Rapid Sampling Protocol of Isoprene Emission Rate of Palm ( Arecaceae ) Species Using Excised Leaves

: The high isoprene emission capacity of palm species can decrease regional air quality and enhance the greenhouse effect when land is converted to palm plantations. Propagation of low-emitting individuals can be a strategy for reducing isoprene emission from palms. However, the identiﬁcation of low-emitting individuals requires large-scale sampling. Thus, we aimed to develop a rapid method in which the isoprene emission rate of leaf segments is observed. We examined the temperature response and effect of incubation length on the isoprene emission rate of palm leaf and found that leaf temperatures at 25 to 30 ◦ C and an incubation length of 40 min are suitable parameters. To further examine the validity of the method, we applied both the enclosure method and this method to the same sections of leaves. High coefﬁcient of determinations (0.993 and 0.982) between the results of the two methods were detected regardless of seasonal temperature. This result proves that the method is capable of measuring the isoprene emission rate under any growth conditions if the incubation temperature is controlled. By using a water bath tank and a tested light source, we can simply implement a uniﬁed environmental control of multiple samples at once, which achieves a higher time efﬁciency than conventional enclosure measurements.


Introduction
Isoprene (2-methyl-1,3-butadiene, C 5 H 8 ) is the most abundant volatile organic compound (VOC) emitted from biogenic sources. It is estimated to constitute approximately 400-700 Tg of annual carbon and to account for 50-70% of total terrestrial biogenic VOC (BVOC) emissions [1,2]. These values are equivalent to those of global methane emissions (410-660 Tg of carbon per year) and are five times larger than global anthropogenic emissions of non-methane VOCs [3,4].
Isoprene can affect the oxidative capacity of the atmosphere and, thus, its chemical balance [5,6]. For instance, the oxidation of isoprene in the presence of nitrogen oxides (NOx ≡ NO + NO 2 ) dominates ozone production in the troposphere [7]. High ground-level ozone concentrations were observed in areas with the interaction of isoprene emission from oil palm plantation and agroindustrial exhaust [8], semi-urban regions with a mixture of biogenic and anthropogenic emissions [9], and cities with rich vegetation sources [10,11]. Ground-level ozone can consequently reduce net ecosystem productivity and, thus, may impact the terrestrial carbon sink [12,13]. Depending on the concentrations of isoprene and NO x , 1-6% of the total mass of isoprene can also be converted to secondary organic aerosols [14,15]. Additionally, isoprene, which is highly reactive, can potentially compete with methane for radicals and consequently alter its lifetime [16][17][18]. Thus, the monitoring of isoprene emission from plants provides critical information for the management and control of these negative effects.
Simulation is the most commonly used method of monitoring isoprene dynamics. The Model of Emissions of Gases and Aerosols from Nature (MEGAN) version 2.1, a widely used simulation model of global-scale BVOC emissions [1], uses the framework of Guenther et al. [19] to estimate isoprene emissions from plants. It calculates the rate by adjusting the isoprene emission rate recorded at or standardized to a photosynthetic photon flux density (PPFD) of 1000 µmol m −2 s −1 and a leaf temperature of 30 • C (i.e., the basal isoprene emission factor) to reflect instantaneous changes in factors such as leaf temperature and light intensity [1]. The basal isoprene emission factor is usually conducted independently by species or group. To obtain the basal isoprene emission factors for a species, a branch/leaf enclosure method is typically recommended [20]. Several studies have used portable gas exchange measuring systems (e.g., LI-6400, Li-Cor Inc., Lincoln, NE, USA) with an external VOC trap and air pump for isoprene emission measurement with accurate leafscale temperature, light control, and better mobility (e.g., [21][22][23][24][25]). However, this method is limited by its short reach (less than 3 m between the platform and branch); in addition, a raised platform is required for plants with high canopy heights. Moreover, the equilibration time of gas exchange in the cuvette/chamber limits the number of leaves sampled. The reason is that, empirically, CO 2 exchange does not become stable in the cuvette for at least 3 min, and the time required is even longer when the light intensity or temperature is adjusted [26][27][28][29]. Moreover, a previous study recorded a period of more than 30 min for the leaves of a shrub (Edgeworthia chrysantha) to reach the stabilized isoprene emission rate in a cuvette [21].
Palms (the Arecaceae family) are monocotyledonous flowering plants with 181 genera and 2600 species [30]; they are distributed in tropical and subtropical areas worldwide [31]. Most species of Arecaceae have tree-like woody trunks which develop by the growth of extremely wide primary stems, progressive lignification, and thickening of the cell wall with age [32]. Tropical plants tend to have a high isoprene emission capacity because isoprene can protect plants from thermal damage [33][34][35]. As tall plants living in tropical and subtropical areas, palm trees encounter transient and continuous heat stresses; thus, palm species can be expected to have a high isoprene emission capacity. Previous studies also found high isoprene emission rates in several palm species (e.g., Calamus gracilis, Elaeis guineensis, Salacca secanda, Euterpe precatoria, and Socratea exorrhiza) [36][37][38]. Palm species are widely planted for agricultural purposes [39]. From 2000 to 2020, the area covered by palms has expanded rapidly, for example, from 10.4 to 28.7 million ha for oil palms, from 10.6 to 11.6 million ha for coconut palms, and from 1.0 to 1.2 million ha for date palms [40]. The high isoprene emission capacity can decrease regional air quality and enhance the greenhouse effect when land is converted to palm plantations. The propagation of low-emitting individuals can be a strategy for reducing isoprene emission from palms. However, the identification of low-emitting individuals requires large-scale sampling, which is difficult using the conventional enclosure measurement. To achieve efficient sampling, this study aimed to develop a rapid method in which the isoprene emission rate of leaf segments is observed. The isoprene emission rate of excised leaves is measured after treatment (i.e., incubation under constant light and temperature). The two main goals of this study are (1) to clarify the appropriate process parameters, including incubation period and temperature and (2) to validate this method by comparing its results to those obtained using an enclosure method.

Plant Materials
Three-to five-year old pot saplings of Butia yatay, Washingtonia filifera (three individuals), Phoenix dactylifera 'Major', and Phoenix dactylifera 'Piarom' were purchased from a local commercial supplier and moved to the campus of the University of Shizuoka, Shizuoka Prefecture, Japan. The trunk heights (vertical length from ground surface to trunk top) of B. yatay, the three W. filifera individuals, P. dactylifera 'Major', and P. dactylifera 'Piarom' were 0.3, 0.3, 0.2, 0.1, and 0.1 m, respectively. The saplings were watered every fourth day. Phoenix canariensis and Trachycarpus wagnerianus previously planted on the University of Shizuoka campus were also studied. Two individuals of P. canariensis grown separately under different conditions (sunlit/shaded) were used. The trunk heights of the sunlit and shaded P. canariensis and T. wagnerianus were 1.2, 1.4, and 0.6 m, respectively.
After 1 November 2021, the pot saplings were moved to an indoor space. A light intensity of~250 µmol m −2 s −1 PPFD at the top of the plants was supplied by horticultural fluorescent lamps from 6:00 to 18:00. The air temperature was fixed at~25 • C.

Measurement of Temperature Response of Isoprene Emission Rate Using Enclosure Method
To clarify the response of the isoprene emission rate of palm leaves to temperature, B. yatay, W. filifera, P. dactylifera 'Major', and P. dactylifera 'Piarom' were used for the measurements (B. yatay: 27 October and 11 November 2021; W. filifera: 26 November and 7 December 2021; P. dactylifera 'Major': 15 November and 22 November, 2021; and P. dactylifera 'Piarom': 12 November and 19 November, 2021). A modified portable photosynthesis measuring system (LI-6400XT, Li-Cor Inc., Lincoln, NE, USA) connected to a proton transfer reaction time-of-flight mass spectrometer (PTR-TOF1000, IONICON Analytik GmbH, Innsbruck, Tyrol, Austria) was used for continuous monitoring of the isoprene emission rate inside the enclosure. The system was assembled using the method described by Tani and Kawawata [22]. The drift tube E/N (where E is the electric field strength, and N is the buffer-gas number density in the drift tube) was kept at 143 Td by maintaining the drift tube voltage, temperature, and pressure at 600 V, 80 • C, and 2.2 mbar, respectively. H 3 O + was used as the primary ion. The m/z (where m is the ion's mass, and z is the charge number of the ion) of ionized isoprene ((C 5 H 8 )H + ) was 69. It was confirmed that leaves of the palm species exhibited no emission of 2-methyl-3-buten-2-ol. Thus, it was deemed that no overlap on the m/z of 69 by its fragment was produced by hydroxide ion transfer. The system was calibrated with a 10 ppb isoprene standard sample, which was prepared by diluting 1 ppm isoprene gas with pure nitrogen gas (purity > 99.99995%). To direct air emitted from the leaf in the cuvette of the photosynthesis measuring system to the spectrometer, the tube between the cuvette and embedded infrared gas analyzer was replaced with a T-junction of polytetrafluoroethylene (PTFE). This modification added an air pathway that was connected to the sample inlet of the spectrometer. The air inflow rates of the cuvette in the photosynthesis measuring system and the spectrometer were set to 500 µmol s −1 and 0.3 L min −1 (~225 µmol s −1 ), respectively. A platinum catalyst heated to 400 • C was used to supply VOC-free air to the photosynthesis measuring system. A blank test was performed before the measurement. During the measurement, the entire plant was placed under a 400 W metal halide lamp (D400, Toshiba Lighting & Technology Corporation, Yokosuka, Japan), and the target leaflet was enclosed in the cuvette. The light intensity and CO 2 concentration in the cuvette were set to 1000 µmol m −2 s −1 and 400 ppm, respectively. The leaf temperature in the cuvette was sequentially set to 20, 25, 30, and 35 • C to observe the effects of the leaf temperature on the rates of isoprene emission, net photosynthesis, and stomatal conductance (g sw ). The isoprene concentration was recorded at 30 s intervals, and the photosynthetic rate was recorded at 1 min intervals.

Isoprene Emission Rate for Different Incubation Periods
A method of measuring the isoprene emitted from excised palm leaves, which included incubation under a constant water temperature and light exposure, was developed. To determine a suitable incubation period, the isoprene emissions from leaves were determined using incubation periods of 10, 40, and 60 min. B. yatay, W. filifera, P. dactylifera 'Piarom', P. dactylifera 'Major', and P. canariensis were tested on 18-21 June and 2 November 2021; 24 November 2021; 25 November 2021; 6 December 2021; and 10 November 2021, respectively; four or five replications were performed for each species. A leaflet was first cut into pieces 1.5-2 cm in length. Then, each piece was sealed in a glass vial (10 mL in volume) with a screw cap having a silicone/PTFE septum. The glass vial was prepared by adding a hard PTFE net as a platform and 5 µL of distilled water to prevent water loss from the leaf. Next, the vial was incubated at a constant temperature for a certain period in a water bath tank (22 cm × 31cm × 14 cm) (Personal-11, Taitec Corporation, Nagoya, Japan), and stable photosynthetic active radiation was provided by a light-emitting diode (LED) light panel (TH2-211X200SW, CCS Inc., Kyoto, Japan). The LED light panel was located 10 cm above the base of the water bath tank. The base of the tank was split into 8 × 8 grids (2.5 cm × 2.5 cm). The PPFD of each grid was measured with a PPFD meter (LI-250A, Li-Cor Inc.) to verify the light intensity distribution; the result is shown in Figure 1. To maximize the number of vials that could be processed simultaneously, grids with a PPFD of 855-875 µmol m −2 s −1 were used, which allows the incubation of eight vials at once. Then, the leaf piece was moved and sealed in another vial and further incubated for 10 min. Note that vial exchange was not performed for measurements with an incubation period of 10 min. Finally, the vial was removed from the incubation system for isoprene collection. During collection, a filter tube was connected to the vial to introduce VOC-free air into the vial, and a collection tube was connected to the vial to capture the isoprene inside the vial. Both tubes were packed with 200 mg of Tenax TA and 100 mg of Carbotrap B (PerkinElmer Inc., Waltham, MA, USA) as adsorbents. Then, a syringe (30 mL) was connected to the other side of the collection tube to draw air from the vial. The collection tube was then analyzed to identify and quantify the VOCs, as described in Section 2.5.
The target leaflet was first measured using the enclosure method while it was attached to the plant. The enclosure measurement method was similar to the method described above in Section 2.2, except that instead of connecting the additional air pathway of the photosynthesis measuring system to the spectrometer, an adsorbent tube was connected to the additional air pathway for each sampling. The light intensity, leaf temperature, and CO 2 concentration in the cuvette were set to 1000 µmol m −2 s −1 , 30 • C, and 400 ppm, respectively. Before isoprene was collected, the leaf was enclosed in the cuvette for approximately 40-60 min until the net photosynthetic rate became steady. During isoprene collection, an adsorbent tube was plugged to the added air pathway of the photosynthesis measuring system, and a micropump (MP-Σ30NII, Sibata Inc., Tokyo, Japan) was connected to the other side of the adsorbent tube to draw air from the cuvette for 5 min at a rate of 0.2 L min −1 ; 1 L of air was passed through the adsorbent tube. After the enclosure measurement, the leaf inside the cuvette was cut for measurement using the process described in Section 2.3. Note that because the leaflet had been stabilized by the cuvette, the incubation period was set to 10 min, and vial exchange was omitted. The water temperature during incubation was set to 30 • C in the first and second experiments and 25 • C in the third and fourth experiments.

Gas Chromatography Analysis
The isoprene samples collected using an adsorbent tube were analyzed by a gas chromatography system. Two-stage desorption was performed by a thermal desorption system (TD-30, Shimadzu Corporation, Kyoto, Japan) to desorb the VOCs from the adsorbent tube, where the desorption, trap, and redesorption temperatures were 280, −20, and 280 • C, respectively. The redesorbed VOCs were then injected into a gas chromatography flame ionization detector system (Nexis GC-30, Shimadzu Corporation) with a SH-I-5MS capillary column (length: 60 m; ϕ: 0.25 mm; ID: 1.0 µm; Shimadzu Corporation) for identification and quantification. This system used helium as a carrier gas; the gas pressure, column flow rate, linear velocity, and split ratio were 125.0 kPa, 1.1 mL min −1 , 21.6 cm s −1 , and 18.7:1, respectively. During analysis, the column temperature was first held at 35 • C for 5 min; then, it was increased to 250 • C at a rate of 5 • C min −1 and held at 250 • C for 10 min. The isoprene quantity (N, nmol) was determined by an internal standard line, where toluene-d8 was used as the standard substance, and the coefficient of determination of the standard line was >0.999.

Calculation of Gas Exchange and Carbon Ratio
The isoprene emission rate (nmol m −2 s −1 ) was determined by different means depending on the measurement method. That of the excised leaves (I excised ) was determined as follows: where A excised (m 2 ) is the area of the excised leaf, and t (s) is the incubation period after vial exchange (approximately 600 s). The isoprene emission rate for the enclosure method (I enclose ) was determined as follows: where C in and C out (nmol mol −1 ) are the inflow and outflow isoprene concentrations of the cuvette, respectively; w in and w out (mol mol −1 ) are the inflow and outflow water vapor concentrations of the cuvette, respectively; F (mol s −1 ) is the air inflow rate of the cuvette (5 × 10 −4 mol s −1 ); and A cuvette and A leaf (m 2 ) are the planar area of the cuvette (0.0006 m 2 ) and actual leaf area inside the cuvette, respectively. C in was assumed to be 0 nmol m −2 s −1 because isoprene was eliminated from the inflow by the platinum catalyst, as described above. C out was determined as follows: where V (L) is the air volume passed through the adsorbent tube, and V m (L mol −1 ) is the molar volume of air at 0 • C (22.4 L mol −1 ). The net photosynthetic rate (Pn, µmol m −2 s −1 ) was determined as follows: where CO 2in and CO 2out (µmol mol −1 ) are the inflow and outflow CO 2 concentrations of the cuvette, respectively. The fraction of carbon emitted as isoprene in the carbon assimilated by photosynthesis (R isoprene , %) is defined as follows: where CN isoprene is the carbon number of isoprene (CN isoprene = 5). Note that the steadystate values of I enclose and Pn were used to calculate each leaf temperature.

Response of Isoprene Emission Rate to Leaf Temperature
The time plots showed clear stepwise increases in I enclose with increasing leaf temperature for all four palm species, B. yatay, P. dactylifera 'Piarom', P. dactylifera 'Major', and W. filifera (Figure 2)   During heating, I enclose generally responded instantaneously to leaf temperature. However, I enclose continued to increase moderately after the leaf temperature stopped increasing and required~40 min, on average, to reach the steady state. The period of moderate increase varied greatly among measurements. The shortest period was that of P. dactylifera 'Major' as leaf temperature increased from 20 to 25 • C (~10 min), and the longest was that of B. yatay as leaf temperature increased from 25 to 30 • C (~90 min).
R isoprene varied widely among the palm species at each temperature; however, overall, it increased nonlinearly with increasing leaf temperature in a pattern similar to that of I enclose (Figure 3). At 20 • C, R isoprene did not exceed 0.5% for any palm species. By contrast, at 35 • C, all the species had R isoprene values larger than 1%, and P. dactylifera 'Major' had an extremely large value (>4%).

Isoprene Emission Rate for Different Incubation Periods
The I excised values of B. yatay, W. filifera, P. dactylifera 'Piarom', P. dactylifera 'Major', and the sunlit P. canariensis individual were measured for incubation periods of 10, 40, and 60 min (Figure 4). The isoprene emission rate of B. yatay increased as the incubation period increased from 10 to 40 min and, then, decreased at an incubation period of 60 min. The I excised values for periods of 10 min (15.6 ± 6.4 nmol m −2 s −1 ) and 60 min (16.5 ± 6.3 nmol m −2 s −1 ) were approximately 65% of that at 40 min (24.0 ± 6.0 nmol m −2 s −1 ). For W. filifera, P. dactylifera 'Piarom', and P. dactylifera 'Major', I excised increased as the incubation period increased from 10 min (2.3 ± 0.6, 1.4 ± 0.2, and 1.6 ± 0.2 nmol m −2 s −1 , respectively) to 40 min (5.9 ± 1.2, 8.3 ± 1.3, and 10.5 ± 5.7 nmol m −2 s −1 , respectively). The value for a period of 60 min was approximately the same level as that of 40 min (6.5 ± 1.1, 8.7 ± 0.9, and 9.8 ± 5.2 nmol m −2 s −1 , respectively). P. canariensis exhibited a constant linear increase in I excised with increasing incubation period during the entire experiment, where I excised was 1.2 ± 0.1, 4.6 ± 1.2, and 6.7 ± 0.7 nmol m −2 s −1 after 10, 40, and 60 min, respectively. I excised was typically low in the initial stage and increased at 40 min for all the palm species; however, the tendency after further incubation depended on the species.  Figure 5 shows the relationship between I excised and I enclose for the palm species. B. yatay showed the highest isoprene emission rates in all the experiments. T. wagnerianus and the shaded P. canariensis individual had the lowest rates. W. filifera, P. dactylifera 'Major', P. dactylifera 'Piarom', and the sunlit P. canariensis individual had similar rates.

Temperature Response of Isoprene Emission Rate of Palm Species
The isoprene emission measurement at different leaf temperatures revealed considerable differences in the isoprene emission rates of the palm species. B. yatay, which was reported for the first time, exhibited extremely high I enclose values under standard conditions, which were comparable to those of species known to be strong emitters, e.g., Populus tremuloides (59 nmol m −2 s −1 ), Quercus alba (79 nmol m −2 s −1 ), and Salix nigra (37 nmol m −2 s −1 ) [41][42][43]. W. filifera and P. dactylifera reportedly showed moderate isoprene emission under standard conditions [35,44], whereas our observation revealed high I enclose under standard conditions. Seasonality might be a factor, as a decreasing trend in I enclose with time (27 October-7 December 2021) was observed. In addition, the lower PPFD in the indoor environment starting on 1 November 2021, might have decreased I enclose , because lower isoprene emission capacity has been reported in plants acclimated to lower light intensity [45][46][47].
For all the palm species (B. yatay, W. filifera, P. dactylifera 'Major', and P. dactylifera 'Piarom'), I enclose clearly depended on temperature. Differences among species were relatively small at 20 • C and increased at higher temperatures. The nonlinear increases in I enclose from 20 to 35 • C for the palm species were generally consistent with the temperature dependence reported in previous studies [48][49][50]. By contrast, the Pn values of the palms exhibited varying responses to leaf temperature, increasing when the leaf temperature increased from 20 to 25 • C but decreasing at higher temperatures. The decrease at high temperatures is attributed mainly to decreasing g sw . R isoprene of the palm species increased significantly with leaf temperature because of the decrease in Pn and substantial increase in I enclose , which is consistent with the pattern of hybrid aspen (Populus tremula × Populus tremuloides) reported by Rasulov et al. [51]. In addition, species such as P. dactylifera 'Major' and P. dactylifera 'Piarom' exhibited extremely high R isoprene (>3%) at a leaf temperature of 35 • C in this study compared to those recorded from hybrid aspen.
Although isoprene emission is not directly related to stomatal status [52,53], water loss at high temperatures is still a concern. Extreme water loss could cause folding of the excised leaves and thus reduce the amount of light they receive, causing large errors in the measured isoprene emission rate.
Our measurements demonstrated that I enclose responded very rapidly to fluctuations in leaf temperature. The relationship was almost the same as that between I enclose and leaf temperature during the initial heating process. However, I enclose continued to increase moderately even after warming stopped. An average of 40 min was required to reach the steady-state value of I enclose . This subsequent increase may be associated with the time required for dimethylallyl diphosphate (DMADP), one of the precursors for isoprene synthesis, to reach equilibrium after a temperature change [54]. This process varied greatly over our observations; the shortest periods were less than 5 min, whereas the longest periods exceeded 90 min.

Determination of Appropriate Incubation Period for Isoprene Measurement Using Excised Palm Leaves
The effect of cutting on the leaves may increase with time. However, the stabilization period must be long enough for the isoprene emission rate of palm leaves to become steady. There should be an optimal incubation period considering the opposing effects of time on stabilization and disturbance. The use of different incubation periods for B. yatay, W. filifera, P. dactylifera 'Major', P. dactylifera 'Piarom', and P. canariensis revealed different trends in I excised . B. yatay exhibited a peak at 40 min. An optimal incubation period for accurate I excised measurement was not identified for W. filifera, P. dactylifera 'Major', and P. dactylifera 'Piarom'. A 10 min incubation period was too short for the excised leaves to adapt to a water temperature of 30 • C and PPFD of 870 µmol m −2 s −1 from an air temperature of 25 • C and PPFD of~200 µmol m −2 s −1 . In addition, B. yatay showed a very large decrease in I excised when the incubation period was increased from 40 to 60 min.
The low isoprene emission rate in the initial stage of incubation can be explained by the time required to increase the DMADP pool. However, the reason for the decrease in emission rate after the peak is not clear. We can only hypothesize that intense water loss after cutting resulted in either lower PPFD received because of deformation or decreased ATP due to inhibited photophosphorylation [55]. The production pathway (the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway) of isoprene-related DMADP is highly dependent on ATP and NADPH produced by the light reactions [56]. Therefore, a smaller DMADP pool can be expected with water loss. Because a few decreases in I excised with time were observed, I excised may be underestimated for long incubation periods. According to our results, 40 min could be a moderately suitable incubation period for obtaining the isoprene emission rate of excised palm leaves.

Suitability of Measurement Using Excised Leaves to Evaluate Isoprene Emission from Palm Leaves
To further examine the validity of the method using excised leaves to obtain the isoprene emission rate, we applied both the enclosure method and this method to the same sections of leaves of B. yatay, W. filifera, P. dactylifera 'Major', P. dactylifera 'Piarom', T. wagnerianus, and P. canariensis. Four experiments conducted under different conditions revealed different ranges of I enclose . A previous observational study of an oak indicated that the isoprene emission rate can be affected by seasonal temperature variation [57]; it was also reported that P. dactylifera exposed to 35 • C for 14 days showed a larger isoprene emission rate than those exposed to 20 • C for 14 days. Our measurements of I enclose also varied with average monthly temperature. The second experiment, with an average monthly temperature of 26.3 • C, yielded the largest I enclose values overall. By contrast, the first and third experiments, with moderate average monthly temperatures (19.6 and 19.9 • C, respectively) revealed the second-largest I enclose values in similar ranges. In the fourth experiment, which had the lowest average monthly temperature (14.5 • C), T. wagnerianus and P. canariensis, which were planted outside, had much lower I enclose values. Although the pot seedlings were moved indoors and exposed to a relatively warm temperature (25 • C), decreases in I enclose were also detected. The reason might be the lower intensity of growing light supplied by horticultural lights (less than 300 µmol m −2 s −1 PPFD), as explained in Section 4.1. Despite the different seasonal patterns, strong correlations between I excised and I enclose were detected regardless of seasonal temperature. For instance, the observed isoprene emission rates were much lower in the fourth experiment than in the third experiment, but the slopes between I excised and I enclose were the same. This result proves that the I enclose value of palm leaves is well correlated with I excised under any growth conditions if the incubation temperature is controlled.
Nevertheless, in the first and second experiments, I excised was significantly larger than I enclose , regardless of the similar temperature setting and lower light intensity in the water bath (~860 µmol m −2 s −1 ) compared to that in the enclosure (1000 µmol m −2 s −1 ). This result is unexpected, because the isoprene emission rate should increase with increasing light intensity because of light-induced growth of the DMADP pool [58]. Although the effect of cutting was not ruled out, another possible reason is that the leaf pieces were above ambient temperature (i.e., the water temperature in the water bath tank) because of non-photochemical quenching [59]. I excised was 1.7 times larger on average at a water temperature of 30 • C compared to that at 25 • C. This increase with temperature seems reasonable but is slightly smaller than the ratio of I enclose at a leaf temperature of 35 • C to that at 30 • C (~1.8 times) in our measurement of the temperature dependence of the enclosure method. Because observations at lower temperatures usually showed much higher values (30 to 25 • C:~2.2 times; 25 to 20 • C:~2.7 times), this result implies that the temperature of the leaf pieces could have been higher than the temperature of the water bath.

Development of Method Using Excised Leaves to Measure Isoprene Emission Rate
Enclosure measurement of VOC emissions from plant leaves has been recommended to obtain an accurate isoprene emission inventory. However, this method is unsuitable for large-scale sampling under controlled conditions because there is a tradeoff between time efficiency and accurate environmental control. Leaf discs have been used for multiple samplings of isoprene emissions with control or treatment in previous studies (e.g., [60][61][62]). However, most of these studies focused on clarifying the effect of isoprene-related factors inferred from changes in isoprene emission rate rather than obtaining an isoprene emission rate inventory. Several studies have reported increased or decreased isoprene emission rates from leaves of a vine (Pueraria lobaia) and grasses (Phragmites australis, Mucuna deeringeniana) resulting from wounds [63,64]. However, studies have also found no significant change after aspen (Populus tremuloides) and hybrid aspen leaves were excised [65,66]. In addition, previous studies indicated that water loss from a leaf disc significantly disturbed the water status of leaves [67,68]. Although the thick cuticle of palm leaves prevents severe water loss from the leaf surface [69,70], it is better to minimize the water loss from the severed part of the leaf.
To reduce water loss, we used rectangular leaflets with a ratio of cut length (cm) to area (cm 2 ) of only 1.0-1.4. By contrast, the ratios of typical leaf discs (1-1.5 cm 2 ) can be as high as 4.2-6.3. In addition, the excised leaves were sealed in a vial containing a drop of water to reduce the vapor pressure deficit between the excised leaf and air in the vial. By using a water bath tank and a tested light source, we can simply implement a unified environmental control of multiple samples at once. Here, the time efficiency was at least eight times higher than that of enclosure measurement. The efficiency could be further improved by increasing the number of water bath systems.

Conclusions
We examined the temperature response and effect of incubation period on the isoprene emission rate of palm leaves to identify the appropriate incubation parameters for the rapid measurement of isoprene emissions from palm leaves. We demonstrated that leaf temperatures of 25 to 30 • C and an incubation period of 40 min were suitable for measuring isoprene emissions from excised leaves of several palm species. After the method was established, the isoprene emission rates obtained by this method were compared with those of a widely used enclosure method. A strong consistent linear correlation was found between the results of these two methods. This result implies that measurement using excised leaves is appropriate for evaluating the isoprene emission capacity of palm species. Moreover, this method enables multiple samplings at the same time using a water bath system and an LED light panel, and, thus, enables highly efficient isoprene measurement. Therefore, this study successfully established a rapid protocol for measuring the isoprene emission rates of palm species.