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.     

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.     

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.     

Hybridization of European oaks (Quercus ilex × Q. robur) results in a mixed isoprenoid emitter type

European oaks have been reported to emit isoprene or monoterpenes derived from recently fixed photosynthetic carbon. The emission type is plant species specific and can be used as chemo‐taxonomic marker. In the present article the isoprenoid biochemical properties of mature Quercus × turneri‘Pseudoturneri’ hybrids resulting from a crossing of a Mediterranean evergreen monoterpene‐emitting species (subgenus Sclerophyllodrys; Quercus ilex L.) and an isoprene‐emitting deciduous oak species (subgenus Lepidobalanus; Quercus robur L.) are described. Both species are compared with respect to the capacity for isoprenoid synthesis and the actual isoprenoid emission pattern of different tree‐types. The analysis showed that the oak hybrid combines properties of both parental species. Furthermore, it could be shown that the enzyme activities of isoprene synthase and monoterpene synthases are reflected in the isoprenoid emission pattern of the hybrids as well as in the observed emission rates.     

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.     

Evolutionary significance of isopreneemission from mosses

Isoprene emission has been documented and characterized from species in all major groups of vascular plants. We report in our survey that isoprene emission is much more common in mosses and ferns than later divergent land plants but is absent in liverworts and hornworts. The light and temperature responses of isoprene emission from Sphagnum capillifolium (Ehrh.) Hedw. are similar to those of other land plants. Isoprene increases thermotolerance of S. capillifolium to the same extent seen in higher plants as measured by chlorophyll fluorescence. Sphagnum species in a northern Wisconsin bog experienced large temperature fluctuations similar to those reported in tree canopies. Since isoprene has been shown to help plants cope with large, rapid temperature fluctuations, we hypothesize the thermal and correlated dessication stress experienced by early land plants provided the selective pressure for the evolution of light-dependent isoprene emission in the ancestors of modern mosses. As plants radiated into different habitats, this capacity was lost multiple times in favor of other thermal protective mechanisms.     

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.     

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.     

Plants utilize isoprene emission as a thermotolerance mechanism

Isoprene is a volatile compound emitted from leaves of many plant species in large quantities, which has an impact on atmospheric chemistry due to its massive global emission rate (5 x 10(14) carbon g year(-1)) and its high reactivity with the OH radical, resulting in an increase in the half-life of methane. Isoprene emission is strongly induced by the increase in isoprene synthase activity in plastids at high temperature in the day time, which is regulated at its gene expression level in leaves, while the physiological meaning of isoprene emission for plants has not been clearly demonstrated. In this study, we have functionally overexpressed Populus alba isoprene synthase in Arabidopsis to observe isoprene emission from transgenic plants. A striking difference was observed when both transgenic and wild-type plants were treated with heat at 60 degrees C for 2.5 h, i.e. transformants revealed clear heat tolerance compared with the wild type. High isoprene emission and a decrease in the leaf surface temperature were observed in transgenic plants under heat stress treatment. In contrast, neither strong light nor drought treatments showed an apparent difference. These data suggest that isoprene emission plays a crucial role in a heat protection mechanism in plants.     

Impact of rising CO2 on emissions of volatile organic compounds: isoprene emission from Phragmites australis growing at elevated CO2 in a natural carbon dioxide spring†

Isoprene basal emission (the emission of isoprene from leaves exposed to a light intensity of 1000 µmol m^?2 s^?1 and maintained at a temperature of 30 °C) was measured in Phragmites australis plants growing under elevated CO₂ in the Bossoleto CO₂ spring at Rapolano Terme, Italy, and under ambient CO₂ at a nearby control site. Gas exchange and biochemical measurements were concurrently taken. Isoprene emission was lower in the plants growing at elevated CO₂ than in those growing at ambient CO₂. Isoprene emission and isoprene synthase activity (IsoS) were very low in plants growing at the bottom of the spring under very rich CO₂ and increased at increasing distance from the spring (and decreasing CO₂ concentration). Distance from the spring did not significantly affect photosynthesis making it therefore unlikely that there is carbon limitation to isoprene formation. The isoprene emission rate was very quickly reduced after rapid switches from elevated to ambient CO₂ in the gas‐exchange cuvette, whereas it increased when switching from ambient to elevated CO₂. The rapidity of the response may be consistent with post‐translational modifications of enzymes in the biosynthetic pathway of isoprene formation. Reduction of IsoS activity is interpreted as a long‐term response. Basal emission of isoprene was not constant over the day but showed a diurnal course opposite to photosynthesis, with a peak during the hottest hours of the day, independent of stomatal conductance and probably dependent on external air temperature or temporary reduction of CO₂ concentration. The present experiments show that basal emission rate of isoprene is likely to be reduced under future elevated CO₂ levels and allow improvement in the modelling of future isoprene emission rates.     

Human-induced changes in US biogenic volatile organic compound emissions: evidence from long-term forest inventory data

Volatile organic compounds (VOCs) emitted by woody vegetation influence global climate forcing and the formation of tropospheric ozone. We use data from over 250 000 re‐surveyed forest plots in the eastern US to estimate emission rates for the two most important biogenic VOCs (isoprene and monoterpenes) in the 1980s and 1990s, and then compare these estimates to give a decadal change in emission rate. Over much of the region, particularly the southeast, we estimate that there were large changes in biogenic VOC emissions: half of the grid cells (1°× 1°) had decadal changes in emission rate outside the range ?2.3% to +16.8% for isoprene, and outside the range 0.2–17.1% for monoterpenes. For an average grid cell the estimated decadal change in heatwave biogenic VOC emissions (usually an increase) was three times greater than the decadal change in heatwave anthropogenic VOC emissions (usually a decrease, caused by legislation). Leaf‐area increases in forests, caused by anthropogenic disturbance, were the most important process increasing biogenic VOC emissions. However, in the southeast, which had the largest estimated changes, there were substantial effects of ecological succession (which decreased monoterpene emissions and had location‐specific effects on isoprene emissions), harvesting (which decreased monoterpene emissions and increased isoprene emissions) and plantation management (which increased isoprene emissions, and decreased monoterpene emissions in some states but increased monoterpene emissions in others). In any given region, changes in a very few tree species caused most of the changes in emissions: the rapid changes in the southeast were caused almost entirely by increases in sweetgum (Liquidambar styraciflua) and a few pine species. Therefore, in these regions, a more detailed ecological understanding of just a few species could greatly improve our understanding of the relationship between natural ecological processes, forest management, and biogenic VOC emissions.     

Effects of environmental conditions on isoprene emission from live oak

Live-oak plants (Quercus virginiana Mill.) were subjected to various levels of CO₂, water stress or photosynthetic photon flux density to test the hypothesis that isoprene biosynthesis occurred only under conditions of restricted CO₂ availability. Isoprene emission increases as the ambient CO₂ concentration decreased, independent of the amount of time that plants had photosynthesized at ambient CO₂ levels. When plants were water-stressed over a 4-d period photosynthesis and leaf conductance decreased 98 and 94%, respectively, while isoprene emissions remained constant. Significant isoprene emissions occurred when plants were saturated with CO₂, i.e., below the light compensation level for net photosynthesis (100 mol m^-2 s^-1). Isoprene emission rates increased with photosynthetic photon flux density and at 25 and 50 mol m^-2 s^-1 were 7 and 18 times greater than emissions in the dark. These data indicate that isoprene is a normal plant metabolite and not — as has been suggested — formed exclusively in response to restricted CO₂ or various stresses.Abbreviation PPFD photosynthetic photon flux density     

Controls on isoprene emission from trees in a subtropical dry forest

Isoprene emission from vegetation is the single most important source of photochemically active reduced compounds to the atmosphere. We present the first controlled-environment measurements of isoprene emission from leaves of tropical forest trees. Our studies were conducted in the Guanica State Forest in Puerto Rico. We report the effects of temperature and light variations on biogenic isoprene emissions during 1995. Maximum emission rates varied among species from 0 to 268 nmol m^?2 s^?1. Values at the upper end of this range of maximum emission rates are 2–3 times higher than values reported from any temperate taxa. Isoprene emission showed strong sensitivity to light and temperature variations. In contrast to temperate plants, whose emissions tend to saturate at a light intensity of ～1000 μmol m^?2 s^?1, emissions from the tropical species increased with light intensity up to 2500 μmol m^?2 s^?1. The temperature optima for emissions from these plants were similar to those previously reported for temperate plants: ～40 °C. The high maximum emission rates and lack of light saturation indicate that estimates of isoprene emission from tropical forests need to be revised upwards.     

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.     

异戊二烯合成酶研究进展

异戊二烯(Isoprene)的排放具有特殊的生物学功能,对大气环境具有重要影响,另外,异戊二烯也是一种具有广泛用途的化合物。在生物体内,异戊二烯是由异戊二烯合成酶(Isoprene synthase,Isps)催化烯丙基二磷酸(Dimethylallyl diphosphate,DMAPP)脱去焦磷酸(Pyrophosphate)而生成的。因此,作为异戊二烯合成过程中的关键酶,Isps在异戊二烯的自然排放和生物合成过程都发挥着重要的作用,对Isps的研究具有非常重要的意义。到目前为止,已经在多种植物中发现了该酶,研究表明,来源于不同生物的异戊二烯合成酶具有保守的结构特征和相似的生化性质。文中就Isps的最新研究进展进行综述,包括比较分析不同生物来源Isps的生化特征和结构特征,探讨催化机制,并对该酶在生物工程领域的应用进行展望。     

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.