How specialized volatiles respond to chronic and short‐term physiological and shock heat stress in Brassica nigra

Brassicales release volatile glucosinolate breakdown products upon tissue mechanical damage, but it is unclear how the release of glucosinolate volatiles responds to abiotic stresses such as heat stress. We used three different heat treatments, simulating different dynamic temperature conditions in the field to gain insight into stress‐dependent changes in volatile blends and photosynthetic characteristics in the annual herb Brassica nigra (L.) Koch. Heat stress was applied by either heating leaves through temperature response curve measurements from 20 to 40 °C (mild stress), exposing plants for 4 h to temperatures 25–44 °C (long‐term stress) or shock‐heating leaves to 45–50 °C. Photosynthetic reduction through temperature response curves was associated with decreased stomatal conductance, while the reduction due to long‐term stress and collapse of photosynthetic activity after heat shock stress were associated with non‐stomatal processes. Mild stress decreased constitutive monoterpene emissions, while long‐term stress and shock stress resulted in emissions of the lipoxygenase pathway and glucosinolate volatiles. Glucosinolate volatile release was more strongly elicited by long‐term stress and lipoxygenase product released by heat shock. These results demonstrate that glucosinolate volatiles constitute a major part of emission blend in heat‐stressed B. nigra plants, especially upon chronic stress that leads to induction responses.     

Changes in photosynthetic rate and stress volatile emissions through desiccation‐rehydration cycles in desiccation‐tolerant epiphytic filmy ferns (Hymenophyllaceae)

Exposure to recurrent desiccation cycles carries a risk of accumulation of reactive oxygen species that can impair leaf physiological activity upon rehydration, but changes in filmy fern stress status through desiccation and rewatering cycles have been poorly studied. We studied foliage photosynthetic rate and volatile marker compounds characterizing cell wall modifications (methanol) and stress development (lipoxygenase [LOX] pathway volatiles and methanol) through desiccation–rewatering cycles in lower‐canopy species Hymenoglossum cruentum and Hymenophyllum caudiculatum, lower‐ to upper‐canopy species Hymenophyllum plicatum and upper‐canopy species Hymenophyllum dentatum sampled from a common environment and hypothesized that lower canopy species respond more strongly to desiccation and rewatering. In all species, rates of photosynthesis and LOX volatile emission decreased with progression of desiccation, but LOX emission decreased with a slower rate than photosynthesis. Rewatering first led to an emission burst of LOX volatiles followed by methanol, indicating that the oxidative burst was elicited in the symplast and further propagated to cell walls. Changes in LOX emissions were more pronounced in the upper‐canopy species that had a greater photosynthetic activity and likely a greater rate of production of photooxidants. We conclude that rewatering induces the most severe stress in filmy ferns, especially in the upper canopy species.     

Ozone‐induced foliar damage and release of stress volatiles is highly dependent on stomatal openness and priming by low‐level ozone exposure in Phaseolus vulgaris

Acute ozone exposure triggers major emissions of volatile organic compounds (VOCs), but quantitatively, it is unclear how different ozone doses alter the start and the total amount of these emissions, and the induction rate of different stress volatiles. It is also unclear whether priming (i.e. pre‐exposure to lower O₃ concentrations) can modify the magnitude and kinetics of volatile emissions. We investigated photosynthetic characteristics and VOC emissions in Phaseolus vulgaris following acute ozone exposure (600 nmol mol^?1 for 30 min) under illumination and in darkness and after priming with 200 nmol mol^?1 O₃ for 30 min. Methanol and lipoxygenase (LOX) pathway product emissions were induced rapidly, followed by moderate emissions of methyl salicylate (MeSA). Stomatal conductance prior to acute exposure was lower in darkness and after low O₃ priming than in light and without priming. After low O₃ priming, no MeSA and lower LOX emissions were detected under acute exposure. Overall, maximum emission rates and the total amount of emitted LOX products and methanol were quantitatively correlated with total stomatal ozone uptake. These results indicate that different stress volatiles scale differently with ozone dose and highlight the key role of stomatal conductance in controlling ozone uptake, leaf injury and volatile release.     

Regulation of Floral Terpenoid Emission and Biosynthesis in Sweet Basil (<Emphasis Type="Italic">Ocimum basilicum</Emphasis>)

Past studies have focused on the composition of essential oil of Ocimum basilicum leaves, but data on composition and regulation of its aerial emissions, especially floral volatile emissions, are scarce. We studied the chemical profile, within-flower spatial distribution (sepals, petals, pistils with stamina, and pedicels), diurnal emission kinetics and effects of exogenous methyl jasmonate (MeJA) application on the emission of floral volatiles by dynamic headspace collection, and identification using gas chromatography-mass spectrometry (GC–MS) and proton-transfer reaction mass spectrometry. We observed more abundant floral emissions from flowers compared with leaves. Sepals were the main emitters of floral volatiles among the flower parts studied. The emissions of lipoxygenase compounds and monoterpenoids, but not sesquiterpene emissions, displayed a diurnal variation driven by light. Response to exogenous MeJA treatment of flowers consisted of a rapid stress response and a longer-term acclimation response. The initial response was associated with enhanced emissions of fatty acid derivatives, monoterpenoids, and sesquiterpenoids without variation of the composition of individual compounds. The longer-term response was associated with enhanced monoterpenoid and sesquiterpenoid emissions with profound changes in the emission spectrum. According to correlated patterns of terpenoid emission changes upon stress, highlighted by a hierarchical cluster analysis, candidate terpenoid synthases responsible for observed diversity and complexity of released terpenoid blends were postulated. We conclude that flower volatile emissions differ quantitatively and qualitatively from leaf emissions, and overall contribute importantly to O. basilicum flavor, especially under stress conditions.     

Detection of plant volatiles after leaf wounding and darkening by proton transfer reaction "time-of-flight" mass spectrometry (PTR-TOF)

Proton transfer reaction-time of flight (PTR-TOF) mass spectrometry was used to improve detection of biogenic volatiles organic compounds (BVOCs) induced by leaf wounding and darkening. PTR-TOF measurements unambiguously captured the kinetic of the large emissions of green leaf volatiles (GLVs) and acetaldehyde after wounding and darkening. GLVs emission correlated with the extent of wounding, thus confirming to be an excellent indicator of mechanical damage. Transient emissions of methanol, C5 compounds and isoprene from plant species that do not emit isoprene constitutively were also detected after wounding. In the strong isoprene-emitter Populus alba, light-dependent isoprene emission was sustained and even enhanced for hours after photosynthesis inhibition due to leaf cutting. Thus isoprene emission can uncouple from photosynthesis and may occur even after cutting leaves or branches, e.g., by agricultural practices or because of abiotic and biotic stresses. This observation may have important implications for assessments of isoprene sources and budget in the atmosphere, and consequences for tropospheric chemistry.     

Release of lipoxygenase products and monoterpenes by tomato plants as an indicator of Botrytis cinerea-induced stress

Changes in emission of volatile organic compounds (VOCs) from tomato induced by the fungus Botrytis cinerea were studied in plants inoculated by spraying with suspensions containing B. cinerea spores. VOC emissions were analysed using on-line gas chromatography–mass spectrometry, with a time resolution of about 1 h, for up to 2 days after spraying. Four phases were delimited according to the starting point and the applied day/night rhythm of the experiments. These phases were used to demonstrate changes in VOC flux caused by B. cinerea infestation. Tomato plants inoculated with B. cinerea emitted a different number and amount of VOCs after inoculation compared to control plants that had been sprayed with a suspension without B. cinerea spores. The changes in emissions were dependent on time after inoculation as well as on the severity of infection. The predominant VOCs emitted after inoculation were volatile products from the lipoxygenase pathway (LOX products). The increased emission of LOX products proved to be a strong indicator of a stress response, indicating that VOC emissions can be used to detect plant stress at an early stage. Besides emission of LOX products, there were also increases in monoterpene emissions. However, neither increased emission of LOX products nor of monoterpenes is specific for B. cinerea attack. The emission of LOX products is also induced by other stresses, and increased emission of monoterpenes seems to be the result of mechanical damage induced by secondary stress impacts on leaves.     

Emission of constitutive isoprene,induced monoterpenes,and other volatiles under high temperatures in Eucalyptus camaldulensis: A 13C labelling study

Eucalypts are major emitters of biogenic volatile organic compounds (BVOCs), especially volatile isoprenoids. Emissions and incorporation of ¹³C in BVOCs were measured in Eucalyptus camaldulensis branches exposed to rapid heat stress or progressive temperature increases, in order to detect both metabolic processes and their dynamics. Isoprene emission increased and photosynthesis decreased with temperatures rising from 30°C to 45°C, and an increasing percentage of unlabelled carbon was incorporated into isoprene in heat‐stressed leaves. Intramolecular labelling was also incomplete in isoprene emitted by heat‐stressed leaves, suggesting increasing contribution of respiratory (and possibly also photorespiratory) carbon. At temperature above 45°C, a drop of isoprene emission was mirrored by the appearance of unlabelled monoterpenes, green leaf volatiles, methanol, and ethanol, indicating that the emission of stored volatiles was mainly induced by cellular damage. Emission of partially labelled acetaldehyde was also observed at very high temperatures, suggesting a double source of carbon, with a large unlabelled component likely transported from roots and associated to the surge of transpiration at very high temperatures. Eucalypt plantations cover large areas worldwide, and our findings may dramatically change forecast and modelling of future BVOC emissions at planetary level, especially considering climate warming and frequent heat waves.     

Synergistic interactions between volicitin,jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays

Plants display differential responses following mechanical damage and insect herbivory. Both caterpillar attack and the application of caterpillar oral secretions (OS) to wounded leaves stimulates volatile emission above mechanical damage alone. Volicitin ( N- 17-hydroxylinolenoyl- l -glutamine), present in beet armyworm (BAW, Spodoptera exigua ) OS, is a powerful elicitor of volatiles in excised maize seedlings ( Zea mays cv. Delprim). We consider some of the mechanistic differences between wounding and insect herbivory in maize by examining the activity of volicitin, changes in jasmonic acid (JA) levels, and volatile emission from both intact plant and excised leaf bioassays. Compared to mechanical damage alone, volicitin stimulated increases in both JA levels and sesquiterpene volatiles when applied to intact plants. In a bioassay comparison, excised leaves were more sensitive and produced far greater volatile responses than intact plants following applications of both volicitin and JA. In the excised leaf bioassay, volicitin applications (10–500 pmol) to wounded leaves resulted in dose dependent JA increases and a direct positive relationship between JA and sesquiterpene volatile emission. Interestingly, volicitin-induced JA levels did not differ between intact and excised bioassays, suggesting a possible interaction of JA with other regulatory signals in excised plants. In addition to JA, insect herbivory is known to stimulate the production of ethylene. Significant increases in ethylene were induced only by BAW herbivory and not by either wounding or volicitin treatments. Using intact plant bioassays, ethylene (at 1 µl l⁻¹ or less) greatly promoted volatile emission induced by volicitin and JA but not mechanical damage alone. For intact plants, wounding, elicitor-induced JA and insect-induced ethylene appear to be important interacting components in the stimulation of insect-induced volatile emission.     

Leaf rust induced volatile organic compounds signalling in willow during the infection

Plants are known to emit volatile organic compounds (VOC) in response to various biotic or abiotic stresses. Although the VOC emission in the case of insect attacks is well described, there is only little known about the impact of pathogens on plant emission. In the present study, we used a willow-leaf rust system to describe the effects of a biotrophic fungal infection on the VOC emission pattern of willow leaves. We detected that isoprene emissions from rust-infected leaves decreased threefold compared to control. The total monoterpene emissions did not change although a stress-signalling compound (Z)-β-ocimene showed an increase in infected plants on several days. The infection also increased the emission of sesquiterpenes and lipoxygenase products (LOX) by factors of 175-fold and 10-fold, respectively. The volatile emission signals showed two clear peaks during the experiment. At 6, 7 and 12 days post-infection (dpi), the relative volatile emission signal increased to about sixfold compared to uninfected plants. These time points are directly connected to rust infection since at 6 dpi the first rust pustules appeared on the leaves and at 12 dpi necrosis had developed around several pustules. We present correlations between LOX and sesquiterpene emission signals, which suggest at least two different steps in eliciting the volatile emission.     

Massive release of volatile organic compounds due to leaf midrib wounding in <Emphasis Type="Italic">Populus tremula</Emphasis>

We investigated the rapid initial response to wounding damage generated by straight cuts to the leaf lamina and midrib transversal cuts in mature aspen (Populus tremula) leaves that can occur upon herbivore feeding. Wound-induced volatile emission time-courses of 24 compounds were continuously monitored by a proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS). After the mechanical wounding, an emission cascade was rapidly elicited, resulting in emissions of key stress volatiles methanol, acetaldehyde and volatiles of the lipoxygenase pathway, collectively constituting ca. 99% of the total emission. For the same wounding magnitude, midrib cuts lead to six-fold greater emissions of volatiles per mm² of surface cut than lamina cuts during the first emission burst (shorter than 7 min), and exhibited a particularly high methanol emission compared to the emissions of other volatiles. This evidence suggests that feeding by herbivores capable of consuming the leaf midrib can result in disproportionally greater volatile release than feeding by smaller herbivores incapable of biting through the major veins.     

The piercing-sucking herbivores Lygus hesperus and Nezara viridula induce volatile emissions in plants

Plant volatiles induced by herbivory are often used as olfactory cues by foraging herbivores and their natural enemies, and thus have potential for control of agricultural pests. Compared to chewing insects and mites, little is known about plant volatile production following herbivory by insects with piercing-sucking mouthparts. Here, we studied factors (insect life stage, gender, the role of salivary glands, and type of bioassay used for volatile induction) that influence the induction of plant volatiles by two agriculturally important hemipterans, Lygus hesperus and Nezara viridula. Feeding on intact cotton by virgin females of L. hesperus induced 2.6-fold greater volatile response compared to that induced by mated females, possibly due to increased feeding activity by virgin females. This plant volatile response was associated with elicitors present in the insect's salivary glands as well as to the degree of mechanical injury. Feeding injury by N. viridula females also increased volatile emissions in intact maize by approximately 2-fold compared to control plants. Maize seedlings injured by N. viridula emitted higher amounts of the monoterpene linalool, the sesquiterpenes (E)-beta-caryophyllene, alpha-trans-bergamotene, and (E,E)-beta-farnesene, and the homoterpene (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, but not amounts of green leaf volatiles, compared to uninjured plants. Emissions from intact maize injured by adult males were lower than those emitted by adult females of the same age and did not differ from those emitted by uninjured plants. Similarly, feeding by virgin female N. viridula followed by excision led to 64% higher quantities of volatiles compared to untreated plants. Volatile emission in excised plants, however, was considerably greater than in intact plants, suggesting that careful consideration must be given to bioassay design in studies of herbivore-induced plant volatiles. Salivary gland extracts of N. viridula led to sesquiterpene emissions approximately 2.5-fold higher than for controls, although no significant differences were observed for green leaf volatiles, monoterpenes, and homoterpenes. These results indicate that L. hesperus and female N. viridula feeding induce volatile production in plants, and that volatile production is affected by gender and life stage of the bug. Although oviposition and mechanical injury by stylets may increase release of volatiles, elicitors from salivary glands of L. hesperus and N. viridula also seem to play a role in the emission of plant volatiles.     

Dynamics of the enhanced emissions of monoterpenes and methyl salicylate, and decreased uptake of formaldehyde, by Quercus ilex leaves after application of jasmonic acid

Jasmonic acid (JA) is a signalling compound with a key role in both stress and development in plants, and is reported to elicit the emission of volatile organic compounds (VOCs). Here we studied the dynamics of such emissions and the linkage with photosynthetic rates and stomatal conductance. We sprayed JA on leaves of the Mediterranean tree species Quercus ilex and measured the photosynthetic rates, stomatal conductances, and emissions and uptake of VOCs using proton transfer reaction mass spectrometry and gas chromatography after a dark-light transition. Jasmonic acid treatment delayed the induction of photosynthesis and stomatal conductance by approx. 20 min, and decreased them 24 h after spraying. Indications were found of both stomatal and nonstomatal limitations of photosynthesis. Monoterpene emissions were enhanced (20-30%) after JA spraying. Jasmonic acid also increased methyl salicylate (MeSa) emissions (more than twofold) 1 h after treatment, although after 24 h this effect had disappeared. Formaldehyde foliar uptake decreased significantly 24 h after JA treatment. Both biotic and abiotic stresses can thus affect plant VOC emissions through their strong impact on JA levels. Jasmonic acid-mediated increases in monoterpene and MeSa emissions might have a protective role when confronting biotic and abiotic stresses.     

Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers

Despite the fact that Arabidopsis is largely self-pollinating, its flowers emit a complex mixture of terpene volatiles consisting predominantly of a large group of over 20 sesquiterpenes. Here we report that only two terpene synthases, encoded by the florally expressed genes At5g23960 and At5g44630, are responsible for the formation of virtually all sesquiterpenes found in the Arabidopsis floral volatile blend. Two independent mutant lines with T-DNA insertions in the previously identified At5g23960 gene lacked the emission of three sesquiterpenes, including the main sesquiterpene volatile (E)-beta-caryophyllene, confirming the previous in vitro functional assignment for this gene. Flowers of a mutant line carrying a T-DNA insertion in gene At5g44630 emitted these three sesquiterpenes, but did not emit any of the remaining sesquiterpene volatiles. An At5g44630 cDNA was expressed in Escherichia coli and the produced protein catalyzed the conversion of farnesyl diphosphate into over 15 sesquiterpenes in similar proportions to those found in the floral volatile blend. At5g23960 and At5g44630 promoter-beta-glucuronidase (GUS) fusion experiments demonstrated that both genes are expressed in several parts of the Arabidopsis flower, with strong At5g23960 promoter-GUS activity in the stigma and strong expression of At5g44630 in intrafloral nectaries. Given the previously reported antimicrobial activity of terpenes, their production in stigmas and nectaries may serve to inhibit microbial infection at these vulnerable sites. A survey of 37 Arabidopsis thaliana ecotypes revealed quantitative, but almost no qualitative, variations of floral monoterpene and sesquiterpene emissions suggesting that floral terpene volatiles must play some significant role in the life of the Arabidopsis plant.     

Flooding induced emissions of volatile signalling compounds in three tree species with differing waterlogging tolerance

To gain insight into variations in waterlogging responsiveness, net assimilation rate, stomatal conductance, emissions of isoprene and marker compounds of anoxic metabolism ethanol and acetaldehyde, and stress marker compounds nitric oxide (NO), volatile products of lipoxygenase (LOX) pathway and methanol were studied in seedlings of temperate deciduous tree species Alnus glutinosa, Populus tremula and Quercus rubra (from highest to lowest waterlogging tolerance) throughout sustained root zone waterlogging of up to three weeks. In all species, waterlogging initially resulted in reductions in net assimilation and stomatal conductance and enhanced emissions of ethanol, acetaldehyde, NO, LOX products and methanol, followed by full or partial recovery depending on process and species. Strong negative correlations between g_s and internal NO concentration and NO flux, valid within and across species, were observed throughout the experiment. Isoprene emission capacity was not related to waterlogging tolerance. Less waterlogging tolerant species had greater reduction and smaller acclimation capacity in foliage physiological potentials, and larger emission bursts of volatile stress marker compounds. These data collectively provide encouraging evidence that emissions of volatile organics and NO can be used as quantitative measures of stress tolerance and acclimation kinetics in temperate trees.     

采用多次顶空固相微萃取分析拟南芥绿叶挥发性物质

顶空固相微萃取作为一种新的挥发性和半挥发性物质分析技术,被广泛应用于植物样品的定性分析。由于进行顶空分析时,挥发性组分间的基质效应以及较为复杂的扩散和吸附过程,定量分析一直是SPME分析应用的难题。目标分析物的量看作是达到吸附平衡后单一萃取的物质量的总和,则无需考虑分析样品在顶空、萃取涂层间的分配,同时可以消除基质效应。在利用标准物质进行校正后只需要一次顶空萃取,即可求出分析物质的总量。首先利用DVB/CAR/PDMS定性得到拟南芥挥发性物质的组成,然后采用CAR/PDMS涂层定量,分析了拟南芥的3种绿叶挥发性物质,优化后萃取条件为40℃萃取20min,相对标准偏差小于12%,在3株植物样品中这些挥发性物质的量为78.6~158.4ng.g-1。     

The influence of intact-plant and excised-leaf bioassay designs on volicitin- and jasmonic acid-induced sesquiterpene volatile release in Zea mays

Induced plant responses to insect attack include the release of volatile chemicals. These volatiles are used as host-location signals by foraging parasitoids, which are natural enemies of insect herbivores. A plant's response to herbivory can be influenced by factors present in insect oral secretions. Volicitin (N-(17-hydroxylinolenoyl)-L-glutamine), identified in beet armyworm (Spodoptera exigua) oral secretions, stimulates volatile release in corn (Zea mays L.) seedlings in a manner similar to beet armyworm herbivory. Volicitin is hypothesized to trigger release of induced volatiles, at least in part, by modulating levels of the wound hormone, jasmonic acid (JA). We compare the sesquiterpene volatile release of damaged leaves treated with aqueous buffer only or with the same buffer containing volicitin or JA. Leaves were damaged by scratching with a razor and test solutions were applied to the scratched area. The leaves were either excised from the plant or left intact shortly after this treatment. Plants were treated at three different times (designated as Evening, Midnight, and Morning) and volatiles were collected in the subsequent photoperiod. JA and volicitin treatments stimulated the release of volatile sesquiterpenes, namely beta-caryophyllene, (E)-alpha-bergamotene, and (E)-beta-farnesene. In all cases, JA stimulated significant sesquiterpene release above mechanical damage alone. Volicitin induced an increase in sesquiterpene volatiles for all excised-leaf bioassays and the Midnight intact plants. Volicitin treatments in the Evening and Morning intact plants produced more sesquiterpenes than the untreated controls, while mechanical damage alone produced an intermediate response that did not differ from either treatment group. Excised leaves produced a 2.5- to 8.0-fold greater volatile response than similarly treated intact plants. Excision also altered the ratio of JA-and volicitin-induced sesquiterpene release by preferentially increasing (E)-beta-farnesene levels relative to beta-caryophyllene. The inducibility of volatile release varied with time of treatment. On average, sesquiterpene release was highest in the Midnight excised leaves and lowest in the Morning intact plants. The duration of induced volatile release also differed between treatments. On average, JA produced a sustained release of sesquiterpenes over time, with over 20% of the combined sesquiterpenes released in the third and final volatile collection period. In contrast, less than 8% of the combined sesquiterpenes induced by volicitin were emitted during this period. The large quantitative differences between intact plants and detached leaves suggest that the results of assays using excised tissues should be cautiously interpreted when considering intact-plant models.     

Growth pattern and carbon allocation to volatile leaf terpenes under nitrogen-limiting conditions in Heterotheca subaxillaris (Asteraceae)

Summary Rosettes of Heterotheca subaxillaris were grown at four levels of nitrate. Individual leaf volatile mono- and sesquiterpene content, leaf nitrogen content, and root and shoot dry weight were measured on individual leaves every two weeks for 18 weeks. Rosettes with the highest nitrate availability had 2.2-fold greater leaf nitrogen levels compared to plants with the lowest availability. As nitrate availability became increasingly limited, carbon allocation to both volatile leaf terpenes and root growht increased. Leaf mono- and sesquiterpene content was greatest in the young leaves of individuals growing at the lowest nitrate availability conditions. Higher levels of carbon-based herbivore-deterring chemicals in nitrate-limited plants may increase net productivity through retention of nitrogen that would otherwise be lost to herbivory.     

Few long-term effects of simulated climate change on volatile organic compound emissions and leaf chemistry of three subarctic dwarf shrubs

Climate change is exposing arctic ecosystems to higher temperature, increased nutrient availability and shading due to the increasing cloud cover and the expanding forests. In this work, we assessed how these factors affect the emissions of biogenic volatile organic compounds (BVOCs) from three subarctic dwarf shrub species in a field experiment after 18 treatment years. Of the studied species the willow Salix phylicifolia L. was the only isoprene-emitter with an emission potential of 16.1 ± 8.4 μg g⁻¹ dw h⁻¹ (at 30 °C and photosynthetic photon flux density of 1000 μmol m⁻² s⁻¹). The dwarf birch Betula nana L. had significant emissions of various reactive BVOCs, including monoterpenes and sesquiterpenes. The evergreen Cassiope tetragona (L.) D. Don emitted high amounts of mono- and sesquiterpenes. Due to chance, the temperature in the warming treatment (employing open-top plastic tents) and the unwarmed treatments was similar at the time of the measurements, and therefore long-term indirect effects of warming could be assessed without interference of temperature differences at the time of measurement. The long-term warming had not altered foliar N, P or condensed tannin concentrations, but it had led to other chemical changes detected in the near-infrared reflectance spectra of the leaves. Nevertheless, there were no significant differences in the BVOC emissions per unit leaf mass measured by the dynamic enclosure method and gas chromatography-mass spectrometry. Annual additions of NPK fertilizer, which mimicked increased nutrient availability, had accumulated P in the leaves of all species. In addition, fertilization marginally increased the leaf N concentration of B. nana. The only significant fertilization effect on BVOC emissions was a stimulation of emission of the sesquiterpene β-selinene from S. phylicifolia. The shading treatment obtained by dome-shaped hessian tents did not cause clear long-term changes in leaf chemistry or BVOC emissions. The only observed change was a marginally significant increase in sesquiterpene emissions from B. nana. When the treatment effects on long-term biomass changes in the different treatments were taken into account by proportioning the BVOC emissions to the biomass of each species in the field treatments, warming led to a significant increase and shading to a decrease in the total BVOC emissions per unit ground area from B. nana. These results highlight the importance of an integrated approach for realistic assessment of responses to climate change.     

Physiological and physicochemical controls on foliar volatile organic compound emissions

Plant leaves emit a broad spectrum of organic compounds that typically play multiple roles in plant protection. Furthermore, most of these compounds actively participate in tropospheric chemistry. There has been rapid progress in understanding how the emission of volatiles is regulated, mostly focusing on the biochemical controls over compound production. However, physicochemical characteristics such as low volatility or diffusion can also control the emissions and interact with physiological limitations. In particular, non-specific leaf storage of less volatile compounds smooths the emission responses to fluctuating environmental conditions, and diffusion through stomata leads to conspicuous emission bursts after stomatal opening and modifications of diurnal emission time courses. Because natural conditions always fluctuate, both physiological and physicochemical controls exert a major influence over plant volatile emissions.     

The biochemical origin of pentenol emissions from wounded leaves

Large releases of 1-penten-3-ol (pentenol) and 1-penten-3-one (pentenone) were recently observed from a variety of leaves subjected to freeze-thaw damage in the presence of oxygen. In order to understand the biochemical origins of these volatiles, soybean leaf extracts were used to determine if the formation of pentenol and pentenone can be explained by known O(2)-dependent lipoxygenase (LOX) reactions. Enzymatic formation of these C5 volatiles was found to be dependent on alpha-linolenic acid or the 13(S)-hydroperoxide of alpha-linolenic acid [13(S)-HPOT] and blocked by LOX inhibitors. Five soybean leaf LOX isozyme genes (VLXA, VLXB, VLXC, VLXD, and VLXE) were then expressed in Escherichia coli and used in in vitro incubations with 13(S)-HPOT to test for volatile formation. Each of the LOX isozymes catalyzed the formation of low levels of pentenol, but not pentenone. It therefore seems likely that the C5,13-cleavage activity of LOX is the direct source of abundant pentenol and the indirect source of pentenone observed upon leaf wounding.