Isoprene measurements to assess plant hydrocarbon emissions and the methylerythritol pathway.

Isoprene is the most abundant non-methane hydrocarbon emitted to the atmosphere and a target of biotechnology as a source of biofuels or chemical feedstock. Measurements of the amount of isoprene or the rate of production of isoprene are important for atmospheric chemistry, evaluating biotechnology processes, and can provide information on the capacity and regulation of the methyl erythritol 4-phosphate pathway found in plants and bacteria. In this chapter we discuss techniques, and their strengths and weaknesses, of methods in common use for measuring isoprene. There are many sources of isoprene for measurements including emissions from leaves and head space analysis of reactions involving recombinant enzymes or bacterial/fungal cultures. Similarly, there are a variety of detection methods including several mass spectrometer methods that are useful for examining rates of labeling of isoprene when carbon isotopes are used.

CO2 -responsiveness of leaf isoprene emission: Why do species differ?

Leaf isoprene emission rate, I, decreases with increasing atmospheric CO2 concentration with major implications for global change. There is a significant interspecific variability in [CO2 ]-responsiveness of I, but the extent of this variation is unknown and its reasons are not understood. We hypothesized that the magnitude of emission reduction reflects the size and changeability of precursor pools responsible for isoprene emission (dimethylallyl diphosphate, DMADP and 2-methyl-erythritol 2,4-cyclodiphosphate, MEcDP). Changes in I and intermediate pool sizes upon increase of [CO2 ] from 400 to 1500 µmol/mol were studied in nine woody species spanning boreal to tropical ecosystems. I varied 10-fold, total substrate pool size 37-fold and the ratio of DMADP/MEcDP pool sizes 57-fold. At higher [CO2 ], I was reduced on average by 65%, but [CO2 ]-responsiveness varied an order of magnitude across species. The increase in [CO2 ] resulted in concomitant reductions in both substrate pools. The variation in [CO2 ]-responsiveness across species scaled with the reduction in pool sizes, the substrate pool size supported and the share of DMADP in total substrate pool. This study highlights a major interspecific variation in [CO2 ]-responsiveness of isoprene emission and conclusively links this variation to interspecific variability in [CO2 ] effects on substrate availability and intermediate pool size.

Role of Stomatal Conductance in Modifying the Dose Response of Stress-Volatile Emissions in Methyl Jasmonate Treated Leaves of Cucumber (Cucumis sativa).

Treatment by volatile plant hormone methyl jasmonate (MeJA) leads to release of methanol and volatiles of lipoxygenase pathway (LOX volatiles) in a dose-dependent manner, but how the dose dependence is affected by stomatal openness is poorly known. We studied the rapid (0-60 min after treatment) response of stomatal conductance (Gs), net assimilation rate (A), and LOX and methanol emissions to varying MeJA concentrations (0.2-50 mM) in cucumber (Cucumis sativus) leaves with partly open stomata and in leaves with reduced Gs due to drought and darkness. Exposure to MeJA led to initial opening of stomata due to an osmotic shock, followed by MeJA concentration-dependent reduction in Gs, whereas A initially decreased, followed by recovery for lower MeJA concentrations and time-dependent decline for higher MeJA concentrations. Methanol and LOX emissions were elicited in a MeJA concentration-dependent manner, whereas the peak methanol emissions (15-20 min after MeJA application) preceded LOX emissions (20-60 min after application). Furthermore, peak methanol emissions occurred earlier in treatments with higher MeJA concentration, while the opposite was observed for LOX emissions. This difference reflected the circumstance where the rise of methanol release partly coincided with MeJA-dependent stomatal opening, while stronger stomatal closure at higher MeJA concentrations progressively delayed peak LOX emissions. We further observed that drought-dependent reduction in Gs ameliorated MeJA effects on foliage physiological characteristics, underscoring that MeJA primarily penetrates through the stomata. However, despite reduced Gs, dark pretreatment amplified stress-volatile release upon MeJA treatment, suggesting that increased leaf oxidative status due to sudden illumination can potentiate the MeJA response. Taken together, these results collectively demonstrate that the MeJA dose response of volatile emission is controlled by stomata that alter MeJA uptake and volatile release kinetics and by leaf oxidative status in a complex manner.

Lethal heat stress-dependent volatile emissions from tobacco leaves: what happens beyond the thermal edge?

Natural vegetation is predicted to suffer from extreme heat events as a result of global warming. In this study, we focused on the immediate response to heat stress. Photosynthesis and volatile emissions were measured in the leaves of tobacco (Nicotiana tabacum cv. Wisconsin 38) after exposure to heat shock treatments between 46 °C and 55 °C. Exposure to 46 °C decreased photosynthetic carbon assimilation rates (A) by >3-fold. Complete inhibition of A was observed at 49 °C, together with a simultaneous decrease in the maximum quantum efficiency of PSII, measured as the Fv/Fm ratio. A large increase in volatile emissions was observed at 52 °C. Heat stress resulted in only minor effects on the emission of monoterpenes, but volatiles associated with membrane damage such as propanal and (E)-2-hexenal+(Z)-3-hexenol were greatly increased. Heat induced changes in the levels of methanol and 2-ethylfuran that are indicative of modification of cell walls. In addition, the oxidation of metabolites in the volatile profiles was strongly enhanced, suggesting the acceleration of oxidative processes at high temperatures that are beyond the thermal tolerance limit.

Responses of Aspen Leaves to Heatflecks: Both Damaging and Non-Damaging Rapid Temperature Excursions Reduce Photosynthesis.

During exposure to direct sunlight, leaf temperature increases rapidly and can reach values well above air temperature in temperate forest understories, especially when transpiration is limited due to drought stress, but the physiological effects of such high-temperature events are imperfectly understood. To gain insight into leaf temperature changes in the field and the effects of temperature variation on plant photosynthetic processes, we studied leaf temperature dynamics under field conditions in European aspen (Populus tremula L.) and under nursery conditions in hybrid aspen (P. tremula × P. tremuloides Michaux), and further investigated the heat response of photosynthetic activity in hybrid aspen leaves under laboratory conditions. To simulate the complex fluctuating temperature environment in the field, intact, attached leaves were subjected to short temperature increases ("heat pulses") of varying duration over the temperature range of 30 °C-53 °C either under constant light intensity or by simultaneously raising the light intensity from 600 µmol m-2 s-1 to 1000 µmol m-2 s-1 during the heat pulse. On a warm summer day, leaf temperatures of up to 44 °C were measured in aspen leaves growing in the hemiboreal climate of Estonia. Laboratory experiments demonstrated that a moderate heat pulse of 2 min and up to 44 °C resulted in a reversible decrease of photosynthesis. The decrease in photosynthesis resulted from a combination of suppression of photosynthesis directly caused by the heat pulse and a further decrease, for a time period of 10-40 min after the heat pulse, caused by subsequent transient stomatal closure and delayed recovery of photosystem II (PSII) quantum yield. Longer and hotter heat pulses resulted in sustained inhibition of photosynthesis, primarily due to reduced PSII activity. However, cellular damage as indicated by increased membrane conductivity was not found below 50 °C. These data demonstrate that aspen is remarkably resistant to short-term heat pulses that are frequent under strongly fluctuating light regimes. Although the heat pulses did not result in cellular damage, heatflecks can significantly reduce the whole plant carbon gain in the field due to the delayed photosynthetic recovery after the heat pulse.

A novel approach for real-time monitoring of leaf wounding responses demonstrates unprecedently fast and high emissions of volatiles from cut leaves.

Wounding is a key plant stress that results in a rapid, within seconds to a few minutes, release of ubiquitous stress volatiles and stored volatiles in species with storage structures. Understanding the timing and extent of wound-dependent volatile elicitation is needed to gain an insight into different emission controls, but real-time monitoring of plant emissions through wounding treatments has been hampered by the need to stop the measurements to perform the wounding, slow stabilization of gas flows upon chamber closure and smearing out the signal by large chambers and long sampling lines. We developed a novel leaf cutter that allows to rapidly perform highly precise leaf cuts within the leaf chamber. The cutter was fitted to the standard Walz GFS-3000 portable gas-exchange system leaf chamber and chamber exhaust air for analysis with a proton transfer reaction time-of-flight mass-spectrometer (PTR-TOF-MS) was taken right at the leaf chamber outlet. Wounding experiments in four species of contrasting leaf structure demonstrated significant species differences in timing, extent and blend of emitted volatiles, and showed unprecedently high emission rates of several stress volatiles and stored monoterpenes. In light of the rapid rise of release of de novo synthesized and stored volatiles, the results of this study suggest that past studies have underestimated the rate of elicitation and maximum emission rates of wound-dependent volatiles.

Alternative Carbon Sources for Isoprene Emission.

Isoprene and other plastidial isoprenoids are produced primarily from recently assimilated photosynthates via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, when environmental conditions limit photosynthesis, a fraction of carbon for MEP pathway can come from extrachloroplastic sources. The flow of extrachloroplastic carbon depends on the species and on leaf developmental and environmental conditions. The exchange of common phosphorylated intermediates between the MEP pathway and other metabolic pathways can occur via plastidic phosphate translocators. C1 and C2 carbon intermediates can contribute to chloroplastic metabolism, including photosynthesis and isoprenoid synthesis. Integration of these metabolic processes provide an example of metabolic flexibility, and results in the synthesis of primary metabolites for plant growth and secondary metabolites for plant defense, allowing effective use of environmental resources under multiple stresses.

Evidence That Isoprene Emission Is Not Limited by Cytosolic Metabolites. Exogenous Malate Does Not Invert the Reverse Sensitivity of Isoprene Emission to High [CO2].

Isoprene is synthesized via the chloroplastic 2-C-methyl-d-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate pathway (MEP/DOXP), and its synthesis is directly related to photosynthesis, except under high CO2 concentration, when the rate of photosynthesis increases but isoprene emission decreases. Suppression of MEP/DOXP pathway activity by high CO2 has been explained either by limited supply of the cytosolic substrate precursor, phosphoenolpyruvate (PEP), into chloroplast as the result of enhanced activity of cytosolic PEP carboxylase or by limited supply of energetic and reductive equivalents. We tested the PEP-limitation hypotheses by feeding leaves with the PEP carboxylase competitive inhibitors malate and diethyl oxalacetate (DOA) in the strong isoprene emitter hybrid aspen (Populus tremula × Populus tremuloides). Malate feeding resulted in the inhibition of net assimilation, photosynthetic electron transport, and isoprene emission rates, but DOA feeding did not affect any of these processes except at very high application concentrations. Both malate and DOA did not alter the sensitivity of isoprene emission to high CO2 concentration. Malate inhibition of isoprene emission was associated with enhanced chloroplastic reductive status that suppressed light reactions of photosynthesis, ultimately leading to reduced isoprene substrate dimethylallyl diphosphate pool size. Additional experiments with altered oxygen concentrations in conditions of feedback-limited and non-feedback-limited photosynthesis further indicated that changes in isoprene emission rate in control and malate-inhibited leaves were associated with changes in the share of ATP and reductive equivalent supply for isoprene synthesis. The results of this study collectively indicate that malate importantly controls the chloroplast reductive status and, thereby, affects isoprene emission, but they do not support the hypothesis that cytosolic metabolite availability alters the response of isoprene emission to changes in atmospheric composition.

Spectacular Oscillations in Plant Isoprene Emission under Transient Conditions Explain the Enigmatic CO2 Response.

Plant isoprene emissions respond to light and temperature similarly to photosynthesis, but CO2 dependencies of isoprene emission and photosynthesis are profoundly different, with photosynthesis increasing and isoprene emission decreasing with increasing CO2 concentration due to reasons not yet understood. We studied isoprene emission, net assimilation rate, and chlorophyll fluorescence under different CO2 and O2 concentrations in the strong isoprene emitter hybrid aspen (Populus tremula × Populus tremuloides), and used rapid changes in ambient CO2 or O2 concentrations or light level to induce oscillations. As isoprene-emitting species support very high steady-state chloroplastic pool sizes of the primary isoprene substrate, dimethylallyl diphosphate (DMADP), which can mask the effects of oscillatory dynamics on isoprene emission, the size of the DMADP pool was experimentally reduced by either partial inhibition of isoprenoid synthesis pathway by fosmidomycin-feeding or by changes in ambient gas concentrations leading to DMADP pool depletion in intact leaves. In feedback-limited conditions observed at low O2 and/or high CO2 concentration under which the rate of photosynthesis is governed by the limited rate of ATP and NADPH formation due to low chloroplastic phosphate levels, oscillations in photosynthesis and isoprene emission were repeatedly induced by rapid environmental modifications in both partly fosmidomycin-inhibited leaves and in intact leaves with in vivo reduced DMADP pools. The oscillations in net assimilation rate and isoprene emission in feedback-inhibited leaves were in the same phase, and relative changes in the pools of photosynthetic metabolites and DMADP estimated by in vivo kinetic methods were directly proportional through all oscillations induced by different environmental perturbations. We conclude that the oscillations in isoprene emission provide direct experimental evidence demonstrating that the response of isoprene emission to changes in ambient gas concentrations is controlled by the chloroplastic reductant supply.

Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen.

Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.

Acclimation of isoprene emission and photosynthesis to growth temperature in hybrid aspen: resolving structural and physiological controls.

Acclimation of foliage to growth temperature involves both structural and physiological modifications, but the relative importance of these two mechanisms of acclimation is poorly known, especially for isoprene emission responses. We grew hybrid aspen (Populus tremula x P. tremuloides) under control (day/night temperature of 25/20 °C) and high temperature conditions (35/27 °C) to gain insight into the structural and physiological acclimation controls. Growth at high temperature resulted in larger and thinner leaves with smaller and more densely packed chloroplasts and with lower leaf dry mass per area (MA). High growth temperature also led to lower photosynthetic and respiration rates, isoprene emission rate and leaf pigment content and isoprene substrate dimethylallyl diphosphate pool size per unit area, but to greater stomatal conductance. However, all physiological characteristics were similar when expressed per unit dry mass, indicating that the area-based differences were primarily driven by MA. Acclimation to high temperature further increased heat stability of photosynthesis and increased activation energies for isoprene emission and isoprene synthase rate constant. This study demonstrates that temperature acclimation of photosynthetic and isoprene emission characteristics per unit leaf area were primarily driven by structural modifications, and we argue that future studies investigating acclimation to growth temperature must consider structural modifications.

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 (Km ) 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.

Elevated atmospheric CO2 concentration leads to increased whole-plant isoprene emission in hybrid aspen (Populus tremula × Populus tremuloides).

Effects of elevated atmospheric [CO2] on plant isoprene emissions are controversial. Relying on leaf-scale measurements, most models simulating isoprene emissions in future higher [CO2] atmospheres suggest reduced emission fluxes. However, combined effects of elevated [CO2] on leaf area growth, net assimilation and isoprene emission rates have rarely been studied on the canopy scale, but stimulation of leaf area growth may largely compensate for possible [CO2] inhibition reported at the leaf scale. This study tests the hypothesis that stimulated leaf area growth leads to increased canopy isoprene emission rates. We studied the dynamics of canopy growth, and net assimilation and isoprene emission rates in hybrid aspen (Populus tremula × Populus tremuloides) grown under 380 and 780 µmol mol(-1) [CO2]. A theoretical framework based on the Chapman-Richards function to model canopy growth and numerically compare the growth dynamics among ambient and elevated atmospheric [CO2]-grown plants was developed. Plants grown under elevated [CO2] had higher C : N ratio, and greater total leaf area, and canopy net assimilation and isoprene emission rates. During ontogeny, these key canopy characteristics developed faster and stabilized earlier under elevated [CO2]. However, on a leaf area basis, foliage physiological traits remained in a transient state over the whole experiment. These results demonstrate that canopy-scale dynamics importantly complements the leaf-scale processes, and that isoprene emissions may actually increase under higher [CO2] as a result of enhanced leaf area production.

Temperature responses of dark respiration in relation to leaf sugar concentration.

Changes in leaf sugar concentrations are a possible mechanism of short-term adaptation to temperature changes, with natural fluctuations in sugar concentrations in the field expected to modify the heat sensitivity of respiration. We studied temperature-response curves of leaf dark respiration in the temperate tree Populus tremula (L.) in relation to leaf sugar concentration (1) under natural conditions or (2) leaves with artificially enhanced sugar concentration. Temperature-response curves were obtained by increasing the leaf temperature at a rate of 1°C min⁻¹. We demonstrate that respiration, similarly to chlorophyll fluorescence, has a break-point at high temperature, where respiration starts to increase with a faster rate. The average break-point temperature (T(RD) ) was 48.6 ± 0.7°C at natural sugar concentration. Pulse-chase experiments with ¹4CO2 demonstrated that substrates of respiration were derived mainly from the products of starch degradation. Starch degradation exhibited a similar temperature-response curve as respiration with a break-point at high temperatures. Acceleration of starch breakdown may be one of the reasons for the observed high-temperature rise in respiration. We also demonstrate that enhanced leaf sugar concentrations or enhanced osmotic potential may protect leaf cells from heat stress, i.e. higher sugar concentrations significantly modify the temperature-response curve of respiration, abolishing the fast increase of respiration. Sugars or enhanced osmotic potential may non-specifically protect respiratory membranes or may block the high-temperature increase in starch degradation and consumption in respiratory processes, thus eliminating the break-points in temperature curves of respiration in sugar-fed leaves.

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.

When it is too hot for photosynthesis: heat-induced instability of photosynthesis in relation to respiratory burst, cell permeability changes and H2O2 formation.

Photosynthesis rate (A(n)) becomes unstable above a threshold temperature, and the recovery upon return to low temperature varies because of reasons not fully understood. We investigated responses of A(n), dark respiration and chlorophyll fluorescence to supraoptimal temperatures of varying duration and kinetics in Phaseolus vulgaris asking whether the instability of photosynthesis under severe heat stress is associated with cellular damage. Cellular damage was assessed by Evans blue penetration (enhanced membrane permeability) and by H2O2 generation [3,3'-diaminobenzidine 4HCl (DAB)-staining]. Critical temperature for dark fluorescence (F(0) ) rise (T(F)) was at 46-48 °C, and a burst of respiration was observed near T(F). However, A(n) was strongly inhibited already before T(F) was reached. Membrane permeability increased with temperature according to a switch-type response, with enhanced permeability observed above 48 °C. Experiments with varying heat pulse lengths and intensities underscored the threshold-type loss of photosynthetic function, and indicated that the degree of photosynthetic deterioration and cellular damage depended on accumulated heat-dose. Beyond the 'point of no return', propagation of cellular damage and reduction of photosynthesis continued upon transfer to lower temperatures and photosynthetic recovery was slow or absent. We conclude that instability of photosynthesis under severe heat stress is associated with time-dependent propagation of cellular lesions.

Temperature response of isoprene emission in vivo reflects a combined effect of substrate limitations and isoprene synthase activity: a kinetic analysis.

The responses of isoprene emission rate to temperature are characterized by complex time-dependent behaviors that are currently not entirely understood. To gain insight into the temperature dependencies of isoprene emission, we studied steady-state and transient responses of isoprene emission from hybrid aspen (Populus tremula × Populus tremuloides) leaves using a fast-response gas-exchange system coupled to a proton-transfer reaction mass spectrometer. A method based on postillumination isoprene release after rapid temperature transients was developed to determine the rate constant of isoprene synthase (IspS), the pool size of its substrate dimethylallyldiphosphate (DMADP), and to separate the component processes of the temperature dependence of isoprene emission. Temperature transients indicated that over the temperature range 25°C to 45°C, IspS was thermally stable and operated in the linear range of its substrate DMADP concentration. The in vivo rate constant of IspS obeyed the Arrhenius law, with an activation energy of 42.8 kJ mol(-1). In contrast, steady-state isoprene emission had a significantly lower temperature optimum than IspS and higher activation energy. The reversible temperature-dependent decrease in the rate of isoprene emission between 35°C and 44°C was caused by decreases in DMADP concentration, possibly reflecting reduced pools of energetic metabolites generated in photosynthesis, particularly of ATP. Strong control of isoprene temperature responses by the DMADP pool implies that transient temperature responses under fluctuating conditions in the field are driven by initial DMADP pool size as well as temperature-dependent modifications in DMADP pool size during temperature transients. These results have important implications for the development of process-based models of isoprene emission.

Evidence that light, carbon dioxide, and oxygen dependencies of leaf isoprene emission are driven by energy status in hybrid aspen.

Leaf isoprene emission scales positively with light intensity, is inhibited by high carbon dioxide (CO(2)) concentrations, and may be enhanced or inhibited by low oxygen (O(2)) concentrations, but the mechanisms of environmental regulation of isoprene emission are still not fully understood. Emission controls by isoprene synthase, availability of carbon intermediates, or energetic cofactors have been suggested previously. In this study, we asked whether the short-term (tens of minutes) environmental control of isoprene synthesis results from alterations in the immediate isoprene precursor dimethylallyldiphosphate (DMADP) pool size, and to what extent DMADP concentrations are affected by the supply of carbon and energetic metabolites. A novel in vivo method based on postillumination isoprene release was employed to measure the pool size of DMADP simultaneously with the rates of isoprene emission and net assimilation at different light intensities and CO(2) and O(2) concentrations. Both net assimilation and isoprene emission rates increased hyperbolically with light intensity. The photosynthetic response to CO(2) concentration was also hyperbolic, while the CO(2) response curve of isoprene emission exhibited a maximum at close to CO(2) compensation point. Low O(2) positively affected both net assimilation and isoprene emission. In all cases, the variation in isoprene emission was matched with changes in DMADP pool size. The results of these experiments suggest that DMADP pool size controls the response of isoprene emission to light intensity and to CO(2) and O(2) concentrations and that the pool size is determined by the level of energetic metabolites generated in photosynthesis.

Postillumination isoprene emission: in vivo measurements of dimethylallyldiphosphate pool size and isoprene synthase kinetics in aspen leaves.

The control of foliar isoprene emission is shared between the activity of isoprene synthase, the terminal enzyme catalyzing isoprene formation from dimethylallyldiphosphate (DMADP), and the pool size of DMADP. Due to limited in vivo information of isoprene synthase kinetic characteristics and DMADP pool sizes, the relative importance of these controls is under debate. In this study, the phenomenon of postillumination isoprene release was employed to develop an in vivo method for estimation of the DMADP pool size and to determine isoprene synthase kinetic characteristics in hybrid aspen (Populus tremula x Populus tremuloides) leaves. The method is based on observations that after switching off the light, isoprene emission continues for 250 to 300 s and that the integral of the postillumination isoprene emission is strongly correlated with the isoprene emission rate before leaf darkening, thus quantitatively estimating the DMADP pool size associated with leaf isoprene emission. In vitro estimates demonstrated that overall leaf DMADP pool was very large, almost an order of magnitude larger than the in vivo pool. Yet, the difference between total DMADP pools in light and in darkness (light-dependent DMADP pool) was tightly correlated with the in vivo estimates of the DMADP pool size that is responsible for isoprene emission. Variation in in vivo DMADP pool size was obtained by varying light intensity and atmospheric CO(2) and O(2) concentrations. From these experiments, the in vivo kinetic constants of isoprene synthase were determined. In vivo isoprene synthase kinetic characteristics suggested that isoprene synthase mainly operates under substrate limitation and that short-term light, CO(2), and O(2) dependencies of isoprene emission result from variation in DMADP pool size rather than from modifications in isoprene synthase activity.

Dark inactivation of ferredoxin-NADP reductase and cyclic electron flow under far-red light in sunflower leaves.

The oxidation kinetics under far-red light (FRL) of photosystem I (PSI) high potential donors P700, plastocyanin (PC), and cytochrome f (Cyt f) were investigated in sunflower leaves with the help of a new high-sensitivity photometer at 810 nm. The slopes of the 810 nm signal were measured immediately before and after FRL was turned on or off. The same derivatives (slopes) were calculated from a mathematical model based on redox equilibrium between P700, PC and Cyt f and the parameters of the model were varied to fit the model to the measurements. Typical best-fit pool sizes were 1.0-1.5 micromol m(-2) of P700, 3 PC/P700 and 1 Cyt f/P700, apparent equilibrium constants were 15 between P700 and PC and 3 between PC and Cyt f. Cyclic electron flow (CET) was calculated from the slope of the signal after FRL was turned off. CET activated as soon as electrons accumulated on the PSI acceptor side. The quantum yield of CET was close to unity. Consequently, all PSI in the leaf were able to perform in cycle, questioning the model of compartmentation of photosynthetic functions between the stroma and grana thylakoids. The induction of CET was very fast, showing that it was directly redox-controlled. After longer dark exposures CET dominated, because linear e- transport was temporarily hindered by the dark inactivation of ferredoxin-NADP reductase.