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.     

异戊二烯合成酶(IspS)在大肠杆菌中的表达及其产异戊二烯的研究

将来源于银白杨的异戊二烯合成酶基因按照大肠杆菌密码子偏爱性进行优化,克隆到表达载体pACYCDu-et-1上,在大肠杆菌BL21(DE3)中异源表达,采用镍柱亲和层析纯化重组蛋白并测定其异戊二烯合成酶活性,通过摇瓶发酵实验对重组菌产异戊二烯进行进一步研究。结果显示:银白杨异戊二烯合成酶在大肠杆菌中能够高效表达,经过镍柱纯化后,电泳检测到特异性表达条带;该重组异戊二烯合成酶能够催化异戊二烯的合成,重组菌的异戊二烯产量可达到60μg/L。     

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.     

Isoprene synthase activity and its relation to isoprene emission in Quercus robur L. leaves

Pedunculate oak ( Quercus robur L.) is known as a strong isoprene (2-methyl-1,3-butadiene) emitter. Diurnal changes in isoprene emission were determined by branch enclosure measurements. In contrast to the diurnal cycle in emission rates, specific isoprene synthase activity in the leaves remained unchanged. Based on in vitro enzyme activity and its temperature dependency, an isoprene synthesis capacity at specific leaf temperatures was calculated. The comparison of these 'leaf temperature-dependent enzyme capacities' and the measured emission rates revealed that the enzyme activity of isoprene synthase is comparable to the observed isoprene emission rates. In addition, variation in the isoprene synthase activity of the leaves due to changes in light intensity during leaf development was investigated. A 50% reduction of light intensity by shading of single branches reduced isoprene synthase activity by ≈ 60% compared with full sunlight. The calculation of isoprene synthesis capacities based on enzymatic data obtained under optimum reaction conditions, corrected for actual leaf temperature and related to leaf surface area, provides a sound basis for predicting the isoprene emission potential of plants.     

High carbon dioxide and sun/shade effects on isoprene emission from oak and aspen tree leaves

Abstract. Isoprene (2-methyl 1, 3-butadiene) is emitted from many plants, especially trees. We tested the effect of growth at high CO₂ partial pressure and sun versus shade conditions on the capacity of Quercus rubra L. (red oak) and Populus tremuloides Michx. (quaking aspen) leaves to make isoprene. Oak leaves grown at high CO₂ partial pressure (65 Pa) had twice the rate of isoprene emission as leaves grown at 40Pa CO₂. However, aspen leaves behaved oppositely, with high CO₂-grown leaves having just 60-70% the rate of isoprene emission as leaves grown in 40 Pa CO₂. Similar responses were observed from 25 to 35 °C leaf temperature during assay. The stimulation of isoprene emission by growth at high CO₂ and the stimulation in high temperature resulted in isoprene emission consuming over 15% of the carbon fixed during photosynthesis in high-CO₂ grown oak leaves assayed at 35 °C. Leaves from the south (sunny) sides of trees growing in natural conditions had rates of isoprene emission double those of leaves growing in shaded locations on the same trees. This effect was similar in both aspen and oak. The leaves used for these experiments had significantly different chlorophyll a/b ratios indicating they were functionally sun (from the sunny locations) or shade leaves (from the protected locations). Because the metabolic pathway of isoprene synthesis is unknown, we are unable to speculate about how or why these effects occur. However, these effects are more consistent with metabolic control of isoprene release rather than a metabolic leak of isoprene from metabolism. The results are also important for large scale modelling of isoprene emission and for predicting the effect of future increases in atmospheric CO₂ level on isoprene emission from vegetation.     

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.     

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.     

Interaction of drought and ozone exposure on isoprene emission from extensively cultivated poplar

The combined effects of ozone (O₃) and drought on isoprene emission were studied for the first time. Young hybrid poplars (clone 546, Populus deltoides cv. 55/56 x P. deltoides cv. Imperial) were exposed to O₃ (charcoal‐filtered air, CF, and non‐filtered air +40 ppb, E‐O₃) and soil water stress (well‐watered, WW, and mild drought, MD, one‐third irrigation) for 96 days. Consistent with light‐saturated photosynthesis (A_sat), intercellular CO₂ concentration (C_i) and chlorophyll content, isoprene emission depended on drought, O₃, leaf position and sampling time. Drought stimulated emission (+38.4%), and O₃ decreased it (?40.4%). Ozone increased the carbon cost per unit of isoprene emission. Ozone and drought effects were stronger in middle leaves (13th–15th from the apex) than in upper leaves (6th–8th). Only A_sat showed a significant interaction between O₃ and drought. When the responses were up‐scaled to the entire‐plant level, however, drought effects on total leaf area translated into around twice higher emission from WW plants in clean air than in E‐O₃. Our results suggest that direct effects on plant emission rates and changes in total leaf area may affect isoprene emission from intensively cultivated hybrid poplar under combined MD and O₃ exposure, with important feedbacks for air quality.     

Use of carbon-11 in Populus shows that exogenous jasmonic acid increases biosynthesis of isoprene from recently fixed carbon

A new approach for pulse labelling of plants using the short-lived positron emitting radioisotope carbon-11 (half-life: 20.4 min) as ¹¹CO₂ is reported together with its application to measuring [¹¹C]isoprene emissions from intact leaves capturing information associated with: (1) rate of emission; (2) the relative contribution of recently fixed carbon to isoprene biosynthesis; and (3) the transit time for tracer movement through the leaf and biochemical pathways associated with isoprene biosynthesis. This approach was applied to study the response of certain Populus species to exogenous treatments of jasmonic acid (JA), a plant hormone implicated in signal transduction linked to defence response against herbivory. Twelve hours after treatment of single intact leaves of aspen (Populus tremuloides) with a 1 m m JA spray, isoprene emissions from those leaves increased 1.5 times the controls from 35.4 ± 2.2 to 53.1 ± 4.8 nmol m⁻² s⁻¹. [¹¹C]Isoprene emissions from the same leaves, reflecting the isoprene that was derived from recently fixed carbon, increased much more, to 2.2 times the controls. This increase coincided with a change in emitted [¹¹C]isoprene from 0.31 to 0.68% of ¹¹C fixed in the leaf tissue, while the tracer transit time remained constant at 12.5 min. These results suggest that JA had no effect on enzyme kinetics involved in isoprene biosynthesis, but did impact the source of recent carbon feeding that pathway. Studies with poplar (Populus nigra clone NC 5271) showed similar trends in systemic emissions (from an untreated leaf on the same plant).     

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.     

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.     

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     

On the relationship between isoprene emission and thermotolerance in Phragmites australis leaves exposed to high temperatures and during the recovery from a heat stress

Thermotolerance induced by isoprene has been assessed during heat bursts but there is little information on the ability of endogenous isoprene to confer thermotolerance under naturally elevated temperature, on the interaction between isoprene-induced thermotolerance and light stress, and on the persistence of this protection in leaves recovering at lower temperatures. Moderately high temperature treatment (38 °C for 1.5 h) reduced photosynthesis, stomatal conductance, and photochemical efficiency of photosystem II in isoprene-emitting, but to a significantly lower extent than in isoprene-inhibited Phragmites australis leaves. Isoprene inhibition and high temperature independently, as well as together, induced lipid peroxidation, increased level of H₂O₂, and increased catalase and peroxidase activities. However, leaves in which isoprene emission was previously inhibited developed stronger oxidative stress under high temperature with respect to isoprene-emitting leaves. The heaviest photosynthetic stress was observed in isoprene-inhibited leaves exposed to the brightest illumination (1500 µmol m⁻² s⁻¹) and, in general, there was also a clear additive effect of light excess on the formation of reactive oxygen species, antioxidant enzymes, and membrane damage. The increased thermotolerance capability of isoprene-emitting leaves may be due to isoprene ability to stabilize membranes or to scavenge reactive oxygen species. Irrespective of the mechanism by which isoprene reduces thermal stress, isoprene-emitting leaves are able to quickly recover after the stress. This may be an important feature for plants coping with frequent and transient temperature changes in nature.     

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.     

The response of isoprene emission rate and photosynthetic rate to photon flux and nitrogen supply in aspen and white oak trees

Isoprene is the primary biogenic hydrocarbon emitted from temperate deciduous forest ecosystems. The effects of varying photon flux density (PFD) and nitrogen growth regimes on rates of isoprene emission and net photosynthesis in potted aspen and white oak trees are reported. In both aspen and oak trees, whether rates were expressed on a leaf area or dry mass basis, (1) growth at higher PFD resulted in significantly higher rates of isoprene emission, than growth at lower PFD, (2) there is a significant positive relationship between isoprene emission rate and leaf nitrogen concentration in both sun and shade trees, and (3) there is a significant positive correlation between isoprene emission rate and photosynthetic rate in both sun and shade trees. The greater capacity for isoprene emission in sun leaves was due to both higher leaf mass per unit area and differences in the biochemical and/or physiological properties that influence isoprene emission. Positive correlations between isoprene emission rate and leaf nitrogen concentration support the existence of mechanisms that link leaf nitrogen status to isoprene synthase activity. Positive correlations between isoprene emission rate and photosynthesis rate support previous hypotheses that isoprene emission plays a role in protecting photosynthetic mechanisms during stress.