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.     

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.     

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.     

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.     

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.     

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.     

Enhanced isoprene emission capacity and altered light responsiveness in aspen grown under elevated atmospheric CO2 concentration

Controversial evidence of CO₂‐responsiveness of isoprene emission has been reported in the literature with the response ranging from inhibition to enhancement, but the reasons for such differences are not understood. We studied isoprene emission characteristics of hybrid aspen (Populus tremula x P. tremuloides) grown under ambient (380 μmol mol^?1) and elevated (780 μmol mol^?1) [CO₂] to test the hypothesis that growth [CO₂] effects on isoprene emission are driven by modifications in substrate pool size, reflecting altered light use efficiency for isoprene synthesis. A novel in vivo method for estimation of the pool size of the immediate isoprene precursor, dimethylallyldiphosphate (DMADP) and the activity of isoprene synthase was used. Growth at elevated [CO₂] resulted in greater leaf thickness, more advanced development of mesophyll and moderately increased photosynthetic capacity due to morphological “upregulation”, but isoprene emission rate under growth light and temperature was not significantly different among ambient‐ and elevated‐[CO₂]‐grown plants independent of whether measured at 380 μmol mol^?1 or 780 μmol mol^?1 CO₂. However, DMADP pool size was significantly less in elevated‐[CO₂]‐grown plants, but this was compensated by increased isoprene synthase activity. Analysis of CO₂ and light response curves of isoprene emission demonstrated that the [CO₂] for maximum isoprene emission was shifted to lower [CO₂] in elevated‐[CO₂]‐grown plants. The light‐saturated isoprene emission rate (I_max,Q) was greater, but the quantum efficiency at given I_max,Q was less in elevated‐[CO₂]‐grown plants, especially at higher CO₂ measurement concentration, reflecting stronger DMADP limitation at lower light and higher [CO₂]. These results collectively demonstrate important shifts in light and CO₂‐responsiveness of isoprene emission in elevated‐[CO₂]‐acclimated plants that need consideration in modeling isoprene emissions in future climates.     

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.     

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

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

Emission of isoprene from salt-stressed Eucalyptus globulus leaves

Eucalyptus spp. are among the highest isoprene emitting plants. In the Mediterranean area these plants are often cultivated along the seashore and cope with recurrent salt stress. Transient salinity may severely but reversibly reduce photosynthesis and stomatal conductance of Eucalyptus globulus leaves but the effect on isoprene emission is not significant. When the stress is relieved, a burst of isoprene emission occurs, simultaneously with the recovery of photosynthetic performance. Later on, photosynthesis, stomatal conductance, and isoprene emission decay, probably because of the onset of leaf senescence. Isoprene emission is not remarkably affected by the stress at different light intensities, CO(2) concentrations, and leaf temperatures. When CO(2) was removed and O(2) was lowered to inhibit both photosynthesis and photorespiration, we found that the residual emission is actually higher in salt-stressed leaves than in controls. This stimulation is particularly evident at high-light intensities and high temperatures. The maximum emission occurs at 40 degrees C in both salt-stressed and control leaves sampled in ambient air and in control leaves sampled in CO(2)-free and low-O(2) air. However, the maximum emission occurs at 45 degrees C in salt-stressed leaves sampled in CO(2)-free and low-O(2) air. Our results suggest the activation of alternative non-photosynthetic pathways of isoprene synthesis in salt-stressed leaves and perhaps in general in leaves exposed to stress conditions. The temperature dependence indicates that this alternative synthesis is also under enzymatic control. If this alternative synthesis still occurs in the chloroplasts, it may involve a thylakoid-bound isoprene synthase.     

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.     

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.     

The effects of high temperature on isoprene synthesis in oak leaves

Isoprene emission from plants is highly temperature sensitive and is common in forest canopy species that experience rapid leaf temperature fluctuations. Isoprene emission declines with temperature above 35 °C but the temperature at which the decline begins varies between 35 and 44 °C. This variability is caused by the rate at which leaf temperature is increased during measurement with lower temperatures associated with longer measurement cycles. To investigate this we exposed leaves of red oak (Quercus rubra L.) to temperature regimes of 35–45 °C for periods of 20–60 min. Isoprene emission increased during the first 10 min of high temperature exposure and then decreased over the next 10 min until it reached steady state. This phenomenon was common at temperatures above 35 °C but was not noticeable at temperatures below that. The response was reversible within 30 min by lowering leaf temperature to 30 °C. Because there is no storage of isoprene inside the leaf, this behaviour indicates regulation of isoprene synthesis in the leaf. We demonstrated that the variability in isoprene decline results from regulation and explains the variability in the temperature response. This is consistent with our theory that isoprene protects leaves from damage caused by rapid temperature fluctuations.     

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).     

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.