A unified mechanism of action for volatile isoprenoids in plant abiotic stress

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1 A unified mechanism of action for volatile isoprenoids in plant abiotic stress Claudia E Vickers, Jonathan Gershenzon, Manuel T Lerdau & Francesco Loreto The sessile nature of plants has resulted in the evolution of an extraordinarily diverse suite of protective mechanisms against biotic and abiotic stresses. Though volatile isoprenoids are known to be involved in many types of biotic interactions, they also play important but relatively unappreciated roles in abiotic stress responses. We review those roles, discuss the proposed mechanistic explanations and examine the evolutionary significance of volatile isoprenoid emission. We note that abiotic stress responses generically involve production of reactive oxygen species in plant cells, and volatile isoprenoids mitigate the effects of oxidative stress by mediating the oxidative status of the plant. On the basis of these observations, we propose a single biochemical mechanism for multiple physiological stressors model, whereby the protective effect against abiotic stress is exerted through direct or indirect improvement in resistance to damage by reactive oxygen species. Owing to their sedentary lifestyle, plants must be capable of coping with a variety of changes in light intensity, temperature, moisture and other abiotic factors in their environments (Fig. 1). When these factors shift out of a certain range, plants are subjected to stress; this can lead to decreased growth rate, reduced reproduction and even death. Beyond the single-plant level, changes in environmental stress can also select for short- and long-term shifts in ecological traits, potentially affecting biological diversity, ecosystem functioning and carbon sequestration. Abiotic stress also results in significant losses in crop yields, the magnitude of which fluctuates from year to year. Sources of abiotic stress include extremes of temperature (including freezing), high light intensity, drought, air pollutants, salinity and mechanical damage. These stresses are rarely experienced singularly: they often occur in combination. The simultaneous occurrences of rapidly rising temperatures, drought and pollutants are among the most striking phenomena associated with global change, and they threaten plants that have not adapted and are not able to rapidly acclimate to these factors 1. Claudia E. Vickers is at the University of Queensland, Australian Institute for Bioengineering and Nanotechnology, St. Lucia, Australia. Jonathan Gershenzon is at the Max Planck Institute for Chemical Ecology, Jena, Germany. Manuel T. Lerdau is in the Environmental Sciences and Biology Departments, University of Virginia, Charlottesville, Virginia, USA. Francesco Loreto is at Consiglio Nazionale delle Ricerche, Istituto di Biologia Agroambientale e Forestale, Monterotondo Scalo (Roma), Italy. c.vickers@uq.edu.au. Published online 17 April 2009; doi: /nchembio.158 Few responses of plants are stress-specific. Most often, stresses elicit generic responses in particular, production of excess reactive oxygen species (ROS) such as singlet oxygen ( 1 O 2 ), superoxide (O 2 ), hydrogen peroxide ( ) and hydroxyl radicals ( OH). ROS are important signaling molecules and also serve to initiate defense responses 2. The cellular balance of ROS is normally kept under tight control 3 ; however, when this control is lost, damage occurs. ROS cause direct damage to plant cells through oxidation of biological components (nucleic acids, proteins and lipids) and can instigate chain reactions resulting in accumulation of more ROS and initiation of programmed cell death 2. Plants have a complex response network of lipid-phase and aqueous-phase antioxidant compounds and enzymes that defend against conditions of excess ROS. Direct reactions to quench and remove ROS occur (for example, Fig. 2), as well as indirect responses including hormone-mediated signaling to upregulate primary defense genes and activate secondary defense genes (reviewed previously 2 4 ). When the antioxidant defense network is overloaded, oxidative stress results. Measuring abiotic stress responses and attributing mechanistic behavior can be problematic because of the complexity of the ROS response network, which makes it difficult to distinguish cause-and-effect relationships. The network is linked between lipid and aqueous phases, so understanding physical compartmentalization is fraught. Many ROS are short-lived, so direct measurements are also difficult; further, many molecules involved in signaling cascades have not been identified and thus cannot be measured. Although most research on plant antioxidants has focused on nonvolatile compounds, certain volatiles belonging to the isoprenoid family have also been implicated in protection against oxidative and other abiotic stresses. The isoprenoids are a very large and extremely diverse group of organic compounds. Isoprenoid carbon skeletons are composed of five-carbon building blocks that may be assembled in a variety of formations and contain many different modifications. In plants, two separate metabolic pathways are responsible for the production of the C 5 building block of isoprenoids: the cytosolic mevalonic acid (MVA) pathway, and the plastidic 2-C-methyl-Derythritol 4-phosphate (MEP) pathway 5 7 (Fig. 3). The two pathways can be linked through exchange of metabolic precursors across the plastid membrane It is generally assumed that later steps in the formation of hemiterpenes (C 5 ), monoterpenes (C 10 ), diterpenes (C 20 ) and tetraterpenes (C 40 ) are present in the plastids, whereas the formation of sesquiterpenes (C 15 ) and triterpenes (C 30 ) takes place in the cytosol. Volatile isoprenoids are generally lipophilic, lowmolecular-weight compounds with masses under 300 (see Fig. 4). Only hemiterpenes (isoprene and methylbutenol), monoterpenes, NATURE CHEMICAL BIOLOGY VOLUME 5 NUMBER 5 MAY

2 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. Na + Cl K + Mg 2+ Ca 2+ sesquiterpenes and some diterpenes have sufficient vapor pressure to volatilize at ordinary biological temperatures 12. Volatility bestows particular properties on these compounds for example, the ability to carry chemical messages away from the sites of synthesis in the plant. Production of volatile isoprenoids represents a substantial investment for the plant in terms of carbon (loss of which in the form of volatiles is irretrievable) and energy. Constitutive emissions in isoprene emitters and strong monoterpene emitters are generally in the range of nmol m 2 s 1 ( load.html). This amount is equivalent to 1 2% of photosynthetic carbon fixation 13. Even when the carbon budget becomes negative under stress conditions and photosynthesis is severely or completely inhibited, isoprenoid emission is often sustained Thislargecost, especially under stressful conditions, suggests the strong possibility that isoprenoid emission also confers benefits to the plant. Though the role of some volatiles in biotic interactions is well established and has been well reviewed 12,17,18, there are many volatiles for which the benefit remains obscure. Higher order (nonvolatile) isoprenoids have a variety of roles in plant cells, including in abiotic stress defense. The mechanisms by which they act are diverse; some are hormonal signals that can carry messages throughout the plant and elicit systemic responses (for example, abscisic acid), whereas others act directly as antioxidants (for example, carotenoids and tocopherols; see Fig. 4). Recent research, which we will highlight here, has revealed that certain volatile isoprenoids also play an important role in abiotic stress responses. Though the bulk of this research has focused historically on isoprene (2-methyl-1,3-butadiene), there is increasing evidence that many (and perhaps all) volatile isoprenoids are involved in abiotic stress responses. Changes in volatile emission patterns under stress conditions originally supplied circumstantial evidence that volatiles are linked with stress responses. Emissions often increase under abiotic stress SO 2 O 3 conditions, particularly under heat and light stress 13,15,19,20.Stressedplantscanmaintain high isoprenoid emissions, and the emission can be transiently enhanced in plants recovering from stresses, particularly in drought-stressed plants 14,21,22.Application of jasmonates, which trigger defense response pathways in both biotic and abiotic stresses, stimulates production of volatile isoprenoids 23,24. Stored carbon can even be mobilized to maintain volatile production under stress conditions 14,25,26. Subsequent experiments have demonstrated that volatile isoprenoids play a protective role under thermal, radiative, oxidative, drought and salt stress. Here we will examine the evidence showing that volatile isoprenoids confer protection against abiotic stress, and we will argue that the common mechanism driving abiotic stress protection is an antioxidant effect of these compounds. It should be noted that other functional possibilities, including balancing of subcellular supplies of phosphoenolpyruvate 27 and dissipation of excess carbon 28 and energy 29 from photosynthesis, have been put forward to explain isoprene emission; however, there are stoichiometric and biochemical arguments against these proposed functions 30, and they have not been extended to volatile isoprenoids in general. Isoprene emissions have also been shown to affect biotic interactions 31,32. The roles of volatile isoprenoids may be multifaceted, and different functional hypotheses are not necessarily mutually exclusive. Here we will focus on the behavior and mechanism of action of volatile isoprenoids in abiotic stress conditions. 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 also causes intracellular water deficit. Figure 1 Plants are exposed to a variety of abiotic stresses. Complex response and protective systems are triggered under these stress conditions (reviewed previously ). 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; soil salinity 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. Volatile isoprenoids protect against abiotic stress Experimental evidence has shown that volatile isoprenoids confer a protective effect to photosynthesis under thermal and oxidative stress conditions. This evidence comes from three types of experiment: (i) the use of fosmidomycin, an antibiotic/herbicide that selectively inhibits the activity of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), thereby inhibiting the MEP pathway 33 (see Fig. 3), (ii) fumigation of non-emitting species with exogenous gaseous isoprenoids, also followed by reconstitution experiments in leaves in which endogenous emission was previously inhibited chemically, and (iii) the use of transgenic plants in which isoprenoid synthesis has been either engineered by insertion of the appropriate terpene synthase genes, or repressed by silencing the same genes. Thermal (high temperature) stress. Theroleofvolatilesinprotection against thermal stress has been relatively well studied. Sharkey and co-workers have been investigating the isoprene effect for over a decade 13,30,34. Under sun-flecking conditions, leaf temperature can fluctuate dramatically, with variations of up to 20 1C occurring in very short time periods; in this scenario, co-incident light andheatstressareexperiencedinapunctuatedfashion.recovery from temperature-induced decreases in photosynthetic efficiency is poorer in isoprene-emitting plants that are treated with fosmidomycin, and fumigation with isoprene can partially restore 284 VOLUME 5 NUMBER 5 MAY 2009 NATURE CHEMICAL BIOLOGY

3 recovery in fosmidomycin-poisoned leaves. These results were confirmed in an independent laboratory However, given that fosmidomycin inhibition of the MEP pathway acts by inhibition of one of the early pathway enzymes, compounds besides isoprene might also be affected (for example, fosmidomycin can affect abscisic acid biosynthesis 38 ). The inhibition studies were followed up using transgenic Arabidopsis thaliana plants bearing heterologously expressed isoprene synthase genes 39,40. Prolonged heat stress experiments confirmed that isoprene emission could confer a thermotolerant growth phenotype relative to non-emitting plants, but protection of photosynthesis could not be tested in this system because control plants did not show inhibition of photosynthesis after heat stress episodes 39. However, these transgenic plants emitted only low levels of isoprene relative to a naturally emitting plant. A second approach was the use of transgenic poplar plants with silenced isoprene synthase expression 41. These plants showed little or no isoprene emission, and demonstrated greater sensitivity of photosynthesis to repeated heat/light stress events. However, interpretation of these experiments is complex because stress sensitivity often occurs in primary-generation transgenic plants due to collateral damage during transformation and tissue culture processes, and can be carried through several transgenic generations 42. Consequently, analysis APX PSII T(OOH) αt(oh) Chloroplast stroma 1 O2 1 O2 O 2.. OH O 2 PSI 1 2 SOD O. 2 2H + GSSG GPX Thylakoid lumen APX Asc MDA NAD(P)H GR GSH MDAR NAD(P) + MDA Asc APX of stress responses in transgenic plants in species with long generation times is fraught with difficulties. To address these issues, transgenic tobacco plants heterologously expressing isoprene synthase have been engineered 43. These lines produced high levels of isoprene, and azygous plants were generated for use as controls. When fourth-generation plants were placed under a repeated heat/light stress regime, photosynthesis recovered better in homozygous emitting plants compared to azygous non-emitting controls. The effect was only distinguishable by longitudinal analysis and was not as strong as that observed in fosmidomycin-poisoned leaves, which suggests that (i) inhibition of other MEP products might play a role in the phenotype observed using fosmidomycin treatments, and/or (ii) species-dependent variation exists. However, these results demonstrated that endogenously emitted isoprene plays a role in thermotolerance of photosynthesis. The subtlety of the effect might explain why it was not observed in the low-emitting transgenic A. thaliana plants. Protection of photosynthesis against high temperature stress is also observed in monoterpene-emitting plants, when emission of monoterpenes inhibited by fosmidomycin is restored with exogenous monoterpene fumigation 19. Monoterpene fumigation can also confer thermotolerance on low-emitting species that are not treated DHA DHAR PL-OH GSH GSSG GR 3 1 O2 PL αc(o. ) αt(oh) PSII PHGPX PL-OOH PL-OO. PRX H 4 2 O 2 TRX-SS TRX-SH NAD(P) + NAD(P)H NTR TRX-SS TRX-SH PRX NTR NAD(P)H NAD(P) + 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 activates 2,70,91. At photosystem II (PSII), O 2,, singlet oxygen ( 1 O 2 ) 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) ( 1 O 2 may also be produced at PSI) (2). O 2 is dismutated by superoxide dismutase (SOD) to hydrogen peroxide ( ). 1 O 2 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 1 O 2 is the major cause of photo-oxidative damage under high light stress 71.Itisquenchedbyb-carotene in the PSII reaction center and a-tocopherol (at(oh)) in the thylakoid membranes; excess 1 O 2 reacts with D1 protein, resulting in degradation of D1 and loss of PSII activity (photoinhibition). Unquenched 1 O 2 also causes lipid peroxidation (3) and can trigger defense gene expression via signal transduction ( 1 O 2 reacts with free radical nitric oxide ( NO) to produce peroxynitrite (ONO 2 ), which acts as a signaling molecule). 1 O 2 and OH can be scavenged by ascorbate (Asc), tocopherols and glutathione (GSH). 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, NADPHthioredoxin reductase; T(OOH), hydroperoxytocopherone; TRX, thioredoxin; TRX-SH, reduced thioredoxin; TRX-SS, thioredoxin disulphide. NATURE CHEMICAL BIOLOGY VOLUME 5 NUMBER 5 MAY

4 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 geranyl diphosphate, 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 wellcharacterized physiological functions in primary metabolism such as growth regulation (for MVA GA3P 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; PP i, pyrophosphate; PQ, plastoquinone. with fosmidomycin 44. Transgenics that over- or underexpress higher order volatile isoprenoids have not yet been studied for resistance to abiotic stress. Oxidative stress. Several studies show that isoprene protects leaf tissues against oxidative stress. Leaves show lower accumulation of ROS, less cellular damage and less damage to photosynthetic processes in response to ozone (O 3 ) fumigation when isoprene is also applied 45. Similarly, fosmidomycin-poisoned leaves show more photosynthetic damage from ozone fumigation than leaves that are producing isoprene 46. Under photo-oxidative stress, singlet oxygen is produced at the photosynthetic membranes, and photosynthetic assimilation PEP PEP Pyruvate HMG-CoA HMGR MVA DMAPP 2x Squalene Sterols Polyprenols Pyruvate Acetyl-CoA DXP DXR MEP IPP MEP GA3P IDI IPP DMAPP(C 5 ) Isoprene (C 5 ) Chloroplast IPP 2x GPP (C 10 ) Monoterpenes (C 10 ) 2 x IPP PQ-9 (C 45 ) (prenyl chain) FPP (C 15 ) GGPP (C 20 ) Diterpenes (C 20 ) +5 IPP Phytyl chains 2x Sesquiterpenes (C 15 ) Chlorophylls Tocopherols Tetraterpenes (C 40 ) Triterpenes (C 30 ) Phylloquinones Carotenoids Phytoalexins Abscisic acid Homoterpenes (C 11, C 30 ) Gibberellins Taxol is inhibited 47. Isoprene fumigation of non-emitting leaves results in protection of photosynthetic processes when singlet oxygen is produced by addition of the photosensitizer Rose Bengal 48 ; fosmidomycin-based inhibition studies support this 49. Experiments using transgenic tobacco plants heterologously expressing isoprene synthase (described above) confirmed that isoprene-emitting plants show much greater resistance to ozone-induced oxidative stress compared to azygous control plants 43. This showed that the protective effect was associated with endogenous isoprene production. As is the case for thermal stress, much less evidence has been collected from monoterpene-emitting plants than from isoprene emitters. However, it has been demonstrated clearly that the 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 (-CH 3 )orhydrogen(-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. Tocopherols R 1 R 2 O HO R 3 Tocotrienols R 1 R 2 O HO R 3 Carotenoids α-carotene β-carotene O HO Violoxanthin O OH Hemiterpenes Isoprene Monoterpenes α-pinene β-pinene Myrcene Limonene Sabinene (E )-β-ocimene (Z )-β-ocimene Sesquiterpenes α-humulene (E )-β-farnesene (E,E)-α-Farnesene (E )-β-caryophyllene δ-cadinene 286 VOLUME 5 NUMBER 5 MAY 2009 NATURE CHEMICAL BIOLOGY

5 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 programmed cell 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). photosynthesis of monoterpene-emitting plants becomes more sensitive to ozone if the emission is inhibited by fosmidomycin, and that, in contrast, photosynthesis becomes less sensitive to ozone in non-emitting plants that are exogenously fumigated with volatile isoprenoids 50. Like isoprene and monoterpenes, many volatile plant sesquiterpenes combine rapidly with ROS 51, and their emission is stimulated by high light and temperature conditions 52 ; thus, these compounds might also be involved in resistance to abiotic stress. Unfortunately, sesquiterpenes have not been well studied owing to difficulties in accurately quantifying emission rates as a consequence of their high reactivity and sensitivity to disturbance 53. Mode of action The mechanisms by which isoprenoids exert their protective effect are as yet undetermined, though two primary mechanistic hypotheses have been put forward in the context of thermal 54 and oxidative 46 stress tolerance. Mechanistic separation in this way assumes a complex multiple mechanisms for multiple stressors model. However, as all environmental stress responses are characterized by the release of dangerous oxidative species, it is parsimonious to argue that the abiotic stress tolerance enhancement conferred by these compounds can be grouped under a common rubric of oxidant protection. Here we propose a single biochemical mechanism for multiple physiological stressors model (Fig. 5) that attempts to unify the diverse empirical studies of volatile isoprenoid compounds and their role in abiotic stress tolerance. Membrane stabilization. Membrane stabilization as a mechanistic explanation for isoprene action was first proposed by Sharkey and coworkers 34. Owing to its lipophilic properties and the site of synthesis (the chloroplast), isoprene is likely to partition into lipid phases of thylakoid membranes. When heat stress occurs, membranes become more fluid, and photosynthetic processes (which are membraneassociated) exhibit a decrease in efficiency. It was therefore proposed that the mechanism of the protective effect is through physical stabilization of hydrophobic interactions (lipid-lipid, lipid-protein Hormones (SA, JA, ET) PCD-associated gene expression ROS 3 RNS 3 Signal transduction HR Environmental stress and/or protein-protein) under increasing temperatures. According to theoretical modeling of molecular dynamics, isoprene does indeed partition into the center of phospholipid membranes 54. This enhances membrane order without a significant change in the dynamic properties of the membrane. Decreased rates of electron transport are also observed in photosynthetic membranes after inhibition by fosmidomycin, which also suggests that the presence of isoprene facilitates photosynthetic processes under heat stress 36. Given that other volatile isoprenoids tend to also be hydrophobic, this mechanism might be generic. However, direct testing of this mechanistic theory in an in vivo system is difficult. Despite this indirect empirical support, there are certain physicochemical problems with membrane stabilization as a mechanism for the isoprene effect. Given that it is a hydrocarbon, isoprene is highly hydrophobic; it is virtually insoluble in pure water, and the Henry s Law constant even in seawater has been estimated at K H B 3.1 (ref. 55). As the site of isoprene production is the chloroplast, it seems reasonable to assume that, even at low emission rates, lipid membranes (particularly chloroplast membranes) must be saturated with isoprene by the time isoprene is measured at the leaf surface. At any given temperature, once membranes are saturated, increasing the production of isoprene must increase the rate that isoprene travels through the membrane rather than the concentration of isoprene in the membrane. If membranes are always isoprene-saturated in emitting species, it follows that fumigation by isoprene should not confer further protection a result that has been observed 56. Interestingly, exogenously supplied isoprene also does not supply protection to isolated membranes under thermal stress 57 ; such membranes presumably have no endogenous isoprene. On the other hand, fumigation with isoprene can partially complement the effects of fosmidomycininduced inhibition of the MEP pathway on photosynthesis under high temperature 30,35. These results are somewhat contradictory and can only be reconciled if we assume that isolated membranes are not physiologically the same as intact plant membranes and do not respond the same way to the presence of isoprene. Partial complementation also suggests that other products from the MEP pathway Ox RES Oxidative damage Accelerated senescence Defenseassociated gene expression Tissue necrosis NATURE CHEMICAL BIOLOGY VOLUME 5 NUMBER 5 MAY

6 (apart from isoprene) contribute to protection of photosynthesis under thermal stress. Finally, if the isoprene effect were purely a physical stabilization of membranes, it should be relatively speciesindependent. However, a species-dependent effect is observed: in species that do not normally emit isoprene, fumigation with isoprene can confer protection in some cases but not in others 30,40,56,58.This variability may arise from differences in membrane properties among species, but such differences have not been identified. The direct antioxidant hypothesis. An alternative mechanistic hypothesis is that isoprene behaves directly as an antioxidant, scavenging ROS by reactions through the conjugated double bond system 45,46,48,49. In the atmosphere, highly reduced isoprenoids react with reactive oxygen and nitrogen species, including ozone, singlet oxygen, hydroxyl radicals and nitrous oxides (NO x ) However, liquidphase chemistry may be different from gas-phase chemistry. In leaves, these reactions potentially occur either in the aqueous environment within the cell (probably at membrane surfaces) or in the humid environment in intercellular spaces and at the boundary layer of the leaf lamina. Lipid-phase reactions may also occur. Under stress conditions, a variety of ROS are produced in plant cells. These ROS cause oxidative damage. The plant responds to the presence of excess ROS through an antioxidant defense system that consists of antioxidant compounds that are either lipophilic (for example, tocopherols and carotenoids) or hydrophilic (for example, ascorbate and glutathione) and enzymes that either mediate regeneration of antioxidants (for example, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) or dismutate toxic ROS to water or less reactive compounds (for example, superoxide dismutase, catalase and peroxidase) 2 (example shown in Fig. 2 for chloroplast ROS response). Lipid- and aqueous-phase oxidative states are closely linked through action of antioxidant enzymes (Fig. 2). Hydrogen peroxide is produced as a byproduct of detoxification of the more dangerous ROS and is involved in initiating stress signaling networks. ROS are also constitutively present in plant cells under nonstress conditions and are involved in a number of cellular responses. For example, production of ROS at the photosynthetic membranes is integral to photosynthetic processes, and ROS signaling is involved in regulation of those processes 2,62,63. The regulation of ROS is normally tightly controlled, but under stress conditions the cellular balance of ROS is often perturbed 2,3. The cellular network of antioxidants and antioxidant enzymes is then required to scavenge the excess ROS and prevent cytotoxic effects. It is well known that nonvolatile isoprenoids such as tocopherols, zeaxanthin and carnosic acid can scavenge ROS directly by reactions through hydroxyl radicals 64. Volatile isoprenoids are typically olefins with reactive conjugated and/or terminal double bonds. We propose that volatile isoprenoids also form part of the non-enzymatic oxidative defense system. This hypothesis rests on the following evidence. Isoprene affects the oxidative status of plants under stress. Inhibition of isoprene emission by fosmidomycin results in greater accumulation of, increased lipid peroxidation levels and increases in antioxidant enzyme activities when plants are placed under thermal stress 35 37,46. Fumigation of fosmidomycin-inhibited leaves with exogenous isoprene partially restores and lipid peroxidation levels to those found in non-inhibited leaves. These results were confirmed in a transgenic tobacco system using plants azygous and heterozygous for isoprene synthase 43. Further, pools of reduced antioxidant (ascorbate) were higher in transgenic isoprene-emitting plants relative to non-emitting plants, which suggests that the emitting plants had a reduced requirement for antioxidant capacity compared to nonemitting plants. These findings all indicate that isoprene plays some role in reducing the oxidizing load under stress conditions. Direct reactions can occur between volatile isoprenoids and oxidizing species. It has been suggested that volatile isoprenoids, especially isoprene, can react directly with ozone, either in planta or at the leaf surface, thus decreasing ozone levels and potentially mitigating oxidative damage caused to the leaf 45,46. In highly oxidizing, humid atmospheric environments, isoprene does react with ozone 65 ;similar conditions might occur at the leaf boundary layer and in the intercellular spaces of the mesophyll tissue when ozone is present. However, only monoterpenes have been experimentally demonstrated to scavenge a significant amount of ozone in the boundary layer 66.The reaction between ozone and isoprene is relatively slow, and direct removal of ozone in this way is insufficient to result in the observed protection of fumigated tissues 66. Furthermore, ozonolysis of isoprene in humid environments results in production of hydrogen peroxide 65 ; this is inconsistent with the lowered hydrogen peroxide levels observed in tissue extracts from ozone-fumigated leaves, which emit isoprene 46. Methacrolein (MACR) and methylvinylketone (MVK) are the first products of isoprene ozonolysis 65 ; reactive electrophile species (RES) such as these are known to activate expression of defense genes 67. Fares et al. observed a surprisingly low production of MACR and MVK from isoprene-emitting leaves that were fumigated with ozone 66 ; this implies that secondary reactions either with ozone or with 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. Scavenging of some or all of these ROS by volatile isoprenoids might help explain the protective effect observed. In aqueous solution, isoprene reacts with hydroxyl radicals to produce 2-methyltetrols 68, and it has been suggested that isoprene might act as a hydroxyl radical scavenger to protect from oxidative damage 69. The presence of conjugated double bounds (delocalized p-electrons) in the isoprene molecule may serve to mediate electron and energy transfers, conferring a ROS-scavenging ability to the molecule 46,48,49. There is strong evidence that isoprene scavenges singlet oxygen. Singlet oxygen is produced (in addition to other ROS) at the thylakoid membranes when absorbed energy is in excess of that used in photosynthesis 70 (see Fig. 2). This may occur because excess light is present (at high light intensities) and/or because use of excitation energy is retarded (for example, under various abiotic stress conditions). Recent research suggests that singlet oxygen is the major cause of photo-oxidative damage under high light stress 71. When production of singlet oxygen at the photosynthetic membranes is exacerbated under light stress using chemical treatments, young non-emitting leaves of isoprene-emitting species that are fumigated with isoprene show higher net assimilation rates than non-fumigated leaves 48. Similar results are observed in mature leaves when isoprene emission is inhibited by application of fosmidomycin 49. In isopreneinhibited leaves, and malonyldialdehyde (MDA) levels were increased compared to uninhibited leaves. MDA is an indicator of lipid peroxidation and is produced at the thylakoid membranes under oxidative stress (see Fig. 2). Changes in signaling responses may also contribute to protective effects. Recent studies show that isoprene may also have indirect effects on oxidative state. Ozone damage is typified by accumulation of hydrogen peroxide followed by biochemical and transcriptional responses similar to those observed during the hypersensitive response in an incompatible plant-pathogen interaction 72. Nitric oxide (NO) is 288 VOLUME 5 NUMBER 5 MAY 2009 NATURE CHEMICAL BIOLOGY

7 involved in the signal cascade that initiates this cellular response; in addition, NO is involved in a number of physiological processes, many of which are stress-related 73,74. NO may also directly scavenge ROS, attenuating the effects of photo-oxidative stress 75. Isoprene-emitting leaves produce less NO compared to fosmidomycin-treated leaves when fumigated with ozone, which leads to the suggestion that isoprene can also quench NO 58. Isoprene can react with oxygenated nitrogen species in the troposphere, but one reaction product is ozone. If this happened also in planta, it would obviously result in further oxidative damage. It therefore seems unlikely that this reaction is occurring in plant cells. The observed decrease in relative NO levels could also be explained if there was a decreased requirement for NO in the presence of isoprene. NO and isoprene may indeed cooperate in protecting leaves against oxidative stresses when they are present at physiological levels 76. Although the mechanism for this effect is unknown, a reduction in the amount of NO might attenuate the induction of stress-induced hypersensitive response, thus avoiding the signal cascade that results in accumulation of and subsequent foliar necrosis. This is supported by the decreased levels of and lipid peroxidation markers observed in isoprene-emitting leaves compared to non-emitting leaves after ozone fumigation 35. If isoprene interacts with NO, then a number of other processes that are initiated by the hypersensitive responses are also likely to be affected, principally the MAPK cascade and the elicitation of jasmonate and salicylate stress signaling pathways. This has yet to be elucidated, and may become a major field of study in stress physiology. Evolution of the stress tolerance function Enzymatic production and emission of volatile isoprenoids occurs in many plants, from bryophytes to highly derived angiosperms. The DOX-MEP pathway appears to be primitive to green plants, and the terpene synthase enzyme that converts the substrate dimethylallyl diphosphate (DMADP) to isoprene has evolved multiple times in mosses, ferns, gymnosperms and angiosperms 77,78. Isoprene biosynthesis appears not to have evolved (or to have been lost) in several groups defined taxonomically or physiologically, most notably in two ancient divisions hornworts (anthocerotophytes) and liverworts (marchantiophytes) and in two physiologically defined groups whose members are much more recently evolved C4 and crassulation acid metabolism (CAM) plants. In the oaks (Quercus spp.), where isoprene emission appears to be a primitive trait, the subgenus that has lost the trait, the European live oaks, has replaced enzymatically controlled light-dependent isoprene emission with enzymatically controlled light-dependent monoterpene emission 79. This offers the strongest phylogenetic evidence of an adaptive significance for volatile diene production and emission by plants, as monoterpenes appear to be more effective in scavenging antioxidants in the gas phase than isoprene 66 and, because of their lower volatility, form larger pools in membranes and intercellular spaces 80,81. In other groups of nonisoprene-emitting taxa, monoterpenes or sesquiterpenes may play the same role as isoprene in protection against abiotic stress. However, much more information about the phylogenetic occurrence of foliar monoterpene and sesquiterpene emission in the plant kingdom is needed to support or reject this conjecture. Analysis of online emission databases ( cnhgroup/download.html) reveals very few patterns between isoprene emission and particular growth forms, ecologies, habitats or phylogenies. However, some strong correlations have been noted. Nearly all isoprene emitters are woody; within taxa that contain both woody and herbaceous groups (for example, the grasses), isoprene emission is far more common in woody groups (for example, bamboos and reeds). Based on the phylogenetic distribution of isoprene emission among nonvascular plants, one may speculate that isoprene emission first developed when plants abandoned the aquatic environment to conquer the land 82. Terrestrial plants may have developed isoprene as a quick and primitive method to cope with rapid, short-term changes of temperatures that occur on land but that do not occur in the more thermally buffered aquatic environment. In support of this, desert ecosystems tend to be lacking in isoprene-emitting taxa; this can be considered as evidence in favor of a role for isoprene in plant tolerance against short-term heat stress (a rare event in desert systems). Exposure to the oxygen-rich terrestrial atmosphere may also have selected for isoprene biosynthesis as an antioxidant mechanism, though this selective pressure does not account for the phylogenetic distribution of the trait or for the sensitivity of isoprene synthesis to light and temperature 83. There is no clear evidence that isoprenoid-emitting species cope better in the long term than non-emitting species in the presence of environmental stresses. However, it should be noted that a wide variety of complex and efficient mechanisms for protection against abiotic stresses can be found in plants. In species where isoprenoid emission has not evolved or has been lost, these mechanisms may be an alternative response. Nonetheless, the potential exists for changes in population structure to occur under global warming conditions, given that (i) emissions specifically increase with increasing temperature, and (ii) oxidative stress (in particular the occurrence of increased ozone pollution) is increasing in parallel 83. On the other hand, rising CO 2, which drives the temperature increase globally, is expected to enhance photosynthesis, thus reducing the oxidative stress in plants. This might negatively feed back on isoprenoid emission. An independent, negative feedback of rising CO 2 on isoprene emission has been observed 84 and may be explained by the insufficient supply of phosphoenolpyruvate (PEP) to isoprene, as PEP is increasingly diverted to oxaloacetate production for anabolic support of mitochondrial respiration under rising CO 2 (ref. 85). Summary and conclusions It is now clear that volatile isoprenoids play an important role in protection 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 of isoprenoids improves the ability of plants to deal with internal oxidative changes regardless of the nature of the external (physiological) stressor. Our single biochemical mechanism for multiple physiological stressors model provides a unified mechanistic explanation for the protection provided by volatile isoprenoids under diverse stress events. Carbon is redirected to volatile production under stress conditions, and the presence of these compounds results in protection, thus justifying the metabolic expense of production. The importance of this defense mechanism is further evidenced by the breadth of taxa that emit volatiles and the apparent repeated evolution of this trait. The mechanism of volatile isoprenoid action is difficult to test directly in planta because current techniques do not allow discrimination between cause and effect. Activity may be mediated through (i) direct reactions of isoprenoids with oxidizing species, (ii) indirect alteration of ROS signaling, and/or (iii) membrane stabilization. Stabilization of lipid membranes also presumably decreases lipid peroxidation, thus directly impacting the oxidative state of the cell; this mechanism might explain a generic oxidative protection that is not necessarily due to direct reactions. The antioxidant behavior of isoprene and other volatiles might be further investigated by searching for specific reaction products from isoprenoid oxidation. NATURE CHEMICAL BIOLOGY VOLUME 5 NUMBER 5 MAY

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