13C labeling reveals chloroplastic and extrachloroplastic pools of dimethylallyl pyrophosphate and their contribution to isoprene formation

Isoprene emitted from plants is made in chloroplasts from dimethylallyl pyrophosphate (DMAPP). Leaves of Populus nigra and Phragmites australis exposed to (13)CO(2) for 15 min emitted isoprene that was about 90% (13)C, but DMAPP isolated from those leaves was only 28% and 36% (13)C, respectively. The labeled DMAPP is likely to represent chloroplastic DMAPP contributing to isoprene emission. A substantial (13)C labeling was also found in both emission and DMAPP pool of low-emitting, young leaves of Phragmites. This confirms that low emission of young leaves is not caused by absence of chloroplastic DMAPP but rather by enzyme characteristics. A very low (13)C labeling was found in the DMAPP pool and in the residual isoprene emission of leaves previously fed with fosmidomycin to inhibit isoprene formation. This shows that fosmidomycin is a very effective inhibitor of the chloroplastic biosynthetic pathway of isoprene synthesis, that the residual isoprene is formed from extra-chloroplastic sources, and that chloroplastic and extrachloroplastic pathways are not cross-linked, at least following inhibition of the chloroplastic pathway. Refixation of unlabeled respiratory CO(2) in the light may explain incomplete labeling of isoprene emission, as we found a good association between these two parameters.     

Isoprene emission rate and intercellular isoprene concentration as influenced by stomatal distribution and conductance

Isoprene emission in relation to stomatal distribution and conductance was determined for the hypostomatous species, aspen and white oak, and the amphistomatous species, cottonwood. For aspen and oak, isoprene emission from the adaxial (nonstomatal) surface was <2% of that from the abaxial (stomatal) surface, even when stomata were closed by addition of abscisic acid (ABA). When treated with ABA, the total flux rate of isoprene emission from leaves of these two hypostomatous species was unchanged, despite decreases in stomatal conductance of over 90%. The lack of control over isoprene emission rate by stomatal conductance, despite the apparent movement of isoprene through the stomatal pores, was due to increases in the intercellular isoprene concentration that compensated for the decreased stomatal conductance and restored the equilibrium between the isoprene synthesis rate and emission rate. This relationship was demonstrated by (a) an experiment in which the decrease in the internal isoprene pool following the imposition of darkness took longer in the presence of ABA than in its absence, and (b) direct measurements of the internal isoprene concentration through vacuum extraction, which revealed substantially higher values in the presence of ABA than in its absence. In the amphistomatous species, cottonwood, isoprene was emitted from both surfaces and addition of ABA caused an increase in isoprene emission from one surface coupled with a decrease from the other surface. The specific surface exhibiting an increase varied among leaves, with some leaves exhibiting an increase from the adaxial surface and other leaves from the abaxial surface. We interpret this as indicating nonuniform stomatal closure with concomitant emission of isoprene at the greatest rate from the surface with the highest stomatal conductance. We also observed an increase in the total isoprene emission rate from cottonwood leaves following treatment with ABA. We interpret this as indicating a stimulation of isoprene synthesis in response to ABA or stomatal closure, with unknown cause.     

Nonradioactive assay for cellular dimethylallyl diphosphate

A sensitive, nonradioactive method was developed to measure cellular levels of dimethylallyl diphosphate (DMAPP), a central intermediate of isoprenoid metabolism in nature. The assay is based on the hydrolysis of DMAPP in acid to the volatile hydrocarbon isoprene (2-methyl-1,3-butadiene), with subsequent analysis of isoprene by headspace gas chromatography with reduction gas detection. In the assay, cell samples are directly acidified with 4 M H(2)SO(4) in sealed reaction vials. Therefore, there is no need to extract metabolites, purify them, and keep them stable prior to analysis, and degradative enzymatic activities are destroyed. DMAPP levels of 23 +/- 4 nmol (g fresh weight)(-1) [ca. 85 nmol (g dry weight)(-1)] and 80 +/- 14 nmol (g fresh weight)(-1) [ca. 296 nmol (g dry weight)(-1)] were measured in dark- and light-adapted leaves of Populus deltoides (Eastern cottonwood), respectively. Evidence is presented to show that DMAPP is the major leaf metabolite giving rise to isoprene following acid hydrolysis. DMAPP levels in Bacillus subtilis and Saccharomyces cerevisiae were determined to be 40.8 +/- 16.7 pmol (OD(600))(-1) [ca. 638 pmol (mg dry weight)(-1)] and 6.3 +/- 3.7 pmol (OD(600))(-1) [ca. 139 pmol (mg dry weight)(-1)], respectively. The method should be suitable for any cell or tissue type and isolated cellular organelles.     

Light-Dependent Isoprene Emission (Characterization of a Thylakoid-Bound Isoprene Synthase in Salix discolor Chloroplasts)

Isoprene synthase is an enzyme that is responsible for the production of the volatile C5 hydrocarbon, isoprene, in plant leaves. Isoprene formation in numerous C3 plants is interesting because (a) large quantities of isoprene are emitted, 5 x 1014 g of C annually, (b) a plant may release 1 to 8% of its fixed C as isoprene, and (c) the function of plant isoprene production is unknown. Because of the dependence of foliar isoprene emission on light, the existence of a plastidic isoprene synthase has been postulated. To pursue this idea, a method to isolate chloroplasts from Salix discolor was developed and shows a plastidic isoprene synthase that is tightly bound to the thylakoid membrane and accessible to trypsin inactivation. The thylakoid-bound isoprene synthase has catalytic properties similar to known soluble isoprene synthases; however, the relationship between these enzymes is unknown. The discovery of a thylakoid-bound isoprene synthase with a stromal-facing domain places it in the chloroplast, where it may be subject to numerous direct and indirect light-mediated effects. Implications for the light-dependent regulation of foliar isoprene production and its function are presented.     

Engineering an Isoprenoid Pathway in Escherichia coli for Production of 2-Methyl-3-buten-2-ol: A Potential Biofuel

2-Methyl-3-buten-2-ol (MBO) is a natural volatile 5-carbon alcohol produced by several pine species that have the potential to be used as biofuel. MBO has a high energy content making it superior to ethanol in terms of energy output, and due to its volatility and lower solubility in water, MBO is easier to recover than ethanol. Pine’s MBO synthase enzyme utilizes the intermediate dimethylallyl pyrophosphate (DMAPP) produced by the methyl-erythritol-4-phosphate isoprenoid pathway for the production of MBO. In this study, we performed metabolic engineering of Escherichia coli to express an alternate mevalonate dependent pathway for production of DMAPP, along with a codon optimized Pinus sabiniana MBO synthase gene. This heterologous expressed pathway carried out the conversion of an acetyl CoA precursor to DMAPP leading to production of MBO.     

Induction of poplar leaf nitrate reductase: a test of extrachloroplastic control of isoprene emission rate

Several recent studies have suggested that control of isoprene emission rate is in part exerted by supply of extrachloroplastic phosphoenolpyruvate to the chloroplast. To test this hypothesis, we altered PEP supply by differential induction of cytosolic nitrate reductase (NR) and PEP carboxylase (PEPC) in plants of Populus deltoides grown with NO3- or NH4+ as the sole nitrogen source. Growth with 8 mM NH4+ produced a high leaf nitrogen concentration, compared with 8 mM NO3-, as well as slightly elevated rates of photosynthesis and significantly enhanced rates of isoprene emission and content of dimethylallyl diphosphate (DMAPP, a precursor to isoprene biosynthesis), chlorophyll (a+b) and carotenoids. Growth with 8 mM NO3- resulted in parallel reductions in both leaf isoprene emission rate and DMAPP. The differential effects of growth with NH4+ or NO3- were not observed when plants were grown with 4 mM nitrogen. The effects of reduced DMAPP availability were specific to isoprene emission and were not propagated to higher isoprenoids, as the correlations between nitrogen content and either leaf chlorophyll (a+b) or total carotenoids were unaffected by nitrogen source. Biochemical analysis revealed significantly higher levels of NR and PEPC activity in leaves of 8 mM NO3- -grown plants, consistent with their fundamental roles in nitrate assimilation. Taken together, these results support the hypothesis that foliar assimilation of NO3- reduces isoprene emission rate by competing for carbon skeletons (mediated by PEPC) within the cytosol and possibly reductant within the chloroplast. Cytosolic competition for PEP is a major regulator of chloroplast DMAPP supply, and we offer a new "safety valve" hypothesis to explain why plants emit isoprene.     

Characterization of an Isoprene Synthase from Leaves of Quercus petraea (Mattuschka) Liebl.

Biogenic emission of isoprene (2-methyl-1,3-butadiene) by many plant species plays an important role in atmospheric chemistry. Its rapid breakdown in the atmosphere substantially affects the oxidation potential of the atmosphere. Leaves of Quercus petraea were found to contain an enzyme which catalyses the conversion of dimethylallyl pyrophosphate (DMAPP) to isoprene. A standard enzyme assay was established and the isoprene synthase activity was characterized in purified leaf extracts. Optimum enzyme activity was observed at pH 8.5. The enzyme had an apparent K_m of 0.97 mM for its substrate DMAPP, but isopentenyl pyrophosphate (IPP), the isomeric form of DMAPP, was not converted to isoprene by the enzyme extract. The temperature optimum of the enzyme activity was 35 °C. Isoprene synthase activity was strictly dependent on the presence of bivalent cations, with magnesium being most effective. Molecular weight determination by FPLC revealed the presence of a single protein with a native molecular weight of approximately 90–100 kDa.     

Isolation and Purification of Protein Responsible for the Conversion of Dimethylallylpyrophosphate from Poplar Leaves into Isoprene

The protein converting dimethylallylpyrophosphate (DMAPP) into isoprene in vitrowas isolated and purified 3000-fold from leaves of berry-bearing poplar (Populus deltoidesMarsh.). As the enzyme was purified, its specific activity increased and at the final stage reached 266 nmol/(min mg protein). The enzyme was eluted by anion-exchange chromatography in a 120–170 mM NaCl gradient and by chromatography on the hydroxyapatite column in 170 mM sodium phosphate. The active molecular weight of the protein determined by gel filtration was 100–110 kD. As the enzyme was purified, the K _Mvalue increased from 2 to 9 mM. A parallelism isoprene emission from DMAPP and an increase in the specific activity of the enzyme as it was purified proved that the enzyme catalyzed isoprene emission.     

Process-based modelling of isoprene emission by oak leaves

The emission rate of the volatile reactive compound isoprene, emitted predominantly by trees, must be known before the level of photo‐oxidants produced during summer smog can be predicted reliably. The emission is dependent on plant species and local conditions, and these dependencies must be quantified to be included in any empirical algorithm for the calculation of isoprene production. Experimental measurements of isoprene emission rates are expensive, however, and existing data are scarce and fragmentary. To overcome these difficulties, it is promising to develop a numerical model capable of precisely calculating the isoprene emission by trees for diverse ecosystems, even under changing environmental conditions. A basic process‐based biochemical isoprene emission model (BIM) has therefore been developed, which describes the enzymatic reactions in leaf chloroplasts leading to the formation of isoprene under varying environmental conditions (e.g. light intensity, temperature). Concentrations of the precursors of isoprene formation, 3‐phosphoglyceric acid and glyceraldehyde 3‐phosphate, are provided by a published light fleck photosynthesis model. Specific leaf and enzyme parameters were determined for the pedunculate oak (Quercus robur L.), so that the BIM is capable of calculating oak‐specific isoprene emission rates as influenced by the leaf temperature and light intensity. High correlation was observed between isoprene emission rates calculated by the BIM and the diurnal isoprene emission rates of leaves measured under controlled environmental conditions. The BIM was even capable of describing changes in isoprene emission caused by midday depression of net photosynthesis.     

Biochemical regulation of isoprene emission

Isoprene (C₅H₈) is emitted from many plants and has a substantial effect on atmospheric chemistry. There are several models to estimate the rate of isoprene emission used to calculate the impact of isoprene on atmospheric processes. The rate of isoprene synthesis will depend either on the activity of isoprene synthase or the availability of its substrate dimethylallyl pyrophosphate (DMAPP). To investigate long‐term regulation of isoprene synthesis, the isoprene emission rate of 15 kudzu leaves was measured. The chloroplast DMAPP level of the five leaves with the highest emission rates and the five leaves with the lowest rates were determined by non‐aqueous fractionation of the bulked leaf samples. Leaves with high basal emission rates had low levels of DMAPP whereas leaves with low basal emission rates had high DMAPP levels in their chloroplasts indicating that the activity of isoprene synthase exerts primary control over the basal emission rate. To investigate short‐term regulation, isoprene precursors were fed to leaves. Feeding dideuterated deoxyxylulose (DOX‐d₂) to Eucalyptus leaves resulted in the emission of dideuterated isoprene. Results from DOX‐d₂ feeding experiments indicated that control of isoprene emission rate was shared between reactions upstream and downstream of the DOX entry into isoprene metabolism. In CO₂‐free air DOX always increased isoprene emission indicating that carbon availability was an important control factor. In N₂, isoprene emission stopped and could not be recovered by adding DOX‐d₂. Taken together, these results indicate that the regulation of isoprene emission is shared among several steps and the relative importance of the different steps in controlling isoprene emission varies with conditions.     

The relationship between the methyl-erythritol phosphate pathway leading to emission of volatile isoprenoids and abscisic acid content in leaves

It was investigated whether the methyl-erythritol phosphate (MEP) pathway that generates volatile isoprenoids and carotenoids also produces foliar abscisic acid (ABA) and controls stomatal opening. When the MEP pathway was blocked by fosmidomycin and volatile isoprenoid emission was largely suppressed, leaf ABA content decreased to about 50% and leaf stomatal conductance increased significantly. No effect of fosmidomycin was seen in leaves with constitutively high rates of stomatal conductance and in plant species with low foliar ABA concentration. In all other cases, isoprene emission was directly associated with foliar ABA, but ABA reduction upon MEP pathway inhibition was also observed in plant species that do not emit isoprenoids. Stomatal closure causing a midday depression of photosynthesis was also associated with a concurrent increase of isoprene emission and ABA content. It is suggested that the MEP pathway generates a labile pool of ABA that responds rapidly to environmental changes. This pool also regulates stomatal conductance, possibly when coping with frequent changes of water availability. MEP pathway inhibition by leaf darkening, and its down-regulation by exposure to elevated CO2, was also associated with a reduction of foliar ABA content. However, stomatal conductance was reduced, indicating that stomatal aperture is not regulated by the MEP-dependent foliar ABA pool, under these specific cases.     

Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.)

Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-d-erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.     

Defoliation effects on isoprene emission from Populus deltoides

Isoprene emission from plants is one of the principal ways in which plant processes alter atmospheric chemistry. Despite the importance of this process, few long-term controls over basal emission rates have been identified. Stress-induced changes in carbon allocation within the entire plant, such as those produced by defoliation, have not been examined as potential mechanisms that may control isoprene production and emission. Eastern cottonwood (Populus deltoides) saplings were partially defoliated and physiological and growth responses were measured from undamaged and damaged leaves 7 days following damage. Defoliation reduced isoprene emission from undamaged and damaged leaves on partially defoliated plants. Photosynthetic rates and leaf carbon and nitrogen pools were unaffected by damage. Photosynthetic rate and isoprene emission were highly correlated in undamaged leaves on undamaged plants and damaged leaves on partially defoliated plants. There was no correlation between photosynthetic rate and isoprene emission in undamaged leaves on partially defoliated plants. Isoprene emission was also highly correlated with the number of source leaves on the apical shoot in damage treatments. Increased carbon export from source leaves in response to defoliation may have depleted the amount of carbon available for isoprene synthesis, decreasing isoprene emission. These results suggest that while isoprene emission is controlled at the leaf level in undamaged plants, emission from leaves on damaged plants is controlled by whole-branch allocation patterns. Received: 12 May 1998 / Accepted: 9 November 1998     

Dimethylallyl diphosphate and geranyl diphosphate pools of plant species characterized by different isoprenoid emissions

Dimethylallyl diphosphate (DMADP) and geranyl diphosphate (GDP) are the last precursors of isoprene and monoterpenes emitted by leaves, respectively. DMADP and GDP pools were measured in leaves of plants emitting isoprene (Populus alba), monoterpenes (Quercus ilex and Mentha piperita), or nonemitting isoprenoids (Prunus persica). Detectable pools were found in all plant species, but P. persica showed the lowest pool size, which indicates a limitation of the whole pathway leading to isoprenoid biosynthesis in nonemitting species. The pools of DMADP and GDP of nonemitting, isoprene-emitting, and monoterpene-emitting species were partially labeled (generally 40%-60% of total carbon-incorporated (13)C) within the same time by which volatile isoprenoids are fully labeled (15 min). This indicates the coexistence of two pools for both precursors, the rapidly labeled pool presumably occurring in chloroplasts and thereby synthesized by the methylerythritol phosphate pathway and the nonlabeled pool presumably located in the cytosol and synthesized by the mevalonic pathway. In M. piperita storing monoterpenes in specialized leaf structures, the GDP pool remained totally unlabeled, indicating either that monoterpenes are totally formed by the mevalonic pathway or that labeling occurs slowly in comparison to the large pool of stored monoterpenes in this plant. The pools of DMADP and GDP increased during the season (from May to July) but decreased when the leaf was darkened or exposed to very high temperature. In the dark, the pool of DMADP of the isoprene-emitting species decreased faster than the pool of GDP. However, after 6 h of darkness, both pools were depleted to about 10% of the pool size in illuminated leaves. This indicates that both the chloroplastic and the cytosolic pools of precursors are depleted in the dark. When comparing measurements over the season and at different temperatures, an inverse correlation was observed between isoprene emission by P. alba and the DMADP pool size and between monoterpene emission by Q. ilex and the GDP pool size. This suggests that the pool size does not limit the emission of isoprenoids. Rather, it indicates that the flux of volatile isoprenoids effectively controls the size of their pools of precursors.     

Biosynthesis of 2-methyl-3-buten-2-ol emitted from needles of Pinus ponderosa via the non-mevalonate DOXP/MEP pathway of isoprenoid formation

The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.     

Functional analysis of genes involved in the biosynthesis of isoprene in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

In comparison to other bacteria Bacillus subtilis emits the volatile compound isoprene in high concentrations. Isoprene is the smallest representative of the natural product group of terpenoids. A search in the genome of B. subtilis resulted in a set of genes with yet unknown function, but putatively involved in the methylerythritol phosphate (MEP) pathway to isoprene. Further identification of these genes would give the possibility to engineer B. subtilis as a host cell for the production of terpenoids like the valuable plant-produced drugs artemisinin and paclitaxel. Conditional knock-out strains of putative genes were analyzed for the amount of isoprene emitted. Differences in isoprene emission were used to identify the function of the enzymes and of the corresponding selected genes in the MEP pathway. We give proof on a biochemical level that several of these selected genes from this species are involved in isoprene biosynthesis. This opens the possibilities to investigate the physiological function of isoprene emission and to increase the endogenous flux to the terpenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate, for the heterologous production of more complex terpenoids in B. subtilis.     

Biochemical characterization and homology modeling of methylbutenol synthase and implications for understanding hemiterpene synthase evolution in plants

2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ~90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K(+), whereas isoprene production is inhibited by K(+) such that, at physiologically relevant [K(+)], little or no isoprene emission should be detected from MBO-emitting trees. The K(m) of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site.     

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.     

Opportunistic emissions of volatile isoprenoids

Isoprene, monoterpenes and sesquiterpenes are synthesized and emitted by some plant species, but not all plant species have this ability. These volatile, nonessential isoprenoid compounds share the same biochemical precursors as larger essential isoprenoids such as gibberellic acids and carotenoids. They have many protective and ecological functions for the plant species that produce them, but plant species that do not produce these compounds also grow and reproduce successfully. Here, we develop an 'opportunist hypothesis' suggesting that (i) volatile isoprenoid production takes advantage of dimethylallyl diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP), which are synthesized primarily to produce essential isoprenoids, and (ii) conditions affecting synthesis of the higher isoprenoids will affect the production and emission of volatile isoprenoids.