Correspondence: T. D.Sharkey UW–Madison Department of Botany, B214 Birge Hall, 430 Lincoln Drive, Madison, WI 53706–1381, USA. Tel: +1 608 262 6802; Fax: +1 608 262 7509; E-mail: firstname.lastname@example.org
Isoprene emission from plants is highly temperature sensitive and is common in forest canopy species that experience rapid leaf temperature fluctuations. Isoprene emission declines with temperature above 35 °C but the temperature at which the decline begins varies between 35 and 44 °C. This variability is caused by the rate at which leaf temperature is increased during measurement with lower temperatures associated with longer measurement cycles. To investigate this we exposed leaves of red oak (Quercus rubra L.) to temperature regimes of 35–45 °C for periods of 20–60 min. Isoprene emission increased during the first 10 min of high temperature exposure and then decreased over the next 10 min until it reached steady state. This phenomenon was common at temperatures above 35 °C but was not noticeable at temperatures below that. The response was reversible within 30 min by lowering leaf temperature to 30 °C. Because there is no storage of isoprene inside the leaf, this behaviour indicates regulation of isoprene synthesis in the leaf. We demonstrated that the variability in isoprene decline results from regulation and explains the variability in the temperature response. This is consistent with our theory that isoprene protects leaves from damage caused by rapid temperature fluctuations.
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Isoprene emission from forest trees is of great interest because of its impact on regional air quality ( Feshenfeld et al. 1992 ; Harley, Monson & Lerdau 1999). In order to predict isoprene emissions, a model was developed which describes the environmental response of isoprene metabolism in leaves ( Guenther et al. 1993 ). This uses a basal emission rate (defined as the isoprene emission rate at 30 °C and 1000 μmol m−2 s−1 PPFD) modified by scaling equations which describe the response to light and temperature; the major environmental factors which control isoprene emission. The parameters of these equations are often determined by non-linear curve fitting to isoprene emission measured from attached leaves as light or temperature is varied.
Although the light-response equation parameters are quite well defined, there is disagreement over the parameters of the temperature-response equation. The three parameters in this equation measure the activation energy (Ha), describing the rate of isoprene emission increase with temperature; deactivation energy (Hd), describing the rate at which isoprene emission declines at high temperature; and falloff temperature (TF), describing the temperature where isoprene emission begins to decline. Although the activation and deactivation energies are relatively constant, TF temperature is quite variable; between 35 and 40 °C ( Guenther et al. 1993 ). Subsequent measurements estimated TF at 44 °C or higher for sun-exposed leaves, but have varied by as much as 10 °C ( Monson et al. 1994 ; Harley, Guenther & Zimmerman 1996). Knowing the correct value of TF temperature is important for simulating isoprene emission on the hottest days ( Singsaas et al. 1999 ); when isoprene is likely to have the most effect on tropospheric ozone ( Feshenfeld et al. 1992 ).
Changes in isoprene emission rate are attributable to changes in the biochemistry of isoprene synthesis. This results from the independence of isoprene emission from photosynthesis and stomatal conductance. Isoprene’s independence from photosynthesis is evident because leaves continue to emit even when they are placed in a stream of CO2-free air ( Loreto & Sharkey 1990). The independence from stomatal conductance results from the fact that isoprene is not stored in the leaf tissues ( Sharkey 1991) and is not metabolized by the plant (P.J. Vanderveer, unpublished observation). Decreasing stomatal conductance causes the isoprene concentration in the leaf intercellular airspaces to increase. Since the driving force for diffusion out of the leaf is proportional to the difference in concentration from the inside to the outside, a decrease in stomatal conductance is matched by an increase in driving force resulting in no net change in isoprene emission rate. Because of its independence, isoprene emission rate equals its synthesis rate, and gas-exchange studies of isoprene emission can yield information about the biochemistry of isoprene synthesis.
In a previous study, it was shown that differences in falloff temperature could be generated by increasing leaf temperature at a slower or faster rate ( Singsaas et al. 1999 ). Our objective in this study was to further investigate this phenomenon using a real-time isoprene analyser ( Hills & Zimmerman 1990) while leaf temperature was varied at different rates. Because of the direct link between isoprene synthesis and emission, this allowed us to make inferences about the biochemical regulation of isoprene synthesis.
MATERIALS AND METHODS
Measurements were made on detached leaves of a red oak tree (Quercus rubra L.) growing outdoors in Madison, WI, USA. The tree is estimated to be 40 years old. Detached leaves from this tree have previously been used in gas exchange and isoprene emission measurements ( Loreto & Sharkey 1990). Measurements were made on leaves from this tree during July–October 1997, and all data were normalized to the emission rate at 30 °C and are reported as relative emission rates unless specified otherwise.
To demonstrate the effect of leaf-heating rate on the temperature response of isoprene emission, leaves were held in a gas-exchange cuvette at 29 °C until the isoprene emission rate was stable. Leaf temperature was then increased in increments of 2 or 3 °C and held at the new temperature for 2, 10, or 40 min before the isoprene emission rate was read. Data from three curves were averaged for each heating rate. The isoprene emission rate was logged continuously during these measurements and an example set of these data is also reported.
The experiments were also designed to investigate the changes in isoprene emission that occur in the minutes to hours following a change in leaf temperature. The leaf was held at a constant temperature, usually 30 °C, until isoprene emission was stabilized. A step change in leaf temperature, usually lasting less than 2 min, was then imposed on the leaf. The leaf was then held at the new temperature and the isoprene emission rate recorded. The leaf temperature could be cycled between the low and high temperature while the isoprene emission rate was recorded continuously. The data from three curves were averaged in all cases.
Gas exchange system
Gas exchange measurements were made using a gas-mixing system that has previously been described ( Tennessen, Singsaas & Sharkey 1994). Whole leaves were enclosed in a 0·6 L cuvette with a 60 cm2 window. This chamber had a 30 s time constant (21 s half-life) for isoprene wash-out which was sufficient for measuring isoprene emission changes occurring over several minutes. Leaf temperature was controlled with six thermoelectric blocks, and was measured with a copper–constantan thermocouple pressed against the abaxial surface of the leaf. Light was provided to 1000 μmol photons m−2 s−1 by a 2·5 kW xenon arc lamp.
Isoprene was measured using a real-time chemiluminescent isoprene detector ( Hills & Zimmerman 1990; Hills, Fall & Monson 1992). The gas flow exiting the leaf cuvette was pumped into a reaction chamber and mixed with a 2·0 L min−1 flow of 2% ozone (Ozone Research Corporation, Phoenix, AZ, USA). The isoprene–ozone reaction produced an activated aldehyde. When the activated electron in the aldehyde fell to the ground state, it released a photon. Photons were counted by a photomultiplier (Model HC135–02; Hamamatsu Corporation, Middlesex, NJ, USA). Data were logged every second on a computer. The isoprene analyser was calibrated daily with a four-point standard drawn from a 5·8 μmol mol−1 isoprene standard in nitrogen (Scott-Marin Specialty Gases, Riverside, CA, USA). The response of the analyser was linear over three orders of magnitude bracketing the concentrations used in these experiments.
Isoprene emission rate was calculated from flow rate, leaf area, and isoprene concentration as described elsewhere ( Singsaas et al. 1997 ). Models were fitted by least squares regression using a data analysis package (Origin version 5·0; Microcal Software, Northampton, MA, USA).
Temperature response of isoprene emission
Isoprene emission increased with increasing leaf temperature up to 38–42 °C, at which point isoprene emission declined ( Fig. 1). For these experiments, leaves were held at 29 °C until the isoprene emission and photosynthesis rates were steady. Leaf temperature was increased in 2–3 °C increments from 30 to 45 °C, and were held at each temperature for 4, 10, or 40 min. Measurements were stopped when leaf temperature reached 45 °C. The isoprene emission rate started to decrease at 38 °C when leaves were held for 40 min at each temperature. When leaf temperature was changed more rapidly, the isoprene emission rate started to decrease at higher temperatures. Although all three curves were identical at temperatures below 37 °C, the decline temperature was sensitive to the length of time between successive measurements.
The apparent temperature sensitivity of isoprene decline is a result of the behaviour of isoprene emission at high leaf temperature. Isoprene emission and leaf temperature were measured continuously during the slowest measurements of those presented in Fig. 1 ( Fig. 2). The time at which single measurements for the 40 min curve in Fig. 1 were recorded are indicated by letters a–d. When leaf temperature was below 35 °C, isoprene emission increased with leaf temperature and then remained steady for the entire 40 min period. At higher temperatures, isoprene emission rate first increased with leaf temperature and then fell to a lower steady-state level (e.g. Fig. 3). Above 38 °C a steady-state rate of isoprene emission was not reached within the 40 min measurement period in the experiment reported in Fig. 2. After 40 min at 40 °C, isoprene emission rate declined to a rate that was lower than at 38 °C (point d on Fig. 2). The numbers (1–4) indicate the isoprene emission rate 10 min after leaf temperature was changed.
Regulation of isoprene emission
To study the regulation of isoprene emission at high leaf temperature, isoprene emission was monitored continuously while the leaves were given a high-temperature treatment. Leaves were held at a temperature of 30 °C until the isoprene emission rate was steady. Leaf temperature was then increased to 35, 37·5, or 40 °C and held for 50 min ( Fig. 3). Within the first 10 min after the temperature of the leaves was increased to 40 °C, isoprene emission increased to nearly 2·5 times the initial rate. In the next 10 min, isoprene emission fell to 1·5 times the initial rate and remained constant at that level for the next 30 min ( Fig. 3). When the temperature of the leaves was increased from 30 to 37·5 °C, the initial peak was smaller and the steady-state level reached was 1·7 times the initial rate ( Fig. 3). When the temperature of the leaves was increased from 30 to 35 °C, the peak level was almost the same as the steady-state level at 1·7-fold over the initial rate ( Fig. 3).
The drop in isoprene emission at high temperature was reversible within 30 min. When leaves were held at 40 °C for 60 min, the isoprene emission rate increased 2·2-fold over the initial rate within 10 min of changing leaf temperature ( Fig. 4). After 40 min, the isoprene emission rate had decreased to 1·2-fold over the initial rate. When leaf temperature was lowered to 30 °C, isoprene emission dropped to 0·5 times the initial rate. If the leaf was held at 30 °C for 30 min, the isoprene emission rate recovered, increasing to the original rate at 30 °C ( Fig. 4a). After the 30 min recovery, the leaf temperature was again increased to 40 °C. Isoprene emission rate behaved in the same manner as during the first 40 °C treatment; increasing to the same peak rate after 10 min and falling to the same plateau rate after 40 min. If the recovery period was less than 30 min, isoprene emission rate did not recover completely ( Fig. 4b). When leaf temperature was increased to 40 °C for a second time, isoprene emission rate peaked at only 1·6-fold over the initial rate. After 40 min, however, isoprene emission rate decayed to 1·2-fold over the initial rate, just as it had during the first 40 °C treatment.
In some cases, isoprene emission rate declined continuously at high temperature rather than reaching a steady-state emission rate. When the temperature of the leaves was increased to 44 °C, isoprene emission increased two-fold over the initial rate and slowly decreased over the next 60 min. This response was also seen at 40 °C in some leaves grown outdoors late in the growing season (first week in October). When this happened, isoprene emission did not recover after 30 min at 30 °C ( Fig. 5). When the temperature of a leaf was increased to 40 °C for 1 h, isoprene emission decreased continuously. After 1 h, leaf temperature was lowered to 30 °C and isoprene emission dropped slightly and recovered. When leaf temperature was again increased to 40 °C, isoprene emission did not recover to its initial rate. In cases like this, isoprene emission rate remained low for several hours after the high temperature treatment.
All data presented in Figs 3, 4 and 5, begin with a temperature jump from 30 °C to either 35, 37·5, or 40 °C. To test whether the isoprene decrease responds to leaf temperature or the magnitude of change in leaf temperature, leaves were held at 25, 30, and 35 °C and increased in temperature by 6 °C for 40 min ( Fig. 6). Although all temperature jumps were equal in magnitude, only the jump from 35 to 41 °C produced the peak in isoprene emission rate that decayed to a lower steady state ( Fig. 6a). Jumps of equal magnitude made at lower temperatures did not produce this response ( Fig. 6b, c).
Most temperature response measurements are made by recording a single isoprene measurement after changing leaf temperature. This can result in misleading information about the temperature response of isoprene emission. If individual measurements are made within 10 min of a temperature change, isoprene emission is likely to be recorded at its peak (1–4 on Fig. 2). If isoprene measurements, are taken 40 min after leaf temperature changes, a lower emission rate is recorded (a–d on Fig. 2). Measurements taken at 1–4 would show isoprene emission increasing at temperatures above 40 °C whereas measurements made at a–d would show isoprene emission decreasing above 38 °C.
Falloff temperature varied across the growing season and through the canopy ( Monson et al. 1994 ; Harley et al. 1996 ). Canopy models estimate isoprene emission based on leaf temperature averaged over an entire forest canopy. This is further averaged over the 30–60 min time interval of the model. Thus, although individual leaf temperatures may be quite high, it is unlikely that average leaf temperature will reach above 35 °C ( Guenther et al. 1996 ). Nevertheless, we recommended simplifying isoprene emission models by removing TF ( Singsaas et al. 1999 ). Three years of field studies have shown activation energy to be constant across three growing seasons and two field sites ( Sharkey et al. 1999 ), indicating no need to consider changes in this parameter of the temperature response. This greatly simplifies isoprene emission models.
Regulation of isoprene synthesis
Since the temperature response of isoprene emission in vivo correlates loosely with the temperature response of isoprene synthase activity in vitro, it has been assumed that the denaturation of isoprene synthase in the leaf controls the temperature response of isoprene emission ( Monson et al. 1992 ). This does not explain why isoprene synthase activity decreases at temperatures above 45 °C while isoprene emission begins to decrease between 35 and 45 °C. Furthermore, the TF temperature varies across the growing season and through the canopy ( Monson et al. 1992 ; Harley et al. 1996 ). Our results suggest that the decline in isoprene emission at high temperature is the result of reversible control. This is evident when isoprene emission rate is monitored continuously ( Fig. 2). The isoprene emission rate increases with each step in leaf temperature, but decreases after 10 min above 35 °C ( Figs 3 & 6). Rather than decreasing continuously, as in the case of enzyme denaturation, isoprene emission decreases to a lower steady-state level ( Figs 3 & 6). This indicates regulation rather than denaturation. This regulation is reversible. Isoprene emission is reduced when the leaf temperature drops back to 30 °C, but recovers to its original rate within 30 min ( Fig. 4).
Enzyme denaturation is one likely explanation when isoprene emission rate was unable to recover after 30 min at a lower temperature. In these cases, isoprene emission never fell to a steady-state level at high temperature, but instead it decreased continuously ( Fig. 5). When measurements were made during July, and leaves experienced relatively hot weather, this response occurred only at temperatures above 44 °C. This response occurred more frequently and at lower temperature for leaves that were sampled in September and October when leaves experienced cooler weather. Since this happened when leaves were brought to the highest temperatures, this non-recovering response is likely to be a result of damage, enzyme denaturation, or irreversible regulation of isoprene synthesis or electron transport. These characteristics of the non-recovering response suggest it is unrelated to the regulation of isoprene synthesis ( Figs 2, 3 & 4).
This regulation of isoprene emission is consistent with the theory that isoprene protects leaves from rapid temperature fluctuations ( Singsaas et al. 1997 ; Singsaas & Sharkey 1998). The highest rate of isoprene production occurs during the first 10 min of a high-temperature episode after which emissions decline. This allows the rapid induction of thermotolerance as leaf temperature rises. However, because isoprene emission is a large carbon sink at high temperatures ( Loreto et al. 1990 ) it becomes an inefficient thermotolerance mechanism during prolonged high temperature episodes. During longer episodes, isoprene emission declines and other thermotolerance mechanisms, such as the production of heat-shock proteins ( Vierling 1991) and changes in xanthophyll epoxidation ( Havaux & Tardy 1996; Havaux et al. 1996 ) become induced after several minutes of elevated temperatures. These mechanisms do not depend on the continuous emission of newly fixed carbon and hence are more carbon-efficient. Thus isoprene works best as a thermotolerance mechanism during short excursions to high temperature whereas these other mechanisms protect leaves from longer excursions.
In the forest canopy, high-temperature excursions usually last from a few seconds to 20 min ( Singsaas et al. 1999 ). This results from leaf flutter during small breezes and the fact that canopy leaves can be exposed to direct sunlight from a sunfleck while being blocked from open sky by other leaves. This reduces the leaf’s ability to radiate heat energy to the open sky. These conditions may be unique to forest canopies. Plants that experience high temperatures continuously, such as those in deserts, would benefit more from long-term thermotolerance mechanisms. This is consistent with the observation that deserts contain few isoprene-emitting species ( Monson, Guenther & Fall 1991) whereas forest and shrub ecosystems contain the vast majority of emitting species ( Guenther et al. 1995 ). Thus, isoprene may be an important thermotolerance mechanism for forest trees although it is irrelevant in plants that do not experience rapid temperature fluctuations.
We thank E. DeLucia and J. Hamilton for their helpful comments on the manuscript. This research was supported by United States National Science Foundation grant IBN-9317900.