily due to direct reactions. The antioxidant behavior ofisoprene and other volatiles might be further investigated by searchingfor specific reaction products from isoprenoid oxidation.

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1. Intergovernmental Panel on Climate Change. Climate Change 2001: The ScientificBasis (Cambridge University Press, Cambridge, UK, 2001).

2. Apel, K. & Hirt, H. Reactive oxygen species: metabolism, oxidative stress, and signaltransduction. Annu. Rev. Plant Biol. 55, 373–399 (2004).

3. Dietz, K.J. Redox control, redox signaling, and redox homeostasis in plant cells.Int. Rev. Cytol. 228, 141–193 (2003).

4. Kwak, J.M., Nguyen, V. & Schroeder, J.I. The role of reactive oxygen species inhormonal responses. Plant Physiol. 141, 323–329 (2006).

5. Rohmer, M. The discovery of a mevalonate-independent pathway for isoprenoidbiosynthesis in bacteria, algae and higher plants. Nat. Prod. Rep. 16, 565–574(1999).

6. Lange, B.M., Rujan, T., Martin, W. & Croteau, R. Isoprenoid biosynthesis: the evolutionof two ancient and distinct pathways across genomes. Proc. Natl. Acad. Sci. USA 97,13172–13177 (2000).

7. Lichtenthaler, H.K. Non-mevalonate isoprenoid biosynthesis: enzymes, genes andinhibitors. Biochem. Soc. Trans. 28, 785–789 (2000).

8. Dudareva, N. et al. The nonmevalonate pathway supports both monoterpene andsesquiterpene formation in snapdragon flowers. Proc. Natl. Acad. Sci. USA 102,933–938 (2005).

9. Laule, O. et al. Crosstalk between cytosolic and plastidial pathways of isoprenoidbiosynthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 100, 6866–6871(2003).

10. Bick, J.A. & Lange, B.M. Metabolic cross talk between cytosolic and plastidialpathways of isoprenoid biosynthesis: unidirectional transport of intermediates acrossthe chloroplast envelope membrane. Arch. Biochem. Biophys. 415, 146–154 (2003).

11. Wu, S.Q. et al. Redirection of cytosolic or plastidic isoprenoid precursors elevatesterpene production in plants. Nat. Biotechnol. 24, 1441–1447 (2006).

12. Dudareva, N., Negre, F., Nagegowda, D.A. & Orlova, I. Plant volatiles: recent advancesand future perspectives. Crit. Rev. Plant Sci. 25, 417–440 (2006).

13. Sharkey, T.D. & Yeh, S. Isoprene emission from plants. Annu. Rev. Plant Physiol. PlantMol. Biol. 52, 407–436 (2001).

14. Brilli, F. et al. Response of isoprene emission and carbon metabolism to drought inwhite poplar (Populus alba) saplings. New Phytol. 175, 244–254 (2007).

15. Loreto, F. & Sharkey, T.D. A gas-exchange study of photosynthesis and isopreneemission in Quercus rubra L. Planta 182, 523–531 (1990).

16. Monson, R.K. & Fall, R. Isoprene emission from aspen leaves: influence of environmentand relation to photosynthesis and photorespiration. Plant Physiol. 90, 267–274(1989).

17. Gershenzon, J. Plant volatiles carry both public and private messages. Proc. Natl.Acad. Sci. USA 104, 5257–5258 (2007).

18. Baldwin, I.T., Halitschke, R., Paschold, A., von Dahl, C.C. & Preston, C.A. Volatilesignaling in plant-plant interactions: ‘‘talking trees’’ in the genomics era. Science 311,812–815 (2006).

19. Loreto, F., Forster, A., Durr, M., Csiky, O. & Seufert, G. On the monoterpene emissionunder heat stress and on the increased thermotolerance of leaves of Quercus ilex L.fumigated with selected monoterpenes. Plant Cell Environ. 21, 101–107 (1998).

20. Loreto, F., Barta, C., Brilli, F. & Nogues, I. On the induction of volatile organiccompound emissions by plants as consequence of wounding or fluctuations of lightand temperature. Plant Cell Environ. 29, 1820–1828 (2006).

21. Sharkey, T.D. & Loreto, F. Water stress, temperature, and light effects on the capacityfor isoprene emission and photosynthesis of kudzu leaves. Oecologia 95, 328–333(1993).

22. Tingey, D.T., Evans, R. & Gumpertz, M. Effects of environmental conditions on isopreneemission from live oak. Planta 152, 565–570 (1981).

23. Filella, I., Penuelas, J. & Llusia, J. Dynamics of the enhanced emissions of mono-terpenes and methyl salicylate, and decreased uptake of formaldehyde, by Quercus ilexleaves after application of jasmonic acid. New Phytol. 169, 135–144 (2006).

24. Martin, D.M., Gershenzon, J. & Bohlmann, J. Induction of volatile terpene biosynthesisand diurnal emission by methyl jasmonate in foliage of norway spruce. Plant Physiol.132, 1586–1599 (2003).

25. Schnitzler, J.P. et al. Contribution of different carbon sources to isoprene biosynthesisin poplar leaves. Plant Physiol. 135, 152–160 (2004).

26. Funk, J.L., Mak, J.E. & Lerdau, M.T. Stress-induced changes in carbon sources forisoprene production in Populus deltoides. Plant Cell Environ. 27, 747–755 (2004).

27. Rosenstiel, T.N., Ebbets, A.L., Khatri, W.C., Fall, R. & Monson, R.K. Induction ofpoplar leaf nitrate reductase: a test of extrachloroplastic control of isoprene emissionrate. Plant Biol. (Stuttg) 6, 12–21 (2004).

28. Sanadze, G.A. Biogenic isoprene (a review). Russ. J. Plant Physiol. 51, 729–741(2004).

29. Magel, E. et al. Photosynthesis and substrate supply for isoprene biosynthesis in poplarleaves. Atmos. Environ. 40 (suppl. 1): 138–151 (2006).

30. Sharkey, T.D., Wiberley, A.E. & Donohue, A.R. Isoprene emission from plants: why andhow. Ann. Bot. (Lond.) 101, 5–18 (2008).

31. Laothawornkitkul, J. et al. Isoprene emissions influence herbivore feeding decisions.Plant Cell Environ. 31, 1410–1415 (2008).

32. Loivamaki, M., Mumm, R., Dicke, M. & Schnitzler, J.P. Isoprene interferes with theattraction of bodyguards by herbaceous plants. Proc. Natl. Acad. Sci. USA 105,17430–17435 (2008).

33. Kuzuyama, T., Shimizu, T., Takahashi, S. & Seto, H. Fosmidomycin, a specific inhibitorof 1-deoxy-D-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathwayfor terpenoid biosynthesis. Tetrahedr. Lett. 39, 7913–7916 (1998).

34. Sharkey, T.D. & Singsaas, E.L. Why plants emit isoprene. Nature 374, 769(1995).

35. Velikova, V., Loreto, F., Tsonev, T., Brilli, F. & Edreva, A. Isoprene prevents the negativeconsequences of high temperature stress in Platanus orientalis leaves. Funct. PlantBiol. 33, 931–940 (2006).

36. Velikova, V., Pinelli, P. & Loreto, F. Consequences of inhibition of isoprene synthesis inPhragmites australis leaves exposed to elevated temperatures. Agric. Ecosyst. Environ.106, 209–217 (2005).

37. Velikova, V. & Loreto, F. On the relationship between isoprene emission and thermo-tolerance in Phragmites australis leaves exposed to high temperatures and during therecovery from a heat stress. Plant Cell Environ. 28, 318–327 (2005).

38. Barta, C. & Loreto, F. The relationship between the methyl-erythritol phosphatepathway leading to emission of volatile isoprenoids and abscisic acid content inleaves. Plant Physiol. 141, 1676–1683 (2006).

39. Loivamaki, M. et al. Arabidopsis, a model to study biological functions of isopreneemission? Plant Physiol. 144, 1066–1078 (2007).

40. Sasaki, K. et al. Plants utilize isoprene emission as a thermotolerance mechanism.Plant Cell Physiol. 48, 1254–1262 (2007).

41. Behnke, K. et al. Transgenic, non-isoprene emitting poplars don’t like it hot. Plant J.51, 485–499 (2007).

42. Molinier, J., Ries, G., Zipfel, C. & Hohn, B. Transgeneration memory of stress in plants.Nature 442, 1046–1049 (2006).

43. Vickers, C.E. et al. Isoprene synthesis protects transgenic plants from oxidative stress.Plant Cell Environ. published online, doi:10.1111/j.1365–3040.2009.01946.x(22 January 2009).

44. Delfine, S., Csiky, O., Seufert, G. & Loreto, F. Fumigation with exogenous mono-terpenes of a non-isoprenoid-emitting oak (Quercus suber): monoterpene acquisition,translocation, and effect on the photosynthetic properties at high temperatures. NewPhytol. 146, 27–36 (2000).

45. Loreto, F. et al. Ozone quenching properties of isoprene and its antioxidant role inleaves. Plant Physiol. 126, 993–1000 (2001).

46. Loreto, F. & Velikova, V. Isoprene produced by leaves protects the photosyntheticapparatus against ozone damage, quenches ozone products, and reduces lipidperoxidation of cellular membranes. Plant Physiol. 127, 1781–1787 (2001).

47. Ledford, H.K. & Niyogi, K.K. Singlet oxygen and photo-oxidative stress management inplants and algae. Plant Cell Environ. 28, 1037–1045 (2005).

48. Affek, H.P. & Yakir, D. Protection by isoprene against singlet oxygen in leaves. PlantPhysiol. 129, 269–277 (2002).

49. Velikova, V., Edreva, A. & Loreto, F. Endogenous isoprene protects Phragmitesaustralis leaves against singlet oxygen. Physiol. Plant. 122, 219–225(2004).

50. Loreto, F. & Fares, S. Is ozone flux Inside leaves only a damage indicator? Clues fromvolatile isoprenoid studies. Plant Physiol. 143, 1096–1100 (2007).

51. Calogirou, A., Larsen, B.R. & Kotzias, D. Gas-phase terpene oxidation products: areview. Atmos. Environ. 33, 1423–1439 (1999).

52. Duhl, T.R., Helmig, D. & Guenther, A. Sesquiterpene emissions from vegetation: areview. Biogeosciences Discuss. 4, 3987–4023 (2007).

53. Helmig, D., Bocquet, F., Pollmann, J. & Revermann, T. Analytical techniques forsesquiterpene emission rate studies in vegetation enclosure experiments. Atmos.Environ. 38, 557–572 (2004).

54. Siwko, M.E. et al. Does isoprene protect plant membranes from thermal shock? Amolecular dynamics study. Biochim. Biophys. Acta 1768, 198–206 (2007).

55. Milne, P.J., Riemer, D.D., Zika, R.G. & Brand, L.E. Measurement of vertical distributionof isoprene in surface seawater, its chemical fate, and its emission from severalphytoplankton monocultures. Mar. Chem. 48, 237–244 (1995).

56. Logan, B.A. & Monson, R.K. Thermotolerance of leaf discs from four isoprene-emittingspecies is not enhanced by exposure to exogenous isoprene. Plant Physiol. 120,821–826 (1999).

57. Logan, B.A., Anchordoquy, T.J., Monson, R.K. & Pan, R.S. The effect of isoprene on theproperties of spinach thylakoids and phosphatidylcholine liposomes. Plant Biol.(Stuttg) 1, 602–606 (1999).

58. Velikova, V. et al. Isoprene decreases the concentration of nitric oxide in leaves exposedto elevated ozone. New Phytol. 166, 419–425 (2005).

59. Thompson, A.M. The oxidizing capacity of the earth’s atmosphere - probable past andfuture changes. Science 256, 1157–1165 (1992).

60. Monson, R.K. & Holland, E.A. Biospheric trace gas fluxes and their control overtropospheric chemistry. Annu. Rev. Ecol. Syst. 32, 547–576 (2001).

61. Pierce, T. et al. Influence of increased isoprene emissions on regional ozone modeling.J. Geophys. Res. Atmos. 103, 25611–25629 (1998).

62. Mullineaux, P.M., Karpinski, S. & Baker, N.R. Spatial dependence for hydrogenperoxide-directed signaling in light-stressed plants. Plant Physiol. 141, 346–350(2006).

63. Mullineaux, P. et al. Are diverse signalling pathways integrated in the regulation ofArabidopsis antioxidant defence gene expression in response to excess excitationenergy? Phil. Trans. R. Soc. Lond. B 355, 1531–1540 (2000).

64. Munne-Bosch, S. & Alegre, L. The function of tocopherols and tocotrienols in plants.Crit. Rev. Plant Sci. 21, 31–57 (2002).

65. Sauer, F., Schafer, C., Neeb, P., Horie, O. & Moorgat, G.K. Formation of hydrogenperoxide in the ozonolysis of isoprene and and simple alkenes under humid conditions.Atmos. Environ. 33, 229–241 (1999).

290 VOLUME 5 NUMBER 5 MAY 2009 NATURE CHEMICAL BIOLOGY

P ERSPECT IVE©

2009

Nat

ure

Am

eric

a, In

c. A

ll ri

gh

ts r

eser

ved

.

66. Fares, S., Loreto, F., Kleist, E. & Wildt, J. Stomatal uptake and stomatal deposition ofozone in isoprene and monoterpene emitting plants. Plant Biol. (Stuttg) 10, 44–54(2008).

67. Almeras, E. et al. Reactive electrophile species activate defense gene expression inArabidopsis. Plant J. 34, 205–216 (2003).

68. Santos, L.S., Dalmazio, I., Eberlin, M.N., Claeys, M. & Augusti, R. Mimicking theatmospheric OH-radical-mediated photooxidation of isoprene: formation of cloud-condensation nuclei polyols monitored by electrospray ionization mass spectrometry.Rapid Commun. Mass Spectrom. 20, 2104–2108 (2006).

69. Zeidler, J.G., Lichtenthaler, H.K., May, H.U. & Lichtenthaler, F.W. Is isopreneemitted by plants synthesized via the novel isopentenyl pyrophosphate pathway?Z. Naturforsch. 52c, 15–23 (1997).

70. Foyer, C., Trebst, A. & Noctor, G. Signaling and integration of defense functions oftocopherol, ascorbate and glutathione. in Photoprotection, Photoinhibition, GeneRegulation, and Environment (eds. Demmig-Adams, B., Adams, W.W. III & Mattoo,A.K.) 241–268 (Springer, Dordrecht, The Netherlands, 2006).

71. Triantaphylides, C. et al. Singlet oxygen is the major reactive oxygen species involved inphotooxidative damage to plants. Plant Physiol. 148, 960–968 (2008).

72. Pasqualini, S. et al. Ozone-induced cell death in tobacco cultivar Bel W3 plants. Therole of programmed cell death in lesion formation. Plant Physiol. 133, 1122–1134(2003).

73. Gould, K.S., Lamotte, O., Klinguer, A., Pugin, A. & Wendehenne, D. Nitric oxideproduction in tobacco leaf cells: a generalized stress response? Plant Cell Environ. 26,1851–1862 (2003).

74. Wilson, I.D., Neill, S.J. & Hancock, J.T. Nitric oxide synthesis and signalling in plants.Plant Cell Environ. 31, 622–631 (2008).

75. Beligni, M.V. & Lamattina, L. Nitric oxide interferes with plant photo-oxidativestress by detoxifying reactive oxygen species. Plant Cell Environ. 25, 737–748(2002).

76. Velikova, V., Fares, S. & Loreto, F. Isoprene and nitric oxide reduce damages in leavesexposed to oxidative stress. Plant Cell Environ. 31, 1882–1894 (2008).

77. Harley, P.C., Monson, R.K. & Lerdau, M.T. Ecological and evolutionary aspects ofisoprene emission from plants. Oecologia 118, 109–123 (1999).

78. Sharkey, T.D. et al. Evolution of the isoprene biosynthetic pathway in kudzu. PlantPhysiol. 137, 700–712 (2005).

79. Harley, P., Guenther, A. & Zimmerman, P. Environmental controls over isopreneemission in deciduous oak canopies. Tree Physiol. 17, 705–714 (1997).

80. Loreto, F. et al. Different sources of reduced carbon contribute to form three classesof terpenoid emitted by Quercus ilex L. leaves. Proc. Natl. Acad. Sci. USA 93,9966–9969 (1996).

81. Niinemets, U., Loreto, F. & Reichstein, M. Physiological and physicochemical controlson foliar volatile organic compound emissions. Trends Plant Sci. 9, 180–186 (2004).

82. Loreto, F. Distribution of isoprenoid emitters in the Quercus genus around the world:chemo-taxonomical implications and evolutionary considerations based on the ecolo-gical function of the trait. Perspect. Plant Ecol. Evol. Syst. 5, 185–192 (2002).

83. Lerdau, M. A positive feedback with negative consequences. Science 316, 212–213(2007).

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

uncouples growth from isoprene emission in an agriforest ecosystem. Nature 421,256–259 (2003).

85. Loreto, F. et al. The relationship between isoprene emission rate and dark respirationrate in white poplar (Populus alba L.) leaves. Plant Cell Environ. 30, 662–669 (2007).

86. Scheel, D. & Wasternac, C. Plant Signal Transduction (Oxford University Press, Oxford,2002).

87. Schilmiller, A.L. & Howe, G.A. Systemic signaling in the wound response. Curr. Opin.Plant Biol. 8, 369–377 (2005).

88. Jenks, M.A. & Hasegawa, P.M. Plant Abiotic Stress (Blackwell, Oxford, 2005).89. Hirt, H. & Shinozaki, K. Plant Responses to Abiotic Stress (Springer, Berlin, 2004).90. Zhu, J.K. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol.

53, 247–273 (2002).91. Krieger-Liszkay, A. Singlet oxygen production in photosynthesis. J. Exp. Bot. 56,

337–346 (2005).

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