Dynamic Balancing of Isoprene Carbon Sources Reflects Photosynthetic and Photorespiratory Responses to Temperature Stress

The volatile gas isoprene is emitted in teragrams per annum quantities from the terrestrial biosphere and exerts a large effect on atmospheric chemistry. Isoprene is made primarily from recently fixed photosynthate; however, alternate carbon sources play an important role, particularly when photosynthate is limiting. We examined the relative contribution of these alternate carbon sources under changes in light and temperature, the two environmental conditions that have the strongest influence over isoprene emission. Using a novel real-time analytical approach that allowed us to examine dynamic changes in carbon sources, we observed that relative contributions do not change as a function of light intensity. We found that the classical uncoupling of isoprene emission from net photosynthesis at elevated leaf temperatures is associated with an increased contribution of alternate carbon. We also observed a rapid compensatory response where alternate carbon sources compensated for transient decreases in recently fixed carbon during thermal ramping, thereby maintaining overall increases in isoprene production rates at high temperatures. Photorespiration is known to contribute to the decline in net photosynthesis at high leaf temperatures. A reduction in the temperature at which the contribution of alternate carbon sources increased was observed under photorespiratory conditions, while photosynthetic conditions increased this temperature. Feeding 2-¹³C]glycine (a photorespiratory intermediate) stimulated emissions of ¹³C_1–5]isoprene and ¹³CO₂, supporting the possibility that photorespiration can provide an alternate source of carbon for isoprene synthesis. Our observations have important implications for establishing improved mechanistic predictions of isoprene emissions and primary carbon metabolism, particularly under the predicted increases in future global temperatures.Many plant species emit isoprene (2-methyl-1,3-butadiene C₅H₈]) into the atmosphere at high rates (Sharkey and Yeh, 2001). With an estimated emission rate of 500 to 750 teragram per year by terrestrial ecosystems (Guenther et al., 2006), isoprene exerts a strong control over the oxidizing capacity of the atmosphere. Due to its high reactivity to oxidants, it fuels an array of atmospheric chemical and physical processes affecting air quality and climate, including the production of ground-level ozone in environments with elevated concentrations of nitrogen oxides (Atkinson and Arey, 2003; Pacifico et al., 2009) and the formation/growth of organic aerosols (Nguyen et al., 2011). At the plant level, isoprene provides protection from stress, through stabilizing membrane processes (Sharkey and Singsaas, 1995; Velikova et al., 2011) and/or reducing the accumulation of damaging reactive oxygen species in plant tissues under stress (Loreto et al., 2001; Vickers et al., 2009b; Velikova et al., 2012). While the mechanism(s) are still under investigation, isoprene may directly or indirectly stabilize hydrophobic interactions in membranes (Singsaas et al., 1997), minimize lipid peroxidation (Loreto and Velikova, 2001), and directly react with reactive oxygen species (Kameel et al., 2014), yielding first-order oxidation products methyl vinyl ketone and methacrolein (Jardine et al., 2012, 2013). The two main environmental drivers for global changes in isoprene fluxes are light and temperature (Guenther et al., 2006). Isoprene production is closely linked to net photosynthesis, and both isoprene emissions and net photosynthesis are controlled by light intensity (Monson and Fall, 1989). There is also a positive correlation between net photosynthesis and isoprene emissions as leaf temperatures increase up to the optimum temperature for net photosynthesis (Monson et al., 1992).Despite the close correlation between photosynthesis and isoprene emissions, plant enclosure observations and leaf-level analyses have both shown that the fraction of net photosynthesis dedicated to isoprene emissions is not constant. During stress events that decrease net photosynthetic rates, isoprene emissions are often less affected or even stimulated; this results in an increase in relative isoprene production from 1% to 2% of net photosynthesis under normal conditions to 15% to 50% under extreme stress (Goldstein et al., 1998; Fuentes et al., 1999; Kesselmeier et al., 2002; Harley et al., 2004). In severe stress conditions such as drought, isoprene emissions can even continue in the complete absence of photosynthesis (Fortunati et al., 2008). An uncoupling of isoprene emissions from net photosynthesis has also been observed in a number of other studies where the optimum temperature for isoprene emissions was found to be substantially higher than that of net photosynthesis; under the high-temperature conditions, isoprene emissions can account for more than 50% of net photosynthesis (Sharkey and Loreto, 1993; Lerdau and Keller, 1997; Harley et al., 2004; Magel et al., 2006).Analyses of carbon sources using ¹³CO₂ leaf labeling have revealed that under standard conditions (i.e. leaf temperature of 30°C and photosynthetically active radiation PAR] levels of 1,000 µmol m^–2 s^–1), isoprene is produced primarily (70%–90%) using carbon directly derived from the Calvin cycle (Delwiche and Sharkey, 1993; Affek and Yakir, 2002; Karl et al., 2002) via the chloroplastic methylerythritol phosphate (MEP) isoprenoid pathway (Zeidler et al., 1997). The relative contributions of photosynthetic and alternate carbon sources for isoprene are now recognized as being variable under different environmental conditions. Changes in net photosynthesis rates under drought stress (Funk et al., 2004; Brilli et al., 2007), salt stress (Loreto and Delfine, 2000), and changes in ambient O₂ and CO₂ concentrations (Jones and Rasmussen, 1975; Karl et al., 2002; Trowbridge et al., 2012) alter their relative contributions. Under heat stress-induced photosynthetic limitation in Populus deltoides (a temperate species), an increase in the relative contribution of alternate carbon sources was also observed (Funk et al., 2004). However, our current understanding of the responses of isoprene carbon sources to changes in temperature and light levels is poor, and the connection(s) of these responses to changes in leaf primary carbon metabolism (e.g. photosynthesis, photorespiration, and respiration) remains to be determined.Studies over the last decade have shown or suggested that potential alternate carbon sources include refixation of respired CO₂ (Loreto et al., 2004), intermediates from the cytosolic mevalonate (MVA) isoprenoid pathway (Flügge and Gao, 2005; Lichtenthaler, 2010), and intermediates from central carbon metabolism, including pyruvate (Jardine et al., 2010), phosphoenolpyruvate (Rosenstiel et al., 2003), and Glc (Schnitzler et al., 2004). Over 40 years ago, it was also proposed that photorespiratory carbon could directly contribute to isoprene production in plants (Jones and Rasmussen, 1975); however, subsequent studies (Monson and Fall, 1989; Hewitt et al., 1990; Karl et al., 2002) have concluded that photorespiration does not contribute to isoprenoid production.In this study, we examined the carbon composition of isoprene emitted from tropical tree species under changes in light and temperature, the two key environmental variables that affect isoprene emissions. Using a novel real-time analytical approach, we were able to observe compensatory changes in carbon source contribution to isoprene during thermal ramping at high temperatures, despite the overall isoprene emissions remaining relatively stable. By conducting leaf temperature curves under variable ¹³CO₂ concentrations and applying 2-¹³C]Gly leaf labeling, we also reopen the discussion on the role of photorespiration as an alternate source of carbon for isoprenoid formation.