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. 2016 Oct 21;172(4):2275–2285. doi: 10.1104/pp.16.01002

Spectacular Oscillations in Plant Isoprene Emission under Transient Conditions Explain the Enigmatic CO2 Response1

Bahtijor Rasulov 1,2,3, Eero Talts 1,2,3, Ülo Niinemets 1,2,3,*
PMCID: PMC5129709  PMID: 27770061

Oscillations in isoprene emission demonstrate that the emission is controlled by chloroplast reductant status.

Abstract

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.

About 500 Tg of carbon fixed by primary producers is lost due to plant isoprene emission, and therefore isoprene is globally the most important plant-generated trace gas with major impacts on atmospheric processes (Guenther et al., 2012; Arneth et al., 2008). As a highly reactive trace gas with the largest source strength, isoprene drives atmospheric chemistry, including formation of ozone and other atmospheric oxidants, from regional to global scales, and further participates in aerosol formation with potential implications for climate (Guenther et al., 2012; Peñuelas et al., 2013; Sharkey and Monson, 2014). Thus, understanding the biological controls on emissions of isoprene is of fundamental importance for predicting isoprene emission fluxes from vegetation.

The emission of isoprene occurs in a light- and temperature-dependent manner similarly to photosynthetic CO2 fixation, and the key enzyme responsible for isoprene synthesis, isoprene synthase, is located in chloroplasts. Therefore, extensive efforts have been made to disentangle the connection between photosynthesis and isoprene emission (Sanadze, 1964; Monson and Fall, 1989; Loreto and Sharkey, 1990; Niinemets et al., 1999; Arneth et al., 2007; Rasulov et al., 2009b; Morfopoulos et al., 2014). However, the divergent CO2 responses of photosynthesis, which increases asymptotically with increasing CO2 concentration, and isoprene emission, which decreases over most of the ambient CO2 concentration range (Sanadze, 1964; Monson and Fall, 1989; Loreto and Sharkey, 1990, 1993), have remained enigmatic.

Since the discovery of chloroplastic 2-C-methyl-d-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate (MEP/DOXP) pathway for chloroplastic isoprenoid synthesis, several competing hypotheses have been put forward to explain the CO2 inhibition of isoprene emission (Rasulov et al., 2009b; Wilkinson et al., 2009; Li and Sharkey, 2013b; Morfopoulos et al., 2014). Given that the synthesis of isoprene as a highly reduced molecule requires more NADPH and ATP per each C than photosynthetic carbon fixation, it has been suggested that isoprene emission is controlled not directly by photosynthesis but by the rate of photosynthetic electron transport (Niinemets et al., 1999; Rasulov et al., 2009b; Morfopoulos et al., 2014). Thus, under conditions of high CO2, photosynthesis is increased because photorespiration is suppressed, but the total electron transport rate is inhibited, ultimately suppressing the rate of isoprene emission. This suppression has lately (Li and Sharkey, 2013b; Banerjee and Sharkey, 2014) been suggested to reflect classic conditions of feedback-inhibited photosynthesis when inorganic phosphate (Pi) levels decrease because of limited capacity of starch and sucrose synthesis reactions releasing Pi; low Pi in turn further inhibits ATP synthesis, and this leads to reduction in the rate of photosynthesis (Sharkey, 1985b). When chloroplastic ATP concentrations decrease, the rate of synthesis of MEP/DOXP pathway phosphorylated intermediates also decreases, leading to suppression of isoprene synthesis (Li and Sharkey, 2013a, 2013b; Banerjee and Sharkey, 2014).

Alternatively, given that pyruvate used in the first condensation step of the MEP/DOXP pathway is likely of cytosolic origin, it has been suggested that reduced pyruvate supply at a higher CO2 concentration curbs the chloroplastic pyruvate level and thereby reduces the MEP/DOXP pathway activity (Rosenstiel et al., 2003; Wilkinson et al., 2009; Potosnak et al., 2014). Both hypotheses converge in predicting that at high CO2, the concentration of the isoprene immediate precursor, dimethylallyl diphosphate (DMADP), pool should decrease, which is consistent with experimental evidence (Rasulov et al., 2009b, 2011; Li et al., 2011; Possell and Hewitt, 2011; Potosnak et al., 2014). However, there is currently no direct experimental support for either of the two hypotheses, and the lack of mechanistic understanding of controls on isoprene emission remains a key weakness in predicting isoprene emissions in future CO2-rich atmospheres and from stressed plants.

Regarding the electron transport/feedback inhibition hypothesis of isoprene emission responses to CO2 concentration, one conspicuous feature of feedback-limited conditions is the induction of photosynthetic oscillations upon increases in ambient CO2 and/or decreases in O2 concentrations (Walker et al., 1983; Sivak and Walker, 1987; Peterson et al., 1988; Laisk et al., 1991). It has been conclusively demonstrated that the oscillatory minima in photosynthesis are caused by reduction of the ATP and NADPH levels due to limited Pi under these transitory conditions (Stitt et al., 1988; Laisk et al., 1991). Were isoprene emission controlled by ATP and/or NADPH supply, oscillations in isoprene emission should occur concurrently with photosynthetic oscillations.

While oscillations in photosynthesis are routinely observed, to our knowledge, oscillations in isoprene emission have not been demonstrated, calling into question the hypothesis of isoprene synthesis being controlled at the level of photosynthetic energy supply. However, isoprene synthase responsible for isoprene synthesis from DMADP has an exceptionally large Km value with respect to its substrate DMADP, two to three orders of magnitude greater than the Km values for mono- and sesquiterpene synthases to their substrates (Köksal et al., 2010). Thus, isoprene-emitting species typically support very high chloroplastic pools of DMADP and upstream metabolites that can sometimes reach impressive magnitudes (Li et al., 2011; Rasulov et al., 2011, 2015; Li and Sharkey, 2013a). In fact, these pools can support isoprene emission in the darkness for extended periods of time (Li et al., 2011; Rasulov et al., 2011, 2015; Li and Sharkey, 2013a). These large pools can potentially buffer changes in photosynthetic energy supply such that oscillations in the MEP/DOXP pathway remain masked in transitory conditions that lead to oscillations in photosynthesis, although certain transitory dynamics in isoprene emission have been sometimes observed (Monson and Fall, 1989; Fall and Monson, 1992). Here, we have developed protocols to study transitory processes in conditions of significantly reduced chloroplastic DMADP pool size in a hybrid aspen (Populus tremula × Populus tremuloides) plant model system (Rasulov et al., 2009a, 2011, 2015). Reduced DMADP pool size was achieved by two different approaches: (1) by partially inhibiting the MEP/DOXP pathway by specific pathway inhibitor fosmidomycin (3-[N-formyl-N-hydroxyamino]propylphosphonic acid; Kuzuyama et al., 1998), and (2) by selection of ambient conditions known to result in feedback inhibition of photosynthesis and in significantly decreased chloroplastic DMADP pool size in intact leaves (e.g. Rasulov et al., 2011; Li and Sharkey, 2013a). We hypothesized that under conditions of reduced DMADP pool size, oscillatory dynamics in photosynthetic characteristics are directly reflected in oscillations in isoprene emissions. Our results demonstrate that the fascinating and conspicuous photosynthetic oscillations induced under transitory conditions, when imbalances in intermediate recycling inhibit the rate of photosynthetic energy and reductant supply (Stitt et al., 1988; Laisk et al., 1991), are accompanied by simultaneous and proportional oscillations in isoprene emission. We argue that the discovery of oscillations in isoprene emissions provides the long-sought explanation for the controversial CO2 dependence of isoprene emission and thus paves the way for development of fully mechanistic models for global isoprene emission and experimental techniques for remote sensing of isoprene release (Peñuelas et al., 2013).

RESULTS

Partial Inhibition of Isoprene Emission by Fosmidomycin

In fosmidomycin inhibition experiments, fosmidomycin is usually applied through the entire experiment at a concentration leading to almost full inhibition of isoprene emission, by 90 to 95% of the initial value. We fed leaves of hybrid aspen with a concentration of 8 μm fosmidomycin solution for a period of approximately 10 min to achieve the target isoprene emission rate of approximately 40% of the initial emission rate (Fig. 1; “Materials and Methods”). Separate experiments demonstrated that under continuous fosmidomycin feeding, isoprene emission was almost fully inhibited in approximately 30 min after the start of feeding (Fig. 1, dashed line). Foliage photosynthetic characteristics remained stable throughout both the partial and full inhibition treatments (data not shown).

Figure 1.

Figure 1.

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Representative sample kinetics of partial inhibition of isoprene emission by the chloroplastic MEP/DOXP pathway inhibitor fosmidomycin and oscillatory transients in isoprene emission following changes in CO2 and O2 concentrations in a hybrid aspen leaf. Partial inhibition of isoprene emission was achieved by feeding the leaf with 8 μm fosmidomycin solution through petiole between 200 and 800 s. At 800 s, the fosmidomycin solution was rapidly replaced by distilled water, and the experimental modifications in ambient gas concentrations were started when a new steady-state level, approximately 40% of the initial value, had been reached at 1,350 s. According to separate measurements, the isoprene emission rate would have been almost completely inhibited (90% to 95% inhibition) were the leaf left in the fosmidomycin solution (extrapolated red dashed line; Rasulov et al., 2011). The measurement window covers 11 transients elicited either by modifications in CO2 or O2 concentration. The time periods when the apparent isoprene emission approached zero at 1,650; 4,450; and 6,350 s correspond to measurements of the reference line (incoming air). The transient within the red square corresponds to the data shown in Figure 3. The inset demonstrates the linear correlation between changes in the isoprene immediate substrate DMADP pool size and the pool size of photosynthetic substrate RUBP (see “Materials and Methods” and Fig. 6 for their estimation) through all the oscillations shown in the figure. For the whole measurement period, leaf temperature was maintained at 30°C and light intensity at 700 μmol m−2 s−1.

In partly fosmidomycin-inhibited leaves, the chloroplastic pool size of the immediate isoprene substrate DMADP determined from the first phase of the dark-decay kinetics of isoprene emission was between 300 and 400 nmol m−2 (Fig. 2), that is approximately 10% to 15% of the DMADP pool size of 1500 to 3000 nmol m−2 in noninhibited leaves. In contrast, the pool size of the upstream intermediate metabolite 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) determined from the kinetics of the second phase of postillumination isoprene release reached values between 10,000 and 15,000 nmol m−2 in partly fosmidomycin-inhibited leaves, which is an approximately 5- to 10-fold enhancement compared with noninhibited leaves (Fig. 2).

Figure 2.

Figure 2.

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Illustration of in vivo kinetic measurements of intermediate pool sizes responsible for isoprene emission in a hybrid aspen leaf partially inhibited by fosmidomycin. The in vivo estimation of intermediate pool sizes relies on monitoring of the postillumination kinetics of isoprene emission (Rasulov et al., 2009a, 2011; Li et al., 2011). After isoprene emission has reached a steady state, light is rapidly switched off (at time 140 s denoted by the arrow), and the release of isoprene emission is measured in the darkness. Isoprene emission during the first 100 to 250 s corresponds to the pool size of primary isoprene substrate DMADP and isopentenyl diphosphate (IDP) that is in equilibrium with DMADP. With depletion of the DMADP pool size in darkness, isoprene emission decreases, but it starts to rise again at approximately 200 s due to dark-activated conversion of the upstream metabolites, presumably MEcDP, to DMADP (Li et al., 2011; Rasulov et al., 2011). Integration of the first and second peak of isoprene emission in the dark provides quantitative estimates of DMADP and MEcDP pool sizes. Partial inhibition of isoprene synthesis (approximately 60%) was achieved by 8 μm fosmidomycin as in Figure 1. Leaf temperature was 30°C through the experiment, and light intensity prior to darkening was 700 μmol m−2 s−1. R denotes measurements of the reference line (incoming air) between 80 and 140 s.

Oscillations in Isoprene Emission Induced under Partial Inhibition by Fosmidomycin

Oscillations in net assimilation rate and chlorophyll fluorescence are typically observed upon rapid changes in ambient gas concentrations or light level, especially in feedback-limited conditions. In partially fosmidomycin-inhibited leaves, all such modifications inducing photosynthetic oscillations also induced oscillations in isoprene emission. Among the environmental modifications resulting in oscillations in isoprene emission were increases in ambient CO2 concentration under normal O2 concentration (Figs. 1 and 3), reduction in O2 concentration under ambient (Fig. 4A) and high (Fig. 4B) CO2 concentrations, and rapidly switching on the light under high CO2 and low O2 concentrations (Fig. 4C). Positive O2 sensitivity of photosynthesis was evident in several experiments (e.g. Figure 4B), suggesting feedback inhibition of photosynthesis.

Figure 3.

Figure 3.

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Representative oscillations in leaf net assimilation, chlorophyll fluorescence, and isoprene emission induced by an increase in ambient CO2 concentration from 400 to 1200 μmol mol−1 (denoted by red arrow at time 35 s) in hybrid aspen. Values for stomatal conductance for water vapor are also shown. The experiment was conducted at ambient O2 concentration of 21%, leaf temperature of 30°C, and light intensity of 700 μmol m−2 s−1. Partial inhibition by 8 μm fosmidomycin (Figs. 1 and 2; approximately 60% inhibition of isoprene emission) was used to reduce the size of the transient pools of the immediate isoprene precursor, DMADP, and the upstream metabolite HMBDP. The dashed lines through oscillations of isoprene and net assimilation rate denote the average rates used to estimate the changes in the metabolite pools during the oscillations (“Materials and Methods”; Fig. 6).

Figure 4.

Figure 4.

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Oscillatory kinetics of isoprene emission and net assimilation rates and chlorophyll fluorescence induced by transfers from ambient oxygen concentration of 21% to 2% at current ambient CO2 concentration of 400 μmol mol−1 (A) and at a high CO2 concentration of 1,200 μmol mol−1 (B) and by a short period (5 s) of darkness (C) in partially fosmidomycin-inhibited leaves of hybrid aspen. Measurement conditions and partial fosmidomycin inhibition as in Figures 1 and 2, and gas concentrations through the experiment as shown at the top of each panel. Ref in B denotes measurement of the reference line.

The oscillations in photosynthesis and isoprene emission were in the same phase, and oscillations in chlorophyll fluorescence in the opposite phase (Figs. 3 and 4). The oscillations were extensive, amounting to ±30% to ±50% of steady-state values through the first oscillatory cycle. After stabilization, new steady-state levels corresponding to changed ambient conditions were reached (the change shown by dashed lines in Fig. 3). No oscillations in stomatal conductance were observed (Fig. 3), and stomatal conductance was not different among partly fosmidomycin-inhibited and non-fosmidomycin-inhibited intact leaves (data not shown), indicating that leaf water status was not altered by fosmidomycin treatment.

Oscillations in Non-Fosmidomycin-Inhibited Leaves

In the second set of experiments without the use of fosmidomycin, we selected environmental conditions known to lead to suppressed DMADP pool size simultaneously with feedback inhibition of photosynthesis due to limited chloroplastic inorganic phosphate concentration (Pi), including low or zero O2 concentration and low or high CO2 concentration (Li et al., 2011; Rasulov et al., 2011; Li and Sharkey, 2013a). Under these conditions, oscillations in photosynthesis, chlorophyll fluorescence, and isoprene emissions were induced in attached intact leaves by either rapid decreases (Fig. 5A) or increases (Fig. 5B) in CO2 concentration similarly to those in partially fosmidomycin-inhibited leaves. In both cases, photosynthesis was feedback-inhibited as the reduction in CO2 concentration resulted in increased net assimilation rate (Fig. 5A), while the increase in CO2 concentration resulted in decreased net assimilation rate (Fig. 5B). The direction of change in isoprene emission through these transients, a major increase upon a decrease of CO2 concentration (Fig. 5A) and a moderate reduction upon increase in CO2 concentration (Fig. 5B), was the same as that for photosynthesis.

Figure 5.

Figure 5.

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Induction of oscillations in isoprene emission, net assimilation rate, and chlorophyll fluorescence in intact, non-fosmidomycin-inhibited leaves of hybrid aspen by decreasing (A) and increasing (B) ambient CO2 concentration. In both cases, the leaf was incubated under the indicated ambient air gas concentrations for at least 10 min prior to the change in CO2 concentration. Leaf temperature was 30°C and light intensity 700 μmol m−2 s−1 through the experiments.

Proportional Oscillations in Isoprene and Photosynthesis through Transients

Analysis of the quantitative changes in isoprene substrate DMADP pool size and photosynthetic primary substrate, presumably ribulose-1,5-bisphosphate (RUBP) pool size (also called assimilatory charge) through oscillations (Fig. 6), demonstrated that they were strongly correlated through the oscillations (Figs. 1, inset, and 7). Furthermore, relative changes in isoprene emission and net assimilation rates were also strongly correlated across all oscillations with the slope of 0.9 for a proportional relationship (Fig. 8).

Figure 6.

Figure 6.

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Estimation of changes in isoprene immediate precursor, DMADP, and key photosynthetic substrate RUBP (also called assimilatory charge) through isoprene and photosynthesis oscillations in a representative experiment with a hybrid aspen leaf partially inhibited by fosmidomycin (the same data as in Fig. 3). First, the zero lines corresponding to average values of isoprene emission Inline graphic and net assimilation Inline graphic rates were estimated through the oscillations (shown by solid horizontal curves), and then crests and troughs above and below the average lines (shaded areas) were integrated with respect to time, yielding the changes in DMADP and RUBP pool sizes corresponding to upward and downward oscillations. The oscillation amplitudes, that is, the maximum changes in isoprene (Im and −Im) and net assimilation (Am and −Am) rates, are also shown for the first full oscillation period.

Figure 7.

Figure 7.

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Changes in the immediate isoprene precursor DMADP pool size and the key photosynthetic intermediate RUBP (also called assimilatory charge) corresponding to the oscillations induced in isoprene emission and net assimilation rate by increasing the ambient CO2 concentration from 400 to 1,200 μmol mol−1 in hybrid aspen (the same experiment as in Fig. 3). The pool sizes were calculated as explained in Figure 6, whereas the pool changes due to upward oscillations relative to the average have positive signs and the changes due to downward oscillations have negative signs. The sums of pool changes due to upward and downward oscillations through the full oscillatory transient are also shown for both processes. The inset demonstrates the correlation between the absolute changes in DMADP pool size and assimilatory charge through all the oscillations.

Figure 8.

Figure 8.

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The relationship among the relative absolute changes in isoprene emission and net assimilation rate through oscillations induced by transfer from ambient (400 μmol mol−1) to high (1,200 μmol mol−1) CO2 concentration, transfer from ambient (21%) to low (2%) O2 concentration at high CO2 concentration or switching off the light at high CO2 concentration (“High CO2”), or induced by transfer from high to ambient CO2 concentration or transfer from ambient to low O2 concentration at ambient CO2 concentration (”Ambient CO2”). Relative change is given as the absolute value of the maximum oscillatory change (amplitude) divided by the corresponding average (Fig. 6 for graphical definitions). Data correspond to oscillations through different replicate experiments (Figs. 2, 3, and 4, B and C, for representative “High CO2” experiments and Figs. 2 and 4A for representative “Ambient CO2” experiments).

DISCUSSION

Oscillations in Isoprene Emission

Conspicuous oscillations in isoprene emission were observed upon changes in ambient gas concentration and upon rapid leaf illumination (Figs. 1 and 3–5). Such oscillations have been characteristically observed for foliage photosynthesis and chlorophyll fluorescence and have been associated with feedback inhibition of photosynthesis (Walker et al., 1983; Sivak and Walker, 1987; Peterson et al., 1988; Laisk et al., 1991). In feedback-inhibited conditions, the reactions of triose-phosphate transport and consumption cannot keep up with the rate of photosynthetic triose phosphate production, and it is well established that such conditions ultimately lead to a reduced rate of ATP and NADPH synthesis due to low stromal Pi (Sharkey, 1985a; Sharkey and Vanderveer, 1989; Sharkey and Vassey, 1989; Lee et al., 2014). Under such conditions, the sensitivity of photosynthesis to changes in CO2 and O2 concentrations decreases, and the photosynthesis might eventually even respond negatively to increases in CO2 concentration and decreases in O2 concentration (Sharkey, 1985a, 1985b, 1986). Indeed, positive O2 sensitivity of photosynthesis (Fig. 4B) or negative CO2 sensitivity of photosynthesis (Fig. 5) was observed in our experiments, confirming the feedback-limited conditions.

While photosynthetic oscillations are frequently observed, we hypothesized that the lack of pronounced experimental evidence of isoprene oscillations in the literature reflects the very high DMADP pool sizes supported in vivo in chloroplasts of isoprene-emitting species (Li et al., 2011; Rasulov et al., 2011; Niinemets et al., 2015). Indeed, the oscillations in isoprene emission were particularly pronounced in partly fosmidomycin-inhibited leaves (Figs. 1, 3, and 4) where the pool size of DMADP was significantly reduced (Fig. 2), but oscillations could be also induced in non-fosmidomycin-inhibited leaves under ambient conditions known to result in a major reduction in DMADP pool size (Fig. 5). During the oscillations, the change in DMADP pool size was between approximately ±50 and ±350 nmol m−2, which is a large change for leaves with reduced DMADP pool size (Fig. 7). However, such a change would constitute only between 2% and 20% of DMADP pool size in noninhibited leaves under current ambient CO2 and O2 concentrations. Although the Km value for DMADP of isoprene synthase is high (Köksal et al., 2010; Rajabi Memari et al., 2013; Rasulov et al., 2014), the rate of isoprene synthesis versus DMADP pool size relationships becomes increasingly nonlinear with rising DMADP pool size (Rasulov et al., 2009a, 2014). Thus, we conclude that the high background of DMADP masks the effects of oscillatory dynamics in the assimilatory charge pool size on the DMADP pool size and on isoprene emission.

Oscillations in Isoprene Emission Are in the Same Phase and Proportional to Photosynthetic Oscillations

The period of oscillations in isoprene emission was about 130 s (Figs. 3 and 4), which is in good agreement with the oscillatory dynamics of photosynthesis and chlorophyll fluorescence demonstrated in previous studies in feedback-inhibited leaves (Walker et al., 1983; Sivak and Walker, 1987; Peterson et al., 1988; Laisk et al., 1991). Furthermore, the oscillations in isoprene emission were in the same phase with oscillations in net assimilation rate, while the oscillations in chlorophyll fluorescence were in the opposite phase (Figs. 3 and 4). Thus, in the declining phase of photosynthesis and isoprene emission, fluorescence was rising, suggesting a reduction in the efficiency of light use in photosynthesis due to progressive inhibition of photosynthetic electron transport as chloroplastic Pi levels start to decline, curbing ATP and ultimately NADPH synthesis. In the rising part of photosynthetic oscillations, chlorophyll fluorescence was decreasing, indicating increasing efficiency of excitation energy use for photosynthetic electron transport as Pi was progressively released, restoring ATP and NADPH synthesis.

Detailed analysis of oscillatory dynamics (Fig. 6) revealed that relative changes in isoprene emission and net assimilation rates were strongly correlated through all oscillations, and the oscillations were proportional with a slope slightly below 1:1 line (Fig. 8). Thus, transitory modifications in net assimilation rates and isoprene emission through changes in ambient gas concentration both occurred in an oscillatory manner driven by fluctuations in the same energy and reductant supply until a new steady-state level was reached (Figs. 3 and 4). At this new steady-state level, either at higher CO2 (Fig. 3) or lower O2 (Fig. 4, A and B), both photosynthesis and isoprene emission operated at a lower energy and reductant supply, although net assimilation was sometimes increased (Figs. 3 and 4A) due to suppression of photorespiration by high CO2 and/or low O2. A slight departure from 1:1 line of relative changes in isoprene emission and net assimilation rate (Fig. 8) might indicate different ATP and NADPH requirements for isoprene emission and net assimilation rate. In fact, the cofactor requirement for DMADP formation can be variable through oscillations, depending on the magnitude of changes in the size of phosphorylated intermediate pools of MEP/DOXP pathway prior to DMADP (see below). In the case of a large pool of phosphorylated intermediates (Fig. 2, dark pool), temporarily, only reductive equivalents are needed for DMADP formation, while when phosphorylated intermediate pools start to deplete, increasingly more ATP is required to maintain DMADP synthesis.

Importantly, in all cases, photosynthesis was slightly (3–5 s) ahead of isoprene emission across oscillations (Figs. 3 and 4). This suggests that photosynthesis is the key sink drawing down chloroplastic ATP and NADPH levels, ultimately leading to reductions in DMADP levels. Furthermore, a direct control of isoprene emission at the level of electron transport is supported by induction of oscillations by short 5-s pulses of darkness in feedback-inhibited leaves (Fig. 4C). This short period of darkness was sufficient to release enough Pi to stimulate ATP and NADPH formation and lead to short-term overshoot of net assimilation and isoprene emission rates.

We argue that these findings constitute direct proof of photosynthetic energy control on the synthesis of isoprene under changing CO2 and O2 concentrations and thus provide the key missing piece of information for development of fully mechanistic isoprene emission models. The possible role of photosynthetic electron transport in controlling the rate of isoprene emission has been suggested in early studies showing a decrease in isoprene release at low O2 concentrations under O2-insensitive photosynthesis (Monson and Fall, 1989; Loreto and Sharkey, 1990, 1993; Monson et al., 1991) and a correlation between isoprene emission and ATP content (Loreto and Sharkey, 1993). Recently, experimental observations of major changes in MEP/DOXP pathway metabolites under different steady-state conditions achieved by manipulations of CO2 and O2 concentrations similar to our study have led to a revival of the hypothesis of the control of isoprene emission by ATP under feedback-limited conditions induced by high CO2 and low O2 (Li and Sharkey, 2013a, 2013b). However, steady-state situations provide only correlative evidence, because changes in metabolite pools can result from either/both changes in carbon availability or/and energy and reductant availability. We argue that the presence of simultaneous and proportional oscillations in isoprene emission and photosynthesis (Figs. 1, 3, 7, and 8) provides the direct experimental evidence of the photosynthetic electron transport control of isoprene emission.

How Could the Photosynthetic Electron Transport Control on Isoprene Emission Operate?

The evidence presented suggests that the redox state of the photosynthetic electron transport chain is the leading factor responsible for the start of oscillations in photosynthesis and isoprene emission. Given that photosynthesis is the main sink for ATP and NADPH, the question is how could such a control operate? First, the whole MEP/DOXP pathway could be inhibited by the lack of ATP in a manner similar to photosynthesis as curbing Pi levels start to reduce phosphorylated intermediate levels, including the key MEcDP pool size (Fig. 9; Li and Sharkey, 2013a, 2013b). While principally possible, MEcDP pool size was large in our experiments (Fig. 2), and major MEcDP pools have been demonstrated in other experiments with feedback-inhibited leaves (Li and Sharkey, 2013a).

Figure 9.

Figure 9.

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Simple scheme of photosynthetic energy-metabolite supply for isoprene synthesis and processes inducing oscillations in photosynthesis and isoprene emission. Photosynthesis is the largest sink for ATP provided by photophosphorylation and for NADPH produced through photosynthetic electron transport. Oscillations in photosynthesis are thought to occur due to feedback inhibition resulting from limited recycling of Pi that is incorporated both in the main photosynthetic intermediate RUBP as well as triose phosphates (AC pool) and is also needed for ATP synthesis. When Pi levels drop because processes downstream of immediate CO2 fixation releasing Pi, starch, and sucrose synthesis, cannot keep up with Pi consumption, ATP production becomes inhibited, in turn inhibiting regeneration of RUBP (Sharkey, 1985a; Stitt et al., 1988; Laisk et al., 1991). Overenergization of thylakoids further feedback inhibits the entire photosynthetic electron transport chain (visible in enhanced fluorescence during oscillations, e.g. Fig. 3), resulting in inhibition of NADPH and Fd formation. Although the chloroplastic MEP/DOXP pathway relies on a relatively small part of photosynthetic electron transport, two key enzymes, HMBDP synthase responsible for HMBDP formation and HMBDP reductase responsible for the immediate isoprene substrate DMADP formation, can accept electrons directly from reduced Fd (Seemann et al., 2006). Thus, fluctuations in the rate of generation of reductive equivalents can lead to oscillatory changes in HMBDP and DMADP pool sizes, resulting in oscillations in isoprene emission as demonstrated in this study. In principle, oscillations in isoprene emission can also result from oscillations in ATP and upstream carbon substrates through changes in MEcDP pool size, but given the extensive MEcDP pools in these experiments with partially fosmidomycin-inhibited leaves (Figs. 2–4) and in noninhibited leaves (Fig. 5; see Rasulov et al., 2011 for estimation of pool sizes under conditions shown here; Li and Sharkey, 2013a), a direct induction of oscillations by ATP concentration seems unlikely.

In fact, current observations are better explained by suppression of reductant supply in feedback-limited conditions and resulting reductions in the activities of two last reactions downstream of MEcDP, that is, 4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBDP) synthase (HDS) responsible for HMBDP formation and HMBDP reductase (HDR) responsible for the immediate isoprene substrate DMADP formation (Fig. 9). Both HDS and HDR can accept electrons directly from reduced ferredoxin (Fd; Seemann et al., 2006), implying that these two last enzymatic steps can most sensitively track changes in reductant availability. Thus, changes in the reductant level are expected to ultimately determine the extent to which MEcDP is converted to DMADP, thereby controlling the pool size of DMADP and resultant rate of isoprene emission. While such a control has been considered to be responsible for the light response of isoprene emission (Rasulov et al., 2009b; Sun et al., 2012; Li and Sharkey, 2013b), we suggest that modifications in chloroplastic redox status upon changes in ambient gas concentrations also underlie the CO2 and O2 responses of isoprene emission.

CONCLUSION

Overall, the oscillations in isoprene emission observed here conclusively demonstrate a direct control of isoprene emission by photosynthetic electron transport chain. Although the total amount of electron flow going into isoprene synthesis is relatively minor, the existence of such a control suggests that the effective Km value of HDS and HDR for reduced Fd is large, leading to a highly sensitive response of the MEP/DOXP pathway to the changes in reductant supply. We argue that resolving the old-standing enigma of how elevation of CO2 concentration inhibits isoprene emission is of fundamental significance for development of fully mechanistic global models of isoprene emission. Furthermore, identification of the mechanism of photosynthetic control explains how the entire MEP/DOXP pathway is regulated in plants.

MATERIALS AND METHODS

Plant Material

Two-year-old hybrid aspen (Populus tremula × Populus tremuloides clone H200) plants were used as in our previous experiments (Rasulov et al., 2009a, 2011, 2015). The plants were grown in 4-liter pots filled with an 1:1 mixture of commercial planting soil and sand with slow-release balanced fertilizer in Fitoclima S600 PLLH climatic chambers (Aralab). The light intensity at plant level was maintained at 600 μmol m−2 s−1 for a 14-h day, day/night temperatures were 25°C/22°C, relative air humidity was 60%, and ambient CO2 concentration 400 μmol mol−1. Plants were watered to field capacity every second day. Fully mature 18- to 20-d-old leaves were used in all experiments.

Experimental System and Measurement Protocol

The measurements of foliage net assimilation, transpiration, and isoprene emission rates were conducted using an ultrafast two-channel custom-made gas-exchange system (system half-time of approximately 0.15 s) specially designed to measure rapid transients and allowing for a full control of measurement air composition, leaf temperature, and light intensity (see Rasulov et al., 2011, 2015 for details). Different gas concentrations could be prepared in the two separate channels and switched rapidly by ASCO SC precision solenoid valves (ASCO Valves), resulting in an almost instant change in measurement gas concentrations. Concentration of CO2 was measured by an LI-6251 infrared gas analyzer (LI-Cor), water vapor concentration by a custom-made psychrometer, oxygen concentration by a N-22M Metek oxygen analyzer (Metek), and isoprene concentration by a proton-transfer reaction quadrupole mass-spectrometer (high-sensitivity version with a response time of approximately 0.1 s; Ionicon Analytik) using the protonated parent ion with mass-to-charge ratio of 69+ (Graus et al., 2004; Rasulov et al., 2014). A standard gas containing all key volatiles (Ionimed) was used to calibrate the proton-transfer reaction quadrupole mass-spectrometer instrument. Light-adapted chlorophyll fluorescence yield, F, was measured using a PAM 101 fluorimeter (Walz) with the measurement light modulated at 100 kHz.

After the leaf enclosure in the measurement cuvette, standard measurement conditions (ambient CO2 concentration of 400 μmol mol−1, oxygen concentration of 2%, relative humidity of 60%, leaf temperature of 30°C, saturating light intensity of 700 μmol m−2 s−1) were established, and the leaf was maintained at these conditions until steady-state gas exchange and isoprene emission rates were reached. Then the leaf was subject to partial inhibition by the chloroplastic MEP/DOXP pathway inhibitor fosmidomycin (3-[N-formyl-N-hydroxyamino]propylphosphonic acid) as described below and conditioned at different combinations of ambient CO2 and O2 concentrations until a new steady-state was reached. Oscillations were induced by rapidly changing the gas concentration or the light level. Analogous experiments were conducted with noninhibited attached leaves conditioned at different combinations of CO2 and O2. The combinations of CO2 and O2 chosen for partially fosmidomycin-inhibited leaves were those known to induce feedback inhibition of photosynthesis (high CO2 and ambient O2, ambient CO2 and low O2, and high CO2 and low O2; Sharkey, 1985b). For noninhibited attached leaves, the conditions used were those demonstrated to lead to reduction in immediate isoprene precursor pool size (low or high CO2 under low O2; Rasulov et al., 2011; Li and Sharkey, 2013a). All kinetic experiments were replicated at least three times, and in all cases reproducible kinetics were observed.

Partial Inhibition of Isoprene Emission by Fosmidomycin

Large pools of immediate isoprene precursor DMADP and further upstream metabolites of MEP/DOXP pathway can accumulate in chloroplasts under steady-state conditions (Rasulov et al., 2009a, 2011; Li et al., 2011; Li and Sharkey, 2013a; Weise et al., 2013). These extensive pools can effectively dampen the responses of isoprene emission to rapid changes in environmental conditions that cause oscillations in photosynthesis with its comparatively smaller intermediate pool sizes. In contrast, in our previous experiments we found that by complete blocking of the MEP/DOXP pathway by fosmidomycin, not only DMADP levels but also isoprene emissions dramatically drop, precluding any kinetic studies (Rasulov et al., 2011, 2015). Therefore, partial inhibition (on average 60% inhibition) of the MEP/DOXP pathway by fosmidomycin was used to reduce the pool size of DMADP (Fig. 1) and create conditions of high responsiveness of isoprene emission to fluctuations in environmental drivers.

The leaves were fed with 8 μm fosmidomycin solution through petiole, and isoprene emission was continuously measured (Fig. 1). When the rate of isoprene emission had been reduced by 20% to 30% relative to the initial value, typically 10 min after the start of fosmidomycin feeding, the feeding was stopped by replacing the fosmidomycin solution by distilled water. After removing the fosmidomycin solution, the rate of isoprene emission continued to decrease for the following 10 to 15 min until a new steady state was reached, characteristically at the level of approximately 40% of the initial emission rate (Fig. 1). Experimental modifications in environmental conditions to induce oscillations were started at this new steady-state level of isoprene emission.

Estimation of Isoprene Precursor Pool Sizes by the in Vivo Kinetic Method

Fosmidomycin is considered a specific inhibitor of deoxyxylulose phosphate reductoisomerase in the beginning of MEP/DOXP pathway (Kuzuyama et al., 1998), but it has also been suggested that fosmidomycin might inhibit simultaneously HMBDP synthase/and or HMBDP reductase (Rasulov et al., 2011). If so, partial inhibition of the MEP/DOXP pathway should reduce both DMADP and HMBDP pool sizes, while the pool size of phosphorylated intermediates, in particular, MEcDP, is expected to increase. In addition, MEcDP pool size is known to increase and DMADP pool size to decrease under conditions of 2% and zero O2 concentrations, especially when CO2 is also low (Rasulov et al., 2011; Li and Sharkey, 2013a) as used in the experiments with non-fosmidomycin-inhibited leaves.

To analyze changes in DMADP and MEcDP pool sizes under the experimental treatments, we used the in vivo method of (Rasulov et al. 2009a, 2011; Li et al., 2011; Weise et al., 2013) that is based on analysis of postillumination kinetics of isoprene release. In short, after a steady-state isoprene emission rate is reached in light, light is rapidly switched off, and the isoprene emission is measured through the biphasic dark kinetics, typically for 10 to 15 min. The first, rapidly decaying part of dark emission release, normally between 100 and 250 s, corresponds to the DMADP pool supporting the given rate of isoprene emission in the light (Fig. 2; Rasulov et al., 2009a; Li et al., 2011). This in vivo estimate is in excellent agreement with separate destructive chemical measurements of the chloroplastic DMADP pool size (Rasulov et al., 2009a; Weise et al., 2013).

While the DMADP pool size synthesized in light is depleted, a second dark-activated isoprene release starts (Fig. 2; Li et al., 2011; Rasulov et al., 2011). This release corresponds to conversion of upstream phosphorylated metabolites to DMADP, and it has been demonstrated to rely on the MEcDP pool (Li and Sharkey, 2013a). Thus, integrating the second dark-induced isoprene release provides an estimate of MEcDP pool size (Fig. 2; Li et al., 2011; Rasulov et al., 2011). Again, direct measurements of MEcDP pool size indicate a strong correlation with in vivo estimates (Li et al., 2011).

Use of this methodology for in vivo quantification of key metabolite pools of the MEP/DOXP pathway indicated that partially fosmidomycin-inhibited leaves had a moderate DMADP pool size and a vast MEcDP pool size (Fig. 2). Analogously, in non-fosmidomycin-inhibited attached leaves conditioned to low O2 (Fig. 5), DMADP pool size was small and MEcDP pool size was large, confirming previous observations (data not shown; Rasulov et al., 2011; Li and Sharkey, 2013a).

Quantification of Changes in Isoprene and Net Photosynthesis Rates and Corresponding Precursor Pool Sizes through Oscillations

Rapid modifications in environmental drivers as used here resulted in damping oscillations that represented transitory processes between the initial steady state and the new steady-state level. To characterize the amplitude of oscillations and changes in substrate pools through the oscillations, a zero line through the upward and downward oscillations that connected the two steady states was fitted through the data using representative polynomial functions and minimizing the sum of squares between the data and the zero line (Fig. 6). The position of the zero line at any moment of time t characterizes the average process rate (Inline graphic(t) for isoprene and Inline graphic(t) for net assimilation), and the maximum and minimum emission rates from the zero line at time t correspond to the oscillation amplitudes (Im(t) and -Im(t) for isoprene and Am(t) and -Am(t) for net assimilation rate; Fig. 6). From these characteristics, the absolute values of relative change corresponding to each oscillation were calculated as abs(Im(t)/Inline graphic(t)) for isoprene and abs(Am(t)/Inline graphic(t)) for net assimilation.

Changes in the immediate isoprene substrate DMADP pool size corresponding to each oscillatory cycle were calculated as the integral of each upward and downward oscillation as shown in Figure 6. Analogously, changes in the pool size of immediate photosynthetic substrates, primarily reflecting RUBP (also called assimilatory charge), were estimated (Fig. 6). The absolute values were used to examine the correlations among changes in DMADP and RUBP pools.

Differences between RUBP and DMADP pool sizes between the initial and final steady-state situations (Figs. 6 and 7) were also calculated to evaluate the metabolic controls under different steady-state conditions. The differences in pool sizes correspond to the sums of pool size changes through each oscillation during the oscillatory process between the two steady-state situations (Fig. 7). For instance, as the result of the negative net change of the DMADP pool size upon increasing CO2 concentration, the new steady-state isoprene emission rate was less than before the transfer (Figs. 3 and 6). In contrast, due to the positive net change in RUBP pool size, the new steady-state net assimilation rate was higher than before the change (Figs. 3 and 6).

Glossary

DMADP

dimethylallyl diphosphate

Fd

reduced ferredoxin

HDR

4-hydroxy-3-methyl-but-2-enyl diphosphate reductase

HDS

4-hydroxy-3-methyl-but-2-enyl diphosphate synthase

HMBDP

4-hydroxy-3-methyl-but-2-enyl diphosphate

MEcDP

2-C-methyl-d-erythritol 2,4-cyclodiphosphate

MEP/DOXP

2-C-methyl-d-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate

RUBP

ribulose-1,5-bisphosphate

References

  1. Arneth A, Monson RK, Schurgers G, Niinemets Ü, Palmer PI (2008) Why are estimates of global isoprene emissions so similar (and why is this not so for monoterpenes)? Atmos Chem Phys 8: 4605–4620 [Google Scholar]
  2. Arneth A, Niinemets Ü, Pressley S, Bäck J, Hari P, Karl T, Noe S, Prentice IC, Serça D, Hickler T, et al. (2007) Process-based estimates of terrestrial ecosystem isoprene emissions: incorporating the effects of a direct CO2-isoprene interaction. Atmos Chem Phys 7: 31–53 [Google Scholar]
  3. Banerjee A, Sharkey TD (2014) Methylerythritol 4-phosphate (MEP) pathway metabolic regulation. Nat Prod Rep 31: 1043–1055 [DOI] [PubMed] [Google Scholar]
  4. Fall R, Monson RK (1992) Isoprene emission rate and intercellular isoprene concentration as influenced by stomatal distribution and conductance. Plant Physiol 100: 987–992 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Graus M, Schnitzler J-P, Hansel A, Cojocariu C, Rennenberg H, Wisthaler A, Kreuzwieser J (2004) Transient release of oxygenated volatile organic compounds during light-dark transitions in grey poplar leaves. Plant Physiol 135: 1967–1975 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Guenther AB, Jiang X, Heald CL, Sakulyanontvittaya T, Duhl T, Emmons LK, Wang X (2012) The Model of Emissions of Gases and Aerosols from Nature version 2.1 (MEGAN2.1): an extended and updated framework for modeling biogenic emissions. Geosci Model Dev 5: 1471–1492 [Google Scholar]
  7. Köksal M, Zimmer I, Schnitzler J-P, Christianson DW (2010) Structure of isoprene synthase illuminates the chemical mechanism of teragram atmospheric carbon emission. J Mol Biol 402: 363–373 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kuzuyama T, Shimizu T, Takashashi S, Seto H (1998) Fosmidomycin, a specific inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis. Tetrahedron Lett 39: 7913–7916 [Google Scholar]
  9. Laisk A, Siebke K, Gerst U, Eichelmann H, Oja V, Heber U (1991) Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle. Planta 185: 554–562 [DOI] [PubMed] [Google Scholar]
  10. Lee S-K, Eom J-S, Voll LM, Prasch CM, Park Y-I, Hahn T-R, Ha S-H, An G, Jeon J-S (2014) Analysis of a triose phosphate/phosphate translocator-deficient mutant reveals a limited capacity for starch synthesis in rice leaves. Mol Plant 7: 1705–1708 [DOI] [PubMed] [Google Scholar]
  11. Li Z, Ratliff EA, Sharkey TD (2011) Effect of temperature on postillumination isoprene emission in oak and poplar. Plant Physiol 155: 1037–1046 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Li Z, Sharkey TD (2013a) Metabolic profiling of the methylerythritol phosphate pathway reveals the source of post-illumination isoprene burst from leaves. Plant Cell Environ 36: 429–437 [DOI] [PubMed] [Google Scholar]
  13. Li Z, Sharkey TD (2013b) Molecular and pathway controls on biogenic volatile organic compound emissions. In Niinemets Ü, Monson RK, eds, Biology, Controls and Models of Tree Volatile Organic Compound Emissions. Springer, Berlin, pp 119–151 [Google Scholar]
  14. Loreto F, Sharkey TD (1990) A gas-exchange study of photosynthesis and isoprene emission in Quercus rubra L. Planta 182: 523–531 [DOI] [PubMed] [Google Scholar]
  15. Loreto F, Sharkey TD (1993) On the relationship between isoprene emission and photosynthetic metabolites under different environmental conditions. Planta 189: 420–424 [DOI] [PubMed] [Google Scholar]
  16. Monson RK, Fall R (1989) Isoprene emission from aspen leaves: influence of environment and relation to photosynthesis and photorespiration. Plant Physiol 90: 267–274 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Monson RK, Hills AJ, Zimmerman PR, Fall RR (1991) Studies of the relationship between isoprene emission rate and CO2 or photon-flux density using a real-time isoprene analyser. Plant Cell Environ 14: 517–523 [Google Scholar]
  18. Morfopoulos C, Sperlich D, Peñuelas J, Filella I, Llusià J, Medlyn BE, Niinemets Ü, Possell M, Sun Z, Prentice IC (2014) A model of plant isoprene emission based on available reducing power captures responses to atmospheric CO₂. New Phytol 203: 125–139 [DOI] [PubMed] [Google Scholar]
  19. Niinemets Ü, Sun Z, Talts E (2015) Controls of the quantum yield and saturation light of isoprene emission in different-aged aspen leaves. Plant Cell Environ 38: 2707–2720 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Niinemets Ü, Tenhunen JD, Harley PC, Steinbrecher R (1999) A model of isoprene emission based on energetic requirements for isoprene synthesis and leaf photosynthetic properties for Liquidambar and Quercus. Plant Cell Environ 22: 1319–1336 [Google Scholar]
  21. Peñuelas J, Marino G, Llusià J, Morfopoulos C, Farré-Armengol G, Filella I (2013) Photochemical reflectance index as an indirect estimator of foliar isoprenoid emissions at the ecosystem level. Nat Commun 4: 2604. [DOI] [PubMed] [Google Scholar]
  22. Peterson RB, Sivak MN, Walker DA (1988) Carbon dioxide-induced oscillations in fluorescence and photosynthesis. Role of thylakoid membrane energization in regulation of photosystem II activity. Plant Physiol 88: 1125–1130 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Possell M, Hewitt CN (2011) Isoprene emissions from plants are mediated by atmospheric CO2 concentrations. Glob Change Biol 17: 1595–1610 [Google Scholar]
  24. Potosnak MJ, Lestourgeon L, Nunez O (2014) Increasing leaf temperature reduces the suppression of isoprene emission by elevated CO₂ concentration. Sci Total Environ 481: 352–359 [DOI] [PubMed] [Google Scholar]
  25. Rajabi Memari H, Pazouki L, Niinemets Ü (2013) The biochemistry and molecular biology of volatile messengers in trees. In Niinemets Ü, Monson RK, eds, Biology, controls and models of tree volatile organic compound emissions. Springer, Berlin, pp 47–93 [Google Scholar]
  26. Rasulov B, Bichele I, Laisk A, Niinemets Ü (2014) Competition between isoprene emission and pigment synthesis during leaf development in aspen. Plant Cell Environ 37: 724–741 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rasulov B, Copolovici L, Laisk A, Niinemets Ü (2009a) Postillumination isoprene emission: in vivo measurements of dimethylallyldiphosphate pool size and isoprene synthase kinetics in aspen leaves. Plant Physiol 149: 1609–1618 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rasulov B, Hüve K, Laisk A, Niinemets Ü (2011) Induction of a longer term component of isoprene release in darkened aspen leaves: origin and regulation under different environmental conditions. Plant Physiol 156: 816–831 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rasulov B, Hüve K, Välbe M, Laisk A, Niinemets Ü (2009b) Evidence that light, carbon dioxide, and oxygen dependencies of leaf isoprene emission are driven by energy status in hybrid aspen. Plant Physiol 151: 448–460 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rasulov B, Talts E, Kännaste A, Niinemets Ü (2015) Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen. Plant Physiol 168: 532–548 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rosenstiel TN, Potosnak MJ, Griffin KL, Fall R, Monson RK (2003) Increased CO2 uncouples growth from isoprene emission in an agriforest ecosystem. Nature 421: 256–259 [DOI] [PubMed] [Google Scholar]
  32. Sanadze GA. (1964) Light-dependent excretion of isoprene by plants. Photosynth Res 2: 701–707 [Google Scholar]
  33. Seemann M, Tse Sum Bui B, Wolff M, Miginiac-Maslow M, Rohmer M (2006) Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG. FEBS Lett 580: 1547–1552 [DOI] [PubMed] [Google Scholar]
  34. Sharkey TD. (1985a) O2-insensitive photosynthesis in C3 plants: its occurrence and a possible explanation. Plant Physiol 78: 71–75 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sharkey TD. (1985b) Photosynthesis in intact leaves of C3 plants: physics, physiology and rate limitations. Bot Rev 51: 53–105 [Google Scholar]
  36. Sharkey TD. (1986) Theoretical and experimental observations on O2 sensitivity of C3 photosynthesis. In R Marcelle, H Clijsters, M Van Poucke, eds, Biological Control of Photosynthesis. Proceedings of a conference held at the 'Limburgs Universitair Centrum', Diepenbeek, Belgium, August 26–30, 1985. Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster, pp 115–125 [Google Scholar]
  37. Sharkey TD, Monson RK (2014) The future of isoprene emission from leaves, canopies and landscapes. Plant Cell Environ 37: 1727–1740 [DOI] [PubMed] [Google Scholar]
  38. Sharkey TD, Vanderveer PJ (1989) Stromal phosphate concentration is low during feedback limited photosynthesis. Plant Physiol 91: 679–684 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sharkey TD, Vassey TL (1989) Low oxygen inhibition of photosynthesis is caused by inhibition of starch synthesis. Plant Physiol 90: 385–387 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sivak MN, Walker DA (1987) Oscillations and other symptoms of limitation of in vivo photosynthesis by inadequate phosphate supply to the chloroplast. Plant Physiol Biochem 25: 635–648 [Google Scholar]
  41. Stitt M, Grosse H, Woo KC (1988) Interactions between sucrose synthesis and CO2 fixation. II. Alterations of fructose 2,6-bisphosphate during photosynthetic oscillations. J Plant Physiol 133: 138–143 [Google Scholar]
  42. Sun Z, Niinemets Ü, Hüve K, Noe SM, Rasulov B, Copolovici L, Vislap V (2012) Enhanced isoprene emission capacity and altered light responsiveness in aspen grown under elevated atmospheric CO2 concentration. Glob Change Biol 18: 3423–3440 [Google Scholar]
  43. Walker DA, Sivak MN, Prinsley RT, Cheesbrough JK (1983) Simultaneous measurement of oscillations in oxygen evolution and chlorophyll a fluorescence in leaf pieces. Plant Physiol 73: 542–549 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weise SE, Li Z, Sutter AE, Corrion A, Banerjee A, Sharkey TD (2013) Measuring dimethylallyl diphosphate available for isoprene synthesis. Anal Biochem 435: 27–34 [DOI] [PubMed] [Google Scholar]
  45. Wilkinson MJ, Monson RK, Trahan N, Lee S, Brown E, Jackson RB, Polley HW, Fay PA, Fall R (2009) Leaf isoprene emission rate as a function of atmospheric CO2 concentration. Glob Change Biol 15: 1189–1200 [Google Scholar]

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