Journal of Experimental Botany, Vol. 70, No. 20 pp. 5827–5838, 2019 doi:10.1093/jxb/erz353 Advance Access Publication August 9, 2019 This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) RESEARCH PAPER Heat stress increases the use of cytosolic pyruvate for isoprene biosynthesis Ana Maria Yáñez-Serrano1,†,‡,*, , Lucas Mahlau1, Lukas Fasbender1, , Joseph Byron2, Jonathan Williams2, , Jürgen Kreuzwieser1, and Christiane Werner1, 1 Institute of Ecosystem Physiology, University Freiburg, Freiburg, Germany Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 2 Atmospheric Chemistry Department, Max-Planck Institute for Chemistry, Mainz, Germany † Present address: CREAF, E08193 Bellaterra (Cerdanyola del Vallès), Catalonia, Spain ‡ Present address: CSIC, Global Ecology Unit, CREAF‐CSIC‐UAB, Bellaterra (Cerdanyola del Vallès), E08193, Spain * Correspondence: ayanezserrano@yahoo.es Received 30 November 2018; Editorial decision 18 July 2019; Accepted 18 July 2019 Editor: Christine Foyer, University of Birmingham, UK Abstract The increasing occurrence of heatwaves has intensified temperature stress on terrestrial vegetation. Here, we inves- tigate how two contrasting isoprene-emitting tropical species, Ficus benjamina and Pachira aquatica, cope with heat stress and assess the role of internal plant carbon sources for isoprene biosynthesis in relation to thermotolerance. To our knowledge, this is the first study to report isoprene emissions from P. aquatica. We exposed plants to two levels of heat stress and determined the temperature response curves for isoprene and photosynthesis. To assess the use of internal C sources in isoprene biosynthesis, plants were fed with 13C position-labelled pyruvate. F. benjamina was more heat tolerant with higher constitutive isoprene emissions and stronger acclimation to higher temperatures than P. aquatica, which showed higher induced isoprene emissions at elevated temperatures. Under heat stress, both isoprene emissions and the proportion of cytosolic pyruvate allocated into isoprene synthesis increased. This repre- sents a mechanism that P. aquatica, and to a lesser extent F. benjamina, has adopted as an immediate response to sudden increase in heat stress. However, in the long run under prolonged heat, the species with constitutive emis- sions (F. benjamina) was better adapted, indicating that plants that invest more carbon into protective emissions of biogenic volatile organic compounds tend to suffer less from heat stress. Keywords: Ficus benjamina, heat stress, isoprene biosynthesis, Pachira aquatica, photosynthesis, pyruvate. Introduction Climate models predict an increase in global ambient air particular are expected to experience extremely large increases temperatures of 1–5 °C by the end of this century (IPCC, in heatwaves (Herold et al., 2017), with their cumulative dur- 2013). Heatwaves, defined as prolonged periods of excessive ations predicted to last up to 120 extra days per season under heat (consecutive days that are excessively hotter than average; the scenario of global warming of 5 °C (Perkins-Kirkpatrick Perkins et al., 2013), are already increasing in frequency and and Gibson, 2017). models predict further increases in the future (Kleist et al., As sessile organisms, plants have evolved a wide variety of 2012; Perkins et al., 2012; IPCC, 2013; Deryng et al., 2014; strategies to thrive in their natural habitats, such as adaptations Perkins-Kirkpatrick and Gibson, 2017). Tropical regions in to heat and drought (Rennenberg et al., 2006; Arab et al., 2016); © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 5828 | Yáñez-Serrano et al. however, when environmental factors shift beyond a certain xylem (Kreuzwieser et al., 2002; Schnitzler et al., 2004). The threshold, plants are subject to stress (Lichtenthaler, 1998; mechanism by which such alternative C enters isoprenoid bio- Wahid et al., 2007). In particular, when stresses persist, meta- synthesis is still not fully understood; the observation that the bolic homeostasis can be disrupted, while an adjustment of the extent of isoprene labelling reflects the labelling of intermedi- metabolic pathways may result in acclimation (Lichtenthaler, ates of the Calvin–Benson cycle may support the view that all 1998; Shulaev et al., 2008). However, excessive heat stress, i.e. carbon in isoprene comes directly out of the cycle (Delwiche a rise in temperature beyond optimal functioning, can cause and Sharkey, 1993; Sharkey and Monson, 2014). However, al- irreversible damage to plant growth and development (Wahid ternative carbon sources for isoprene biosynthesis might be et al., 2007).The severity of heat stress depends on the intensity cytosolic phosphoenolpyruvate (PEP) or pyruvate entering the and duration as well as species-specific adaptations to tolerate MEP pathway (Rosenstiel et al., 2004) or other compounds elevated temperatures. In order to cope with heat, plants have that are directly channeled into the Calvin–Benson cycle after developed numerous mechanisms, such as scavenging of the uptake into the chloroplasts, for example as triose-phosphates reactive oxygen species (ROS) that are induced by excess heat via Pi-transporters (Anoman et al., 2016; de Souza et al., 2018). through the biosynthesis of antioxidants (Wahid et al., 2007; Under stress conditions that cause reduction of photosynthesis, Dietz et al., 2016). Another important mechanism to alleviate the portion of leaf internal carbon sources can indeed increase Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 the consequences of heat stress is the maintenance of thylakoid (Kreuzwieser et al., 2002; Funk et al., 2004; Trowbridge et al., membrane stability, which helps protect the photosynthetic 2012). apparatus and allows photosynthetic CO2 fixation to continue In the present study, we selected two species, Pachira aquatica at elevated temperatures (Rennenberg et al., 2006; Zhang and (Malabar chestnut) from the Malvaceae family and Ficus Sharkey, 2009; Niinemets, 2018). Volatile isoprenoids such as benjamina (weeping fig) from the Moraceae family, that ori- isoprene and monoterpenes are believed to constitute an im- ginate from contrasting habitats. P. aquatica is a woody evergreen portant component in the plant’s arsenal against oxidative stress wetland species native to a region stretching from southern (Holopainen and Gershenzon, 2010). These volatiles reduce Mexico to north-eastern Brazil (Oliveira et al., 2000; Lorençon the levels of damaging ROS within the leaf and thus increase et al., 2016; Cheng et al., 2017) and is abundant in mangrove thermotolerance (Loreto and Velikova, 2001; Affek and Yakir, areas (Infante-Mata et al., 2014). In contrast, F. benjamina 2002; Loreto and Fares, 2007; Sharkey et al., 2008), although is native to mixed tropical and sub-tropical deciduous for- this comes at a very high carbon and energetic cost (Harvey ests in Australia and South-East Asia (Veneklaas et al., 2002), et al., 2015). In addition, volatiles such as isoprene may con- and is also abundant in disturbed areas (Pakkad et al., 2001). tribute to the stabilization of cell membranes and integrated According to their natural habitats, P. aquatica is highly tolerant membrane proteins (Singsaas et al., 1997; Sharkey and Yeh, to flooding, drought, and salt stress (Infante-Mata et al., 2014) 2001; Peñuelas and Munné-Bosch, 2005; Behnke et al., 2007; whilst F. benjamina shows high tolerance to drought and heat Velikova et al., 2011), although it has been shown more re- stress (Cokuysal et al., 2006; Qados, 2015). Both species possess cently that the isoprene content in thylakoid membranes may a very active secondary metabolism, and store large amounts of be too low to affect the membrane dynamics (Harvey et al., chemical defense compounds in different tissues (Lansky et al., 2015). A fascinating role of isoprene as signalling compound 2008). For example, P. aquatica accumulates high contents of has been proposed by (Harvey and Sharkey (2016) as this it the fungitoxin isohemigossypolone in the outer bark of the seems to modulate plant gene expression (Zuo et al., 2019). below-ground part of the swollen trunk (Shibatani et al., 1999) Isoprene is produced in chloroplasts via the 2-C-methyl-D- and it has been found to be a weak isoprene emitter (Taylor erythritol 4-phosphate (MEP) pathway (Schwender et al., 1996; et al., 2018). Ficus species contain numerous secondary plant Lichtenthaler et al., 1997; Lichtenthaler, 1999; Dudareva et al., metabolites such as alkaloids (Baumgartner et al., 1990; Khan 2006), which forms the precursor of isoprenoids, dimethylallyl et al., 1993; Novelli et al., 2014) and in contrast to P. aquatica they diphosphate (DMAPP) and its isomer isopentenyl diphosphate have been found to be strong isoprene emitters (Wyche et al., (IPP). The MEP pathway involves the formation of 1-deoxy- 2014). Many bioactive constituents such as cinnamic acids, lac- d-xylulose 5-phosphate (DXP), which is derived from tose, naringenin, quercetin, caffeic acid, and stigmasterol as well glyceraldehyde-3-phosphate (GA-3P) and the decarboxyl- as 28 different alkaloids have been identified in F. benjamina ation of pyruvate (Schwender et al., 1997; Sharkey et al., 2008). (Novelli et al., 2014). Because these compounds are products of the Calvin–Benson The aim of the study was to investigate how these two cycle, isoprenoid biosynthesis in plastids is directly coupled to contrasting species would cope with a prolonged 36-d heat- photosynthesis. Due to this close connection, environmental wave: with global warming of 3 °C, heatwaves in the tropics factors that influence photosynthesis also exert control over are predicted to be as long as 40–50 d, Perkins-Kirkpatrick isoprenoid biosynthesis. The main drivers for isoprene emis- and Gibson, 2017). We tested the hypotheses that the strong sion are therefore solar radiation and temperature (Monson isoprene emitter F. benjamina is better adapted to heat than et al., 1992; Harley et al., 1996). Consistent with this view, the weaker isoprene emitter P. aquatica, and that prolonged 13 CO2 labelling experiments have demonstrated that under exposure to heat stimulates the use of internal C sources for normal, non-stress conditions, ~80% of the carbon emitted as isoprene biosynthesis due to reduced rates of photosynthetic isoprene is directly derived from recently fixed CO2, while the CO2 fixation. We further hypothesized that both plants would remaining ~20% may be provided from ‘alternative’ C sources induce higher isoprene emissions under heat stress as long as internal to the leaf, such as starch or sugars transported in the carbon sources were available to maintain biosynthesis. Heat stress increases use of cytosolic pyruvate for isoprene biosynthesis | 5829 Material and methods Temperature dependence of isoprene emission To characterize the temperature dependence of isoprene emission of Plant material and growth conditions control and heat-stressed plants, six plants of F. benjamina and four plants Individual plants of Ficus benjamina L. and Pachra aquatica Aubl. were pur- of P. aquatica were used. Plants of each species were kept under control chased from a garden center and were grown in 3-l pots filled with 2/3 (25 °C day/night) and heat stress (33 °C day/night) for 5 d. Following soil multiplication substrate (Floragard, Oldenburg, Germany) and 1/3 this, branches were placed in the cuvettes the night before the experi- sand. Since the plants were purchased from a single source, a high level of mental treatments to ensure stabilization of conditions. At 2 h before genetic homogeneity is likely. All plants were supplied with 50 ml Wuxal the beginning of the photoperiod the chamber temperature was set to fertilizer solution (AGLUKON Spezialdünger, Duesseldorf, Germany) 20 °C. From the start of the photoperiod, the chamber temperature was once per week. The plants were watered according to their demand be- increased in 5 °C steps every 2 h. The temperature steps were set to 20, tween 100–250 ml three times a week. They were grown in a climate- 25, 30, 35, and 40 °C, after which the temperature was abruptly set back controlled chamber (ThermoTec Weilburg, Germany) under conditions to 20 °C. As it took ~15 minutes for each new temperature level to be of 25 °C day/night air temperature at 60% relative humidity, with a 12-h attained in the climate chamber, only the last data points for each tem- photoperiod at a photon flux density of ~600 μmol m−2 s−1. The number perature step were used for analysis. of replicates used for the treatments in each experiment is shown in Supplementary Fig. S1 at JXB online. Labelling method Plants of P. aquatica and F. benjamina were subjected to either 25 °C or Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 33 °C for 5 d and measurements were taken at these temperatures.To en- Experimental set-up sure stabilized conditions, branches were placed into cuvettes the evening The cuvette system used for the measurement of volatiles consisted of before the experimental treatments. To characterize the rates of iso- 600-ml cuvettes made of either borosilicate glass (Kummer, Freiburg, prene emission of the intact plants, each branch was measured for 5 min. Germany) for F. benjamina, or of FEP foil (PFTE Spezialvertrieb, Following this, the branches were carefully cut without changing their Germany) for P. aquatica. Two different types of cuvettes were necessary position in the cuvette. To avoid xylem embolism, the branches were due to the very different shapes of twigs and leaves of the two species. In immediately recut under water and the cut end was placed in deionized the P. aquatica cuvettes, a small ventilator (Conrad Electronic, Germany) water. To feed 13C-labelled pyruvate to the leaves via the transpiration was used to improve air mixing. Branches of both species were inserted stream, after 10 min the deionized water was replaced by a solution of into the bottomless cuvettes which were then closed tightly at the stem 10 mM sodium pyruvate (pH 6.5) with the pyruvate 99% labelled with with FEP foil, taking care not to damage the stem surface. Hydrocarbon- 13 C at the [1-C]- or [2,3-C]- position (Cambridge Isotope Laboratories, free, humidified, and CO2-controlled (400 ppm) air was generated using MA, USA). The concentration was selected so as not to induce any a specially constructed ‘zero air’ generator (Fasbender et al., 2018).The air changes in the metabolism (Priault et al., 2009), as the pyruvate pools flow at the cuvette inlets was regulated by mass-flow controllers (Omega is small but with a high turnover rate. It should be noted that this la- Engineering, Stanford, USA) and kept constant at 500 ml min−1, which belling approach is sufficient for tracing the carbon flow through dif- resulted in a calculated residence time of 60 s in the cuvettes. The outlet ferent metabolic pathways but it is not expected to fully label the internal air flow was split between the different analytical devices (see below). pyruvate pool. Emissions of the three isoprene isotopologues (m/z 69.07, The response-time of air leaving the cuvettes and reaching the analytical 70.07, and 71.07 for 0, 1, and 2 13C-atoms, respectively) were followed for devices was less than 10 s. In operational mode, the analytical instruments 60 min after applying the 13C-label. The amount of pyruvate uptake did were supplied with an air flow of ~400 ml min−1 resulting in a slight not differ significantly between the two species and the two heat regimes overpressure in the cuvettes. To ensure chemical inertness, all the parts of (F. benjamina took up 0.25±0.08 g h−1 and 0.27±0.08 g h−1 for control the cuvette system were made of glass or FEP, and the outlet lines were and heat conditions, respectively; and P. aquatica took up 0.50±0.17 g h−1 isolated and continuously heated to 60 °C to prevent adsorption of bio- and 0.43±0.25 g h−1, respectively). genic volatile organic compounds (BVOCs) or water condensation. The cuvette air temperature increased by 2 °C (from 25±0.5 °C to 27±0.5 °C) compared to the growth chamber during the light period, which is an in- Determination of leaf area herent property of cuvette systems and was taken into account in the data The leaf area of the branches in each cuvette was determined after each analysis. Due to the lack of temperature sensors, the actual leaf temper- experiment using a CanoScan LiDE 110 scanner (Canon) and the GSA atures were unknown. In all experiments one cuvette type for each plant Image Analyser software v.4.09. Emission rates are presented on the basis species was kept empty as a reference and these were always measured of m–2 leaf area. for 10 min every hour for the heat-stress and temperature-dependence experiment, and for 10 min at the beginning and the end of labelling experiments. Analytical methods Online isoprene measurements were conducted using a 4000 ultra Experimental design proton-transfer-reaction–time-of-flight–mass spectrometer (PTR- Heat stress TOF-MS) (Ionicon Analytic, Innsbruck, Austria). The PTR-TOF-MS Six plants of each species were kept under normal growth conditions as was operated at 2.7 mbar drift pressure, 600 V drift voltage, at an E/N detailed above at 25 °C day/night for 90 d to determine control rates of of 120 Td, and drift tube heated to 80 °C. More detailed information net CO2 assimilation and isoprene emission. Thereafter, all plants were can be found in Fasbender et al. (2018). We measured 12C-labelled iso- exposed to a temperature of 33 °C for 15 d and then to 38 °C for a prene (m/z 69.07) and its isotopologues containing one 13C-atom (m/z further 21 d. Throughout the experiment, the plants were watered ac- 70.07, hereafter referred to as the single-labelled isotopologue) and cording to their demand to avoid the occurrence of drought stress, par- two 13C-atoms (m/z 71.07, hereafter referred to as the double-labelled ticularly during the period of the heat treatment. During the experiment, isotopologue). Humidity-dependent calibration of isoprene was done F. benjamina grew by 5–10 cm while P. aquatica did not show any signs with a multicomponent calibration gas standard (1000 ppb ±5%, Ionicon of growth. Moreover, F. benjamina developed many new leaves during Analytic) using a Liquid Calibration Unit (LCU, Ionicon Analytic). the 33 °C heat treatment, which became significantly larger than those Isoprene emissions were calculated as: grown at 25 °C. In contrast, P. aquatica exhibited clear signs of leaf sen- (1) e = (ui /s) × (co ci ) escence at 38 °C. Consequently, the heat intensity was limited to a max- imum of 33 °C in subsequent labelling experiments because we were Where ui is the molar flux into the cuvette (mol s−1), s is the leaf area in more interested in following carbon allocation during acclimation rather the cuvette in m2, co is the mixing ratio at the outlet of the cuvette, and ci than after irreversible damage. is the mixing ratio at the cuvette inlet (both mol mol−1). 5830 | Yáñez-Serrano et al. Photosynthetic gas exchange was quantified using a differential infrared temperature-dependency curve under control conditions, six replicates gas analyser (LI-7000 CO2/H2O Analyzer; LI-COR) as the differences of were used for F. benjamina and four for P. aquatica. Under heat conditions, CO2 and H2O concentrations between empty and plant-containing cu- four replicates for each species were used. For the labelling experiment, vettes. Transpiration, assimilation rate, and stomatal conductance to water a minimum of three replicates for each species were used for each of vapor were calculated according to von Caemmerer and Farquhar, (1981). the different treatments. For statistical analysis, Student’s t-tests were per- 13 CO2 flux (nmol m−2 s−1) was quantified by accounting for the dif- formed using the Sigma plot 2017 software (Systat, USA). ference in CO2 isotopic composition and CO2 concentration between empty and plant-containing cuvettes as analysed by a Delta Ray Isotope Ratio Infrared Spectrometer (IRIS, ThermoFisher Scientific). The ana- lyser operated at a sample flow rate of 80 ml min−1 with a temporal Results resolution of 1 s and an analytical precision of δ 13CO2 of 0.15‰. More Effect of heat duration on gas exchange and isoprene information about operation and calculation of net fluxes of 13CO2 can be found in Fasbender et al. (2018). emission Exposure to heat (15 d at 33 °C and followed by 21 d at Cross-validation of identification of isoprene 38 °C) produced strong differences between the two species. The PTR-TOF-MS method is a mass-selective technique, and hence Net CO2 assimilation in F. benjamina was relatively constant at isomers could interfere with the measured masses: isoprene measure- 3.6±0.42 µmol m−2 s−1 at 33 °C and only decreased slightly Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 ments could be affected by another compound with the same mass, i.e. to 2.74±0.36 µmol m−2 s−1 by the end of the period of severe prenol. Therefore, thermodesorption–GC-MS (TD-GC-MS) was used to verify the results of PTR-TOF-MS, as this technique is able to distin- stress, although this was not significant (Fig. 1A). Furthermore, guish between compounds with the same mass. Samples were collected F. benjamina seemed to acclimate to higher temperatures, using adsorbent tubes filled with two different sorbents: 180 mg Tenax which was reflected in initial, transient decreases in assimila- TA 60/80 (35 m2 g−1) followed by 130 mg of Carbograph 1 (90 m2 g−1). tion when temperature was increased from 25 °C to 33 °C More information can be found in Kesselmeier et al. (2002). The ad- and from 33 °C to 38 °C followed by a clear recovery within 2 sorbent tubes were analysed using an Agilent 7890B Gas-Chromatograph (GC) using an INSIGHT® flow modulator (SepSolve Analtical, UK).The d. In contrast, P. aquatica suffered more from heat stress. It was GC was coupled with a time-of-flight-mass-spectrometer (BenchTOF able to maintain CO2 assimilation at a relatively constant level Select, MARKES International, UK) as a detector. Samples were de- at 33 °C, but declined rapidly at 38 °C to only 0.3±0.2 µmol sorbed into the GC system using a two-stage automated thermal de- m−2 s−1; however, this indicated that net photosynthesis was still sorber (TD100-xr, MARKES International). The carrier gas used was counterbalancing respiratory losses during the day. Chlorosis of helium. The sample was then removed from the adsorbent tube at a tem- perature of 250 °C and a flow of 50 ml min−1 for 10 min, and was leaves was apparent by day 12, during the 33 °C period. pre-concentrated onto a cold trap (general purpose carbon, MARKES In both species there was a significant (P<0.001) tran- International) at 30 °C. The cold trap was then purged with helium for sient increase in isoprene emission when the temperature in- 1 min with a flow of 50 ml min−1 before being rapidly heated to 300 °C. creased from 25 °C to 33 °C, and this was more pronounced The sample was removed from the cold trap with a flow of 3.5 ml min−1 in P. aquatica than in F. benjamina (Fig. 1B). From day 6 there and injected into the column. The separation of the sampled compounds was achieved in the first was a continuous decline in emissions in P. aquatica that became dimension using a 25-m MEGA-DEX DMT-Beta column with 0.1 mm internal diameter and a film thickness of 0.15 μm. The separation in the second dimension was achieved using a 4-m mid-polarity BPX50 column with an internal diameter of 0.25 mm. A 22.5-cm sample loop with an internal diameter of 0.53 mm was used to temporarily store the sample components that had already been separated in the first dimension before introduction into the second column. A 4-m deactivated bleed line with an internal diameter of 0.1 mm was also connected to the modulator plate. A modulation time of 3 s was used. The temperature program used was as follows: 40 °C for 5 min, then 40 °C to 150 °C at 3 °C min−1 and 150 °C to 200 °C at 30 °C min−1. Following the separation of the com- pounds, identification was achieved using the BenchTOF operating in tandem ionization mode at 70 eV and 14 eV so that two chromatograms were obtained per sample for each ionization energy. The common ion fragment m/z 67 of isoprene and prenol was first extracted from the total ion count (TIC) of the 70-eV chromatogram to uncover the peaks of iso- prene and prenol.Their mass spectra were then compared with the NIST 70 eV electron ionization library. For further confirmation, the headspace of a liquid standard of isoprene was collected onto an adsorbent tube to confirm its retention time. The signal peaks belonging to isoprene and prenol were found in each of the samples. The integrated area of iso- prene was found to be a factor of ~100 greater than the integrated area of prenol. Therefore, it was concluded that since the area of the peak of prenol remained relatively low compared to that of isoprene, the prenol emission by the two species could be considered negligible. Fig. 1. Effects of prolonged exposure to increasing levels of heat in Ficus AQ1 benjamina and Pachira aquatica. Plants were grown at 25 °C for 90 d (indicated by dark shading), and then the temperature was increased to Statistics 33 °C (medium shading) and then to 38 °C (light shading). (A) Effects on The number of replicates varied among the different experiments. For net CO2 assimilation rate and (B) on isoprene emission rate. Data are mean heat-stress duration, six replicates for each species were used. For the midday values (±SE). Heat stress increases use of cytosolic pyruvate for isoprene biosynthesis | 5831 more pronounced when the temperature was further increased Incorporation of 13C into isoprene depends on the to 38 °C. Interestingly, however, the decrease in isoprene emis- labelled position of the pyruvate applied sion was not as rapid as that observed for net CO2 assimilation. In contrast, F. benjamina seemed to acclimate to the conditions, In order to evaluate the use of alternative carbon sources for and an initial drop in isoprene emission at 38 °C was followed by isoprene biosynthesis, branches of control and heat-stressed recovery to levels that were not significantly lower than at 33 °C. F. benjamina and P. aquatica plants were fed with position-specific [1-13C]- or [2,3-13C]-labelled pyruvate. For the plants used in this experiment, heat exposure increased isoprene emission in Heat stress modifies the temperature dependence of F. benjamina from 0.49±0.3 nmol m−2 s−1 to 2.8±0.4 nmol m−2 isoprene emission s−1 in control and heat-stressed plants, respectively, whilst in P. aquatica fluxes remained relatively constant at 2.9±0.8 nmol Net CO2 assimilation of F. benjamina subjected to control con- m−2 s−1 to 2.4±0.2 nmol m−2 s−1, respectively.. Net CO2 as- ditions (25 °C) showed a temperature optimum at ~27 °C (Topt) similation remained relatively constant during the course of of 5.5±0.3 µmol m−2 s−1 (Fig. 2A, left). Exposure to 33 °C for 5 the experiment (Fig. 3) suggesting that the branches were not d altered the shape of the temperature response curve (Fig. 2A, appreciably influenced by cutting or application of pyruvate at Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 right); although the maximum CO2 assimilation rate remained least for P. aquatica. unaffected, net assimilation was greater at higher temperatures When the carboxyl group (C1, first carbon in the pyruvate compared to control plants. For example, at 42 °C assimila- molecule) was decarboxylated from pyruvate at the metabolic tion of F. benjamina plants that had been exposed to heat was branching point, a considerable increase in 13CO2 emission 3.8±0.7 µmol m−2 s−1, which was ~2.5-fold higher than in was detected when [1-13C]-pyruvate was applied (Fig. 3). control plants measured at this temperature. Isoprene emission Remarkably, we observed a 2-fold (F. benjamina) to 3-fold of F. benjamina showed a Topt at 37 °C for plants grown under (P. aquatica) increase in release of 13CO2 under heat conditions control conditions, with a rate of 6.4±0.9 nmol m−2 s−1 (Fig. in both species: i.e. day-respiration was stimulated by heat, 2B, left). Exposure to heat shifted the maximum to at least resulting in higher 13CO2 release from the plant. Release of 13 42 °C and increased the maximum emission rate to at least CO2 occurred at higher rates in F. benjamina than in P. aquatica 10.2±3.2 nmol m−2 s−1 (Fig. 2B, right). despite the latter taking up more labelled pyruvate during the Exposure to heat exerted similar effects on P. aquatica plants, course of the experiment (F. benjamina control 0.07±0.02 mg although assimilation and isoprene emission rates were lower s−1, heat 0.08±0.02 mg s−1; P. aquatica control 0.14±0.05 mg than for F. benjamina. The Topt for net CO2 assimilation was s−1, heat 0.12±0.07 mg s−1). In contrast, release of 13CO2 was 27 °C with a rate of 3.6±0.4 µmol m−2 s−1. Following ex- considerably lower for both control and heat-treated plants if posure to heat conditions, both Topt and the maximum as- they were labelled with [2,3-13C]-pyruvate. similation rate remained in a comparable range. Similarly, there Incorporation of 13C into isoprene strongly depended on was no pronounced heat effect on the Topt of isoprene emis- the form of labelled pyruvate applied in both species (Figs 4, 5). sion; however, the emission rate increased significantly from Under [1-13C]-labelling, the isotopologue containing one 13C 2.1±0.5 nmol m−2 s−1 in control plants to 7.1±0.8 nmol m−2 atom (m/z 70) always increased more than the double-labelled s−1 in plants that had been exposed to heat. 13 C isotopologue (m/z 71.07). In P. aquatica, the emission of Fig. 2. Temperature dependence of (A) net CO2 assimilation rate and (B) isoprene emission rate in Ficus benjamina and Pachira aquatica plants exposed to either control conditions (25 °C) or to heat stress of 33 °C for 5 d. Data are means (±SE) of six and four replicates for controls of F. benjamina and P. aquatica, respectively, and of four replicates for each species under heat conditions. 5832 | Yáñez-Serrano et al. Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 Fig. 3. Effect of heat stress on net CO2 assimilation rates and 13CO2 release in plants of Ficus benjamina and Pachira aquatica. Plants were Fig. 4. Temporal dynamics of fluxes of unlabelled and 13C-labelled isoprene exposed to either control conditions (25 °C) or to heat stress at 33 °C for isotopologues in plants of Pachira aquatica exposed to heat stress. Plants 5 d. Measurements were taken after feeding excised twigs with position- were exposed to either control conditions (25 °C) or to heat stress at 33 °C specific labelled pyruvate, namely [1-13C]-pyruvate and [2,3-13C]-pyruvate for 5 d. Measurements of isoprene isotopologues containing no 13C atoms (time of feeding indicated by arrows). Data are means (±SE) of 4–5 replicates. (m/z 69), one 13C atom (m/z 70), and two 13C atoms (m/z 71) were taken after feeding excised twigs with position-specific labelled pyruvate, namely the single-labelled isotopologue increased by ~2-fold from [1-13C]-pyruvate and [2,3-13C]-pyruvate (time of feeding indicated by 0.3±0.1 nmol m−2 s−1 (pre-label) to 0.7±0.1 nmol m−2 s−1 (post- arrows). Data are means (±SE) of 4–5 replicates. label) under heat conditions, whereas it remained constant at 0.06±0.03 nmol m−2 s−1 in control plants (Fig. 4).The double- emissions of the double-labelled isotopologue (m/z 71) were labelled isotopologue (m/z 71) was only detectable in trace greater. However, there were no clear differences between con- amounts if [1-13C]-pyruvate was applied in control plants, but trol and heat-stressed plants between the rates of increase be- was ~10-fold higher in heat stressed plants (0.02±0.002 nmol tween the single- and double-labelling. Notably, total isoprene m−2 s−1). In contrast, the effects of [1-13C]-pyruvate were less emissions (sum of all isotopologues including m/z 69.07) were pronounced in F. benjamina, although the levels of emission significantly increased in response to heat in both species. of the single-labelled isotopologue (m/z 70) were still ~2-fold higher in heat-stressed plants compared to controls (Fig. 5). Heat affects carbon partitioning into isoprene in a There were no really clear differences in the emissions for the species-dependent manner double-labelled isotopologue (m/z 71). Feeding [2,3-13C]-pyruvate in P. aquatica resulted in rela- Heat affected the fraction of assimilated carbon released as tively small increases in emissions of the single-labelled isoprene in a species-dependent manner (Fig. 6). The fraction isotopologue (m/z 70) whereas emissions of the double- was not affected by heat in F. benjamina, where it amounted labelled isotopologue (m/z 71) were increased more con- to ~4.2% in plants grown under both treatments (25 °C and siderably, particularly in heat-stressed plants (Fig. 4). Feeding 33 °C). In contrast, it significantly increased in response to heat [2,3-13C]-pyruvate in F. benjamina (Fig. 5) produced broadly in P. aquatica, from ~2.5% to ~5.5%. The fraction of assimi- similar results to P. aquatica in that emissions of the single- lated carbon emitted as isoprene in P. aquatica exceeded that labelled isotopologue (m/z 70) increased only slightly and of F. benjamina at 33 °C, although the levels of emission were Heat stress increases use of cytosolic pyruvate for isoprene biosynthesis | 5833 Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 Fig. 6. Effects of heat stress on net CO2 assimilation, isoprene emissions, and the carbon released as isoprene as a portion of the CO2 fixed in photosynthesis for plants of Pachira aquatica and Ficus benjamina exposed to heat stress. Plants were exposed to either control conditions (25 °C) or to heat stress at 33 °C for 5 d. Data are means (±SE) of six Fig. 5. Temporal dynamics of fluxes of unlabelled and 13C-labelled isoprene replicates. Significant differences between means were determined using isotopologues in plants of Ficus benjamina exposed to heat stress. Plants Student’s t-test: *P<0.001. were exposed to either control conditions (25 °C) or to heat stress at 33 °C for 5 d. Measurements of isoprene isotopologues containing no 13C atoms P. aquatica. Under control conditions, the optimum tempera- (m/z 69), one 13C atom (m/z 70), and two 13C atoms (m/z 71) were taken ture (Topt) for isoprene emission was ~37 °C for F. benjamina after feeding excised twigs with position-specific labelled pyruvate, namely and ~39–40 °C for P. aquatica (Fig. 2), which for both species [1-13C]-pyruvate and [2,3-13C]-pyruvate (time of feeding indicated by arrows). Data are means (±SE) of 4–5 replicates. exceeded the Topt of net CO2 assimilation by 12–15 °C, similar to responses reported previously for Quercus robur (Schnitzler the same in both species at this temperature. In contrast, both et al., 2004) and Phoenix dactylifera (Arab et al., 2016). This pat- the fraction and the level emitted at 25 °C were significantly tern seems to reflect acclimation of a plant to the prevailing lower in P. aquatica. environmental conditions. Higher Topt of isoprene emission than of net CO2 assimilation enables the plant to respond quickly to short-term increases in leaf temperature, for ex- ample during periods of high irradiation. It has been demon- Discussion strated that isoprene quickly mediates thermotolerance for the Using two tropical species, we tested the hypothesis that plants photosynthetic apparatus by modulation of membrane protein with high isoprene emission are better adapted to prolonged dynamics (Harvey et al., 2015) and/or ROS quenching (Loreto heatwaves than plants with lower emission, and we also in- and Velikova, 2001; Affek and Yakir, 2002; Sharkey et al., 2008), vestigated the role of internal carbon sources for isoprene thereby allowing net CO2 assimilation to be maintained even biosynthesis with regards to thermotolerance. Under control at temperatures clearly exceeding Topt of photosynthesis. conditions (25 °C), F. benjamina emitted isoprene at a rate three After 5 d of acclimation to 33 °C, the temperature response times higher rates that of P. aquatica (6.8 nmol m−2 s−1 versus of isoprene emission of both species changed (Fig. 2). The Topt 2.7 nmol m−2 s−1; Fig. 1), which is in agreement with pre- of emission in F. benjamina increased from 37 °C to at least vious reports for F. benjamina (0.03–25 µg C g−1 h−1; Sun and 42 °C whilst the emission capacity also increased, as indicated Leu, 2004; Carvalho et al., 2005; Geron et al., 2006). There by higher emission rates. An increase in emission rates was also have been no previous reports for isoprene emission rates in observed for P. aquatica, but in this case the Topt of emission did 5834 | Yáñez-Serrano et al. not change. These results were in good agreement with pre- of leaf internal C sources used for isoprene biosynthesis is in- vious data for date palm where both the capacity and Topt of creased (Kreuzwieser et al., 2002; Funk et al., 2004). It is gener- isoprene emission have been shown to increase when plants are ally assumed that under non-stress conditions up to 80% of C cultivated at elevated air temperature (Arab et al., 2016). Thus, used for isoprene biosynthesis is provided from instantaneously our results demonstrated that the higher isoprene emissions in- fixed CO2. The remaining ~20% C might be provided by al- duced by elevated temperature in both species was most likely ternative C sources (Delwiche and Sharkey, 1993; Karl et al., a response to acclimate the leaves to heat. However, since the 2002b; Affek and Yakir, 2003; Schnitzler, 2004) such as xylem constitutive isoprene emission was already high in F. benjamina sap sugars (Kreuzwieser et al., 2002), starch (Monson et al., 1994; under control conditions, the portion of induced emission was Schnitzler, 2004), or cytosolic sources (de Souza et al., 2018). lower than in P. aquatica. The latter showed very low consti- This alternative carbon might be imported into the chloroplast tutive emission under control conditions, but highly induced as pyruvate, PEP, or as glyceraldehyde-3-phosphate (GA-3P) emission in response to heat stress. This is consistent with pre- by specific transporters such as triose-phosphate transporters vious findings that have demonstrated constitutive BVOC (Bamberger et al., 1975; Hoefnagel et al., 1998) pyruvate trans- emissions from healthy, non-stressed plants (Holopainen, 2004) porters (Furumoto et al., 2011), and PEP transporters (Fischer and additional, stress-induced emissions due to stimulated de et al., 1997; Flügge, 1999). The observation from 13C-labelling Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 novo biosynthesis of volatile compounds (Paré and Tumlinson, experiments that intermediates of the Calvin–Benson cycle 1997; Paré et al., 2005). The high constitutive isoprene emis- are labelled similarly to isoprene (Delwiche and Sharkey, 1993) sion capacity of F. benjamina might underlie its high tolerance may support the view of a contribution of GA-3P uptake into to short-term increases in temperature. plastids. Our position-specific pyruvate 13C-labelling experi- Interestingly, we found that net CO2 assimilation of ments were able to shed new light on the question of alter- F. benjamina exposed to 33 °C was significantly higher than in native C sources and potential changes in contributions to control plants grown at 25 °C (Fig. 3), which clearly highlights isoprene biosynthesis under heat stress, as they enabled us to its considerable acclimation potential to higher temperatures. follow the fate of the 13C atoms of pyruvate into isoprene. This finding supports a thermoprotectant function of isoprene Feeding cytosolic [1-13C]- and [2,3-13C]-pyruvate caused for the photosynthetic apparatus, as high isoprene-emitting production and emission of 13C-labelled isoprene in both species generally display higher rates of photosynthesis under plant species (Figs 4, 5). As expected, the resulting isoprene heat stress, and also the role of isoprene in fast recovery after isotopologues differed depending on the form of pyruvate ap- heat stress (Velikova and Loreto, 2005). In contrast, no such plied. If [1-13C]-pyruvate was fed to plants under control con- effect was observed for P. aquatica, which seemed to be more ditions (25 °C), we observed considerable emissions of single 13 sensitive to temperature increases and probably reached its C-labelled isoprene (m/z 70) and only traces of the double 13 upper temperature limit at 33 °C: further exposure to higher C labelled isoprene (m/z 71). The occurrence of single 13 temperature (38 °C) resulted in detrimental stress effects. This C-labelled isoprene most likely resulted from re-fixation of 13 was further reflected under prolonged exposure to elevated CO2 within the leaf (Kreuzwieser et al., 2002; Tcherkez et al., temperatures (Fig. 1). Pachira aquatica was not able to maintain 2017; Fasbender et al., 2018) from the decarboxylation of the isoprene biosynthesis over extended periods even at 33 °C, as C1 carboxylic group of pyruvate. Thus, the substantial emis- evidenced by some individuals that had a midday assimilation sions of 13CO2 during [1-13C]-pyruvate labelling (Fig. 3) may rate of 1.4 µmol m−2 s−1 at the beginning of the 33 °C treat- have been used in the Calvin–Benson cycle, hence contributing ment, dropping to 0.04 µmol m−2 s−1 after 19 d of exposure to the formation of intermediates that are used as precursors (data not shown); this occurred simultaneously with a drop in of the MEP pathway. On the other hand, if pyruvate is con- isoprene emissions. verted to GA-3P and channeled into the chloroplasts via the Under such stress conditions, isoprene emission seemed to triose-phosphate transporter (via reversible conversion to di- become decoupled from net CO2 assimilation, resulting in hydroxyacetone phosphate), it might enter the MEP pathway an impaired leaf C budget, i.e. reduced photosynthetic CO2 directly. In contrast, if [2,3-13C]-pyruvate was applied, double 13 fixation but increased C loss via isoprene emission. Previous C-labelled isoprene was the main volatile product; although studies have demonstrated that C losses via isoprene can single 13C-labelled isoprene appeared as well. Emission of double 13 account for up to 25% of the C fixed in photosynthesis if plants C-labelled isoprene can be explained by transport of pyruvate are stressed (Sharkey et al., 1996; Brüggemann and Schnitzler, into the chloroplasts where it acts as the precursor of the MEP 2002a, 2002b), which greatly exceeds the loss in leaves under pathway, thus leading to formation of double 13C-labelled iso- non-stressed conditions (0.2–2%; Kesselmeier et al., 2002). prene (Figs 4–6).Transport of cytosolic pyruvate into plastids can Ficus benjamina maintained high levels of photosynthetic CO2 either be direct, as mediated by a sodium-pyruvate transporter fixation under prolonged periods of exposure to 33 °C. A fur- (Furumoto et al., 2011), or indirect, after conversion of pyruvate ther increase in temperature to 38 °C only had a relatively to PEP, by a PEP transporter located in the chloroplast mem- small effect on net CO2 assimilation in F. benjamina (Fig. 1), and brane (Fischer et al., 1997; Flügge, 1999). Alternatively, abun- isoprene emissions under these conditions were maintained at dant pyruvate in the cytosol could be converted into GA-3P a high level, thus, contributing to the acclimation of the plant or other triosephosphates and be channeled into the Calvin– to heat stress. Benson cycle after uptake into the chloroplast, which can be Maintenance of high levels of isoprene emission under con- mediated by bi-directional phosphate translocators that have ditions of reduced net CO2 assimilation implies that the portion been described previously (Bamberger et al., 1975; Hoefnagel Heat stress increases use of cytosolic pyruvate for isoprene biosynthesis | 5835 et al., 1998). A diagram of the different responses to heat of the In both species, feeding [1-13C]-pyruvate caused strong processes involved in isoprene biosynthesis is shown in (Fig. 7). release of 13CO2 from the leaves in the light (Fig. 3). This It should be noted that the amount of 13C incorporated process was considerably enhanced when the plants were ex- into isoprene upon 13C-pyruvate feeding was relatively low, posed to heat stress. Importantly, although it is well known and therefore it did not reflect the ~20% of C expected to that heat increases mitochondrial respiration in plants, we de- be derived from alternative (other than CO2) sources. This duce a more significant contribution to enhanced 13CO2 re- can be explained by the low amount of pyruvate fed to the lease from other metabolic processes. We conclude this from leaves and the assumed dilution of labelled pyruvate within the the fact that the plants showed only slightly enhanced dark cytosolic and plastidic pyruvate pools. The pyruvate pool has a respiration (F. benjamina: control –0.4±0.01 µmol m−2 s−1, heat fast turnover and 13C-lablled pyruvate is quickly (within min- –1±0.02 µmol m−2 s−1; P aquatica: control –0.4±0.01 µmol utes) incorporated into several chloroplastic volatile organic m−2 s−1, heat –0.6±0.05 µmol m−2 s−1) and, more import- compounds (VOCs) such as monoterpenes or benzenoids antly, from the lower amount of 13CO2 emitted from each (Fasbender et al., 2018). Consequently, the 13C-signal in VOCs species after feeding with [2,3-13C]-pyruvate as compared quickly vanishes when the labelled solution is exchanged with to feeding with [1-13C]-pyruvate . With high respiration water (Jardine et al., 2014). Thus, whilst our results are efficient rates, turnover in the tricarboxylic acid (TCA) cycle would Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 in tracing the metabolic fluxes within mesophyll cells, they are be high, resulting in a greater release of 13CO2 from the C2– not sufficient to provide quantification of the fluxes. Moreover, C3 moiety of [2,3-13C]-pyruvate. Our results instead suggest C from other alternative sources (e.g. sugars) may well have that the TCA cycle was inhibited under light conditions, sug- contributed to isoprene biosynthesis, hence further diluting gesting a light-dependent inhibition of the TCA cycle, which the 13C-pyruvate signal. is similar to previous findings in other species (Priault et al., When both species were exposed to heat for 5 d, the rela- 2009; Sweetlove et al., 2010; Araújo et al., 2012; Cheung et al., tive (%) increases in emissions of 13C-labelled isoprene did not 2014; Tcherkez et al., 2017). Consequently, other processes change compared to controls. However, because in P. aquatica must have contributed to 13CO2 release after [1-13C]-pyruvate total isoprene emissions increased due to the heat increase feeding. Numerous reactions are known to cause pyruvate from 25 °C to 33 °C (Fig. 1) and the portion of 13C-labelled decarboxylation in plant metabolism, such as the those cata- isoprene remained the same as in controls (data not shown), we lysed by pyruvate dehydrogenase (PDH; Sharkey et al., 1991) conclude that the rate of pyruvate transport from the cytosol and pyruvate decarboxylase (PDC; Kreuzwieser et al., 1999; into chloroplasts and the use of pyruvate for isoprene biosyn- Karl et al., 2002a, 2002b), and other enzymatic reactions thesis were increased in this species to the same extent as iso- that use pyruvate as a substrate for decarboxylation (e.g. see prene formation increased (Fig. 6). In conclusion, uptake of KEGG, https://www.genome.jp/kegg). The relative contribu- pyruvate into chloroplasts seems to be controlled by pyruvate tions of each of these processes to overall emissions of 13CO2 consumption in the organelles. cannot be determined from our present data. To identify these Fig. 7. Diagram illustrating the differential effects of heat stress on the processes involved in isoprene biosynthesis. Control conditions are represented in blue and heat-stress conditions in red. The thickness of the lines indicates the magnitude of the flux, and dashed lines indicate the action of transporters. Black arrows represent processes that have not been elucidated with the data from this study. 5836 | Yáñez-Serrano et al. enzymatic decarboxylation processes more detailed analysis of within the plants for isoprene biosynthesis. Under heat stress, 13 C-labelled metabolites such as fatty acids, amino acids, and both isoprene emissions and the proportion of cytosolic pyru- others would be required. vate allocated into isoprene synthesis increased. This represents Our comparison of the two tropical plants F. benjamina and a mechanism that P. aquatica, and to a lesser extent F. benjamina, P. aquatica demonstrated that they have very different responses has evolved as an immediate response to sudden increases in to heat stress. These differences might be a result of adapta- heat stress. However, in the long run under prolonged heat, the tion to their different natural habitats, namely well-watered species with higher constitutive emissions (F. benjamina) was tropical forests with relatively constant temperature regimes better adapted, indicating that plants that invest more carbon for P. aquatica and sites that are probably drier and hotter into protective BVOC emissions tend to suffer less from heat sites for F. benjamina (Imran et al., 2014; Infante-Mata et al., stress. These different strategies in carbon allocation need to be 2014). The more heat-tolerant F. benjamina seems to constitu- considered in environmental models if they are to have a better tively invest more C for isoprene biosynthesis than P. aquatica. predictive capability of the effects of increasing heatwaves in Consequently, the photosynthetic apparatus of F. benjamina the future. is better thermoprotected and net CO2 assimilation can be maintained at higher levels during heat stress, similar to other Downloaded from https://academic.oup.com/jxb/article/70/20/5827/5545358 by guest on 29 June 2021 isoprene-emitting tropical species (Taylor et al., 2019). In con- Supplementary data trast, P. aquatica seems to follow a different strategy by investing Supplementary data are available at JXB online. less C into isoprene as a photo-protective mechanism during Fig. S1. Diagrams showing the designs of the experiments. non-stress conditions; however, under short-term heat stress it greatly activates its capacity for isoprene biosynthesis to pro- tect its leaves. This strategy might be advantageous as C is only Acknowledgements invested when it is actually needed (Spinelli et al., 2011) and is otherwise used for other processes such as biomass production The authors acknowledge funding from the ERC project VOCO2 (i.e. growth processes, Schippers et al., 2015). However, under (647008) and DFG (WE 2681/10-1). We are also very grateful to Michael Rienks and David Dubbert for their logistical help. 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