Isoprene (2-methyl-1,3-butadiene, C5
) is a reactive hydrocarbon gas emitted in large amounts to the atmosphere by many plants [1
]. Approximately 500 Tg C year−1
is released as isoprene by vegetation [3
]. Tropical rainforests are an important source of isoprene to the atmosphere and estimates suggest that they are responsible for 80% of global isoprene emissions (Is
]. Isoprene has an important role in atmospheric chemistry involving air quality and climate [5
]. Due to its high chemical reactivity with respect to photooxidation, isoprene impacts the atmospheric concentrations of ozone, methane, and secondary organic aerosols, resulting in strong effects on the radiation balance of the Earth [6
]. Despite great advances in our knowledge on the roles of plant Is
on atmospheric chemistry, much less is known about the physiological roles that isoprene plays in plants [9
]. The emerging view is that isoprene production can protect photosynthesis during abiotic stress through mechanisms including excess photosynthetic energy consumption, physical stability of biological membranes, and direct roles as an antioxidant through reactions with reactive oxygen species that accumulate under stress conditions [12
]. However, recent work has noted that protection of photosynthesis through isoprene via the physical stabilization of membranes may not be possible and that the mechanisms of protection from oxidative stress are still unclear [14
Isoprene is synthesized in the chloroplast by the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway [15
]. The synthesis is initiated by the condensation of two primary precursors; pyruvate and glyceraldehyde 3-phosphate (G3P), a direct product of the Calvin–Benson cycle [15
]. The fact that isoprene cannot be stored in the leaves results in Is
from production, with the majority of carbon derived from recent photosynthesis [21
]. Under steady-state conditions of light and temperature (1000 µmol m−2
and 30 °C) ~70%–90% of the carbon used for isoprene synthesis is produced from recently assimilated atmospheric CO2
]. It was observed that plants exposed to 13
rapidly incorporate 13
C into isoprene molecules [21
]. Despite this rapid incorporation of 13
C, ~10%–30% of the carbon atoms in the isoprene molecules emitted have been reported to remain unlabeled, even when the leaves are continuously exposed to 13
]. This indicates a contribution of carbon sources other than the recently assimilated CO2.
Those alternative carbon sources are important under stress conditions [27
] and increase under high temperatures, while net photosynthesis (Pn
) is reduced at the expense of photorespiration [25
]. Therefore, under stressful situations that reduce the uptake of atmospheric CO2
due to partial stomatal closure while stimulating internal sources of CO2
(e.g., respiration and photorespiration), Is
can be increased even if Pn
is substantially reduced [28
Several studies have identified that potential alternative carbon sources for Is
can be greater when environmental conditions are limiting Pn
, including xylem-transported carbohydrates [26
], starch degradation [26
], pyruvate [33
], stored carbon pools [17
], and extrachloroplastic intermediates [34
]. In addition, the incorporation of CO2
released by intercellular decarboxylations, e.g., during mitochondrial respiration, photorespiration or decarboxylation of pyruvate during formation of MEP was suggested [25
]. For example, photorespiratory CO2
release is strongly stimulated under high temperatures and stomatal closure due to the decrease of atmospheric CO2
uptake and the relative increase of O2
in relation to CO2
. Therefore, under stressful conditions that promote stomatal closure, such as drought and high temperature, an increase in the release of photorespiratory CO2
inside the leaf could provide alternative carbon sources for isoprene production [35
]. However, the identity and quantitative importance of alternative carbon sources for isoprene synthesis in tropical species under abiotic stress is still unclear.
In this study, we investigated the incorporation of CO2 from decarboxylation process into isoprene molecules by quantifying Is under CO2-free reference air in temperature- and light response curves from a neotropical specie Inga edulis Mart. Taking off the CO2 from the leaf chamber, we stimulated the photorespiration process and the release of internal CO2 in the leaf. We then compared leaf Is in CO2-free reference air with Is in ambient CO2 under standard conditions of light and temperature. We further investigated the potential for the reassimilation of CO2 release by internal decarboxylation processes using an inhibitor of photosynthesis under CO2-free reference air. Finally, we directly evaluated the potential of internal leaf CO2 to be incorporated into Is by providing 13C-sodium bicarbonate (NaH13CO3) solutions to detached leaves under CO2-free reference air. We suggest that CO2 released by internal decarboxylation processes is a quantitatively important source of alternative carbon for isoprene formation. Together with photorespiration, isoprene production may be a key tolerance mechanism under plant stress that leads to a decrease in stomatal conductance and CO2 uptake. Under these conditions, isoprene could still be synthesized using the CO2 released by photorespiration, offering a protective mechanism by consuming excess photosynthetic energy and reducing equivalents. These results deepen our understanding about Is by tropical species under different environmental conditions commonly experienced in the tropics, such as high irradiance and temperature, and contributes to the modeling of Is from terrestrial ecosystems in the tropics under climate change.
2. Materials and Methods
2.1. Plant Material and Experimental Design
Six I. edulis
trees with a height ranging from 5 to 10 m were used in this study. The experimental trees occur naturally near the Laboratory of Plant Physiology and Biochemistry at the National Institute for Amazonian Research (INPA) campus III in Manaus, Brazil. This tropical species was selected because of its high reported Is
and for its ability to maintain high transpiration rates for many hours following branch detachment from the tree [25
]. From October 2014 to February 2016 Pn
rates were quantified from healthy mature leaves, located in the upper third of the canopy. For each day of the study, one branch near the top of one of the plants was cut and placed in tap water before being recut and transported to the laboratory. The leaf gas exchange measurements occurred between 9:00 AM and 5:00 PM, with the branch exposed to ambient light and temperature conditions.
2.2. Isoprene Emissions and Net Photosynthesis
rates were quantified from I. edulis
leaves using a portable open gas exchange system (IRGA) (LI-6400XT, LI-COR Inc., Lincoln, NE, USA) with an artificial light source 6400-02B Red Blue. The flow rate entering the LI-6400XT leaf chamber was set to 537 mL min−1
. A fraction of air exiting the leaf chamber was used to determine Is
using two methods: quantified in real-time using a high sensitivity quadrupole proton transfer reaction-mass spectrometry (PTR-MS, Ionicon Analytik, Innsbruck, Austria) or thermal desorption (TD) gas chromatography–mass spectrometry (GC-MS, 5975C series, Agilent Technologies, Santa Clara, CA, USA). Using four-way junction fitting, air exiting the leaf chamber was delivered or to the PTR-MS (40 mL min−1
) or to the TD tube (100 mL min−1
when collecting), with the remainder of the flow diverted to the vent/match valve within the LI6400XT. The excess flow entering the vent/match valve was maintained to at least 200 mL min−1
]. Background measurements were performed with an empty leaf chamber, at the beginning and at the end of each experiment.
2.3. Light and Temperature Response of Isoprene
For each I. edulis leaf, either a light or temperature response curve was generated. Leaves were evaluated for their response of Pn and Is to changes in PPFD (Photosynthetic Photon Flux Density) at 0, 25, 50, 75, 100, 250, 500, 750, 1000, 1500, and 2000 µmol m−2 s−1 under constant leaf temperature (30 °C) in both a CO2-free atmosphere (0 µmol mol−1, 4 leaves) and ambient CO2 (450 µmol mol−1, 7 leaves). To achieve a CO2-free atmosphere inside the LI-6400XT leaf chamber, the CO2 mixer was turned off and the soda lime was put to fully scrub. Leaf temperature response curves (25→ 30→ 35→ 40 °C) were measured under constant PPFD (1000 µmol m−2 s−1), again in both CO2-free atmosphere (n = 3) and ambient CO2 (n = 4). Before each series of measurements, the leaves were acclimated for ~15 min in the chamber, until the stomatal conductance and photosynthesis were stable.
To quantify the ratio of Is between CO2-free atmosphere (0 µmol mol−1 of CO2, 8 leaves) and ambient CO2 concentrations (450 µmol mol−1 of CO2, 8 leaves), gas exchange was measured with leaves of I. edulis under standard conditions of light and leaf temperature (1000 µmol m−2 s−1 PPFD, 30 °C leaf temperature).
2.4. Isoprene Emission under Limiting Conditions for Net Photosynthesis
To evaluate Is
under limiting conditions for Pn
we measured gas exchange under two limiting situations: (a) using a specific photosynthesis inhibitor, DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) or (b) in the dark. DCMU blocks photosynthetic electron flow from photosystem II to the plastoquinone pool [36
], inhibiting photosynthesis.
For the inhibitor experiment, a 180 µM DCMU solution, was prepared and adjusted to pH 7.5. A leaflet was cut from a leaf and placed in the DCMU solution for two hours, until the inhibitor took effect. This inhibition time was determined during previous experiments to be sufficient to decrease Pn to near 0 µmol m−2 s−1 in leaflets under standard conditions (i.e., with CO2, PPFD and leaf temperature at 450 µmol mol−1, 1000 µmol m−2 s−1, and 30 °C, respectively). Following uptake of the DMCU solution by the transpiration stream, a light response curve was measured under CO2-free atmosphere and constant leaf temperature (30 °C).
For the dark experiment, a temperature response curve was established in the absence of light. During the measurements the leaves were kept under CO2
-free atmosphere and zero PPFD. The samples were collected in TD tubes, using the LI-6400XT/GC-MS methodology [25
2.5. 13C-labeling of Leaf Isoprene Emissions Using Sodium Bicarbonate 13C Delivered through the Transpiration Stream
In order to observe possible reassimilation of internal CO2 by photosynthesis through isoprene labeling molecules, measurements of Is under CO2-free atmosphere were carried out on detached fully expanded I. edulis leaflets in a solution of 13C labeled sodium bicarbonate (NaH13CO3) (n = 5). Fresh solutions of sodium bicarbonate 13C (20, 60, 120 mM) were prepared with the pH adjusted to 7.0. For each concentration five leaflets were cut and recut in the sodium bicarbonate 13C solution, and leaf gas exchange measurements were initiated. In sequence, the measurements were made for two hours, under CO2-free atmosphere at constant PPFD (1000 µmol m−2 s−1) and temperature (30 °C) using the LI-6400XT/GC-MS methodology. Isoprene 13C-labeling is reported as the 13C/12C isotope ratio of [13C1]isoprene/[12C5]isoprene, [13C2]isoprene/[12C5]isoprene, [13C3]isoprene/[12C5]isoprene, [13C4]isoprene/[12C5]isoprene, and [13C5]isoprene/[12C5]isoprene by calculating the peak area ratios (7.1 min retention time) of m/z 69/68, 70/68, 71/68, 72/68, and 73/68, respectively.