Special Issue: Induced biogenic volatile organic compounds from plants
Abiotic stresses and induced BVOCs Francesco Loreto1 and Jo¨rg-Peter Schnitzler2 1
Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione delle Piante (IPP), Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy 2 Karlsruhe Institute of Technology (KIT), Institute of Meteorology and Climate Research (IMK-IFU), Kreuzeckbahnstraße 19, 82467 Garmisch-Partenkirchen, Germany
Plants produce a wide spectrum of biogenic volatile organic compounds (BVOCs) in various tissues above and below ground to communicate with other plants and organisms. However, BVOCs also have various functions in biotic and abiotic stresses. For example abiotic stresses enhance BVOCs emission rates and patterns, altering the communication with other organisms and the photochemical cycles. Recent new insights on biosynthesis and eco-physiological control of constitutive or induced BVOCs have led to formulation of hypotheses on their functions which are presented in this review. Specifically, oxidative and thermal stresses are relieved in the presence of volatile terpenes. Terpenes, C6 compounds, and methyl salicylate are thought to promote direct and indirect defence by modulating the signalling that biochemically activate defence pathways. The emission of BVOCs: few biochemical pathways but many compounds emitted Biosynthesis of the main BVOCs Plants produce a wide spectrum of BVOCs in various tissues above and below ground. Most BVOCs are largely lipophilic and have enough vapour pressure to be released into the atmosphere in significant amounts. The availability of new methods of head-space sampling (such as solid phase micro-extraction) in combination with gas chromatography–mass spectroscopy and new techniques for online analysis (proton transfer reaction-mass spectrometry)  has led, in the last 15 years, to a significant expansion of our knowledge on the occurrence and temporal and spatial distribution of BVOCs emissions. At present, about 1700 substances have been found to be emitted from plants . Nearly all organs from vegetative parts, as well as flowers  and roots  emit these compounds. Many BVOCs are emitted constitutively and the emissions can be observed throughout the life cycle of the plant or, more often, at specific developmental stages (e.g. leaf and needle maturation, senescence, flowering, and fruit ripening). The emission is biosynthetically controlled by abiotic factors such as light and/or temperature, atmospheric CO2 concentration, or nutrition. Other BVOCs are induced after wounding and herbivore feeding or after environmental stresses. Stresses may induce change of constitutive BVOCs, either stimulating or quenching the emissions (e.g. ) or may induce de novo synthesis and emission Corresponding author: Loreto, F. ([email protected]
of BVOCs. Induced emissions may occur in a systemic way, i.e. away from the site of damage . The biosynthesis of most BVOCs can be assigned to the following three major pathways: terpenes (= isoprenoids), oxylipins, and shikimate and benzoic acid [7,8]. Low molecular weight, C1 and C2, compounds, such as methanol, ethanol, formaldehyde, and acetaldehyde can be synthesized via other biosynthetic routes . Two other BVOCs are methane and ethylene. Emission of non-microbial methane by vegetation has been discovered recently  and research on this important topic is still in its infancy. Recent isotope labelling studies provided evidence that methane can be generated from methoxyl groups deriving from breakdown of plant pectins . In this review of the impact of abiotic factors on the induction of BVOCs emissions, we will mostly concentrate on volatile terpenes which are the most important compounds for plant biology  and atmospheric chemistry  because of their role in plant protection (e.g. in the protection of photosynthesis against thermal and oxidative stresses, and in direct and indirect defence against herbivores), as well as in the chemical properties of the atmosphere (e.g. entering the cycle of photochemical production/ destruction of ozone, aerosols, and particles). The emission of volatile terpenes is estimated to account for more than half of the total emission of BVOCs  and is constitutively ten times higher than other emissions, as heavy emitters can release isoprene at rates of 50–100 nmol m 2 s 1, representing up to 2–5% of the photosynthetic net carbon uptake in tree species. Volatile terpene emissions are far more sustained than the emission of other induced volatiles, for which emissions are transient by nature and limited to specific periods after stress, depending on the damage experienced and on the activation of the biosynthetic pathways producing the volatiles. Biosynthesis of volatile terpenes Terpenes are constitutively formed in some plant families that store them in massive amounts in internal or external structures (e.g. the resin ducts of conifers or the glandular cells of Lamiaceae leaves). They may also be induced in response to wounding or herbivory attack. The emission of terpenes from storage structures is generally uncoupled from photosynthesis as it may occur, for example, at night . Direct emission of isoprene or monoterpenes from the mesophyll is common in some tree species (Figure 1), in
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Figure 1. Origin of volatile terpene emissions from different leaf types. In deciduous leaves of many tree species (e.g. oaks and poplars) with no specific storage structures for terpenes, isoprene and monoterpene, BVOCs emissions originate from mesophyll cells in a light- and temperature-dependent manner. In conifer needles (e.g. Picea abies - Norway spruce) light-dependent terpene emission stems from photosynthetic tissue and is superposed by a temperaturedependent volatilization of terpenes from resin ducts. Lamiaceae (e.g. Ocimum basilicum basil) leaves release temperature-dependent volatile terpenes from external glandular cells. Confocal laser scanning microscopic images were taken from cross-sections of Quercus robur (top), Picea abies (middle) and leaf surface of Ocimum basilicum (bottom).
particular from the Fagaceae and Salicaceae (oaks and poplars) [16,17]. These emissions are light-dependent [18,19] and are closely linked to the availability of photosynthetic intermediates [19,20]. The investigation of volatile terpene biosynthesis is a very active area of plant research, especially since the discovery and complete elucidation of the methylerythritol (MEP) pathway, responsible for the formation of the basic C5 units isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) in many bacteria and in the plastids of all organisms from phototrophic phyla . In plants, IDP and DMADP are formed via two alternative pathways: (i) in the cytosolic mevalonic acid (MVA) pathway from acetyl-CoA, and (ii) in the plastidic MEP pathway from pyruvate and glyceraldehyde-3-phosphate . Generally, the MEP pathway provides IDP and DMADP for hemiterpene and monoterpene biosynthesis, while the MVA pathway provides the C5 units for sesquiterpene formation. However, a very recent report has revealed that the MEP pathway also contributes to sesquiterpene formation . In addition, some metabolic
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crosstalk between both biosynthetic routes is possible  particularly (via IDP) in the direction from chloroplasts to the cytosol . Prenyl transferases catalyze the condensation of IDP and DMADP to form geranyl diphosphate (GDP) and farnesyl diphosphate (FDP) . Finally, the conversion of DMADP, GDP, and FDP, into volatile hemi-, mono-, and sesquiterpenes, respectively, is catalyzed by terpene synthases (TPS), a large family of enzymes, encoded by closely related genes . Isolation and characterization of prenyl transferase  and terpene synthase genes  is now giving new insights into the evolutionary origin [30,31] and genetic and biochemical regulation of terpene biosynthesis. Isoprene and 2-methyl-3-buten-2-ol (MBO), a hemiterpene common only in American western pines,  are biochemically synthesized in chloroplasts by isoprene  and MBO synthase  from DMADP. Due to the importance of isoprene for atmospheric processes and plant functions, isoprene synthase (ISPS) became one of the best studied TPS . A positive correlation between ISPS activity and basal standard emission capacity was found in different isoprene-emitting species [36,37]. Up to now, five ISPS genes from different poplar (Populus) species or poplar hybrids have been described . The only ISPS gene cloned so far from another genus, Pueraria montana (kudzu) , shows only 52% identity with the poplar protein sequences, although the structure of poplar and kudzu genes are similar (six introns and seven exons) . All ISPS genes belong to the subgroup b of the class 1 plant TPS-family which includes monomeric mono-, sesqui-, and diterpene synthases, grouped in six subgroups (Tspa–TspfTpsa–Tpsf?) . All known ISPS enzymes have a 10–100-fold higher Michaelis constant (kM) for its substrate DMADP (in the millimolar range) than monoterpene synthases for GDP  or prenyltransferases for DMADP . The low kM of prenyltransferases may control the metabolic flux within the MEP pathway because downstream reactions leading to monoterpene and non-volatile terpene biosynthesis are favoured over isoprene biosynthesis. Based on this finding, it was suggested that isoprene emission occurs only when plants’ need for ‘essential’, higher terpenes (hormones, e.g. ABA and gibberellins; tocopherol; phytosterols; and photosynthetic pigments) are satisfied . Interest in genetic regulation and biochemical properties of TPS other than ISPS mostly focused on those TPS involved in the formation in storage structures (e.g. in gymnosperms and Lamiaceae) of those terpenes with defensive or attractive functions  (see Dicke and Baldwin, this issue). In light-dependent monoterpene emitters, the information about TPS regulation is scant. Differences in monoterpene emission pattern of chemotypes and oak hybrids , the seasonal development of monoterpene emission [44,45], and the dependence of monoterpene emission on atmospheric CO2  all appear to be controlled by TPS activities. However, our knowledge about the underlying genes is scarce; in oak, only two mono-TPS genes, a bmyrcene synthase  and a multiproduct a-pinene synthase  have been isolated and functionally characterized up to now. 155
Review The commonly used model plant Arabidopsis thaliana (Arabidopsis) is a good example of how modern technologies can improve knowledge of BVOCs biosynthesis and functions. In the past classified as ‘non-emitting’ species, analysis of the Arabidopsis genome revealed the existence of over 30 putative genes belonging to the multigene family of TPS [49,50]. Most of these genes are almost exclusively expressed in flowers [50,51], but low constitutive terpene emissions from leaves and siliques [50,52] and even emissions from roots (namely 1,8-cineole, ) could be detected. Isoprene synthase overexpression in Arabidopsis allowed verification of a ‘thermo-protective’ activity of isoprene [53,54], see below, and indicated an ecological function of this hemiterpene in plant–insect interactions as a repelling cue . The expression of b-caryophyllene was also successfully engineered in this plant (J. Gershenzon, personal communication) and may help assess functions and fate of sesquiterpenes. Sesquiterpenes are not emitted in large amounts constitutively, but their biosynthesis can be induced by biotic stresses, and may be important as an indirect defence mechanism (see Dicke and Baldwin, this issue). Even at low concentrations sesquiterpenes are also important as nucleation factors, eventually leading to particle formation in the atmosphere . Poplar is another seminal model system to study regulation and function of plant volatiles. Besides ISPS , only one TPS gene [( )-germacrene-D synthase]  is characterized. However, full length cDNAs , cDNA microarrays [59,60], and a largely sequenced genome of Populus trichocarpa  are now available, and these tools will certainly allow for quick progress in the understanding of BVOCs formation in a near future. Biosynthesis of C1 and C2 oxygenated compounds Volatile terpenes are certainly the family of compounds that contributes the majority of BVOCs . However, short-chained oxygenated compounds (especially methanol and acetaldehyde) are important components of constitutive and induced emissions of many plants  with a large presence globally . Methanol is predominantly emitted because of degradation and formation of cell wall pectins, e.g. (i) during cell expansion in all types of plant tissue and seeds, and (ii) during leaf abscission, senescence, and seed maturation (Figure 2). The formation of methanol is catalyzed by pectin methylesterases (PME) which, among the other functions, demethoxylates pectin [63,64]. To a minor extent, methanol emissions originate from protein methyltransferase and protein repair reactions , or from tetrahydrofolate metabolism . Emission of methanol can be induced by mechanical wounding  or herbivore feeding, due to an upregulation of PME expression. This finding has stimulated a discussion about whether methanol might act as a signal in plant–plant communication . The metabolic origin of acetaldehyde emitted by forest trees is still a matter of debate [69,70]. This compound seems to be predominantly induced by stresses. It is known that acetaldehyde emission correlates with root flooding [71,72] and with xylem sap ethanol concentrations [73,74]. Ethanol formed under anoxic conditions in roots is trans156
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Figure 2. Simplified scheme of the subcellular origin and biosynthesis of volatile organic compounds upon abiotic stress. Abbreviations: CH4, methane; DMADP, dimethylallyl diphosphate; DXS, 1-deoxy-D-xylulose 5-phosphate synthase (EC 126.96.36.199); FDP, farnesyl diphosphate; GDP, geranyl diphosphate; HMGR, 3-hydroxy3-methylglutaryl-CoA reductase (EC 188.8.131.52); 13-HPOT, 13S-hydroperoxy9(Z),11(E),15(Z)-octadecatrienoic acid; IDP, isopentenyl diphosphate; ISPS, isoprene synthase (EC 184.108.40.206); a-LeA, a-linolenic acid; 13-LOX, 13-lipoxygenase (EC 220.127.116.11); MeOH, methanol; MTS, monoterpene synthase (e.g. myrcene EC 18.104.22.168); PDC, pyruvate decarboxylase (EC 22.214.171.124); PEP, phosphoenolpyruvate; PME, pectine methylesterase (EC 126.96.36.199); PYR, pyruvate; STS, sesquiterpene synthase (e.g. epi-aristolochene EC 188.8.131.52); 13-HPL, 13-hydroxyperoxide lyase (not listed in enzyme classification); TP, triose phosphate. The broken arrow indicates a proposed, yet unidentified acetaldehyde emission path from chloroplasts.
ported to leaves by the transpiration stream, where it is oxidized to acetaldehyde by alcohol dehydrogenase (ADH). However, only a small portion of acetaldehyde is emitted while the bulk is further metabolized by aldehyde dehydrogenase (ALDH) to acetate and acetyl-CoA. In some tree species, strong transient acetaldehyde bursts during light–dark transitions have been reported [70,72,75]. These acetaldehyde bursts are thought to be the result of a ‘pyruvate overflow mechanism’ . In the proposed mechanism pyruvate decarboxylase (PDC) acts as a metabolic regulator converting excess cytosolic pyruvate into acetaldehyde, which is subsequently oxidized to acetate. Such an excess of cytosolic pyruvate may be the result of transiently decreased transport rates of pyruvate equivalents [i.e. phosphoenolpyruvate (PEP)] into organelles, or reduced pyruvate utilization in leaf cells immediately after darkening . However, emission of acetaldehyde may also derive from cleavage of moieties of C6 aldehydes, which emissions are also transiently stimulated during light–dark transitions . In this case, acetaldehyde emission is independent of cytosolic pyruvate and is part of the leaf response to wounding that also includes biosynthesis of C6 compounds (see below). Biosynthesis of C6 aldehydes and alcohols Wounding induces the release of ‘green leaf volatiles’ (GLV) as can be easily sensed in the odour of fresh hay. In most wounded plants GLV are C6 aldehydes, C6 alcohols, and their derivatives, often collectively called C6- or LOX-products . Physiologically these compounds have
Review antibiotic properties inhibiting the invasion of damaged tissue , and can serve a signalling function within plants to induce or prime defence . GLV are derived from oxidized polyenoic fatty acids (PUFA), collectively called oxylipins. The initial formation is catalyzed by lipoxygenases (LOX), a large gene family of non-haeme iron containing fatty acid dioxygenases  (Figure 2). LOXs (13-LOX, classified with respect to their positional specificity of oxidation at carbon-13 of the fatty acid hydrocarbon backbone) initiate the octadecanoic pathway by adding O2 stereospecifically to unsaturated fatty acids [e.g. linoleic acid (18:2) and a-linolenic acid (18:3)] generating 13-(S)-hydroperoxides. Subsequently, 13-(S)-hydroperoxide lyase (HPL) catalyzes the cleavage between C12 and C13 releasing n-hexanal (from linoleic acid) and (Z)-3-hexenal (from a-linolenic acid) which are the parent compounds for all other aldehydes and alcohols, and enzymatically acetylated compounds such as hexyl acetate and (Z)-3-hexenyl acetate . Analysis of C6 compounds is complicated by their chemical instability and the transient nature of their formation after wounding. Thanks to modern on-line techniques like proton transfer reaction mass spectrometry (PTR-MS) that avoid pre-concentration of the samples on adsorbent phases, the rapid and transient emission of C6 upon various stimuli, such as physical damage , light–dark transitions, herbivory, or senescence processes, can be easily monitored [70,79]. Plants and abiotic stresses: how stresses affect BVOCs biosynthesis and emissions Abiotic stresses affect primary and secondary metabolism in different ways. Stresses generally inhibit photosynthesis by reducing CO2 uptake and diffusion inside leaves to the site of fixation into carbohydrates or by altering the photochemical or biochemical reactions of the photosynthetic cycle . The impact of stresses on the secondary metabolisms that produces BVOCs is more controversial, as some of the pathways may be elicited by stresses. In the case of volatile terpenes, the uncoupling of photosynthesis and BVOCs emission is surprising, because of the wellknown high requirement of photosynthetic carbon for terpene biosynthesis . The stimulation of terpene production is made possible by the activation of sources of carbon which are alternatives to freshly fixed photosynthates and not yet fully identified. Some labelling studies have provided evidence that isoprene may also be formed from xylem-transported glucose and chloroplastic starch . However, under stress conditions, other 13C labelling studies point out that, because of starch depletion, extra-chloroplastic sources of carbon may be activated and feed carbon to volatile terpenes . Stresses may also induce damage that elicits or induces the synthesis of other volatiles with important consequences for plant protection, directly against the environmental constraint  or indirectly, against possible, associated bursts of pathogens and herbivores (see Dicke and Baldwin, this issue). The impact of the main environmental factors of stress on the biosynthesis and emission of volatiles will be reviewed in the following sections (see also Figure 3).
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Figure 3. Schematic overview of long-term and short-term regulation of terpene emissions upon various abiotic stresses. Long-term adaptation of volatile terpene emission (predisposition and/or priming) relates to differential gene expression resulting in upregulation (green circles) and/or downregulation (red circles) of specific enzymes and changed emission pattern and emission rates. Short-term regulation reflects rapid changes in metabolic fluxes and enzyme kinetic properties (e.g. temperature, pH and ion shifts, and redox/energy status) due to abiotic factors like atmospheric CO2 concentration, light and temperature. Orientation of arrows in the green and red circles indicate strength of upregulation and downregulation. Abbreviations: h, hours; min, minutes; s, seconds.
Temperature BVOCs partition between the gas and liquid phase in the plants according to their Henry’s law constant (kH), which is generally very high (e.g. kH 7500 Pa m3 mol 1 at 25 8C for isoprene) . The equilibrium between gas and aqueous phases is indeed determined by the temperature and therefore it is expected that more BVOCs enter the gas phase and are emitted at rising temperatures. This direct effect of temperature on BVOCs emission is, however, modulated by diffusion resistances encountered from the sites of synthesis inside the leaf to the atmosphere. BVOCs that are stored in specialized structures (ducts or glands) reach very high concentrations and are tightly separated by the surrounding cells by an impermeable cell layer, generally made by sub-cuticular cells . This is because high concentrations of BVOCs, which have probably important ecological functions as deterrents of herbivores and as anti-bacterial and anti-mycotic substances, could be auto-toxic for plant cells . Temperature affects the evaporation and release of a minimal part of the pools of BVOCs that leaks out the impermeable cell layer. Indeed plants with large storage pools are moderate emitters [15,17] unless the pools are opened up, e.g. by herbivory , strong winds, or forest fires . High humidity may also lead to a swelling and to the consequent explosion of the structures containing the pools. In this case strong emissions of terpenes may even be sensed, e.g. walking on a pine forest at sunrise. On the other hand, plants that do not store BVOCs into specialized structures have small temporary pools in the leaf mesophyll that freely diffuse out of the leaf driven by a concentration gradient. The only important constraint to this diffusion is at the stomatal conductance to gas 157
Review exchange. High temperatures often affect stomatal behaviour, either per se or because this stress is generally associated with a drought stress. Stomatal opening under transient heat stress is an important mechanism to dissipate latent heat through transpiration of water and to uncouple the leaf temperature from air temperature. On the other hand, stomatal closure improves instantaneous water use efficiency (the ratio between net CO2 assimilation and transpiration) and avoids excessive loss of water driven by increasing transpiration. Stomatal movements do not affect the steady-state diffusion rates of gases with high kH, such as isoprene and monoterpenes . In the case of stomatal closure, these compounds build up transiently significant partial pressures inside leaves that compensate for the increasing resistance at stomatal level [89,90]. However, diffusion of gases that are mainly partitioned into liquid phase, such as oxygenated VOCs (methanol, C6 aldehydes, and alcohols) might be strongly restrained by stomatal closure . Temperature has a strong and immediate influence on the activity of the enzymes that catalyze the synthesis of many BVOCs. Emissions of volatile terpenes typically have a Q10 = 2–4, at temperatures variable between 20 and 40 8C . Thus the main effect of rising temperature is a direct increment of the terpenes formed through enzymatic reactions. However, when a heat stress occurs with temperature above the optimal enzyme temperature (around 40–45 8C for enzymes in the MEP pathway and most TPS), then a very rapid inhibition of terpene emission is observed . This is probably due to downregulation or impairment of primary metabolism and to the consequently insufficient supply of photosynthetic metabolites into the MEP pathway [92,93]. However, there are cases in which the emission is not rapidly re-established upon heat stress removal, and in these cases a heat-induced denaturation of the TPS is also likely to occur . Interestingly, when the heat-induced inhibition of volatile terpenes occurs, then the emission of other BVOCs is highly enhanced . This is particularly evident in the case of methanol and C6 compounds, pinpointing that the inhibition of volatile terpene emission coincides with the occurrence of damage to cell walls and membranes, respectively . The emission of C6 compounds is sustained for the whole period of heat stress and may continue for a long time after temperatures go back to physiological levels . Significant fluxes of methyl-butenol, ethanol, and acetaldehyde were found in North American conifers exposed to high temperature , and methanol and acetone emissions were also observed from bare agricultural soils following a heat wave episode . Thus soil microorganisms may also contribute to the temperaturedependent emission of oxygenated BVOCs. Finally, temperature seems to have an important role in determining emissions of methyl salicylate (MeSA), a signalling molecule whose induction is frequent in response to biotic stresses [74,96]. One study  reported induction of MeSA only after spraying plants with jasmonic acid, another important signalling molecule activating the induction of genes involved in hypersensitive responses, both after biotic and abiotic stresses. In another study a significant induction of MeSA, with fluxes comparable to 158
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those of monoterpenes was observed in plants exposed to night chilling temperatures  and the induction of this compound correlated to the difference in temperature between day and night that was experienced by walnut (Juglans californica Juglans regia) plants. Drought and salt The impact of drought and salt on BVOCs emission is surprising. These abiotic stresses directly affect stomatal conductance and produce diffusive and biochemical limitations of photosynthesis . Both the reduction of photosynthesis and the stomatal closure are expected to negatively impact on BVOCs emission by altering the carbon supply into the MEP pathway and by increasing resistance to their emission. In fact, the emission of volatile terpenes is resistant to these stresses and is often elicited by stress occurrence. The original observation that isoprene is not reduced by increasing drought stress until the stress becomes heavy and almost completely inhibits photosynthesis  has been repeatedly made, also with experiments in which the drought stress has been controlled differently, e.g. [90,100]. The sustained emission of isoprene to salt and drought [101,102] is made possible by the induction of carbon sources alternative to photosynthesis, possibly related to respiration  or starch breakdown . Labelling experiments with 13CO2 [35,83] demonstrate clearly the preference of ‘old’ unlabeled (12C) carbon skeletons over recently fixed, 13C enriched photosynthetic intermediates when photosynthesis (which is heavily reduced by drought and salt stress) [83,101] and isoprene biosynthesis become uncoupled. This may also explain why in some instances re-establishment of photosynthetic metabolism, by re-watering plants, results in a burst of isoprene emission . However, it has been shown  that the alternative carbon sources rapidly cease to feed carbon into the MEP pathway once the water status of plants is reintegrated. A recent observation indicates that in plants exposed to severe drought stress and in those recovering from drought stress the temperature dependency of isoprene emission is lost for at least several weeks, if not permanently . The mechanism behind this observation is still unknown, but it is likely that the ISPS protein is slowly or incompletely re-synthesized after the stress . Climate-change impact on isoprene emission has been mainly attributed to positive long-term (enzymatic) and short-term (substrate) feedback of rising temperature [104,105], implying that future emissions of isoprene will also increase . However, as drought is often associated with heat waves and summer climate, the finding that drought suppresses temperature-dependency of isoprene emission may have consequences for trees’ thermal and ozone tolerance in regions (e.g. the Mediterranean basin) which will be plagued by climate-changeinduced droughts associated with rising temperatures. Laboratory measurements of the impact of drought on monoterpene emissions are missing, but in field experiments monoterpene emissions are inhibited by drought stress [46,107,108]. Interestingly, the inhibition is particularly evident only when drought stress is severe (e.g. the water potential is less than –2 MPa) again suggesting that
Review biosynthesis of volatile terpenes is resistant to mild drought stress . It is unclear whether alternative carbon sources can be used to generate monoterpenes under these conditions. As for temperature, heavily drought-stressed oak plants lose the capacity to respond to other environmental factors that are known to modulate the emission, such as CO2 (see below) . Drought seems to have a different effect on pools of monoterpenes stored in specialized structures or nonstored in the mesophyll. In conifers, if the drought event occurs in winter, when the biosynthesis of terpenes is restrained by temperature, the pools appear to be negatively affected. However, summer droughts can further enrich the monoterpene pools. In non-storing species, summer drought depletes the pools of terpenes in the mesophyll which are under direct control of photosynthetic carbon via the MEP pathway . As described above, the emission of oxygenated BVOCs depends on stomatal opening . Stomatal closure in response to drought and salt stress is therefore expected to reduce particularly the emission of these compounds. However, morning peaks of acetone and methanol emissions may be very high in drought-stressed leaves, because the temporary opening of stomata during times of higher humidity allows the release of large pools of oxygenated BVOCs that were built inside the leaf mesophyll [111,112]. Thus, drought stress does not inhibit per se the biosynthesis of oxygenated BVOCs. Moreover, if the stress reaches levels that are able to damage membranes and cell walls, further increments of the emission of C6 compounds and methanol must be expected. However, emission of these compounds has not been reported generally in response to drought and salt . Indeed, C6 emissions occur in bursts, immediately after the damage to cellular structures has occurred [5,113]. A recent experiment has established a correlation between the emission of C6 volatiles and the damage to membranes, as assessed by the ion leakage, under a developing drought . Ozone and other oxidants In the atmosphere, volatile terpenes perform a dual action, depending on the presence of anthropogenic pollutants. When these compounds are absent, volatile terpenes cleanse the atmosphere of ozone. In the presence of NOx, however, these BVOCs initiate reactions leading to increased ozone formation . The chemical reactivity of terpenes in the atmosphere led to the idea that volatile terpenes play a similar dual action inside the leaves, before they are released into the atmosphere [115,116]. In general, terpenes have been demonstrated to reduce ozone damage and to quench ozone and reactive oxygen species (ROS) [115–118]. The mechanism(s) by which this protective effect occurs is still under investigation and the prevalent theories will be discussed below. Here we concentrate on the impact of oxidative stresses on the emission of BVOCs. If isoprene reacts with ozone and other oxidative species then it is expected that it disappears, concurrently with the appearance of its reaction products. In the atmosphere, these products are mainly methyl-vinyl-ketone and methacrolein, two compounds that are indeed found in chamber studies in which isoprene-emitting trees were
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fumigated with ozone . In plants exposed to ozone either a reduction [113,120] or a stimulation of isoprene and monoterpene emissions is observed. The stimulation of the emission is more evident in response to acute and heavy doses of ozone [121–123] (e.g. 150–300 ppb) whereas it is often absent when plants are exposed to low doses of ozone above background [122,124]. Ozone-enhanced emission of isoprene is due to a higher expression of the ISPS mRNA which probably upregulates the protein and the activity of the enzyme . Interestingly, such an upregulation is more evident in leaves that develop under enriched ozone and that build up a better resistance to pollutants, as well as in new leaves that develop above those that have been ozonated . Evidently, impairment of photosynthetic activities prevents older ozonated leaves from enhancing the secondary metabolism, leading to volatile terpene formation. When plants are exposed to low or moderate and chronic doses of ozone, such an induction of the volatile terpene pathway is absent, and the expression of ISPS mRNA and the level of ISPS protein may even be reduced [120,124]. Clearly, the signals that activate the biochemistry of terpene formation, and which are unknown at present, are not released under these conditions. It has been hypothesized  that the induction of volatile terpenes in response to ozone follows a hormetic dose–response relationship, i.e. that the volatile terpenes are increasingly induced at low but growing doses of ozone until an ozone threshold is reached after which the biosynthesis of terpenes is repressed. The experiments, however, reveal a more complex picture, with biosynthesis of volatile terpenes being induced only when ozone dose overcomes a threshold that marks cellular damage but at which photosynthesis is not yet so heavily suppressed to be unable to supply enough substrate for volatile terpenes. On the basis of these findings a long-term induction of isoprene biosynthesis, and the consequent evolutionary hypothesis that high terpene emitters will be favoured in a future more oxidative atmosphere  may not hold true. Ozone is a very damaging pollutant for plant cells, and one of the first recognized ozone effects is the denaturation of the lipids in cellular membranes . It is therefore expected that volatiles that are associated with lipid peroxidation are also more emitted in ozone-stressed leaves. Accordingly, bursts of C6 compound emissions were observed in ozone-stressed leaves and the lag time with which these compounds were emitted was proportional to the ozone dose absorbed by the leaves  and to the ozone-induced injuries . These experiments clearly show that C6 compounds are in vivo indicators of membrane denaturation and damage as already indicated also in response to drought . In addition, these studies [113,128] also revealed transient pulses of methanol and MeSA. While methanol emission has been mainly attributed to demethylation of cell wall pectins, and is therefore likely to be another indicator of ozone damage, the induction of MeSA is more intriguing. MeSA emissions showed a weak association with high levels of ozone recorded in nonmanipulative field experiments , but acute exposures to ozone and UV light are apparently also able to induce bursts of this compound (Velikova et al., personal com159
Review munication). Emissions of MeSA induced by oxidative and other environmental stresses may further contribute to biogenic secondary organic aerosols [129,130] and may alter the network of plant communication with other organisms which is mediated by chemical messengers . Other climate change factors: UV-B radiation Studies of the effect of UV-B radiation (wavebands of 290– 320 nm) on BVOCs emissions are rare, despite the fact that UV-B radiation is known (i) in moderate doses (environmentally relevant) as a priming abiotic agent, triggering the formation of UV screening pigments and modulating plant growth; and (ii) in higher doses (e.g. as a result of the stratospheric ‘ozone hole’ and indeed transiently present in certain regions surrounding the South Pole) as an agent causing severe damage to the photosynthetic machinery, nucleic acids, and proteins . The impact of UV-B radiation on terpenes seems to indicate a stimulation of their biosynthesis and emission. In one study with European oak  the higher emission of isoprene under UV-B radiation was attributed to a higher biomass density rather than to a higher instantaneous photosynthetic rate. A more recent study  again showed increased isoprene emission rates of subarctic peatlands when irradiated with increasing levels of UV-B radiation, and explained the rising emission as a consequence of oxidative damage to membranes and to the induction of the terpene defensive antioxidant pathway . UV damage to cellular structure may also induce emission of other BVOCs. Emission of methane under aerobic conditions originating from plant material  is a controversial topic in recent plant and atmospheric research, e.g. [134,135]. Plant-mediated transport of methane originating from methanogenic soil microorganisms through the aerenchyma and out of the leaves of wetland plants, e.g. rice  has been known from many years. However, there is growing evidence that UV radiation can mediate non-microbial methane release from cell wall material, in particular pectin. In the initial work  it was suggested that the methoxyl groups of pectin can be one, albeit not the only, source of aerobic methane, e.g. [137,138]. It was recently demonstrated [137–139] that the release of methane from structural cell wall components of fresh and dried leaf tissue was UV-B dependent. In line with this observation, the authors  also showed that the removal of methoxyl groups interrupted methane emission from UV-irradiated pectin. Very recently it was demonstrated  that the emission of methane from cell wall material is mediated by UV-generated ROS [hydroxyl radicals (OH) and singlet oxygen; but not hydrogen peroxide or superoxide radicals]. Environmental stresses as well as cellular signalling processes involve the formation of ROS. Therefore it might be speculated that aerobic methane formation is a common, yet overlooked part of plant stress responses complementing the transient burst of BVOCs, i.e. methanol, acetaldehyde, and C6 alcohols and aldehydes [67,79], and the long-lasting stimulation of terpene biosynthesis. Support for this assumption is given by recent work demonstrating increased emissions of methane upon bacterial 160
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infection, chemical generation of ROS  or physical injury . Other climate change factors: atmospheric CO2 Rising CO2 concentration at global level is dramatically affecting plant life. The primary effect is the increased availability of substrate for Rubisco, and the consequent enhancement of photosynthesis . However, CO2 is one more factor that uncouples terpene metabolism and emissions from photosynthesis. As early as 1964, G. Sanadze  demonstrated that at low CO2 concentration photosynthesis is reduced, but isoprene emission increases. In a more recent laboratory study the highest emission of isoprene was detected at an intercellular CO2 concentration (Ci) of around 150–200 ppmv, not far from the Ci experienced in nature by leaves of tree species , and then progressively decreased at higher Ci. Since then, many studies on different plant species have shown an inhibition of isoprene biosynthesis under higher than ambient CO2 concentrations [144–148]. For example elevated CO2 in a natural CO2 spring reduces ISPS activity and isoprene emission from common reed (Phragmites australis) . Compared to isoprene, less is known about the influence of CO2 on light-dependent monoterpene emission. A study on holm oak (Quercus ilex) growing under CO2 concentration double than ambient in open top chambers  showed a parallel downregulation of mono-TPS activities and monoterpene emission. However, studies conducted in holm oak plants growing for their entire life in natural CO2 springs under very high but not steady CO2 levels did not reveal substantial inhibition of the emitted monoterpenes , probably because the treatment was also associated with recurrent drought stress episodes that could also affect the emission (see above). While a downregulation of ISPS and mono-TPS is generally associated with lower emission of isoprene and monoterpenes grown under elevated CO2 , the enzyme properties are not necessarily the factors controlling the emissions. This control may be exerted primarily by reduced substrate availability (DMADP and GDP) resulting from a downregulation of the MEP pathway. Although this assumption has no direct support from the literature, a simultaneous decrease in isoprene emission and DMADP content was observed . This reduction in isoprene (and also monoterpene) emission under elevated CO2 might result from enforced higher consumption rates of cytosolic PEP (phosphoenolpyruvate) through PEP carboxylase activity, thus lowering the rates of PEP transport into the chloroplast, where PEP, in its dephosphorylated form (pyruvate) feeds into the MEP pathway. Based on this work  and earlier suggestions by G. Sanadze [143,150] a double carboxylation hypothesis was proposed [148,151] for C3 plants (to which all of isoprene emitters belong) with cytosolic PEP carboxylase and plastidic RuBP carboxylase as antithetic precursors controlling the flow of carbon to plastidic terpene metabolism (as well as to the shikimic acid pathway and fatty acid biosynthesis) in response to changes in CO2 concentration. This idea gets support from a study  showing an inverse relationship of isoprene biosynthesis and PEP carboxylase activity
Review when cottonwoods (Populus deltoides) were grown on high nitrate concentration, a condition favouring cytosolic organic acids synthesis. In line with this scheme, mitochondrial respiration can constitute a growing sink for cytosolic PEP under rising CO2, thus competing with the import of this substrate in the chloroplast for terpene biosynthesis . Recent experiments with Free Air CO2 Enhancement (FACE) facilities, however, indicated that the inverse relationship between isoprene and respiration was not straightforward under elevated CO2, and that competition occurs only when oxaloacetate production from PEP for anabolic support of respiration is strong, such as in young, expanding leaves . A positive CO2 effect on isoprene  and monoterpene  emissions may be occasionally observed. Moreover, no effect on isoprene and monoterpenes was reported on poplar [125,153] or on Scots pine (Pinus sylvestris) . Currently we do not know whether the CO2 impact on light-dependent isoprene and monoterpene emission is a general effect and which fundamental biochemical mechanisms are responsible. If the biosynthesis of volatile terpenes is ubiquitously enhanced at CO2 lower than ambient, then stress conditions in which a lower intercellular CO2 concentration is set by increasing resistances to CO2 diffusion can generically lead to higher emissions. Why is emission of volatile terpenes induced by abiotic stress? Whereas oxygenated BVOCs are mainly catabolic products of the denaturation of cellular walls and membranes, isoprene and monoterpenes are produced through a dedicated metabolic pathway that is stimulated by several abiotic stresses (see above). This tightly regulated terpene biosynthesis and the observation that emission of volatile terpenes represents a significant loss of photosynthetic carbon, led to the proposition that these compounds play important physiological and ecological roles in the protection of plants from environmental constraints. However, the debate is still ongoing whether one or more ecological actions should be attributed to volatile terpenes, and which are the physiological mechanisms that allow terpenes to exert their protective action. Two ‘metabolic’ hypotheses suggest that isoprene acts as a kind of ‘safety valve’ which allows quenching energy or metabolites (Figure 4). In particular, one study  considers isoprene biosynthesis as a pathway for dissipation of excess photosynthetic energy, whereas a more recent one  postulates that isoprene biosynthesis prevents the overflow of chloroplastic DMADP. We consider as a ‘metabolic’ hypothesis also the ‘opportunistic hypothesis’  which suggests that volatile terpenes are somehow alternative to ‘essential’ terpenes (such as carotenoids, also formed through the MEP pathway, and for which an important antioxidant role is clearly established). The same pool of carbon may generate volatile and/or essential terpenes according to the need to face different constraints. This may be true also within the class of volatile terpenes, as it was recently demonstrated that isoprene decreases when monoterpenes are synthesized in poplar leaves attacked by beetles . A second group of hypotheses establishes a more distinctive functional role for volatile terpenes in plant
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protection against abiotic stressors (Figure 4). The main hypothesis is that isoprene, as well as monoterpenes, are thermoprotective molecules, able to stabilize chloroplast membranes during high temperature events  therefore protecting the photosynthetic apparatus. There is ample experimental support for this idea. In brief: (i) experiments in which plants have been fumigated with terpenes or in which the biosynthesis of these compounds have been chemically blocked have shown that the photosynthetic apparatus of isoprene-emitting plants is better protected against heat stress [158–160] and in particular against rapid temperature changes . The interesting idea behind the latter observation is that isoprene acts as a rapid mechanism of protection before plants can synthesize more complex molecules (including non-volatile terpenes) that improve thermal stabilization. (ii) An in silico experiment has demonstrated that isoprene may indeed partition into the phospho-lipid bilayer of membranes and maintain their stability, in particular during exposure to high temperature . (iii) Transgenic plants that have been engineered to emit isoprene or monoterpenes, or in which isoprene biosynthesis has been repressed are now available. Isoprene-emitting transgenic plants are more resistant to heat stress than wild types [53,54,163]. Volatile terpenes also appear to have a relevant antioxidant action. Again several lines of evidence support this hypothesis: (i) isoprene and monoterpenes have been shown to reduce the damage caused by ozone [115,122] and ROS [117,133,164]. (ii) Evidence of the reaction between isoprene and monoterpenes and ozone or other ROS has been produced by observing the appearance of the reaction products and the disappearance of the reagents [120,165]. In plants exposed to oxidative stresses isoprene has also been shown to quench nitrogen reactive species (namely NO) that can also have an important role as messenger molecules of the hypersensitive response to stress [166,167]. (iii) Transgenic tobacco (Nicotiana tabacum) plants that have been engineered to emit isoprene are more resistant to ozone toxicity than non-emitting wild types , and the ozone sensitivity of poplar clones is inversely related to their capacity to emit isoprene . However, both functions of isoprene have been challenged by studies in which the authors were not able to reproduce improved resistance to the stresses, especially when using artificial systems or transgenic plants. For instance, photosynthesis analysis showed that transgenic Arabidopsis plants do not benefit from isoprene when a transient heat stress occurs . In transgenic, isopreneemitting tobacco plants, tolerance of photosynthesis against transient high-temperature episodes could only be observed in lines emitting high levels of isoprene. Moreover, this effect was very mild and could only be identified over repetitive stress events . Finally, grey poplar (Populus canescens) plants in which isoprene emission was efficiently repressed were not more sensible to oxidative stresses . There may be good reasons that explain why the protection offered by volatile terpenes is not observed in specific cases: (i) the protection offered by these molecules may not be achieved when photosynthesis is already heavily impaired by prolonged or acute stresses, 161
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Figure 4. Schematic overview of the proposed physiological functions of volatile terpenes: (a) In the ‘Metabolic overflow’ hypothesis cytosolic PEPC activity plays a central role dividing the PEP pool into fractions (red line indicates the negative impact of PEPC activity on isoprene emission) available for isoprene biosynthesis and mitochondrial metabolism or carbon metabolism (e.g. amino acid biosynthesis) (cellular scheme adapted from ) explaining the downregulation of isoprene emission under enhanced CO2 . (b) The thermoprotective function of isoprene is evident when comparing the net CO2 assimilation in wild type and non-isoprene emitting poplars  when short periods of heat were applied. (c) The property of isoprene to quench NO accumulation and ozone injury is demonstrated with transgenic tobacco leaves modified in isoprene emission potential . (d) Interactions of isoprene with biomembranes are indicated by an in silico model study postulating a stabilizing effect of isoprene under high temperature . (Microscopic images of NO staining are provided by V. Velikova; photos from ozone-stressed tobacco leaves are provided by V. Velikova and C. Vickers).
or when the stresses are too mild to affect photosynthesis. Probably a window of stress exists in which these molecules substantially protect the photosynthetic apparatus. (ii) In some transgenic plants, especially Arabidopsis [53,54], the achieved emission of isoprene may be too low to induce any significant physiological effect. (iii) Downregulation or repression of the emission of isoprene in natural emitters may cause a large induction of other antioxidant molecules (e.g. ascorbate and tocopherol ). This is actually another strong indication that compensatory mechanisms are operated to replace the antioxidant action of volatile terpenes. The observation that volatile terpene emission is resistant and is often induced by stress conditions, including stress other than heat and strong oxidants (e.g. drought  and salt ), has led to the hypothesis that a unique mechanism, mostly related to the antioxidant capacity of volatile terpenes, exists . As for higher terpenes which are known for their antioxidant function (i.e. tocopherols, carotenoids, and sesquiterpenes), the antioxidant action of volatile terpenes may be due to the presence of conjugated double bounds . Indeed, it might be that the role of isoprene and monoterpenes in thermal protection also comes from their antioxidant properties. Hydrogen peroxide may also be produced by enhanced 162
photorespiration under moderately high temperatures. The generation of ROS under heat stress has been reported often [170,171]; however, there are many heat-tolerance mechanisms in plants . Probably, ROS scavenging would not be sufficient to protect leaves from heavy heat stress which directly affects thylakoid structure and the functions associated with thylakoid intactness, namely photochemical reactions. Thylakoid functionality would be explained better by a mechanism counteracting thylakoid leakiness and the consequent increase of cyclic electron flow around photosystem I. Xanthophylls, a group of non-volatile terpenes also originating from the MEP pathway, may indeed render the thylakoid membranes more resistant to heat , and this function may also be carried out by volatile terpenes. The localization of ISPS in the stromal side of thylakoidal membranes [35,174] and the hydrophobic nature of isoprene are expected to assist with its partition into photosynthetic membranes . Lipophilic isoprene partitioned into membranes can also prevent the formation of water channels responsible for the membrane leakiness at high temperature [158,175]. Isoprene could also enhance hydrophobic interactions within thylakoids and thereby stabilize interactions between lipids and/or membrane proteins during episodes of heat-shock or high temperature stress conditions .
Review Preliminary research using circular dichroism spectroscopy, a valuable tool for probing the molecular architecture of the complexes and supercomplexes and their macro-organization in the membrane system  confirms that a higher thermal stability of thylakoid membranes is induced in transgenic plants that are able to emit isoprene (Fortunati et al., personal communication). Conclusions and future directions We have seen that abiotic stresses may induce the emission of multiple BVOCs. Many of the emitted compounds are synthesized from the degradation of cellular structures and may be used as reliable indicators of cell wall degradation (methanol and methane) or membrane denaturation (C6 volatiles). In the case of volatile terpenes the induced emissions reflect the elicitation of the MEP pathway, revealing important function(s) of these compounds in the protection against stresses. The physiological and ecological functions of volatile terpenes are well established; however, more studies are needed to reveal the molecular and biochemical mechanisms that oversee the protective role of volatile terpenes. Induction or alteration of these BVOCs emissions by abiotic stresses and other climate change factors may also contribute to modify the communication of plants with other organisms, namely herbivores and carnivores that use BVOCs emissions as olfactory cues to retrieve hosts suitable both as food and shelter. Technical advances have made it possible to detect induced emissions of MeSA not only in response to biotic stresses but also in plants subjected to abiotic stresses. This volatile is therefore emerging as a central molecule for the signalling of stresses and for the consequent activation of systemic acquired resistance and hypersensitive responses. Acknowledgments We thank I. Zimmer for critical reading of the manuscript and C. Vickers and V. Velikova for providing us with tobacco images. The work was supported by the ESF Project Volatile Organic Compounds in the Biosphere-Atmosphere System (VOCBAS). The German Research Foundation (DFG; SCHN653/4 to J.-P.S.) supported the research within the German joint research group ‘Poplar – a model to address tree-specific questions’.
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