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.     

Effect of temperature on postillumination isoprene emission in oak and poplar

Isoprene emission from broadleaf trees is highly temperature dependent, accounts for much of the hydrocarbon emission from plants, and has a profound effect on atmospheric chemistry. We studied the temperature response of postillumination isoprene emission in oak (Quercus robur) and poplar (Populus deltoides) leaves in order to understand the regulation of isoprene emission. Upon darkening a leaf, isoprene emission fell nearly to zero but then increased for several minutes before falling back to nearly zero. Time of appearance of this burst of isoprene was highly temperature dependent, occurring sooner at higher temperatures. We hypothesize that this burst represents an intermediate pool of metabolites, probably early metabolites in the methylerythritol 4-phosphate pathway, accumulated upstream of dimethylallyl diphosphate (DMADP). The amount of this early metabolite(s) averaged 2.9 times the amount of plastidic DMADP. DMADP increased with temperature up to 35°C before starting to decrease; in contrast, the isoprene synthase rate constant increased up to 40°C, the highest temperature at which it could be assessed. During a rapid temperature switch from 30°C to 40°C, isoprene emission increased transiently. It was found that an increase in isoprene synthase activity is primarily responsible for this transient increase in emission levels, while DMADP level stayed constant during the switch. One hour after switching to 40°C, the amount of DMADP fell but the rate constant for isoprene synthase remained constant, indicating that the high temperature falloff in isoprene emission results from a reduction in the supply of DMADP rather than from changes in isoprene synthase activity.     

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.     

Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-d-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-d-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg²⁺ was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.     

Growth condition controls on G-93 parameters of isoprene emission from tropical trees

Despite its major role in global isoprene emission, information on the environmental control of isoprene emission from tropical trees has remained scarce. Thus, in this study, we examined the relationship between parameters of G-93 isoprene emission formula (C_T1, C_T2, and α), growth temperature and light intensity, photosynthesis (?, P_max), isoprene synthase (IspS) level, and 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway metabolites using sunlit and shaded leaves of four tropical trees. The results showed that the temperature dependence of isoprene emission from shaded leaves did not differ significantly from sunlit leaves. In contrast, there was a lower saturation irradiance in shaded leaves than in sunlit leaves, the same as temperate plants. The photosynthesis rate of shaded leaves showed lower saturation irradiance, similar to the light dependence of isoprene emission. In most cases, the concentration of MEP pathway metabolites was of lower tendency in shaded leaves versus in sunlit leaves, whereas no significant difference was noted in IspS level between sunlit and shaded leaves. Correlation analysis between these parameters found that C_T1 of the G-93 parameter was positively correlated with the concentration of DXP and DMADP, whereas C_T2 correlated with the concentration of MEP and the average air temperature for the past 48 h. Similarly, α closely associated with the initial slope (?) of photosynthesis rate, and the basal emission factor is also linked to the photon flux of past days. These results suggest that growth conditions may control the temperature dependence of isoprene emission from tropical trees via the changes in the profiles of MEP pathway metabolites, causing alteration in the parameters of the isoprene emission formula.

     

Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase

Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).     

Evolution of the isoprene biosynthetic pathway in kudzu

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.     

Isoprene synthesis in plants: lessons from a transgenic tobacco model

Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves.     

Isoprene emission from plants: why and how

BACKGROUND: Some, but not all, plants emit isoprene. Emission of the related monoterpenes is more universal among plants, but the amount of isoprene emitted from plants dominates the biosphere-atmosphere hydrocarbon exchange. SCOPE: The emission of isoprene from plants affects atmospheric chemistry. Isoprene reacts very rapidly with hydroxyl radicals in the atmosphere making hydroperoxides that can enhance ozone formation. Aerosol formation in the atmosphere may also be influenced by biogenic isoprene. Plants that emit isoprene are better able to tolerate sunlight-induced rapid heating of leaves (heat flecks). They also tolerate ozone and other reactive oxygen species better than non-emitting plants. Expression of the isoprene synthase gene can account for control of isoprene emission capacity as leaves expand. The emission capacity of fully expanded leaves varies through the season but the biochemical control of capacity of mature leaves appears to be at several different points in isoprene metabolism. CONCLUSIONS: The capacity for isoprene emission evolved many times in plants, probably as a mechanism for coping with heat flecks. It also confers tolerance of reactive oxygen species. It is an example of isoprenoids enhancing membrane function, although the mechanism is likely to be different from that of sterols. Understanding the regulation of isoprene emission is advancing rapidly now that the pathway that provides the substrate is known.     

Rapid regulation of the methylerythritol 4-phosphate pathway during isoprene synthesis

More volatile organic carbon is lost from plants as isoprene than any other molecule. This flux of carbon to the atmosphere affects atmospheric chemistry and can serve as a substrate for ozone production in polluted air. Isoprene synthesis may help leaves cope with heatflecks and active oxygen species. Isoprene synthase, an enzyme related to monoterpene synthases, converts dimethylallyl diphosphate derived from the methylerythritol 4-phosphate pathway to isoprene. We used dideuterated deoxyxylulose (DOX-d(2)) to study the regulation of the isoprene biosynthetic pathway. Exogenous DOX-d(2) displaced endogenous sources of carbon for isoprene synthesis without increasing the overall rate of isoprene synthesis. However, at higher concentrations, DOX-d(2) completely suppressed isoprene synthesis from endogenous sources and increased the overall rate of isoprene synthesis. We interpret these results to indicate strong feedback control of deoxyxylulose-5-phosphate synthase. We related the emission of labeled isoprene to the concentration of labeled dimethylallyl diphosphate in order to estimate the in situ K(m) of isoprene synthase. The results confirm that isoprene synthase has a K(m) 10- to 100-fold higher for its allylic diphosphate substrate than related monoterpene synthases for geranyl diphosphate.     

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.     

Controls of the quantum yield and saturation light of isoprene emission in different‐aged aspen leaves

Leaf age alters the balance between the use of end‐product of plastidic isoprenoid synthesis pathway, dimethylallyl diphosphate (DMADP), in prenyltransferase reactions leading to synthesis of pigments of photosynthetic machinery and in isoprene synthesis, but the implications of such changes on environmental responses of isoprene emission have not been studied. Because under light‐limited conditions, isoprene emission rate is controlled by DMADP pool size (S_DMADP), shifts in the share of different processes are expected to particularly strongly alter the light dependency of isoprene emission. We examined light responses of isoprene emission in young fully expanded, mature and old non‐senescent leaves of hybrid aspen (Populus tremula x P. tremuloides) and estimated in vivo S_DMADP and isoprene synthase activity from post‐illumination isoprene release. Isoprene emission capacity was 1.5‐fold larger in mature than in young and old leaves. The initial quantum yield of isoprene emission (α_I) increased by 2.5‐fold with increasing leaf age primarily as the result of increasing S_DMADP. The saturating light intensity (Q_I90) decreased by 2.3‐fold with increasing leaf age, and this mainly reflected limited light‐dependent increase of S_DMADP possibly due to feedback inhibition by DMADP. These major age‐dependent changes in the shape of the light response need consideration in modelling canopy isoprene emission.     

Induction of a longer term component of isoprene release in darkened aspen leaves: origin and regulation under different environmental conditions

After darkening, isoprene emission continues for 20 to 30 min following biphasic kinetics. The initial dark release of isoprene (postillumination emission), for 200 to 300 s, occurs mainly at the expense of its immediate substrate, dimethylallyldiphosphate (DMADP), but the origin and controls of the secondary burst of isoprene release (dark-induced emission) between approximately 300 and 1,500 s, are not entirely understood. We used a fast-response gas-exchange system to characterize the controls of dark-induced isoprene emission by light, temperature, and CO(2) and oxygen concentrations preceding leaf darkening and the effects of short light pulses and changing gas concentrations during dark-induced isoprene release in hybrid aspen (Populus tremula × Populus tremuloides). The effect of the 2-C-methyl-D-erythritol-4-phosphate pathway inhibitor fosmidomycin was also investigated. The integral of postillumination isoprene release was considered to constitute the DMADP pool size, while the integral of dark-induced emission was defined as the "dark" pool. Overall, the steady-state emission rate in light and the maximum dark-induced emission rate responded similarly to variations in preceding environmental drivers and atmospheric composition, increasing with increasing light, having maxima at approximately 40 °C and close to the CO(2) compensation point, and were suppressed by lack of oxygen. The DMADP and dark pool sizes were also similar through their environmental dependencies, except for high temperatures, where the dark pool significantly exceeded the DMADP pool. Isoprene release could be enhanced by short lightflecks early during dark-induced isoprene release, but not at later stages. Fosmidomycin strongly suppressed both the isoprene emission rates in light and in the dark, but the dark pool was only moderately affected. These results demonstrate a strong correspondence between the steady-state isoprene emission in light and the dark-induced emission and suggest that the dark pool reflects the total pool size of 2-C-methyl-d-erythritol-4-phosphate pathway metabolites upstream of DMADP. These metabolites are converted to isoprene as soon as ATP and NADPH become available, likely by dark activation of chloroplastic glycolysis and chlororespiration.     

ISOPRENE SYNTHASE GENES FORM A MONOPHYLETIC CLADE OF ACYCLIC TERPENE SYNTHASES IN THE TPS‐B TERPENE SYNTHASE FAMILY

Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)‐β‐ocimene synthases form a monophyletic group within the Tps‐b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps‐b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps‐b genes indicates that isoprene emission from non‐rosid angiosperms likely arose independently.     

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.     

Regulation of isoprene emission in Populus trichocarpa leaves subjected to changing growth temperature

The hydrocarbon isoprene is emitted in large quantities from numerous plant species, and has a substantial impact on atmospheric chemistry. Temperature affects isoprene emission at several levels: the temperature at which emission is measured, the temperature at which leaves develop, and the temperatures to which a mature leaf is exposed in the days prior to emission measurement. The molecular regulation of the response to the last of these factors was investigated in this study. When plants were grown at 20 degrees C and moved from 20 to 30 degrees C and back, or grown at 30 degrees C and moved from 30 to 20 degrees C and back, their isoprene emission peaked within 3 h of the move and stabilized over the following 3 d. Trees that developed at 20 degrees C and experienced 30 degrees C episodes had higher isoprene emission capacities than did leaves grown exclusively at 20 degrees C, even 2 weeks after the last 30 degrees C episode. The levels and extractable activities of isoprene synthase protein, which catalyses the synthesis of isoprene, and those of dimethylallyl diphosphate (DMADP), its substrate, alone could not explain observed variations in isoprene emission. Therefore, we conclude that control of isoprene emission in mature leaves is shared between isoprene synthase protein and DMADP supply.     

Diurnal variation of dimethylallyl diphosphate concentrations in oak (Quercus robur) leaves

In the present work a rapid and sensitive non-radioactive assay for the determination of cellular dimethylallyl diphosphate (DMADP) was developed and used for the analysis of the diurnal variation of DMADP levels in oak leaves. The method is based on the acid-catalysed hydrolysis of DMADP to isoprene, which subsequently is determined by gas chromatography. Diurnal variation of cellular DMADP levels in oak leaves of young saplings was measured on 6 days in 1998 and 1999, showing a 2 to 3-fold light-dependent increase from approximately 15 pmol mg⁻¹ DW in the night to 31–75 pmol mg⁻¹ DW around noon. The leaf DMADP contents showed a significant positive correlation with net assimilation and isoprene emission rates, indicating that the availability of cellular DMADP might be an important regulatory factor of leaf isoprene emission.