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.

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.

Elevated [CO2] magnifies isoprene emissions under heat and improves thermal resistance in hybrid aspen.

Isoprene emissions importantly protect plants from heat stress, but the emissions become inhibited by instantaneous increase of [CO2], and it is currently unclear how isoprene-emitting plants cope with future more frequent and severe heat episodes under high [CO2]. Hybrid aspen (Populus tremula x Populus tremuloides) saplings grown under ambient [CO2] of 380 µmol mol(-1) and elevated [CO2] of 780 µmol mol(-1) were used to test the hypothesis that acclimation to elevated [CO2] reduces the inhibitory effect of high [CO2] on emissions. Elevated-[CO2]-grown plants had greater isoprene emission capacity and a stronger increase of isoprene emissions with increasing temperature. High temperatures abolished the instantaneous [CO2] sensitivity of isoprene emission, possibly due to removing the substrate limitation resulting from curbed cycling of inorganic phosphate. As a result, isoprene emissions were highest in elevated-[CO2]-grown plants under high measurement [CO2]. Overall, elevated growth [CO2] improved heat resistance of photosynthesis, in particular, when assessed under high ambient [CO2] and the improved heat resistance was associated with greater cellular sugar and isoprene concentrations. Thus, contrary to expectations, these results suggest that isoprene emissions might increase in the future.

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.

Ecosystem-scale biosphere-atmosphere interactions of a hemiboreal mixed forest stand at Järvselja, Estonia.

During two measurement campaigns, from August to September 2008 and 2009, we quantified the major ecosystem fluxes in a hemiboreal forest ecosystem in Järvselja, Estonia. The main aim of this study was to separate the ecosystem flux components and gain insight into the performance of a multi-species multi-layered tree stand. Carbon dioxide and water vapor fluxes were measured using the eddy covariance method above and below the canopy in conjunction with the microclimate. Leaf and soil contributions were quantified separately by cuvette and chamber measurements, including fluxes of carbon dioxide, water vapor, nitrogen oxides, nitrous oxide, methane, ozone, sulfur dioxide, and biogenic volatile organic compounds (isoprene and monoterpenes). The latter have been as well characterized for monoterpenes in detail. Based on measured atmospheric trace gas concentrations, the flux tower site can be characterized as remote and rural with low anthropogenic disturbances. Our results presented here encourage future experimental efforts to be directed towards year round integrated biosphere-atmosphere measurements and development of process-oriented models of forest-atmosphere exchange taking the special case of a multi-layered and multi-species tree stand into account. As climate change likely leads to spatial extension of hemiboreal forest ecosystems a deep understanding of the processes and interactions therein is needed to foster management and mitigation strategies.

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.

Heat sensitivity of photosynthetic electron transport varies during the day due to changes in sugars and osmotic potential.

In water-stressed leaves, accumulation of neutral osmotica enhances the heat tolerance of photosynthetic electron transport. There are large diurnal and day-to-day changes in leaf sugar content because of variations in net photosynthetic production, respiration and retranslocation. To test the hypothesis that diurnal and day-to-day variations in leaf sugar content and osmotic potential significantly modify the responses to temperature of photosynthetic electron transport rate, we studied chlorophyll fluorescence rise temperatures (i.e. critical temperatures at break-points in fluorescence versus temperature response curves, corresponding to enhanced damage of PSII centers and detachment of pigment-binding complexes) in the dark at a background of weak far-red light (T(FR)) and under actinic light (T(L)), and responses of foliar photosynthetic electron transport rate to temperature using gas-exchange and chlorophyll fluorescence techniques in the temperate tree Populus tremula L. Sucrose and sorbitol feeding experiments demonstrated strong increases of fluorescence rise temperatures T(FR) and T(L) with decreasing leaf osmotic potential and increasing internal sugar concentration. Similar T(FR) and T(L) changes were observed in response to natural variation in leaf sugar concentration throughout the day. Increases in leaf sugar concentration led to an overall down-regulation of the rate of photosynthetic electron transport (J), but increases in the optimum temperature (Topt) of J. For the entire dataset, Topt varied from 33.8 degrees C to 43 degrees C due to natural variation in sugars and from 33.8 degrees C to 52.6 degrees C in the sugar feeding experiments, underscoring the importance of sugars in modifying the response of J to temperature. However, the correlations between the sugar concentration and fluorescence rise temperature varied between the days. This variation in fluorescence rise temperature was best explained by the average temperature of the preceding 5 or 6 days. In addition, there was a significant year-to-year variation in heat sensitivity of photosynthetic electron transport that was associated with year-to-year differences in endogenous sugar content. Our data demonstrate a diurnal variation in leaf heat tolerance due to changes in sugar concentration, but they also show that this short-term modification in heat tolerance is super-imposed by long-term changes in heat resistance driven by average temperature of preceding days.

Reductive titration of photosystem I and differential extinction coefficient of P700+ at 810-950 nm in leaves.

We describe a method of reductive titration of photosystem I (PSI) density in leaves by generating a known amount of electrons (e-) in photosystem II (PSII) and measuring the resulting change in optical signal as these electrons arrive at pre-oxidized PSI. The method complements a recently published method of oxidative titration of PSI donor side e- carriers P700, plastocyanin (PC) and cytochrome f by illuminating a darkened leaf with far-red light (FRL) [V. Oja, H. Eichelmann, R.B. Peterson, B. Rasulov, A. Laisk, Decyphering the 820 nm signal: redox state of donor side and quantum yield of photosystem I in leaves, Photosynth. Res. 78 (2003) 1-15], presenting a nondestructive way for the determination of PSI density in intact leaves. Experiments were carried out on leaves of birch (Betula pendula Roth) and several other species grown outdoors. Single-turnover flashes of different quantum dose were applied to leaves illuminated with FRL, and the FRL was shuttered off immediately after the flash. The number of e- generated in PSII by the flash was measured as four times O2 evolution following the flash. Reduction of the pre-oxidized P700 and PC was followed as a change in leaf transmittance using a dual-wavelength detector ED P700DW (810 minus 950 nm, H. Walz, Effeltrich, Germany). The ED P700DW signal was deconvoluted into P700+ and PC+ components using the abovementioned oxidative titration method. The P700+ component was related to the absolute number of e- that reduced the P700+ to calculate the extinction coefficient. The effective differential extinction coefficient of P700+ at 810-950 nm was 0.40+/-0.06 (S.D.)% of transmittance change per micromol P700+ m(-2) or 17.6+/-2.4 mM(-1) cm(-1). The result shows that the scattering medium of the leaf effectively increases the extinction coefficient by about two times and its variation (+/-14% S.D.) is mainly caused by light-scattering properties of the leaf.