Significantly enhanced production of isoprene by ordered coexpression of genes dxs, dxr, and idi in Escherichia coli

We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-d-xylulose 5-phosphate synthase (dxs), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg?g^?1?h^?1 (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.     

Microbial oxidation of isoprene, a biogenic foliage volatile and of 1,3-butadiene, an anthropogenic gas

Nocardia strains that were able to degrade isoprene were isolated from several locations using enrichment cultures with isoprene or 1,3-butadiene as the sole carbon and energy source. Specific growth rates of representative isolates on isoprene and 1,3-butadiene ranged from 0.05 to 0.2 h⁻¹. The initial oxygenation of both 1,3-butadiene and isoprene was mediated by mono-oxygenases which converted these alkadienes into the respective epoxyalkanes.     

Fractionation of Carbon Isotopes during Biogenesis of Atmospheric Isoprene

The stable carbon isotope composition of isoprene emitted from leaves of red oak (Quercus rubra L.) was measured. Isoprene was depleted in ¹³C relative to carbon recently fixed by photosynthesis. The difference in isotope composition between recently fixed carbon and emitted isoprene was independent of the isotopic composition of the source CO₂. β-Carotene, an isoprenoid plant constituent, was depleted in ¹³C relative to whole leaf carbon to the same degree as isoprene, but fatty acids were more depleted. Isoprene emitted from leaves fed abscisic acid was much less depleted in ¹³C than was isoprene emitted from unstressed leaves. We conclude that isoprene is made from an isoprenoid precursor that is derived from acetyl-CoA made from recent photosynthate. The carbon isotope composition of isoprene in the atmosphere is likely to be slightly more negative (less ¹³C) than C₃ plant material but when plants are stressed the isotopic composition could vary.     

Thermotolerance of Leaf Discs from Four Isoprene-Emitting Species Is Not Enhanced by Exposure to Exogenous Isoprene

The effects of exogenously supplied isoprene on chlorophyll fluorescence characteristics were examined in leaf discs of four isoprene-emitting plant species, kudzu (Pueraria lobata [Willd.] Ohwi.), velvet bean (Mucuna sp.), quaking aspen (Populus tremuloides Michx.), and pussy willow (Salix discolor Muhl). Isoprene, supplied to the leaves at either 18 μL L⁻¹ in compressed air or 21 μL L⁻¹ in N₂, had no effect on the temperature at which minimal fluorescence exhibited an upward inflection during controlled increases in leaf-disc temperature. During exposure to 1008 μmol photons m⁻² s⁻¹ in an N₂ atmosphere, 21 μL L⁻¹ isoprene had no effect on the thermally induced inflection of steady-state fluorescence. The maximum quantum efficiency of photosystem II photochemistry decreased sharply as leaf-disc temperature was increased; however, this decrease was unaffected by exposure of leaf discs to 21 μL L⁻¹ isoprene. Therefore, there were no discernible effects of isoprene on the occurrence of symptoms of high-temperature damage to thylakoid membranes. Our data do not support the hypothesis that isoprene enhances leaf thermotolerance.     

Identification of novel isoprene synthases through genome mining and expression in Escherichia coli

Isoprene is a naturally produced hydrocarbon emitted into the atmosphere by green plants. It is also a constituent of synthetic rubber and a potential biofuel. Microbial production of isoprene can become a sustainable alternative to the prevailing chemical production of isoprene from petroleum. In this work, sequence homology searches were conducted to find novel isoprene synthases. Candidate sequences were functionally expressed in Escherichia coli and the desired enzymes were identified based on an isoprene production assay. The activity of three enzymes was shown for the first time: expression of the candidate genes from Ipomoea batatas, Mangifera indica, and Elaeocarpus photiniifolius resulted in isoprene formation. The Ipomoea batatas isoprene synthase produced the highest amounts of isoprene in all experiments, exceeding the isoprene levels obtained by the previously known Populus alba and Pueraria montana isoprene synthases that were studied in parallel as controls.     

Endogenous isoprene protects Phragmites australis leaves against singlet oxygen

The possible protective role of endogenous isoprene against oxidative stress caused by singlet oxygen (¹O₂) was studied in the isoprene‐emitting plant Phragmites australis. Leaves emitting isoprene and leaves in which isoprene synthesis was inhibited by fosmidomycin were exposed to increasing concentrations of ¹O₂ generated by Rose Bengal (RB) sensitizer at different light intensities. In isoprene‐emitting leaves, photosynthesis and H₂O₂ and malonyldialdehyde (MDA) contents were not affected by low to moderate ¹O₂ concentrations generated at light intensities of 800 and 1240 µmol m^?2 s^?1, but symptoms of damage and reactive oxygen accumulation started to be observed when high levels of ¹O₂ were generated by very high light intensity (1810 µmol m^?2 s^?1). A dramatic decrease in photosynthetic performance and an increase in H₂O₂ and MDA levels were measured in isoprene‐inhibited RB‐fed leaves, but photosynthesis was not significantly inhibited in leaves in which the isoprene leaf pool was reconstituted by fumigating exogenous isoprene. The inhibition of photosynthesis in isoprene‐inhibited leaves was linearly associated with the light intensity and with the consequently formed ¹O₂. Hence, physiological levels of endogenous isoprene may supply protection against ¹O₂. The protection mechanisms may involve a direct reaction of isoprene with ¹O₂. Moreover, as it is a small lipophilic molecule, it may assist hydrophobic interactions in membranes, resulting in their stabilization. The isoprene‐conjugated double bond structure may also quench ¹O₂ by facilitating energy transfer and heat dissipation. This action is typical of other isoprenoids, but we speculate that isoprene may provide a more dynamic protection mechanism as it is synthesized promptly when high light intensity produces ¹O₂.     

Structure of Isoprene Synthase Illuminates the Chemical Mechanism of Teragram Atmospheric Carbon Emission

The X-ray crystal structure of recombinant PcISPS (isoprene synthase from gray poplar hybrid Populus × canescens) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg²⁺ and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at 2.8 Å resolution. Analysis of these structures suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate-leaving group serves as a general base. This chemical mechanism is responsible for the annual atmospheric emission of 100 Tg of isoprene by terrestrial plant life. Importantly, the PcISPS structure promises to guide future protein engineering studies, potentially leading to hydrocarbon fuels and products that do not rely on traditional petrochemical sources.     

The role of isoprene in insect herbivory

Several hypotheses have previously been proposed to explain the function of isoprene in plants, including its ability to protect the leaf metabolic machinery from transient high temperature^1,2 and from oxidative stress.³ Isoprene may also serve as a metabolic overflow mechanism for carbon or photosynthetic energy^4–6 and may promote flowering in neighbouring plants.⁷ We have reported recently that isoprene can be detected by a herbivore, Manduca sexta, and that it directly deters them from feeding, with an isoprene emission threshold level of <6 nmol m⁻² s⁻¹.⁸ We demonstrated this using both in vivo experiments, using isoprene-emitting transgenic tobacco plants (Nicotiana tabacum cv. Samsun) and non-emitting azygous control plants, and in vitro experiments, using an artificial (isoprene-emitting and non-emitting control) diet. Here we discuss the potential role of isoprene in plant-herbivore interactions and the possibility that isoprene actually serves multiple purposes in plants.Key words: multiple functions, deterrence, signal, Manduca sextaIsoprene (C₅H₈; 2-methyl 1,3-butadiene) is a volatile organic compound that is produced in many, but not all, plant species.⁹ Because of its high volatility, once produced, isoprene is rapidly released from the leaf surface into the atmosphere, the chemical and physical properties of which can be altered due to the high chemical reactivity and large mass flux rate of isoprene.^10,11 While isoprene production in plants consumes considerable amounts of energy,¹² its role in plants has not yet been fully explained. Isoprene production and emission may confer significant benefits for plants that balance or outweigh its high production costs,¹³ but why only some plants produce the compound and why several beneficial effects have been demonstrated is not clear.Isoprene has recently been reported to directly deter M. sexta caterpillars from feeding. This is supported both by in vivo and in vitro studies and we propose that isoprene may confer competitive advantage to plant against herbivory.⁸ The reason why isoprene functions as a deterrent is not clear. The very obvious explanation is that isoprene is a harmful substance acting, for example as a digestibility reducing substance or toxin, and that it might operate together with other types of plant defences. However, another possibility is that isoprene might be a harmless deterrent as, for example, in the case of the alkaloid gramine in grasses. A grasshopper (Locusta migratoria) normally avoids this compound, but when force fed, habituation occurs and chronic intake does not affect herbivore fitness.¹⁴ This type of substance may be a chemical mimic or be associated with other more toxic compounds.¹⁵ It is also possible that the avoidance behaviour towards harmless deterrents may not be associated at all with toxicity but rather with the avoidance of non-host plants,¹⁶ or avoidance of unsuitable food sources to reduce the risk of feeding on a toxic plant.¹⁷Using nutritional indices, it is possible to investigate the effect of a compound on food consumption and utilisation.¹⁸ We therefore investigated the effect of isoprene on food consumption and utilization by M. sexta using nutritional indices. In this experiment, third instar caterpillars were force fed on isoprene-emitting or non-emitting artificial diets for 24 hour. There were no significant differences (p > 0.05, n = 27) in relative growth rate, consumption index, approximate digestibility, efficiency of conversion of ingested food to body mass and efficiency of conversion of digested food to body mass between the caterpillars that fed on the isoprene emitting-diet and those that fed on the non-emitting diet (Fig. 1). This indicates that isoprene does not directly influence food uptake and utilization by the caterpillars, and that the observed avoidance behaviour⁸ may be related to avoidance of isoprene behaving as a ‘non-host plant’ signal, or isoprene might act in association with other defence mechanism in plants.Open in a separate windowFigure 1Quantification of food consumption and utilization of third instar M. sexta larvae. Larvae fed on isoprene-emitting (55 nmol m⁻² s⁻¹) or non-emitting artificial diet for 24 hours. Larval weight gained, frass produced and leaf weight consumed were measured and analysed according to Waldbauer nutritional indices (1968). ANOVA was used to analyse the data at 95% confidence and no significant confidence was found (n = 27). RGR, relative growth rate; CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food to body mass; ECD, efficiency of conversion of digested food to body mass.It should be noted that this experiment was only performed over 24 hours and it therefore does not probe the longer term effect of isoprene on caterpillar fitness. The function of isoprene in plant-herbivore interactions should therefore be investigated further by extending the experimental period and measurement of caterpillar development, both by using in vitro (isoprene-emitting and non-emitting artificial diet) and in vivo (isoprene-emitting and non-emitting transgenic plants) model systems. The recent availability of transgenic plants, including (a) one that does not normally emit isoprene but is induced to produce isoprene^19,20 and (b) one that normally produces isoprene but has its isoprene emission suppressed,² can facilitate this research and provide insights into the function of isoprene in planta.Various adaptation strategies for reducing the production costs of terpenes have been proposed, including sharing biosynthetic enzymes among multiple pathways, minimizing enzyme turnover rate, using a single enzyme to generate multiple products and using products for more than one function.²¹ The possibility of isoprene being a compound with multiple functions has been hypothesized,⁸ as isoprene has been shown, by inhibitor studies and genetic manipulation, to be able to defend plants from high temperature episodes^1,2 and against ozone³ and now also to protect against herbivory.⁸ It has also been shown to promote flowering in neighboring plants.⁷ Further investigations are, however, required to determine if isoprene emission can deter herbivore insects from a wide range of plant species, or whether this and other effects are specific to plant species. The multiple functions of isoprene may explain how the benefits of isoprene production by plants can outweigh the costs of its production, and why some, but not all, plants produce this reactive and volatile compound.     

Biochemical Characterization of Stromal and Thylakoid-Bound Isoforms of Isoprene Synthase in Willow Leaves

Isoprene synthase is the enzyme responsible for the foliar emission of the hydrocarbon isoprene (2-methyl-1,3-butadiene) from many C₃ plants. Previously, thylakoid-bound and soluble forms of isoprene synthase had been isolated separately, each from different plant species using different procedures. Here we describe the isolation of thylakoid-bound and soluble isoprene synthases from a single willow (Salix discolor L.) leaf-fractionation protocol. Willow leaf isoprene synthase appears to be plastidic, with whole-leaf and intact chloroplast fractionations yielding approximately equal soluble (i.e. stromal) and thylakoid-bound isoprene synthase activities. Although thylakoid-bound isoprene synthase is tightly bound to the thylakoid membrane (M.C. Wildermuth, R. Fall [1996] Plant Physiol 112: 171–182), it can be solubilized by pH 10.0 treatment. The solubilized thylakoid-bound and stromal isoprene synthases exhibit similar catalytic properties, and contain essential cysteine, histidine, and arginine residues, as do other isoprenoid synthases. In addition, two regulators of foliar isoprene emission, leaf age and light, do not alter the percentage of isoprene synthase activity in the bound or soluble form. The relationship between the isoprene synthase isoforms and the implications for function and regulation of isoprene production are discussed.     

Rapid appearance of 13C in biogenic isoprene when 13CO2 is fed to intact leaves

Biogenic isoprene substantially affects atmospheric chemistry, but it is not known how or why many plants, especially trees, make isoprene. We fed ¹³CO₂ to leaves of Quercus rubra and monitored the incorporation of ¹³C into isoprene by mass spectrometry. After feeding ¹³CO₂ for 9 min we found all possible labelling patterns from completely unlabelled to fully labelled isoprene. By 18 min, 84% of the carbon atoms in isoprene were ¹³C. Labelling of the last 20% of the carbon atoms was much slower than labelling of the first 80%. The rate of labelling of isoprene was similar to that reported for phosphoglyceric acid indicating that there is a close linkage between the carbon source for isoprene synthesis and the photosynthetic carbon reduction pathway.     

Plant isoprenoid biosynthesis via the MEP pathway: In vivo IPP/DMAPP ratio produced by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase in tobacco BY-2 cell cultures

Feeding tobacco BY-2 cells with [2-¹³C,4-²H]deoxyxylulose revealed from the ¹³C labeling that the plastid isoprenoids, synthesized via the MEP pathway, are essentially derived from the labeled precursor. The ca. 15% ²H retention observed in all isoprene units corresponds to the isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) ratio (85:15) directly produced by the hydroxymethylbutenyl diphosphate reductase, the last enzyme of the MEP pathway. ²H retention characterizes the isoprene units derived from the DMAPP branch, whereas ²H loss represents the signature of the IPP branch. Taking into account the enantioselectivity of the reactions catalyzed by the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase, the IPP isomerase and the trans-prenyl transferase, a single biogenetic scheme allows to interpret all labeling patterns observed in bacteria or plants upon incubation with ²H labeled deoxyxylulose.     

Combinational biosynthesis of isoprene by engineering the MEP pathway in Escherichia coli

As an important feedstock in petrochemistry, isoprene is used in a wide range of industrial applications. It is produced almost entirely from petrochemical sources; however, these sources are being progressively depleted. A reliable biological process for isoprene production utilizing renewable feedstocks would be an industry-redefining development. There are two biosynthetic pathways producing isoprene: the mevalonate (MVA) pathway and the methyl erythritol 1-phosphate (MEP) pathway. In this study, the MEP pathway was modified in Escherichia coli BL21 (DE3) to produce isoprene. The isoprene synthase (IspS) gene chemically synthesized from Populus alba after codon optimization for expression in E. coli was heterologously expressed. The endogenous genes of 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) were over-expressed. The isopentenyl pyrophosphate isomerase (Idi) gene from Streptococcus pneumoniae was exogenously over-expressed, and farnesyl diphosphate synthase (ispA) was weakened to enhance the yield. The control strain harboring empty plasmids did not emit any isoprene. The over-expression of the DXR gene only had little impact on the yield of isoprene. Idi from S. pneumoniae played a significant role in the improvement of isoprene production. The highest yield was achieved by an ispA-weakened DXS-IDI-IspS recombinant with 19.9 mg/l isoprene, which resulted in a 33-fold enhancement of the isoprene yield from the IspS recombinant.     

Characterization of the hexahydropolyprenols of Aspergillus fumigatus fresenius

The isolation and properties of a group of alcohols from the mycelium of Aspergillus fumigatus Fresenius are described. Mass-, nuclear-magnetic-resonance- and infrared-spectrometric studies coupled with evidence from ozonolytic degradation and chromatography show the mixture to contain hexahydroprenols-18, -19, -20, -21, -22, -23 and -24. Each contains a saturated `hydroxy-terminal' isoprene residue, a saturated ω-terminal isoprene residue and a saturated ζ-isoprene residue (adjacent to the ω-residue). The presence of only two trans-isoprene residues is also a feature of the series of alcohols, but the precise position of these in each molecule is not known.     

A model of isoprene emission based on energetic requirements for isoprene synthesis and leaf photosynthetic properties for Liquidambar and Quercus

We present a physiological model of isoprene (2-methyl-1,3-butadiene) emission which considers the cost for isoprene synthesis, and the production of reductive equivalents in reactions of photosynthetic electron transport for Liquidambar styraciflua L. and for North American and European deciduous temperate Quercus species. In the model, we differentiate between leaf morphology (leaf dry mass per area, M_A, g m ^{? 2}) altering the content of enzymes of isoprene synthesis pathway per unit leaf area, and biochemical potentials of average leaf cells determining their capacity for isoprene emission. Isoprene emission rate per unit leaf area ( μ mol m ^{? 2} s ^{? 1}) is calculated as the product of M_A, the fraction of total electron flow used for isoprene synthesis ( ? , mol mol ^{? 1}), the rate of photosynthetic electron transport (J) per unit leaf dry mass (J_m, μ mol g ^{? 1} s ^{? 1}), and the reciprocal of the electron cost of isoprene synthesis [mol isoprene (mol electrons ^{? 1})]. The initial estimate of electron cost of isoprene synthesis is calculated according to the 1-deoxy- D -xylulose-5-phosphate pathway recently discovered in the chloroplasts, and is further modified to account for extra electron requirements because of photorespiration. The rate of photosynthetic electron transport is calculated by a process-based leaf photosynthesis model. A satisfactory fit to the light-dependence of isoprene emission is obtained using the light response curve of J, and a single value of ? , that is dependent on the isoprene synthase activity in the leaves. Temperature dependence of isoprene emission is obtained by combining the temperature response curves of photosynthetic electron transport, the shape of which is related to long-term temperature during leaf growth and development, and the specific activity of isoprene synthase, which is considered as essentially constant for all plants. The results of simulations demonstrate that the variety of temperature responses of isoprene emission observed within and among the species in previous studies may be explained by different optimum temperatures of J and/or limited maximum fraction of electrons used for isoprene synthesis. The model provides good fits to diurnal courses of field measurements of isoprene emission, and is also able to describe the changes in isoprene emission under stress conditions, for example, the decline in isoprene emission in water-stressed leaves.     

Isoprene Emissions from Downy Oak under Water Limitation during an Entire Growing Season: What Cost for Growth?

Increases in the production of terpene- and phenolic-like compounds in plant species under abiotic stress conditions have been interpreted in physiological studies as a supplementary defense system due to their capacity to limit cell oxidation. From an ecological perspective however, these increases are only expected to confer competitive advantages if they do not imply a significant cost for the plant, that is, growth reduction. We investigated shifts of isoprene emissions, and to a lesser extent phenolic compound concentration, of Quercus pubescens Willd. from early leaf development to leaf senescence under optimal watering (control: C), mild and severe water stress (MS, SS). The impact of water stress was concomitantly assessed on plant physiological (chlorophyll fluorescence, stomatal conductance, net photosynthesis, water potential) functional (relative leaf water content, leaf mass per area ratio) and growth (aerial and root biomass) traits. Growth changes allowed to estimate the eventual costs related to the production of isoprene and phenolics. The total phenolic content was not modified under water stress whereas isoprene emissions were promoted under MS over the entire growing cycle despite the decline of P_n by 35%. Under SS, isoprene emissions remained similar to C all over the study despite the decline of Pn by 47% and were thereby clearly uncoupled to Pn leading to an overestimation of the isoprene emission factor by 44%. Under SS, maintenance of isoprene emissions and phenolic compound concentration resulted in very significant costs for the plants as growth rates were very significantly reduced. Under MS, increases of isoprene emission and maintenance of phenolic compound concentration resulted in moderate growth reduction. Hence, it is likely that investment in isoprene emissions confers Q. pubescens an important competitive advantage during moderate but not severe periods of water scarcity. Consequences of this response for air quality in North Mediterranean areas are also discussed.     

Dolichols, ubiquinones, geranylgeraniol and farnesol as the major metabolites of mevalonate in Phytophthora cactorum

Farnesol, geranylgeraniol, dolichols and ubiquinones were the main radioactive components of the unsaponifiable lipid recovered from Phytophthora cactorum grown in aerated cultures containing [2-¹⁴C]mevalonate. The ¹⁴C recovered in each of these components was in the approximate proportion 2:4:3:5. When the culture was not aerated no radioactive ubiquinone was recovered. Most of the ¹⁴C recovered in the dolichols was found in dolichol-15 (37%), with decreasing amounts in dolichol-14 (30%) and -13 (14%) and only a little (5%) in dolichol-16, whereas the major components, by weight, of the mixture (13μg/g of damp-dry tissue) were dolichol-14, -15 and -16 in the approximate proportion of 1:3:1. Radioautography of appropriate chromatograms indicated the presence also of traces of radioactivity in dolichol-9, -10, -11, -12 and -17. Most (80%) of the ¹⁴C recovered in the ubiquinones was associated with ubiquinone-9, the rest being in ubiquinone-8. Most (80%) of the weight of ubiquinones (19μg/g of damp-dry tissue) was also ubiquinone-9. The identification of these compounds was by chromatographic methods and, for the ubiquinones and dolichols, was confirmed by mass spectrometry. In addition, the incorporation of 4R- and/or 4S-³H from [4-³H]-mevalonates showed the expected stereochemistry of biosynthesis, namely that farnesol, geranylgeraniol and ubiquinones were biogenetically all trans and the dolichols each contained three biogenetically trans isoprene residues, the remaining residues being biogenetically cis. The distribution of ¹⁴C in the components of the whole lipid of the fungus was consistent with 97% of both the farnesol and geranylgeraniol being present as the fatty acid ester. The corresponding value for dolichols was 37%. The observation by other workers, that this fungus does not form either squalene or sterol, was confirmed.     

Relationships among Isoprene Emission Rate, Photosynthesis, and Isoprene Synthase Activity as Influenced by Temperature

Isoprene emissions from the leaves of velvet bean (Mucuna pruriens L. var utilis) plants exhibited temperature response patterns that were dependent on the plant's growth temperature. Plants grown in a warm regimen (34/28°C, day/night) exhibited a temperature optimum for emissions of 45°C, whereas those grown in a cooler regimen (26/20°C, day/night) exhibited an optimum of 40°C. Several previous studies have provided evidence of a linkage between isoprene emissions and photosynthesis, and more recent studies have demonstrated that isoprene emissions are linked to the activity of isoprene synthase in plant leaves. To further explore this linkage within the context of the temperature dependence of isoprene emissions, we determined the relative temperature dependencies of photosynthetic electron transport, CO₂ assimilation, and isoprene synthase activity. When measured over a broad range of temperatures, the temperature dependence of isoprene emission rate was not closely correlated with either the electron transport rate or the CO₂ assimilation rate. The temperature optima for electron transport rate and CO₂ assimilation rate were 5 to 10°C lower than that for the isoprene emission rate. The dependence of isoprene emissions on photon flux density was also affected by measurement temperature in a pattern independent of those exhibited for electron transport rate and CO₂ assimilation rate. Thus, despite no change in the electron transport rate or CO₂ assimilation rate at 26 and 34°C, the isoprene emission rate changed markedly. The quantum yield of isoprene emissions was stimulated by a temperature increase from 26 to 34°C, whereas the quantum yield for CO₂ assimilation was inhibited. In greenhouse-grown aspen leaves (Populus tremuloides Michaux.), the high temperature threshold for inhibition of isoprene emissions was closely correlated with the high temperature-induced decrease in the in vitro activity of isoprene synthase. When taken together, the results indicate that although there may be a linkage between isoprene emission rate and photosynthesis, the temperature dependence of isoprene emission is not determined solely by the rates of CO₂ assimilation or electron transport. Rather, we propose that regulation is accomplished primarily through the enzyme isoprene synthase.     

Competition between isoprene emission and pigment synthesis during leaf development in aspen

In growing leaves, lack of isoprene synthase (IspS) is considered responsible for delayed isoprene emission, but competition for dimethylallyl diphosphate (DMADP), the substrate for both isoprene synthesis and prenyltransferase reactions in photosynthetic pigment and phytohormone synthesis, can also play a role. We used a kinetic approach based on post‐illumination isoprene decay and modelling DMADP consumption to estimate in vivo kinetic characteristics of IspS and prenyltransferase reactions, and to determine the share of DMADP use by different processes through leaf development in Populus tremula. Pigment synthesis rate was also estimated from pigment accumulation data and distribution of DMADP use from isoprene emission changes due to alendronate, a selective inhibitor of prenyltransferases. Development of photosynthetic activity and pigment synthesis occurred with the greatest rate in 1‐ to 5‐day‐old leaves when isoprene emission was absent. Isoprene emission commenced on days 5 and 6 and increased simultaneously with slowing down of pigment synthesis. In vivo Michaelis–Menten constant (K_m) values obtained were 265 nmol m^?2 (20 μm ) for DMADP‐consuming prenyltransferase reactions and 2560 nmol m^?2 (190 μm ) for IspS. Thus, despite decelerating pigment synthesis reactions in maturing leaves, isoprene emission in young leaves was limited by both IspS activity and competition for DMADP by prenyltransferase reactions.     

Increased thermostability of thylakoid membranes in isoprene-emitting leaves probed with three biophysical techniques

Three biophysical approaches were used to get insight into increased thermostability of thylakoid membranes in isoprene-emittingplants.Arabidopsis (Arabidopsis thaliana) plants genetically modified to make isoprene and Platanus orientalis leaves, in which isoprene emission was chemically inhibited, were used. First, in the circular dichroism spectrum the transition temperature of the main band at 694 nm was higher in the presence of isoprene, indicating that the heat stability of chiral macrodomains of chloroplast membranes, and specifically the stability of ordered arrays of light-harvesting complex II-photosystem II in the stacked region of the thylakoid grana, was improved in the presence of isoprene. Second, the decay of electrochromic absorbance changes resulting from the electric field component of the proton motive force (ΔA₅₁₅) was evaluated following single-turnover saturating flashes. The decay of ΔA₅₁₅ was faster in the absence of isoprene when leaves of Arabidopsis and Platanus were exposed to high temperature, indicating that isoprene protects the thylakoid membranes against leakiness at elevated temperature. Finally, thermoluminescence measurements revealed that S₂Q_B⁻ charge recombination was shifted to higher temperature in Arabidopsis and Platanus plants in the presence of isoprene, indicating higher activation energy for S₂Q_B⁻ redox pair, which enables isoprene-emitting plants to perform efficient primary photochemistry of photosystem II even at higher temperatures. The data provide biophysical evidence that isoprene improves the integrity and functionality of the thylakoid membranes at high temperature. These results contribute to our understanding of isoprene mechanism of action in plant protection against environmental stresses.Vegetation is a source of large quantities of biogenic volatile organic compounds emitted into the atmosphere, of which isoprene is the most abundant (Guenther et al., 2006). Owing to the high reactivity and fast oxidation by hydroxyl radicals in the atmosphere, isoprene can increase the formation of atmospheric ozone (Monson and Holland, 2001; Kleindienst et al., 2007), organic nitrates (O’Brien et al., 1995), and peroxyacylnitrates (Sun and Huang, 1995). Isoprene is also a significant factor in secondary aerosol formation, with direct and indirect effects on the global radiation balance of the atmosphere (Claeys et al., 2004; Matsunaga et al., 2005; Kroll et al., 2006; Ervens et al., 2008; Paulot et al., 2009).Besides having significant influences on atmospheric chemistry, many biogenic volatile organic compounds play an important role in plant biology. Experimental evidence shows that isoprene protects photosynthesis under thermal and oxidative stress conditions (for review, see Vickers et al., 2009a; Loreto and Schnitzler, 2010). It was demonstrated that leaves in which isoprene biosynthesis was blocked by the methyl erythritol pathway inhibitor fosmidomycin, were more sensitive to high temperature and ozone exposure, and developed stronger oxidative damage, compared to isoprene-emitting leaves (Loreto and Velikova, 2001; Sharkey et al., 2001; Velikova and Loreto, 2005). Plants fumigated with isoprene suffer less damage when exposed to oxidative stresses (Loreto et al., 2001) and recover more rapidly from heat stress than untreated controls (Singsaas et al., 1997; Sharkey et al., 2001; Velikova et al., 2006). Genetic approaches to develop isoprene-emitting species from nonemitting wild types (Sharkey et al., 2005; Loivamäki et al., 2007; Sasaki et al., 2007; Vickers et al., 2009b) or to suppress isoprene synthesis in strong emitters (Behnke et al., 2007) have also been used to clarify the role of isoprene in plant protection. Studies with genetically modified plants mostly confirmed improved tolerance associated with the capacity to form and emit isoprene. Recently important compensatory responses, such as the activation of alternative defensive biochemical pathways, e.g. phenolics biosynthesis, to cope with stressful conditions, were also highlighted (Fares et al., 2010), especially when isoprene biosynthesis is knocked out (Behnke et al., 2009).Several hypotheses have been put forward to explain the physiological mechanism(s) by which isoprene protects the photosynthetic apparatus (Loreto and Schnitzler, 2010). The oldest and most widely accepted idea is that isoprene stabilizes chloroplast membranes (Sharkey and Singsaas, 1995). Thylakoid membranes become leaky at high temperature (Pastenes and Horton, 1996; Bukhov et al., 1999; Schrader et al., 2004; Zhang et al., 2009). It was suggested that the positive effect of isoprene might be due to the hydrophobic nature of the molecule, the localization of isoprene synthase enzyme near the thylakoid membranes (Wildermuth and Fall, 1998; Schnitzler et al., 2005), and the high octanol/water partitioning coefficient (Copolovici and Niinemets, 2005). Lipophilic isoprene partitioned into membranes might prevent the formation of water channels responsible for the membrane leakiness at high temperature, or may prevent the formation of nonlamellar aggregates, or help stabilize the photosynthetic complexes embedded in thylakoid membranes (Singsaas et al., 1997; Sharkey et al., 2001). 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 (Sharkey and Yeh, 2001). Based on molecular dynamics simulations of phospholipid bilayers with and without isoprene, Siwko et al. (2007) suggested that isoprene enhances the packing of lipid tails. The authors suggested that the role of isoprene as a membrane stabilizer can be related to the fact that it fits well into the available pockets of the free volume inside the membrane, and adds cohesiveness, amplifying membrane packing, while not affecting the dynamics of phospholipid bilayers (Siwko et al., 2007). However, Logan and Monson (1999) working with reconstituted liposomes were not able to prove that isoprene improves the thermal stability of membranes.Another hypothesis to explain the generally positive role of isoprene in plant metabolism is based on the high reactivity of volatile isoprenoids with radicals and other reactive compounds. Loreto et al. (2001) first suggested that isoprene might operate as a volatile molecule, scavenging reactive oxygen species in the intercellular spaces of the leaf mesophyll. More recently, it was proven that isoprene also removes reactive nitrogen species from the mesophyll (Velikova et al., 2008). Vickers et al. (2009a) reviewed the possible mechanisms of isoprene function and suggested that the molecule may have a general antioxidant role.A demonstration of effects of isoprene on biophysical measurements of thylakoid function at high temperature could help in working toward a resolution of the primary mechanism of isoprene action. Here three well-known techniques in biophysical studies of thylakoid membrane function were tested for the effect of isoprene at high temperature. Two plant systems were used. Arabidopsis (Arabidopsis thaliana), which does not normally make isoprene, was engineered with an isoprene synthase gene from kudzu (Pueraria lobata) so that wild-type plants (nonemitting) could be compared to the transformed plants that do make isoprene. Leaves of Platanus (plane tree) normally do make isoprene but this was inhibited by fosmidomycin so that emitting (water-fed) and nonemitting (fosmidomycin-fed) leaves could be compared. The thermal stability of the thylakoid membranes was characterized with biophysical approaches not previously used in isoprene studies, namely circular dichroism (CD) spectrosocopy, electrochromic absorbance transients (ΔA₅₁₅), and thermoluminescence (TL). These measurements revealed that in the presence of isoprene, the macroorganization of the pigment-protein complexes in the membranes were more stable to elevated temperature, the membranes were better able to maintain a light-induced transmembrane electric field at elevated temperatures, and the recombination of the PSII donor and acceptor side charges occurred up to higher temperature.