ORIGINAL RESEARCH published: 14 August 2020 doi: 10.3389/fpls.2020.01242 Heat Waves Change Plant Carbon Allocation Among Primary and Secondary Metabolism Altering CO2 Assimilation, Respiration, and VOC Emissions Christiane Werner 1*, Lukas Fasbender 1, Katarzyna M. Romek 1, Ana Maria Yáñez-Serrano 1,2,3 and Jürgen Kreuzwieser 1 Edited by: Uwe Rascher, 1 Ecosystem Physiology, University of Freiburg, Freiburg, Germany, 2 Center of Ecological Research and Forest Applications Helmholtz Association of German (CREAF), Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Global Ecology Unit CREAF-CSIC-UAB, Cerdanyola del Research Centers (HZ), Germany Vallès, Barcelona, Spain Reviewed by: Toshihiro Obata, Processes controlling plant carbon allocation among primary and secondary metabolism, University of Nebraska-Lincoln, United States i.e., carbon assimilation, respiration, and VOC synthesis are still poorly constrained, Raquel Esteban, particularly regarding their response to stress. To investigate these processes, we University of the Basque Country, Spain simulated a 10-day 38°C heat wave, analysing real-time carbon allocation into primary *Correspondence: and secondary metabolism in the Mediterranean shrub Halimium halimifolium L. We Christiane Werner traced position-specific 13C-labeled pyruvate into daytime VOC and CO2 emissions and c.werner@cep.uni-freiburg.de during light-dark transition. Net CO2 assimilation strongly declined under heat, due to Specialty section: three-fold higher respiration rates. Interestingly, day respiration also increased two-fold. This article was submitted to Decarboxylation of the C1-atom of pyruvate was the main process driving daytime CO2 Functional Plant Ecology, release, whereas the C2-moiety was not decarboxylated in the TCA cycle. Heat induced a section of the journal Frontiers in Plant Science high emissions of methanol, methyl acetate, acetaldehyde as well as mono- and Received: 17 November 2019 sesquiterpenes, particularly during the first two days. After 10-days of heat a Accepted: 29 July 2020 substantial proportion of 13C-labeled pyruvate was allocated into de novo synthesis of Published: 14 August 2020 VOCs. Thus, during extreme heat waves high respiratory losses and reduced assimilation Citation: Werner C, Fasbender L, Romek KM, can shift plants into a negative carbon balance. Still, plants enhanced their investment into Yáñez-Serrano AM and Kreuzwieser J de novo VOC synthesis despite associated metabolic CO2 losses. We conclude that heat (2020) Heat Waves Change Plant stress re-directed the proportional flux of key metabolites into pathways of VOC Carbon Allocation Among Primary and Secondary Metabolism Altering biosynthesis most likely at the expense of reactions of plant primary metabolism, which CO2 Assimilation, Respiration, might highlight their importance for stress protection. and VOC Emissions. Front. Plant Sci. 11:1242. Keywords: carbon allocation, day respiration, temperature stress, 13C position-specific labeling, photosynthesis, doi: 10.3389/fpls.2020.01242 pyruvate, dark, volatile organic compounds (VOC) Frontiers in Plant Science | www.frontiersin.org 1 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes INTRODUCTION resistance, most probably by stabilizing membranes, in particular the thylakoid membranes (Singsaas et al., 1997; Global climate change will markedly alter environmental conditions Sharkey and Yeh, 2001; Behnke et al., 2007; Sharkey et al., for plant growth and functioning. However, potential changes 2008). Recent reports have shown that this may be an indirect between the magnitudes of plant carbon sequestration by effect mediated via modulating membrane bound proteins of the photosynthesis relative to the rate of growth, respiration and thylakoid membranes (Harvey et al., 2015). Moreover, VOCs can secondary metabolism are highly uncertain. There is a marked aid to enhance the photosynthetic quenching of excess energy in disparity between the process-based formulations of biochemical the thylakoid membranes (Penuelas and Munne-Bosch, 2005; models of photosynthetic CO2 assimilation (Farquhar et al., 1980), Pollastri et al., 2014), which is particularly critical to protect the and models of respiration (Sweetlove et al., 2013; O’Leary et al., photosynthetic apparatus (Rennenberg et al., 2006; Niinemets, 2019), allocation (Brüggemann et al., 2011), or those describing the 2018). Consequently, many plant species enhance the release of response of the secondary metabolism to resources availability (e.g., isoprene and monoterpenes under heat stress (Loreto and Larbat et al., 2016). C-allocation is thought to be driven by the C Schnitzler, 2010), which can be mediated by enhanced de novo balance between supply (source) and demand (sink), however, the biosynthesis and/or increased vapour pressure of stored dominating factor is still a matter of debate (Körner, 2013; Fatichi compounds (Peñuelas and Staudt, 2010; Holopainen et al., et al., 2014). Indeed, plants exhibit a higher than expected plasticity to 2018). It must be denoted, that severe heat stress has also been adjust both source and sink capacities in response to environmental shown to reduce both de novo and total emissions of mono- and changes (e.g., Wegener et al., 2015b), or carbon allocation into sesquiterpenes (Kleist et al., 2012). primary and secondary metabolism (e.g., Heinrich et al., 2015). In De novo synthesis of VOCs depends on the availability of particular, knowledge on plant internal readjustment of the carbon, as well as energy provided by primary metabolism. metabolic carbon fluxes in response to environmental stresses for Therefore, the availability of building blocks has a major impact synthesis of defence compounds such as volatile organic compounds on the concentration of any secondary metabolite, demonstrating (VOCs) is still limited (e.g., Huang et al., 2019). In the light of extreme the high degree of connectivity between primary and secondary climatic events, such as drought spells and heat waves, the plant’s metabolism (Dudareva et al., 2013). Carbon partitioning occurs at ability to rapidly adjust its metabolism and protect the the metabolic branching points of the respiratory pathways and photosynthetic machinery against these stresses will be decisive for secondary metabolism. The relevant pathways are linked via a its persistence. Plant species, particularly from hot and arid number of key metabolites including the central intermediates environments, have evolved multiple structural and functional pyruvate (Werner and Gessler, 2011), phosphoenolpyruvate adaptations to withstand environmental stress (Werner et al., (PEP) or malate (Souza et al., 2018). Notably, pyruvate is a 1999; Werner et al., 2002). However, heat waves, i.e., the rapid known substrate in a large array of secondary pathways leading to occurrence of excessive temperatures over prolonged periods, can the biosynthesis of many VOCs, such as isoprenoids, some expose plants and ecosystems to stress levels beyond their oxygenated VOCs, aromatics as well as fatty acid oxidation acclimation capacity (Teskey et al., 2015; Tatarinov et al., 2016). products, which can be emitted by plants (Jardine et al., 2014). Over the past decades, record-breaking monthly temperature However, elevated temperatures also increase respiratory losses extremes and heat waves have increased (Coumou et al., 2013; (Atkin, 2003) and reduce carbon assimilation, which may limit the IPCC, 2014), strongly impacting ecosystem carbon balances (Ciais availability of carbon skeletons for secondary metabolism including et al., 2005; Bastos et al., 2013). As heat waves are predicted to further biosynthesis of volatile compounds. increase in frequency and intensity (Perkins-Kirkpatrick and In general, leaf day respiration is recognised to play a central Gibson, 2017) it is imperative to understand how they will affect role to provide carbon backbones for several metabolic processes plant physiological processes and growth (Teskey et al., 2015). (Tcherkez et al., 2012). Due to the light-inhibition of the Plants have evolved a plethora of mechanism to cope with respiratory pathways (Atkin et al., 1998), day respiration provides heat stress, which causes production of reactive oxygen species a low-flux though highly dynamic metabolic pathway that (ROS) in plant cells (Jajic et al., 2015; Dietz et al., 2016; Suzuki interacts with photosynthesis and environmental conditions and Katano, 2018; Waszczak et al., 2018). Scavenging of (Tcherkez et al., 2017). While the mitochondrial respiratory ROS can be achieved through elevated levels of antioxidants, tricarboxylic acid (TCA) cycle is partially inhibited in the light accumulation and adjustment of solutes, specialized kinase (e.g., Sweetlove et al., 2010; Tcherkez et al., 2012), there are several cascades, as well as chaperone signalling (see review by Dietz metabolic processes both in the cytosol and organelles causing et al., 2016). Another mechanism to mitigate the effects of partial decarboxylation of pyruvate. Thus, while the C2-C3 moiety oxidative stress under high temperatures is through the of pyruvate fuels anabolic processes such as fatty acid and secondary production of volatile isoprenoids (Vickers et al., 2009), which compound synthesis, the carboxyl group at the C1 position is have been shown to reduce the levels of damaging ROS within released as CO2 (Werner and Gessler, 2011). However, few studies the leaf (Loreto and Velikova, 2001; Sharkey and Yeh, 2001; have investigated carbon allocation into day respiration in response Affek and Yakir, 2002; Affek and Yakir, 2003; Sharkey, 2005). to rising temperature and VOC emissions (Fares et al., 2011) and Elevated temperatures increase the fluidity of membranes and even less is known regarding the response to heat extremes. can induce leakiness. Volatile isoprenoids such as isoprene and/ We hypothesise that heat stress leads to enhanced carbon or monoterpenes have often been shown to increase heat allocation into secondary metabolism in particular VOC Frontiers in Plant Science | www.frontiersin.org 2 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes biosynthesis for stress-protection and that such metabolic re- 2015a) were grown from seeds in a greenhouse for three years. adjustment may result in higher CO2 release in the light. Plants that reassemble a genetic variability were grown under Moreover, we assume a trade-off between carbon investment into similar conditions in a semi-controlled greenhouse, with natural protective functions such as VOC synthesis and maintenance of a light and additional illumination (12 h); temperatures were positive carbon balance under severe stress, particularly if stress regulated by heating during winter. Plants were cultivated in 3 levels exceed the acclimation potential during extreme events. L plastic pots on a potting soil/sand (2/1, v/v) mixture. They were To test this hypothesis, we selected the highly stress-adapted fertilised weekly with ¼-strength modified Hoagland’s fertilizer Mediterranean shrub, Halimium halimifolium L. (Zunzunegui solution (Peperkorn et al., 2005). Four weeks before the et al., 2000; Peperkorn et al., 2005; Werner et al., 2010), a member experiments, plants were subdivided into two groups and of the Cistaceae family, which is widely spread in Mediterranean placed into two walk-in chambers to allow for acclimation at shrublands (Thanos et al., 1992). Species of this family are day/night conditions of 25/20°C, 60/60% relative humidity and particularly suitable to test our hypothesis since they are 14/10 h light/dark with 900/0 μmol m-2 s-1 photosynthetic active characterized by a rich secondary metabolism forming an photon flux density. Plants were watered according to their immense quantity and diversity of plant secondary metabolites demand. To simulate the heat wave, on day 1 the temperature including a rich volatile emission blend (Kesselmeier and Staudt, of one chamber was stepwise increased in 2°C/h steps over 8 h up 1999; Peñuelas and Llusià, 2001; Owen et al., 2002; Rivoal et al., to 40°C and thereafter kept constant at 38°C during day and 2010; Bracho-Nunez et al., 2013; Papaefthimiou et al., 2014; night for ten days of continuous measurements and subsequent Haberstroh et al., 2018; Yá ñez-Serrano et al., 2018). In addition, labeling experiments. H. halimifolium shows large amplitudes in daytime d13CO2 release Branches of six plants were placed into self-constructed (Werner et al., 2009; Wegener et al., 2010; Wegener et al., 2015a), 600 ml borosilicate glass cuvettes (Kummer, Freiburg, which most likely are connected to processes of plant secondary Germany) two days before the heat treatment. The bottomless metabolism (Priault et al., 2009; Werner and Gessler, 2011; cuvettes were closed with PTFE-foil at the plant stem and Lehmann et al., 2016). Such properties suggest that members of ventilated by small fans; an additional empty cuvette was kept the Cistaceae family possess a high capability to cope with heat by as reference. The cuvette temperature increased by 0.40 ± 0.12°C up-regulating processes of the secondary metabolism, thus, during the light period compared to the dark period. The cuvette redirecting cellular C fluxes. Cistaceae possess trichomes which position was adjusted to maintain the natural orientation of the secrete a resinous exudate (Papaefthimiou et al., 2014) and can store branches. Absolut humidity of the air stream was maintained substantial amounts of VOCs in secretarial trichomes (Gulz et al., constant but due to the increase in temperature during the heat 1996). A study in France has shown that local VOC emissions from treatment relative humidity in the cuvettes decreased at 38°C. Mediterranean shrublands can be in the same order of magnitude of Fluxes of VOCs, H2O and CO2 and their isotopic composition non-methane VOC emissions from anthropogenic sources, and were continuously recorded prior and during the heat treatment. thus can exert a significant impact on air quality (Simon et al., 2006). For labeling experiments, six plants were placed into cuvettes Cistus species are predicted to be favoured by climate change due to an evening before the measurements. A total of 36 plants were their high plasticity to cope with environmental stresses (Correia used; half of them were kept under control temperatures, half and Ascensão, 2017). However, extreme climatic events, such as were consecutively exposed to a 10-day heat treatment. After 10- sudden heat waves or severe drought, may exceed the acclimation days of heat or control temperature, branches were carefully cut potential and induce cascading effects on species and ecosystem and re-cut immediately under water to avoid xylem embolism. functioning (Caldeira et al., 2015). Such extreme climatic events are The deionized water was replaced by a 10 mM 13C-labeled predicted to increase both in intensity and frequency in pyruvate solution after 10 min. We applied position-specific 13 Mediterranean regions (Seneviratne et al., 2012). C-labeled pyruvate 99% 13C-enriched at the C1 or the C2 The objective of our study was to investigate potential position (Cambridge Isotope Laboratories, Andover, MA, USA). changes in leaf internal carbon allocation, i.e., carbon Continuous measurements of the isotopic composition of VOCs assimilation, respiration and investment into VOC under and 13CO2 as well as photosynthetic gas exchange fluxes were extreme heat stress. In particular, we aimed to investigate recorded for 60 min in the light, after which the plants were whether plants under severe stress would still invest into de darkened for 30 min. labeling procedure and concentrations had novo synthesis of VOCs and used position-specific 13C-labeled been established for H. halimifolium by Priault et al. (2009) and pyruvate experiments to trace 13C-allocation into VOCs versus Lehmann et al. (2016) to ensure that no changes in physiological CO2 in light and dark. To explore these effects, we subjected H. processes occurred. Plants, which did respond with stomatal halimifolium to an extreme 10-day heat wave of 38°C. closure at any stage of the treatment, were excluded from the analysis. MATERIAL AND METHODS On-Line Measurement System for Plant VOC, 13CO2, and H2O Emissions Plant Material and Labeling Experiments The measuring system is described in detail in Fasbender et al. Mediterranean drought-adapted, semi-deciduous Halimium (2018). Briefly, it consists of (i) a zero air generator, providing halimifolium L. plants (Zunzunegui et al., 2002; Wegener et al., ultra-pure hydrocarbon free air with controlled CO2 and H2O Frontiers in Plant Science | www.frontiersin.org 3 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes concentrations, (ii) the cuvette system to capture trace gas Net fluxes of 13CO2 (e13CO2) were calculated per projected leaf emissions of plants, and (iii) the analytical section for real-time area (s) as: detection of VOC, CO2 and H2O fluxes and their 13C isotopes uin 13 consisting of a proton transfer reaction time-of-flight mass e13CO2 = ∗ ( cout −13 cin ) spectrometer (4000 ultra, PTR-TOF-MS, Ionicon Analytic, s Innsbruck, Austria), an isotope ratio infrared spectrometer Where uin is the molar flow of incoming air calculated as: (IRIS, Thermo Fisher Scientific, Bremen, Germany), and an infrared gas analyser IRGA (LI-7000 CO2/H2O Analyser; LI- V p ∗ uin = COR, Lincoln, NE, USA). t R∗T The inlet flow of the plant cuvettes was controlled by mass where: V - gas volume, t - time, p - gas pressure, R - ideal gas flow controllers (Omega Engineering, Stamford, CT, USA) at constant, T - temperature, and 13cout/13cin is the molar fraction of 500 ml min‑1 resulting in a calculated residence time in the 13 CO2 at the inlet and outlet of the cuvette calculated as: cuvette of 4.8 min. The response-time of air leaving the cuvette    13  0 1 and reaching the PTR-TOF-MS and IRIS/IRGA was less than 4 d 13 CO2 1000 + 1 ∗ 12 cc and 10 s, respectively. All cuvette parts were made of glass or PFA, 13 c = @    13  VPDB  A ∗ ctotal d 13 CO2 and outlet lines were continuously isolated and heated (60°C) 1000 + 1 ∗ 12 cc +1 VPDB to prevent VOC adsorption or water condensation. where: ctotal - mole fraction of total CO2 in the cuvette and VOC Detection by PTR-TOF-MS (13C/12C)VPDB - isotope ratio of the international standard The PTR-TOF-MS was operated at 2.7 mbar drift pressure, Vienna Pee Dee Belemnite. 600 V drift voltage, at an E/N of 120 Td, and drift tube heated Calibration for isotopic composition was performed to 80°C. H2O+ and O2+ ions in the drift tube were kept lower automatically whenever any significant change in the CO2 than 5% with respect to the hydronium ions. The mass resolution mixing ratio in the sample gas occurred, e.g., during the was 2,000 ± 500 m/Dm depending on the compound. Non-13C- change between plant and blank cuvette. Furthermore, labeled VOCs were detected at m/z 45.03 for acetaldehyde, 75.04 automatic calibration for concentration dependency of the for methyl acetate, 137.13 for monoterpenes and 205.20 for analyser was conducted nightly. sesquiterpenes; their isotopologues containing one 13C-atom Transpiration (E) and assimilation rates (A) were calculated were detected at m/z +1. Calibration of the assigned VOCs was according to Caemmerer and Farquhar (1981). The IRGA was done using the Liquid Calibration Unity (LCU, Ionicon Analytic, calibrated weekly with synthetic air (0 and 408 ppm; Messer, Bad Innsbruck, Austria) either with a multicomponent calibration Soden, Germany) for CO2 and right before the experiment for gas standard for acetaldehyde and monoterpenes (1,000 ppb ± H2O by a manufacture calibrated GFS3000 (Walz GmbH, 5%, Ionicon Analytic, Innsbruck, Austria) or liquid standards for Effeltrich, Germany). sesquiterpenes and methyl acetate (Sigma-Aldrich, Taufkirchen, Germany). For acetaldehyde and monoterpenes humidity Chlorophyll a Fluorescence dependent calibrations were performed. For methyl acetate Chlorophyll a fluorescence was measured with the MINI-PAM II liquid calibrations were carried out with water-based solutions fluorometer (Walz, Effeltrich, Germany). The maximum while for sesquiterpenes hexane-based solutions were used. PTR- quantum yield of PSII was measured after dark-adapting leaves TOF-MS data were post-processed by correction for non- with leaf clips for 20 min (Werner et al., 2002). A saturation pulse extending and extending dead times as well as the correction of 10.000 μmol m-² s-1 was applied; measuring light intensity, for Poisson statistics (Titzmann et al., 2010), iterative residual gain and damping factors were adjusted to allow optimal pulse analysis and cumulative peak fitting (Müller et al., 2013) using resolution. Three leaves per plant were measured on 20 control the PTR-TOF Data Analyser software (version 4.49). Data was and 20 heat stressed plants (after 10 days of heat exposure). In a normalized to primary ions and water, background subtracted, few cases, not all three readings per plant could be used due to and calibration factors applied. Data from PTR-TOF-MS was leaf damage or too low fluorescence signals. synchronized with IRIS and IRGA data. Emission rates were calculated considering the background corrected VOC Statistics concentrations in the cuvettes, leaf area and flow rates. Data were tested for equal variance and normality distribution by a Shapiro-Wilk test. A repeated measurement ANOVA with a 13 Holm-Sidak post-hoc test was applied to test for significant CO2 Detection by IRIS and IRGA 13 CO2 fluxes [nmol m-2 s-1] were calculated from the differences changes in total net carbon assimilation, nocturnal respiratory in 13CO2 isotopic composition and their concentrations between losses and net carbon balance during the 10-day heat waves. empty and plant-containing cuvettes derived from IRIS. The IRIS measures the 12CO2 and 13CO2 fluxes continuously. Calculating the net 13CO2 flux enables to trace both, the RESULTS natural 13C- isotope discrimination during photosynthetic CO2 uptake and the 13 CO 2 release from metabolic processes Exposure to continuous heat (38°C) for 10 days considerably after labeling. reduced net CO2 assimilation compared to pre-treatment rates, Frontiers in Plant Science | www.frontiersin.org 4 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes while nocturnal respiration increased significantly (p < 0.001, Beside lower stomatal conductance and enhanced respiration, Figure 1). The strongest depression of net CO2 assimilation also a direct inhibition of the photosynthetic light use efficiency occurred during the first two days. Thereafter, net assimilation was observed because the maximum quantum yield of decreased slowly, but steadily, further lowering carbon gain photosystem II declined from 0.752 ± 0.05 under control during the light period compared to the respiratory losses at conditions to 0.57 ± 0.12 (n=20) after 10 days of heat. night. Notably, the amplitude between rates of CO2 release Plant were not able to maintain a positive net carbon balance during night and uptake during day remained widely constant during the 10-day heat exposure (Figure 2). Diurnal net (Figure 1), due to an immediate 3.2-fold increase in nocturnal assimilation was significantly reduced by 44% and 84% during respiration. Interestingly, mean nocturnal respiration slowly the first and second day of heat, respectively. Moreover, nocturnal decreased with increasing stress duration, being only 2.3-fold respiration increased significantly 3.3-fold. Therefore, total plant higher than under control conditions at the end of the heat stress. carbon balance turned negative after day three (Figure 2). Even Internal CO2 concentrations increased in all plants under heat though total nocturnal respiratory CO2 losses decreased with stress from 256 ± 14 to 354 ± 57 ppm. stress duration, it could not counterbalance the concomitant There was a striking phenotypic heterogeneity among plant decline in daily net assimilation. Thus, plants exhibited a individuals: while some individuals showed a strong stress negative carbon balance throughout the rest of the heat response resulting in negative net CO2 balances within the first treatment (Figure 2). two days (reddish colours in Figure 1), others still maintained The strong phenotypic variability between plant individuals positive assimilation rates during the day throughout the whole was also reflected in the heat response of VOC emissions period (two individuals had to be replaced on day 6). Mean regarding both, intensity and chemo-diversity. Overall, the transpiration slightly increased in response to heat treatment, but strongest emissions occurred as an immediate stress response again showing high variability between plant individuals (Figure within the first days of heat exposure. Large fluxes were observed 1). Though plants were kept well-watered, they responded with for methyl acetate (Figure 3A), methanol (Figure 3B), stomatal closure during the heat period (data not shown) most acetaldehyde (Figure 3C), as well as monoterpenes (Figure probably in response to a drop in relative humidity due to the 3D) and sesquiterpenes (Figure 3E). Sesquiterpenes and to a higher temperatures. Surprisingly, nocturnal transpiration lesser extent monoterpenes showed the most pronounced seemed to be enhanced in heat stressed plants. reaction upon the initial increase in temperature, resulting in FIGURE 1 | Diurnal courses of air temperature in the climate chamber, net CO2 assimilation, and transpiration rates (upper, mid and lower panel respectively). Plants were continuously measured under controlled conditions (day -1) and 10 days in the heat (38°C). Mean values of 6 plants (solid blue line) and standard error and their individual fluxes are shown. Individual fluxes are colour-coded from reddish to dark blue, ranking individuals from the most to the least affected plants by heat, respectively. Two dying individuals had to be replaced on day 6. Data gaps are due to an instrument failure on day 3 and a calibration routine on day 8. Gray shading indicates night periods. Frontiers in Plant Science | www.frontiersin.org 5 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes To elucidate to what extend VOC emissions were sustained by de novo synthesis or release from storage pools, we conducted position-specific 13 C-labeling experiment by feeding the branches with pyruvate labeled at the C1 or C2 position (Figure 4, left and right hand panels, respectively) in control plants and after 10 days of heat exposure (blue and red lines, respectively). The y-axes scales in Figure 4 are set to reflect proportional changes in the parent mass (left y-axes) and the first heavy isotopologue (right y-axes) for each compound. Very clearly, neither heat stressed nor control plants did incorporate detectable amounts of the 13C1-atom of pyruvate into methyl acetate (Figure 4A, left panel). In contrast, the 13C2 atoms of pyruvate were efficiently used for biosynthesis of methyl acetate, which is visible by an increase in 13C-emission (Figure 4A, right panel). Moreover, heat stressed plants used almost 5-times more 13 C2-pyruvate than non-stressed control plants (compare red with blue lines, Figure 4A). We also observed very interesting labeling patterns for acetaldehyde. Similar to methyl acetate, the FIGURE 2 | Total daily CO2 exchange during the heat treatment: total diurnal net CO2 assimilation (dark red); total nocturnal respiratory CO2 losses (yellow); heat-stressed and control plants did show little 13 C1- and total daily net carbon balance (orange) for the pre-treatment conditions incorporation of pyruvate for acetaldehyde biosynthesis (left (day -1, 25°C) and during 10-day continuous heat exposure (38°C). panel, Figure 4B). However, heat stressed plants very strongly Measurements on day three are lacking due to an instruments failure. Mean incorporated the 13C2-atom into acetaldehyde, an effect not seen of n = 6 and SE of plants shown in Figures 1. in control plants (right panels, Figure 4B). Even more surprising was the much stronger 13C2-use after darkening for acetaldehyde large fluxes during the first two days. Subsequent low formation than compared to incorporation during the light. sesquiterpene emission rates may indicate that sesquiterpene Moreover, non-stressed plants showed a strong dark burst of pools became depleted or biosynthesis decreased. Monoterpene acetaldehyde immediately upon light-dark transition, with emission first decreased but increased again after four days of strong incorporation of 13C from 13C2-pyruvate. Interestingly, stress exposure, even though total monoterpene emissions were this was not the case in heat stressed plants. Such patterns and relatively low in this species. Only plant individuals with very low strong dark emission of acetaldehyde in heat stressed plants have net CO2 assimilation (red lines Figures 1 and 3) maintained never been described before. higher sesquiterpene fluxes during the following days but 13 C-incorporation patterns into mono- and sesquiterpenes without a clear light-dependent pattern. were very similar (Figures 4C, D). In non-stressed control Interestingly, methyl acetate, methanol, and acetaldehyde plants, the C1-atom of pyruvate was not used for biosynthesis emissions exhibited pronounced morning peaks upon stomatal of any of these compounds. However, heat stress stimulated use opening during the initial days of the heat wave (Figures 3A–C). of the C1-atom for terpenoid formation. In contrast to the C1- During the course of the experiment, methyl acetate emissions atom, the C2-atom of pyruvate was used for terpenoid remained high during the day and even an elevated night-time biosynthesis both, in control and heat stressed plants. Notably, flux could be observed. Surprisingly, besides the morning burst, heat stressed plants incorporated 3- to 5-times more 13C2- highest acetaldehyde fluxes were always observed during night in pyruvate than plants of the control group (Figures 4C, D). the heat treatment, while fluxes were negligible prior to stress Unfortunately, labeling patterns of methanol were application (Figure 3C). inconclusive, as a reliable peak separation for the mass of the Large phenotypic plasticity between plant individuals was first isotopologue of methanol was not possible. Thus no increase also reflected in a pronounced chemodiversity regarding their in 13C-labeled methanol could be detected, which might be emission pattern: for example, the individual with the highest expected as it is a known product from cell wall formation and methyl acetate flux (Figure 3B, light blue line) was a low mono- degradation. However, as data were inconclusive due to these and sesquiterpene-emitter. Strongly stressed plants as seen from methodological constrains they are not shown. negative CO2 balances (Figures 1 and 3, red lines) revealed Simultaneous measurements of the isotopic composition of higher VOC emission rates, particularly for mono- and emitted CO2 enabled us to follow the release of 13CO2 from the 13 sesquiterpenes but also for methyl acetate. Still, the highest C-pyruvate labeled plants. As expected, application of 13C1- monoterpene emitter was among the plant individuals pyruvate resulted in strong release of 13CO2 into the atmosphere maintaining highest net CO2 assimilation rates under heat due to decarboxylation of the C1 position (Figure 5). stress (Figures 1B and 3E; thin black line). Thus, these Importantly, heat stress triggered enhanced release of 13CO2 continued real-time emission pattern of CO 2 and VOCs both under light and dark conditions. In contrast, if 13C2- indicated a high dynamic response to heat exposure, as well as pyruvate was applied, 13CO2 release from control plants was a high chemodiversity of emission blends between individuals. negligible. Even under heat stress, only small amounts of 13CO2 Frontiers in Plant Science | www.frontiersin.org 6 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes A B C D E FIGURE 3 | (A–E) Diurnal courses of VOC fluxes prior and during the heat stress of methyl acetate (A), methanol (B), acetaldehyde (C), monoterpenes (D), and sesquiterpenes (E). Individual fluxes are colour-coded as in Figure 1 from reddish to dark blue, ranking individuals from the most to the least affected plants by heat, respectively. were emitted in the light. These results indicate that the pyruvate DISCUSSION fed to the plants was not fully decarboxylated in metabolic pathways, i.e., it was not fully respired in the light. Regulation of plant internal carbon allocation is still poorly Noteworthy, day time CO2-emissions from the C1-position of understood, particularly in response to severe environmental pyruvate more than tripled during heat stress, and so did light- stresses. Here we investigate the impact of a 38°C heat wave on enhanced pulse of dark-respiration during light-dark transitions carbon allocation among primary and secondary metabolism in (Figure 5), indicating that these processes were strongly the Mediterranean species H. halimifolium. We applied position- upregulated during heat stress. specific 13C-pyruvate to trace the fate of the 13C-atom at the Frontiers in Plant Science | www.frontiersin.org 7 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes A B C D FIGURE 4 | 13C-VOC labeling by positions specific 13C-pyruvate during light-dark transitions. Emissions of the parent (12C-isotopologue, solid line, left scale) and the first isotope (13C-isotopologue, open symbols, right scale) of (A) methyl acetate, (B) acetaldehyde, (C) monoterpenes, and (D) sesquiterpenes. Plant of the control (blue) and 10-day heat treatment (red) fed with 13C1-pyruvate (left panels) or 13C2-pyruvate (right panels) for 1 h after which plants were darkened for 30 min. N = 4-8, standard errors are given. Please note that scales differ for each compound; left and right hand scales are set to match the proportional changes in the 12C and 13C-isotoplogues for each compound, i.e., the 13C-scale is set to reflect the changes in the first natural 13C- isotopologue abundance (m/z+1) proportional to the parent (mz), which is an order of magnitude higher. An increase in the 13C-isopologue emission compared to the parent does therefore indicate label incorporation into this compound. metabolic branching points linking VOC synthesis with Continuous day/night exposure to heat stress over ten days respiratory pathways. We could confirm our hypothesis that in induced highly dynamic responses in H. halimifolium: while net spite of a negative carbon balance under severe heat stress (i) photosynthesis significantly declined, respiratory CO2 release enhanced carbon allocation into de novo synthesis of particular during both, day and night, markedly increased. VOCs occurred even though total terpenoid emissions declined Simultaneously, a strong pulse of VOC emissions occurred in H. halimifolium under prolonged heat stress and (ii) that it during the first days (Figure 3). Sudden exposure to elevated was associated with enhanced CO2 losses in the light, as temperatures exceeded the acclimation potential of discussed below in detail. H. halimifolium and net photosynthesis was further impaired Frontiers in Plant Science | www.frontiersin.org 8 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes FIGURE 5 | 13CO2 emissions from plants of the control (blue) and heat treatment (red) fed with 13C1-pyruvate or 13C2-pyruvate for 1 h in the light and 30 min in the dark (shaded area). N= 4–8, standard errors are given. The 13CO2 flux is given in nmol m-2 s-1; for calculation see material and methods. Measurements were conducted simultaneously with volatile organic compounds (VOCs) emissions shown in Figure 4. with increasing stress duration, exhibiting a reduced efficiency of under elevated night-time temperatures has been reported in photochemical energy conversion of PSII. Elevated respiratory many species, including the Mediterranean shrubs Heteromeles CO2 losses affected the net carbon balance of the branches, which arbutifolia and Lepechinia fragans (Villar et al., 1995), and many turned negative already from day 3 onwards, i.e., leaves suffered crops, e.g., soybean (Bunce, 2005), rice (Kanno and Makino, from a net carbon loss as nocturnal CO2 emissions exceeded 2010; Mohammed et al., 2013) and cotton (Loka and Oosterhuis, diurnal CO2 uptake by 78% (Figure 2). 2010). However, even though there is evidence that night-time Even though plants were well watered, transpiration did not temperatures may be of particular importance, contrasting effects increase to an extend which would help facilitate the dissipation have been observed (Atkin et al., 2005). For example, night of latent heat (Loreto and Schnitzler, 2010), since stomatal temperatures between 28 and 42.5 °C resulted in substantially conductance declined during the heat treatment, probably as increased respiration rates in cotton (Salvucci and Crafts- response to lower relative air humidity in the cuvettes and/or Brandner, 2004). Therefore, high night temperatures have even high ci. On the one hand, H. halimifolium is a Mediterranean been considered one of the main environmental factors species, well adapted to seasonally hot and dry environments contributing to lowered cotton yields (Loka and Oosterhuis, with strong evolutionary pressure to control excessive water loss 2010). In contrast, nocturnal temperature increase from 17 to during these stress periods (Diaz Barradas et al., 1999; Werner 34°C lead to only minor increases in respiration in rapidly et al., 2010; Wegener et al., 2015b). On the other hand, reduced growing soybean, lettuce, and tomato and no effect on net assimilation and high day respiration and subsequent cumulative carbon gain was found even after 20 d of treatment increased ci values might have counterbalanced the need to (Frantz et al., 2004). These contrasting findings indicate that open stomata. Even though 38°C can be frequently reached in further research on the impact of nocturnal temperature on the the Mediterranean summer, continuous exposure to 38°C during carbon balance of plants is needed. This is particularly relevant in day and night might have impaired the photosynthetic the light of global warming, where night temperatures are machinery as suggested from declined photochemical efficiency. predicted to increase faster than day temperatures in several Day and night-time temperatures were kept constant during parts of the world (IPCC, 2014). However, while the thermal the heat wave to allow the assessment of VOC fluxes as well as sensitivity of night respiration can potentially reduce biomass in day and night respiration at the same temperature. However, it a warmer climate, acclimation processes have to be taken into must be noted that this implies a larger increase in night-time account at longer time scales (Atkin and Tjoelker, 2003; temperatures during the heat treatment, and thus nocturnal Peraudeau et al., 2015). metabolism might have been more strongly affected. This may High temperatures can significantly inhibit photosynthetic at least partially explain the significant increase in nocturnal efficiency in multiple ways, e.g., through impacts on electron respiration (Figure 1). Such an increase in respiration rates transport activity, or changed ratio of Rubiscos oxygenase/ Frontiers in Plant Science | www.frontiersin.org 9 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes carboxylase activity resulting in higher photorespiration and/or H. halimifolium is a strong methyl acetate emitter (Jardine reduced efficiency of Rubisco activase (Havaux, 1993; Law and et al., 2014) with high intra-specific variability (Fasbender et al., CraftsBrandner, 1999; Salvucci and Crafts-Brandner, 2004; 2018, Figure 3). So far, not many species are characterised as Carmo-Silva et al., 2015). In the present study, this was strong methyl acetate emitters (Davison et al., 2007; Jardine et al., reflected in declined maximum quantum yield of photosystem 2010), though H. halimifolium emits methyl acetate at ca. 10-fold II to 0.570 ± 0.12 after 10 days of heat. higher rates than monoterpenes. Here, we demonstrate for the Whereas several studies demonstrated the effects of heat on first time that methyl acetate emissions markedly increased with isoprene emission (Sharkey et al., 2008; Pollastri et al., 2014; heat stress (Figure 3). It is assumed that methyl acetate Niinemets and Sun, 2015; Bamberger et al., 2017), less biosynthesis starts from pyruvate, which is decarboxylated in a information is available on the dynamic response of other first reaction to produce acetaldehyde that is further oxidized to VOC fluxes toward prolonged heat stress (Fares et al., 2011; acetate (Figure 6). Either acetate or its activated form acetyl-CoA Kleist et al., 2012; Kask et al., 2016). H. halimifolium is not a reacts with methanol to eventually form methyl acetate. Because strong isoprene emitter (Yá ñez-Serrano et al., 2018), similar to of this assumed pathway, we expected similar emission patterns what has been reported for other members of the Cistaceae for methyl acetate and acetaldehyde, which were partially family (Kesselmeier and Staudt, 1999; Bracho-Nunez et al., observed. Both compounds showed enhanced emission in heat 2013). It exhibits a diverse blend of different VOCs, in stressed plants, particularly during night-time. To our particular, methyl acetate (Jardine et al., 2014), mono-, sesqui- knowledge, this is the first report showing a heat dependent, and diterpenes (Yá ñez-Serrano et al., 2018) and a variety of rise in nocturnal acetaldehyde emission which exceeded daytime oxygenated and aromatic VOCs (Fasbender et al., 2018). Here we fluxes by far. Though we did not label the plants during the night, focus on the compounds showing a strong response to heat, darkening the leaves for 30 min revealed a substantial increase in namely methyl acetate, acetaldehyde, methanol, as well as mono- de novo synthesis by 13C-incorporation of 13C2-pyruvate (Figure and sesquiterpenes and compare these with similar observations 4). This suggest that night-time emissions were also driven by de from literature. novo synthesis. This may have fostered the morning burst of acetaldehyde which were more than one order of magnitude Dynamic Response of Oxygenated VOC higher in heat stressed plants than in the control group. Further Emissions to Heat Stress research is needed to investigate the role of nocturnal We observed a marked increase in methyl acetate, methanol and acetaldehyde synthesis during prolonged heat stress in different acetaldehyde emissions upon heat stress. However, in contrast to species. Similar to our results, Behnke et al. (2013) observed heat- terpenoids, none of these compounds showed an immediate induced acetaldehyde production (during 4 h 40°C) in excised increase upon rising temperatures and strongest emissions poplar leaves. However, they suggested that acetaldehyde might occurred between day 2 and 3. Thus a direct physical effect, originate from fatty acid peroxidation reactions after such as higher volatility due to increased temperature, cannot be accumulation of ROS (Jardine et al., 2009) during membrane the reason for such pattern. Since emission of these compounds breakage in damaged tissue (Fall et al., 1999; Loreto et al., 2006; increased when the carbon balance turned negative, biosynthesis Brilli et al., 2011). Indeed, heat-induced membrane dysfunction might be related to a metabolic readjustment of cellular pathways can result in the release of various compounds such as aldehydes in response to stress. The fast 13C-incorporation of 13C2- or ethane (Nanaiah and Anderson, 1992). However, we observed pyruvate into methyl acetate and acetaldehyde, indicates high emission in intact branches of well-watered plants exposed already abundant enzyme activities to ensure fast biosynthesis to heat, and, thus, desiccation effects can be excluded. Though we of these compounds (Fasbender et al., 2018). Enhanced emission cannot exclude direct effects of heat or ROS inducing membrane of oxygenated VOCs was particularly significant during the dysfunction, strong 13 C-incorporation from 13 C2-labeled morning bursts (e.g., for methanol) when stomata opened pyruvate provides clear evidence that acetaldehyde bursts also upon illumination (Figure 3), suggesting that these emissions derive from substantial de novo synthesis. Although the source of are under tight stomatal regulation. Such polar compounds acetaldehyde emission is still a matter of debate (Graus et al., accumulate within leaf internal aqueous phases at night 2004), it seems to be induced by stress (Loreto and Schnitzler, (Kreuzwieser, 1999a; Jardine et al., 2014); hence, the extent of 2010), e.g., during wounding or desiccation under high morning bursts is directly associated with the compounds’ ability temperature (Holzinger et al., 2000; Loreto et al., 2006; Brilli to build up liquid pools during night (Niinemets and Reichstein, et al., 2011) and after root flooding (Kreuzwieser, 1999a; 2003), which are then released when stomata are opening in the Kreuzwieser et al., 2001). Morning bursts could be indicative morning. In contradiction to these observations, in the present of root produced acetaldehyde transported to the leaves when work, heat stressed plants showed high emissions at night. transpiration increases after sunrise (Kreuzwieser et al., 1999b). Considering the slightly increased nocturnal transpiration, this Upon darkening, we found another burst of acetaldehyde might indicate that heat stress either prevented full nocturnal during light‐to‐dark transition under control-conditions (Figure stomatal closure or it induced enhanced biosynthesis of these 4, blue lines), which has often been described (Holzinger et al., oxygenated compounds, raising leaf internal VOC partial 2000; Karl et al., 2002; Brilli et al., 2011; Ghirardo et al., 2011; pressures and inducing diffusion even through mostly closed Jardine et al., 2012; Jud et al., 2016). Pyruvate labeling indicated stomata (Kreuzwieser et al., 1999b). that the dark-burst of acetaldehyde originated from de novo Frontiers in Plant Science | www.frontiersin.org 10 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes FIGURE 6 | Simplified metabolic network indicating major pathways of volatile organic compound (VOC) synthesis and associated CO2 decarboxylation processes in the light. Dark red arrows indicate potential sources of upregulated day CO2 flux during heat stress (from C1-pyruvate), blue denotes CO2 decarboxylation from C2-pyruvate. Dashed lines: low flux in the heat and under light. Pathway names in grey, enzymes in italic. Acetyl-CoA, acetyl coenzyme-A; AA, amino acids; DMAPP, dimethylallyl diphosphate; GA3P, glyceraldehyde-3-phosphate; GPP, geranyl diphosphate; GPP, geranyl diphosphate; IPP, isopentenyl pyrophosphate; LOX, lipoxygenase; MEP, MEP,2-C-methyl-D-erythritol 4-phosphate; MVA, mevalonate; OAA, oxaloacetate; PDH, pyruvate dehydrogenase; mPDH, mitochondrial PDH; cPDH, chloroplastic PDH; PDC, pyruvate decarboxylase; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; Phe, phenylalanine; PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle. Information partially derived from (Dudareva et al., 2006; Dudareva et al., 2013; Tcherkez et al., 2017; Fasbender et al., 2018). synthesis from the acetyl-CoA moiety of pyruvate (Figure 6). In suggest that cell wall degradation might be the primary source of agreement with Jud et al. (2016) and Ghirardo et al. (2011), dark large methanol emission, particularly in highly impacted plants burst of acetaldehyde was reduced or even absent in stress‐ during heat stress. affected plants (see Figure 4B, red lines). As mentioned above, biosynthesis of methyl acetate may also Dynamic Response of Terpenoid depend on the availability of methanol as a precursor. Heat Emissions to Heat Stress strongly enhanced methanol emission when temperatures were In contrast to oxygenated VOCs, both mono- and sesquiterpene stepwise increased, though with a time-lag of a few hours (Figure emissions increased instantaneously with higher temperatures 3), and highest emissions occurred during the morning bursts on probably due to increased volatility of these terpenoids. They days 2 and 3 of the heat wave. Methanol is known to be exhibited highest emissions during the first 48 h of heat exposure predominantly emitted from degradation (and formation) of and thereafter declined. Isoprene and monoterpene emissions cell wall pectins, e.g., during senescence and leaf abscission are expected to stabilize thylakoid membranes and proteins, (Harriman et al., 1991; Loreto and Schnitzler, 2010; thereby enhancing the plant’s thermotolerance (Singsaas et al., Niederbacher et al., 2015). To a minor extent, methanol 1997; Singsaas, 2000; Sharkey, 2005). Biophysical evidence emissions might also originate from protein methyltransferase indicates that isoprene improves the integrity and functionality and protein repair reactions (Kreuzwieser et al., 1999b), which of the thylakoid membranes at high temperature, i.e., though could be enhanced in response to stress. Methanol emissions are stabilisation of the ordered arrays of light harvesting complex also found in response to mechanical wounding or other stresses and photosystem II in the thylakoid grana (Velikova et al., 2012). due to damage of the cell wall (Dorokhov et al., 2018), or with Further mechanisms are protection of the thylakoid membrane increasing temperatures (Schade et al., 2011). Interestingly, against leakiness to maintain the proton-motive force and emissions from wounded plants can induce defence reactions enhance the efficient primary photochemistry of PSII (Velikova in intact leaves of the same and neighbouring plants, by et al., 2011). Recent studies have shown that this is most likely activating methanol-inducible genes that regulate plant mediated by isoprene binding to proteins, and by modulating the resistance to biotic and abiotic factors (Dorokhov et al., 2018). dynamics of thylakoid-embedded proteins that affect membrane Unfortunately, we did not find a clear labeling signal for stabilization (Harvey et al., 2015). methanol due to difficulties in peak separation for the 13C- However, here it is evident that the sudden increase by +13°C isotopologue of methanol. Ghirardo et al. (2011) reported obviously exceeded the capacity of heat acclimation in H. significant 13C-labeling of methanol emitted from mature halimifolium, even though typical stress markers like green leaf leaves and apices of shoots if plants were fed with 13C-Glc, but volatiles were not emitted in large amounts (data not shown). not with 13CO2. However, in absence of further evidence, we Interestingly, even though the heat wave might have depleted the Frontiers in Plant Science | www.frontiersin.org 11 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes terpene pools within the first 48 h, 13C2-pyruvate incorporation Serrano et al., 2019) (Figure 4). It has recently been shown that into both, mono- and sesquiterpenes (Figures 4C, D) clearly there is substantial elasticity in the MEP pathway, strongly related indicated de novo synthesis. This finding corroborates that to the availability of reducing power and ATP from photochemical terpene emissions in H. halimifolium are driven by both, reactions (Rasulov et al., 2015; Rasulov et al., 2018). Souza et al. temperature dependent release of stored compounds (i.e., (2018) proposed that depending on the availability photochemical storage pools for b-caryophyllene and farnesene, see Yá ñez- energy supply and the limitations imposed by either Rubisco Serrano et al., 2018) and de novo biosynthesis (Fasbender et al., activity or RuBP regeneration (under high and low CO2 and 2018). Remarkably, even though the absolute emission rates of temperature) multiple alternative carbon sources can fuel the monoterpenes and particularly sesquiterpenes declined after 10- MEP pathway. They conclude that the use of alternative carbon days of heat (Figure 3), the proportion of de novo synthesis of sources is greater under photosynthesis-limiting conditions, which mono- and sesquiterpenes was strongly enhanced during heat is in line with our results, showing that the use of cytosolic pyruvate (Figure 4) despite limited carbon availability. This might be for de novo synthesis is strongly enhanced under heat stress and underlining the importance of carbon investment into these limited photosynthetic carbon fixation (Yáñez-Serrano et al., 2019) compounds in order to enhance stress tolerance in plants. (Figure 4). However, there could also be alternative pathways Pyruvate is a precursor for both, monoterpenes and which are currently not understood. sesquiterpenes. However, whereas monoterpenes are generally synthesised via the chloroplastic methylerythrol-phosphate Day and Dark Respiration (MEP) pathway, sesquiterpenes are produced via the cytosolic The strong increase of dark respiration in response to heat was mevalonate (MVA) pathway (Dudareva et al., 2006). Therefore, mirrored by enhanced decarboxylation during day (Figure 5). for the use of the applied labeled 13C-pyruvate in the MEP Interestingly, this enhanced metabolic day respiration was not pathway it needs to be transported into this organelle. It has been evenly fuelled from all C-positions of the pyruvate molecule: in reported that pyruvate can be shuttled into the chloroplast either the light, CO2 was mostly emitted from the C1-position of pyruvate directly via a pyruvate transporter, after conversion to PEP via a and only upon darkening, substantial decarboxylation of the C2 PEP transporter , via GA3P, or via a malate shuttle (Dudareva position, most probably from up-regulation of the TCA-cycle in the et al., 2006; Linka and Weber, 2010; Dudareva et al., 2013; Souza mitochondria, occurred. The low decarboxylation of the C2 et al., 2018). In both pathways, the C1 of pyruvate is then position of pyruvate in the light indicates down-regulation of the decarboxylated and released as CO2 and only the C2-C3 TCA-cycle activity (Tcherkez et al., 2005; Werner et al., 2009). It is moiety is further used for biosynthesis (Figure 6). It was, assumed that the TCA-cycle undergoes a major re-organization therefore, unexpected to find incorporation of the 13C1 of during illumination (Sweetlove et al., 2010; Sweetlove et al., 2013), in pyruvate in both terpene classes in heat stressed plants. We order to supply carbon skeletons, e.g., for amino acid synthesis could assume that photosynthetic re-fixation of day respired (Tcherkez et al., 2017). Thus, high decarboxylation from the C1- 13 CO2 occurred; in consistence with this view, incorporation of position of pyruvate occurred when carbon skeletons are used for the 13C1 of pyruvate was more pronounced in mono- than in processes such as synthesis of many secondary compounds sesquiterpenes, since monoterpene biosynthesis is directly including fatty acids and VOCs. Remarkably, during heat stress fuelled from fresh photosynthates formed in the chloroplasts. we observed that such uneven decarboxylation of the C1 and C2 of The 13C1-incorporation into sesquiterpenes, however, requires pyruvate was even enhanced as heat caused a three-fold increase in export of 13C-labeled metabolites from the chloroplast. This can decarboxylation of C1: this unexpected finding of low C2-pyruvate be either an export of labeled triose phosphates or it points decarboxylation led us hypothesize that the CO2 released from heat toward substantial crosstalk between MEP- and MVA-pathway stressed plants in the light is mainly derived from cytosolic and (Souza et al., 2018). The fact that none of the cytosolic chloroplastic reactions and not from the TCA-cycle (Figure 6). In oxygenated VOCs did show significant incorporation of 13C1- accordance with studies suggesting that pyruvate uptake into label could be interpreted as an export of IPP via an postulated mitochondria, as well as the activities of mitochondrial pyruvate IPP transporter (Arigoni et al., 1997; Hemmerlin et al., 2003). dehydrogenase, malic enzyme, isocitrate dehydrogenase and 2- However, recent work has shown little evidence of a large ketoglutarate dehydrogenase of the TCA cycle are light inhibited exchange of IPP (Rasulov et al., 2018; Souza et al., 2018). (Tovar-Mendez et al., 2003; Tcherkez et al., 2005; Sweetlove et al., Moreover, it must be denoted that a strong imprint of 13CO2- 2010; Tcherkez et al., 2017). The fact that very low decarboxylation refixation would result in an equal distribution of 13C-label at all of C2-pyruvate in the light was observed might be an indication, carbon positions. As triose phosphates are exported from the Calvin that under extreme heat stress and limiting carbon availability, also cycle and fuel cytosolic processes, a similar labeling pattern in all de the non-cyclic pathways of the TCA-cycle, which supplement the novo synthesised molecules would be expected, if re-fixation would organic carbon pool (Tcherkez et al., 2017), might have been down- play a dominant role. Furthermore, 13CO2 decarboxylation was regulated. However, further work is needed to substantiate significantly higher from 13C1-pyruvate than from 13C2-pyruvate functioning of the TCA cycle under prolonged heat. (Figure 5), and thus cells were exposed to higher 13CO2 It has been hypothesized that high day respiration may compete concentrations from 13C1-pyruvate decarboxylation. Therefore, with isoprene biosynthesis for pyruvate or PEP, because decreased the impact of refixation would be expected to be higher from isoprene emissions were found in heat-acclimated plants or in 13 C1-pyruvate feeding, which was clearly not the case (Yáñez- plants grown under elevated CO2 (Loreto et al., 2007). Other work Frontiers in Plant Science | www.frontiersin.org 12 August 2020 | Volume 11 | Article 1242 Werner et al. Heat Changes VOC and CO2-Fluxes suggests that the partitioning of PEP and pyruvate between importance for plant protection and survival, though for many mitochondrial respiration and chloroplastic VOC synthesis is poorly studied compounds their physiological role in stress controlled in a way that retains the balance in substrate demand protection is not yet fully resolved. Moreover, the fact that (Loreto et al., 2007). However, our position-specific labeling clearly extreme heat waves may shift plants into a negative carbon demonstrates that VOC synthesis contributes, rather than balance and that assimilation at temperatures exceeding the competes, to enhanced daytime CO2 emissions due to metabolic optimal condition is more inhibited than respiration, pose partitioning of the pyruvate precursor (CO2-releasing pathways important interrogatives on the role of plant carbon balance in during VOC-synthesis under heat stress are indicated in Figure 6): future scenarios of increasing temperatures and extreme events the synthesis of the universal precursors for terpenoids, isopentenyl (Loreto et al., 2007). pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP), requires three acetyl-CoA moieties in the MVA pathway. These are provided by decarboxylation of three pyruvate molecules and, thus, results in the release of 3 CO2 molecules. Thus, synthesis of one mole DATA AVAILABILITY STATEMENT sesquiterpenes (i.e., 15 carbon skeletons) via the MVA pathway is The datasets generated for this study are available on request to associated with release of 9 mol CO2. In contrast, in the MEP the corresponding author. pathway only one mole CO2 is released per mole IPP as after decarboxylation pyruvate reacts with the C1-aldehyde group of GA3P derived from the Calvin cycle (Rohmer et al., 1996; Arigoni et al., 1997). Hence, monoterpene synthesis in the MEP pathway AUTHOR CONTRIBUTIONS should provide a lower ratio of CO2 decarboxylation and, therefore CW design the research. LF and KR planned and conducted the a lower incorporation of 13C2-label compared to precursors experiments. AY-S and JK performed the data analysis. CW and synthesised via the MVA pathway. However, as pointed out JK interpreted data. CW wrote the manuscript with substantial above, we found similar labeling patterns of monoterpenes and input of JK, and revision by all authors. All authors contributed sesquiterpenes (Figure 4), which might be another strong to the article and approved the submitted version. indication for substantial crosstalk between MVA and MEP pathway as discussed above. FUNDING CONCLUSIONS This work was financed by the ERC consolidator grant of CW In summary, these results show that the sudden exposure to a (VOCO2; 647008). heat wave can exceed the acclimation potential even of a Mediterranean species, resulting in a marked depression of net photosynthesis, and enhanced respiratory losses inducing a ACKNOWLEDGMENTS negative carbon balance in plants. Nevertheless, marked investment into de novo synthesis of several VOC was found, The authors gratefully acknowledge the help of Michael Rienks even at the expense of further respiratory losses in the light. constructing the Zero-Air-Generator and David Dubbert, Given the fact that plants invest carbon in a plethora of VOCs Monika Eiblmeier and Susanne Mult for help on technical even under stress conditions is a clear indication for their aspects and IRMS analysis. REFERENCES Atkin, O. K., Bruhn, D., Hurry, V. M., and Tjoelker, M. G. (2005). The hot and the cold: unravelling the variable response of plant respiration to temperature. Affek, H. P., and Yakir, D. (2002). Protection by isoprene against singlet oxygen in Funct. Plant Biol. 32 (2), 87–105. doi: 10.1071/FP03176 leaves. Plant Physiol. 129, 269–277. doi: 10.1104/pp.010909 Atkin, O. (2003). Thermal acclimation and the dynamic response of plant Affek, H. P., and Yakir, D. (2003). 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Bot. 70 (20), 5827–5838. doi: potential conflict of interest. 10.1093/jxb/erz353 Zunzunegui, M., Diaz Barradas, M. C., and Garcı́a Novo, F. (2000). Different Copyright © 2020 Werner, Fasbender, Romek, Yáñez-Serrano and Kreuzwieser. This phenotypic response of Halimium halimifolium in relation to groundwater is an open-access article distributed under the terms of the Creative Commons availability. Plant Ecol. 148, 165–174. doi: 10.1023/A:1009847628243 Attribution License (CC BY). The use, distribution or reproduction in other forums is Zunzunegui, M., Diaz Barradas, M. C., Aguilar, F., Ain Lhout, F., Clavijo, A., and permitted, provided the original author(s) and the copyright owner(s) are credited and Garcı́a Novo, F. (2002). Growth response of Halimium halimifolium at four that the original publication in this journal is cited, in accordance with accepted sites with different soil water availability regimes in two contrasted academic practice. No use, distribution or reproduction is permitted which does not hydrological cycles. Plant Soil 247, 271–281. doi: 10.1023/A:1021598112037 comply with these terms. Frontiers in Plant Science | www.frontiersin.org 17 August 2020 | Volume 11 | Article 1242