An automated system for trace gas flux measurements from plant foliage and other plant compartments

Plant shoots can act as sources or sinks of trace gases including methane and nitrous oxide. Accurate measurements of these trace gas fluxes require enclosing of shoots in closed non-steady state chambers. Due to plant physiological activity, this type of enclosures, however, lead to CO2 depletion in the enclosed air volume, condensation of transpired water, and warming of the enclosures exposed to sunlight, all of which may bias the flux measurements. Here, we present PlasTraGAS ::::::::::::: ShoTGa-FluMS, a novel measurement system designed for continuous and automated measurements of trace gas 5 and volatile organic compound (VOC) fluxes from plant shoots. The system uses transparent shoot enclosures equipped with Peltier cooling elements and automatically replaces fixated CO2, and removes transpired water from the enclosure. The system is designed for measuring trace gas fluxes over extended periods, capturing diurnal and seasonal variations and linking trace gas exchange to plant physiological functioning and environmental drivers. Initial measurements show daytime CH4 emissions two pine shoots of 0.056 and 0.089 nmol g−1 foliage d.w. h−1 or 7.80 and 13.1 nmolm−2 h−1. Simultaneously measured CO2 10 uptake rates were 9.2 and 7.6 mmolm−2 h−1 and transpiration rates of 1.24 and 0.90 molm−2 h−1. Concurrent measurement of VOC emissions demonstrated that potential effects of spectral interferences on CH4 flux measurements were at least tenfold smaller than the measured CH4 fluxes. Overall, this new system solves multiple technical problems that so far prevented automated plant shoot trace gas flux measurements, and holds the potential for providing important new insights into the role of plant foliage in the global CH4 and N2O cycles. 15 Copyright statement. TEXT

Shoot chambers (Fig. 2a) were custom built by Toivo Pohja Tmi (Juupajoki, Finland). The chambers' inner dimensions are 12 x 24 x 4 cm and each chamber encloses a volume of 1.15 L. The bottom and the rear plate of the chamber are constructed from aluminium, the other sides from UV-transparent acrylic glass covered with FEP tape on the inside of the chamber. UV transparency of the cover was confirmed by UV-VIS spectroscopy (Perkin-Elmer Lambda25; Fig. 2b). The connection between 85 the removable cover and aluminium base of the chamber is sealed with a thin (1mm) foam gasket placed in a groove in the cover against the bottom of the base and in a groove in the aluminium rear plate against the rear end of the cover. The seal can be further improved by applying vacuum grease (Sigma Aldrich) to the gasket. The cover is attached to the base with eight screws; six against the bottom and two against the rear plate. To seal the opening for the shoot in the rear plate, the shoot is buffered with a pressure-sensitive adhesive (Blu-tack, Bostik S.A.) wrapped in PTFE tape at the chamber opening. The needles 90 or leaves are held in place inside the chamber by means of a fishing line bed.
The bottom of each chamber is equipped with a Peltier cooling element. One fan is located inside each chamber, a second fan was placed outside below each Peltier element on a finned radiator. Each chamber is further equipped with a Pt 100 temperature stem of the plant being measured goes through an opening in the cover, which is then also sealed with the pressure-sensitive adhesive described above.

Static chamber module for trace gas flux measurements
Trace gas fluxes are measured in a closed loop setup where air is recirculated between a shoot or soil chamber and one or more online gas analysers. In principle, any flow-through trace gas analyser or combination of analysers can be used with 105 this setup given that it can (a) completely recirculate the analysed air into the enclosure chambers, and (b) the analyser does not emit the analysed trace gas or interfering volatile compounds (e.g. from pump membranes). At minimum one analyser capable of measuring CO 2 mixing ratios is required. Since our initial measurements were focused on CH 4 fluxes, we used a Picarro G2301 (CH 4 / CO 2 / H 2 O) or a Picarro G2201i ( 12/13 CH 4 / 12/13 CO 2 / H 2 O) cavity ring-down spectroscopic analyser equipped with a KNF oil-free membrane vacuum pump. Analysers with low flow rates (e.g. the Picarro G2201i) 110 require a bypass loop with a membrane pump (e.g. Nitto Kohki GMBH, model DP0140-A1111) to accelerate gas transport between a chamber and the analyser and thus reduce the lag between mixing ratio change occurring in the chamber and that being observed by the analyser. Analysers without an internal pump require an external pump to circulate the air between a chamber and the analyser.
2.1.3 Temperature, CO 2 , and humidity control Temperature control The enclosure temperature is controlled through the Peltier elements located beneath each shoot chamber. The Peltier element is activated when the temperature inside the shoot chamber exceeds ambient temperature (measured through an additional temperature sensor) by 2°C and deactivated when the temperature inside the chamber drops 1°C below the ambient temperature. Homogeneous temperature inside the chamber is ensured by the fan directing the air flow straight onto the area where the Peltier element is connected. This also minimises water condensation on the cooled area.

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Humidity control. To avoid moisture build-up from transpired water during static chamber measurements, a membrane dryer (Nafion MD-050-12S-2) placed in the return line from the analyser to the soil and shoot chambers. The dryer is either flushed with dry air in counter-stream or evacuated with a vacuum pump (Gardner-Thomas, model 1410V).
CO 2 control To maintain CO 2 mixing ratios in the closed loop mode, CO 2 removed due to photosynthesis is replaced by CO 2 injections regulated by a mass flow control unit (MFC1; 0-50 mL/min, Bürkert GmbH, type 8715). We initially injected 125 a 1% CO 2 in N 2 gas mixture utilising a PID algorithm to keep the CO 2 level stable. These injections, however, diluted the chamber air and decreased the trace gas mixing ratios. CH 4 mixing ratios in typical operation, for example, decreased by 100-300 ppb (5-15%) below ambient mixing ratios. Under these circumstances, small diffusion leaks can lead to an increase in the trace gas mixing ratios over time during chamber closures, which can be mistaken for shoot emissions.
After initial tests, we therefore changed the system to inject pure CO 2 . In addition, we changed the injection algorithm to 130 inject a fixed amount of CO 2 (0.14 mL, corresponding to approximately 400 ppm CO 2 in a shoot chamber) whenever the CO 2 mixing ratio falls below a configurable threshold value (set to 400 ppm). With this method, CO 2 injections have only a minimal effect on trace gas mixing ratios (e.g. <10 ppbv CH 4 ), and injections can be easily identified and corrected for. To facilitate rapid mixing of the injected CO 2 into the sample stream, we placed a hand-crafted flap in the fitting that connects that MFC to the main sample loop to force the sample air to flow through the throat of the MFC controlling the injections. valve on the outlet of the Li-850 regulates the flow rate generated by its internal pump, such that the air flow pulled from the enclosure chamber by the analyser(s) matches the air flow pushed into the chamber via MFC2. In addition, VOC mixing ratios are measured by a proton transfer quadrupol mass spectrometer (PTR-QMS; Ionicon, Innsbruck, Austria).

Chamber flushing with ambient air
To keep the conditions in shoot chambers close to ambient between the flux measurements, shoot chambers are constantly 150 flushed with ambient air. Initially, this was achieved by placing an opening to ambient air into the gas lines just upstream the shoot chambers. This inlet is protected by a check valve that only allows air inflow into the shoot chamber when the pressure differential between the chamber and ambient air is more than -50 mbar. Shoot chambers were flushed by connecting separate membrane pumps (Nitto Kohki GMBH, model DP0140-A1111) to each shoot chamber via the switching board. This way, inactive shoot chambers are flushed with ambient air at a flow rate of 750-1000 mL min −1 . Initial tests showed that the brushes 155 in these flush pumps burn out easily. We therefore changed the systems such that the chambers were flushed by pressing pressurized air into the chambers. In this setup, the opening was moved downstream of the shoot chamber, and a check valve was inverted, such that it allows air outflow but not inflow.

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An additional 3-way solenoid valves (SMC VX3114K-01N-5G1-B) is used to switch between chamber and bypass air in the dynamic chamber module. Both setups allow the connection of one chamber to static chamber module while another chamber can be connected to the dynamic chamber module; chambers not connected to either modules are operated in flush mode. In addition, each system was equipped with a sampling inlet to analyze trace gas mixing ratios in ambient air.
2.1.7 Control software and data recording 170 Both measurement systems are operated by Koppi, a custom made software written in Python. The software allows for the automatic switching between chambers and the measurement modes, regulates the CO 2 injections and Peltier coolers in response to CO 2 and temperature data, and records the instrument configuration and all measurement data at 0.2 Hz frequency.
The volume of injected CO 2 is recorded at 0.1 Hz due to the slow response time of the MFC, and it interpolated to 0.2 Hz frequency prior to data analysis. PTR-MS data is recorded separately and synchronized with the main measurement dataset 175 during data processing.

Data analysis and calculations
Data was processed in four steps from raw data to a time series of flux and auxiliary measurements that have been scaled to shoot measures where appropriate. All data processing was conducted in R version 3.6.3 (R Development Core Team, 2015).
In step one, raw data from the main operating software and auxiliary datasets (e.g., raw data recorded by internal dataloggers 180 in the analysers) are imported, synchronized, and combined into a single data set. In addition, individual closures are identified with their start and end times, and the volume of injected CO 2 was interpolated to 0.2 Hz frequency.
Step two comprises corrections conducted at the raw data level. Most importantly, measurements in closed loop mode were corrected for the effects of CO 2 injections. For this, we modelled the mixing of CO 2 with chamber air after each injection.
The model contained two elements, (a) mixing of injected CO 2 with air returning to the shoot chamber, and (b) mixing of air 185 in the shoot chamber and air in the analyser loop.
For (a), mixing of air released by the MFC into the return air stream was described by an exponential decay function (eq. 1), where J ef f ective (t) stands for the effective flux at time point t (t = 0 at the start of the modelled chamber closure), a stands for an empirically fitted constant (2.5 for the test measurements presented in this study), f conv for the gas-specific conversion 190 factor for thermal conductance based on mass flow measurements (0.7 for CO 2 ), t stands for a time point prior to t during the same closure, J M F C (t ) for the injection flux recorded by the mass flow control unit at that time, and τ for a fitted exponential decay constant (90 sec) for the data analysed in this study) that is empirically fitted to describe the data in a given setup. After installing the metal flap at the tee-connector between MFC and return air flow, this component was not necessary anymore and instantaneous mixing (i.e., Q ef f ective (t) = Q M F C (t)) could be assumed.

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For (b), the system was conceptualized as the combination of the main chamber and a single tube with a volume equivalent to the total volume of all tubing and analyzers in the system. The tube was further modeled as consisting of n elements, each holding a volume equivalent to the flow rate per time step (5 sec). At each time step, air was moved from tube element n to tube element n+1, the first tube element was filled with chamber air and the last tube element was emptied into the chamber.
Measurements assumed to be conducted in tube element n/2-1, injections in tube element n/2. The flow rate was assumed based 200 on the specifications of the analyser (400 mLmin −1 ), while the number of tube elements (n=5) were fitted to the data.
We confirmed the validity of this model by applying it to CO 2 injections during nocturnal leakage tests (see below), when CO 2 injections were conducted at set intervals rather than triggered by a mixing ratio threshold, and when the CO 2 emissions from foliar respiration were well characterized. We then calculated the corrected CO 2 and CH 4 mixing ratios according to equations 2 and 3, where [CO 2 ] corr (t) and [CH 4 ] corr (t) stand for the corrected dry CO 2 and CH 4 mixing ratios at time point t, and [CH 4 ] raw (t) for the measured dry mixing ratios, and [CO 2 ] inj (t) for the mixing ratio of injected CO 2 at time point t.
This correction could be avoided when analysing data from PlaSTraGAS-clim2 :::::::::::: ShoTGa-clim2 and when flux rates were 210 sufficiently high to reduce the effective time of static chamber closures. Instead of correcting for the effect of CO 2 injections, we identified such injections as local maximums of the CO 2 mixingratio) were identified and time periods during which the CO 2 mixing ratio change was affected by the injection were removed. We then treated the time periods between the injections as separate subclosures (see below). This approach was not possible in PlaSTraGAS-gh7 ::::::::::: ShoTGa-gh7 due to the relatively long tube length between chambers and switching board (2x10m) which caused a relatively long delay until full mixing was reached 215 after each injection.
Other corrections during this processing step included converting CO 2 mixing ratios conducted by the Li-850 to mixing ratios in dry air. In the test experiments presented herein, we also had to apply a a 6-minute running average filter on CO 2 mixing ratios to remove an oscillation of measured values due to an instrument malfunction. Raw data from PAR measurements (in mV) was converted to PAR (in µmol m −2 sec −1 ) using the calibration equations provided by the manufacturer.
For dynamic chamber closures, the mixing ratio in air leaving the chamber (C out ) was calculated as the average mixing ratio measured from 180 sec after closure start to 60 sec before closure end. Similarly, the mixing ratio in air entering the chamber 230 (C in ) was calculated as the average mixing ratio from 180 sec after closure start to 60 sec before closure during bypass periods.
Auxiliary measurements (PAR, temperature) were averaged over the entire closure time.
Step four consisted of calculating gas fluxes and normalizing them to sample size (e.g., foliage dry weight or leaf area). For static chamber closures, dC / dt was then used to calculate the flux rate per leaf area (Q A ) or leaf dry weight (Q m ) according to eqs. 4 and 5, where A and m stands the leaf area and leaf dry weight of the enclosed branch, V for the chamber volume including analyser loop, and V mol molar volume, which is calculated from pressure p, temperature T, and ideal gas constant R.
During initial tests, we also quantified L taking advantage of the initial measurements where a 1% CO 2 in N 2 mixture was used to replace the photosynthesized CO 2 . These injections decreased the mixing ratio inside the shoot chamber (C chamber ) by 5-10 % (to 1.8-1.9 ppmv), while the C ambient remained constant (∼ 2.0 ppm). We used these variations in chamber CH 4 mixing ratios to calculate L as the regression between the change in the mixing ratio of CH 4 over time (dC/dt) and C ambient − C chamber , assuming that any CH 4 exchange between shoot and chamber air is not affected by C chamber .

Blank tests
Our initial tests were focused on the ability of the chamber systems to accurately measure shoot CH 4 emissions. We therefore evaluated the system blank for CH 4 exchange from shoot chamber, but not for other greenhouse gases or the soil chamber. To quantify the system blank, all openings of the shoot chamber were closed and the systems were operated in the same way as for plant shoot measurements. These measurements were either conducted before and after each experiment 270 (PlaSTraGAS-clim2 :::::::::::: ShoTGa-clim2) or during the experiment with chambers left empty for blank control (PlaSTraGAS-gh7 :::::::::: ShoTGa-gh7).
We furthermore calculated the system detection limits for individual chamber closures as equal to three times the standard deviation of the blank measurements.

Test measurements with Scot's pine shoots
Test measurements were conducted with the PlaSTraGAS-gh7 :::::::::: ShoTGa-gh7 system and a two year old Scots pine (Pinus 275 sylvestris L.) sapling. The sapling was obtained from a commercial grower in Fall 2019, potted in a 20 L pot, and stored outdoors in the University of Helsinki's Viikki greenhouse facility over the winter. In late January 2020, the tree was transferred into a greenhouse compartment and allowed to acclimatize for three weeks prior to the measurement campaign (Feb 22-25). The ambient temperature in the compartment was between 15 and 18°C during nighttime and warmed to 22 to 32°C during daytime, depending on weather conditions. The trees were watered weekly, and received additional light from a high 280 pressure sodium lamp resulting in 250-400 µmol m −2 sec −1 photosynthesis active radiation (PAR). In addition, we placed 6 UV-A lamps (Q-lab UVA-340) approximately 20cm above the measured shoot to stimulate aerobic CH 4 production. Both PAR and UV lighting followed 12h day/night cycles (7am to 7pm).
We installed a total of four automated shoot chambers into the system. Chambers 1 and 4 were kept empty as blank controls, while chambers 2 and 3 were placed on separate branches of the sapling. As exposure to sunlight was low, we decided not to 285 cool the chambers with the Peltier cooling system to keep the experiment more simple. The system was programmed to place connect each shoot chamber to a Picarro G2301 analysis via static chamber module for 24 minutes followed by measuring ambient air for 3 minutes. To explore the effects of CO 2 injections on CH 4 flux measurements, CO 2 injections were deactivated during every second closure cycle (Fig. 5). This cycle was restarted every two hours. Only 'daytime' measurements (i.e., artificial lighting on; 7am-7pm) were included in the presented data, while the results of the temporal trends (e.g., diurnal 290 cycles) will be published separately. We obtained a total of 25-26 measurements per chamber.
Concurrent with each static chamber closure, a different chamber was connected for 12 minutes to the Li-850 and PTR-QMS analysers, followed by analyzing the in-going pressurized air for 15 minutes (Fig. 6). For simplicity, only three molecular massto-charge ratios were monitored: 33 (methanol), 59 (acetone), and 137 (monoterpenes). The PTR-QMS was calibrated with a gas standard containing methanol, acetone, α-pinene, as well as other VOCs not measured in this study. Data processing was 295 conducted as described previously (Taipale et al., 2008).
After the experiment, the enclosed shoots were cut from the tree and the (projected) needle leaf area was quantified by scanning an subset of the needles and scaling to the whole branch by weight. The needle dry weight was quantified after drying for 48h at 80 C.
We state our main measurement result -CH 4 fluxes -as mean and 95% confidence interval because our focus here the overall 300 uncertainty associated with the average flux found in these measurement. Results from auxiliary measurements -temperature, PAR, CO 2 and water fluxes -are presented as means and standard deviation, because we primarily present these results to document the conditions under which the trace gas measurements were conducted.
The measurements CH 4 fluxes were close to the detection limit and measurements of both empty and pine shoot chambers had long-tailed distributions (i.e., contained likely outliers). To test for differences in apparent CH 4 fluxes between the shoot 305 chambers, we therefore applied the non-parametric Kruskal-Wallis test and Nunn post-hoc tests as normal distribution could not be assumed.

Assessment of measurement uncertainties
We identified three potential sources of inaccuracy in the measurements; chamber leakage, CO 2 injection modelling, and spectral interference by volatile organic compounds. We assessed the impact of these potential errors by propagating the 310 uncertainty caused by these processes onto measured CH 4 fluxes. All estimates were scaled based on chamber closure times (24 min), leaf areas (0.02 m 2 ), and foliar dry weights (3g) in this study. To evaluate the impact of gas exchange with ambient air due to chamber leakage, we assumed a mixing ratio difference between chamber and ambient air of 10 ppbv and a chamber leakage rate L=1.5%. For the effect of inaccuracy of the CO 2 injection model, we assumed a 250 ppmv inaccuracy in the mixing ratio of CO 2 in the injection model and a CH 4 mixing ratio of 2 ppmv. Finally, to evaluate the potential effect for 315 spectral interferences by co-emitted VOCs, we assumed methanol, acetone, and monoterpene emission rates based on the average emission rates found in this study (1.54, 2.55, and 2.33 nmol g −1 d.w. h −1 . respectively). Based on these emission rates, we estimated the mixing ratio of plant-emitted methanol, acetone, and monoterpenes reached at the end of static chamber closures as 28.5, 47.4, and 43.3 ppbv, respectively. We note that this approach likely overestimates the final VOC mixing ratios as increasing headspace VOC mixing ratios often lead to a decrease in emission rates and even net-uptake of VOCs by foliage 320 (??) ::::::::::::::::::::::::::::::::::::: (Cojocariu et al., 2004;Cappellin et al., 2017). Nevertheless, we consider them a good conservative estimate for assessing the potential impact of VOC emissions on CH 4 flux measurements. We converted these VOC mixing ratios to apparent CH 4 mixing ratios based on our recent quantification of upper limits to the spectral interference of various VOC in methane mixing ratio measurements with the Picarro G2301 and other methane analysers (Kohl et al., 2019a), using conservative uncertainty limits (±0.4 ppbv apparent CH 4 ppmv −1 methanol and ±0.2 ppbv apparent CH 4 ppmv −1 monoterpenes). Since the spectral 325 interference of acetone was not quantified by Kohl et al. (2019a), we applied the higher values value derived from methanol.

Temperature control
Initial tests of PlaSTraGAS-clim2 ::::::::::: ShoTGa-clim2 showed that cooling was not necessary as the enclosure chambers do not warm significantly compared to the ambient (cabinet) temperature due to the low thermal energy emitted by the LED based lighting 330 system. In a test consisting of 1311 closures with pine seedlings in the chamber, the mean difference in temperatures between lights on and lights off was found to be 1.06 ± 0.03°C, and the median change in chamber temperature during measurement was 6x10 −6 C s −1 .
Temperature measurements with PlaSTraGAS-gh7 ::::::::::: ShoTGa-gh7 conducted in August 2019 showed that uncooled shoot chambers can heat to 10°C and more above ambient temperature during summer conditions in northern Europe. Cooling 335 allowed us to keep the difference between ambient and chamber temperature below 2°C (Fig 4). In the test measurements with pine shoots conducted in the greenhouse in February 2020, uncooled chambers warmed to 3-4°C above the ambient temperature when the room lighting was on (Tab. 5), indicating that moderate cooling is required for experiments under greenhouse conditions even during winter months.

H 2 O control 340
The membrane dryer was capable of reducing the moisture in an empty shoot chamber connected to the static chamber module to <10% relative humidity within <5 minutes (Fig 5a). During the measurements with pine shoots in the chamber, the membrane drier removed sufficient water from the chamber to prevent condensation of transpired water in the system and hold the relative humidity in the shoot chamber between 40 and 50%.

CO 2 control 345
Photosynthesis by the enclosed pine shoots depleted CO 2 in the enclosed volume to <100 ppm within 2-3 minutes. In the test experiments with pine shoots, an injection corresponding to approx. 400 ppm CO 2 was triggered once every 10 minutes. These injections allowed to sustain the CO 2 between 400 and 700 ppm (Fig. 7) for extended periods of time (tested for up to 2 hours).
While maintaining more constant CO 2 mixing ratios is possible with this system, pulsed injections make it easier to correct trace gas mixing ratios for dilution by the injected CO 2 .

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To evaluate the performance of the CO 2 injection model, we evaluated 20 leak test measurements. In these nighttime measurement, shoot CO 2 emissions and leakage were well characterized, such that the effect of CO 2 injections on measured CO 2 mixing ratios could be studied in isolation of other processes (Fig. 3). The model generally predicted CO 2 mixing ratios within 250 ppmv. Assuming a CH 4 mixing ratio of 2 ppmv, the propagated error of CH 4 mixing ratios due to this uncertainty is <0.5 ppbv.

Chamber leakage
Initial tests showed relatively high leakage rates of up to 1-2% per minute (2). Over time, we made improvements to the chamber seal (e.g., application of vacuum grease to contact surfaces, testing seal with a hand held pressure meter while closing the shoot chamber.) This resulted in lower leakage rates, <0.15% min −1 in the climate chamber system and <0.5 % min −1 in the greenhouse system. This leakage rate has negligible effects on flux measurements when the analyte gas's initial initial 360 mixing ratio in the shoot chamber is close to its mixing ratio in the ambient air surrounding the shoot chamber (cabinet air in the case of the climate chamber). It is currently not common to report leakage rates in static chamber studies, and we are therefore unable to compare these rate literature values. However, we hope that this reporting becomes more common to allow for such a comparison in the future.
Chamber leakage becomes a more serious issue when the analyte gas's mixing ratio inside the chamber (C c ) differs signifi-365 cantly from its mixing ratio in ambient air (C a ). This is relevant in two cases: (a) when the CO 2 injections strongly dilute the analyte gas inside the shoot chamber, or (b) when the analyte gas's mixing ratio inside the climate chamber cabinet increase due to strong emissions from the plant or soil. We observed, for example, elevated CH 4 mixing ratios in the cabinet air when a Betula nana plant growing in water saturated peat was placed in the cabinet. In these cases, an apparent flux of L · (C a − C i ) occurs, and needs to be corrected for during data analysis.

System blank and method detection limit
Average system blanks, that is, the apparent CH 4 flux in an empty control chamber, were <0.3 nmol h −1 in both systems, corresponding to a mixing ratio change of <1.8 ppbv CH 4 during a 24 minute chamber closure. Method detection limits (MDL) for CH 4 emissions from plant shoots were <0.15 nmol g −1 d.w. h −1 in the climate chamber system and <1.5 nmol g −1 d.w. h −1 in the greenhouse system (assuming 3 g d.w. foliar biomass per chamber; Table 2). This method detection limit is defined for 375 a single closure measurement and further decreases with √ n in the case of repeated measurements. It is thus easy to reach a MDL well below reported plant methane emissions rates (e.g., 0.75 -55 nmol g −1 d.w. h −1 ; (Keppler et al., 2006)).
3.6 Test measurements with Scot's pine shoots 3.6.1 Auxiliary measurements The two enclosed shoots contained needles with a total dry weight of 2.61 and 3.92 g dry weight and leaf areas of 0.019 and 380 0.027 m 2 , respectively. During the included test (i.e., daytime) measurements, average temperature and PAR were 24.1C (SD 3.4; range 16.5 to 31.8) and 328 µmol m −2 sec −1 (SD 104; range 62 to 620). As mentioned above, the measured temperatures inside the shoot chambers were higher than ambient temperature, on average by 3.3°C (SD 1.8; range -2.6 to 10.3).
Temperature and PAR values of individual chambers are summarized in Table 5.
The average measured CO 2 mixing ratio (1 SD) of air entering the shoot enclosure in dynamic chamber mode was 384.8±5.5 385 ppmv (Fig. 8). After passing through empty chambers, CO 2 mixing ratios were on average slightly elevated (390.6±5.8 and 391.1±5.7 ppmv, respectively), whereas CO 2 was significantly depleted after air passed through shoot chambers (295.9±18.3 and 308.6±17.3 ppmv). The average carbon uptake by pine shoots, calculated as the difference between shoot and empty chamber, were 7.63±1.39 and 9.16±1.93 mmol CO 2 m −2 leaf area h −1 ( Table 3).
These emission rates are comparable to field measurements (e.g. Tarvainen et al., 2005).

Methane flux measurements
The apparent CH 4 emission rates and their 95% confidence intervals were 0.700±0.137 and 1.106±0.170 nmol h −1 in chambers with pine shoots, and 0.279 ±0.134 and 0.445±0.111 nmol h −1 in empty chambers (Fig 7a). Apparent emission rates in 405 chambers with pine shoots were significantly different from the empty chambers and from each other, whereas fluxes from the two empty chambers were not significantly different from each other (Kruskal-Wallis χ 2 = 52.8, p<0.001). Apparent CH 4 production rates of pine shoots were significantly lower for closures with CO 2 injections compare to closures without injections (Fig 7b), representing the dilution of CH 4 by the injected CO 2 . However, apparent CH 4 production rates were near identical to those measured from the same shoot without CO 2 injections when CH 4 mixing ratios were corrected for this dilution. This 410 demonstrates the correction of CH4 mixing ratios successfully compensated for effects of CO 2 injections. It also indicates that there was not short-term response of CH 4 emissions rates to the inhibition of CO 2 fixation rates due to low CO 2 mixing ratios.
Scaled and blank-corrected CH 4 fluxes were 0.130±0.062 and 0.190±0.047 nmol g −1 foliar d.w. h −1 or 18.1±8.7 and 28.0±7.2 nmol g −1 m −1 leaf area h −1 (Table 3). These values are approximately five-fold below the lowest values reported by Keppler et al. (2006) for living plant tissues, but 5-10 times higher than fluxes measured from shoots of mature Scots pine 415 trees (Machacova et al., 2016) (median 3.13 nmol m −2 leaf area h −1 ). A number of reasons may have led to these relatively low emissions rates compared to experiments by Keppler et al. (2006), including the timing of our measurements during the early growing season and the relatively low PAR irradiation provided in our experiments. Conversely, the higher emissions in our experiment compared to field measurements of the same species might have resulted form the augmented UVA irradiation or the fact that Machacova et al. (2016) conducted measurements during cloudy days only to avoid the overheating of their 420 manual shoot enclosure. Regardless, these measurements demonstrate that our system is capable of detecting and quantifying CH 4 emissions at or below the levels observed in many laboratory and field conditions.

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We developed an automated system to measure trace gas fluxes from plant shoots and other plant compartments while controlling the temperature, CO 2 mixing ratio, and humidity in the plant chamber. Initial tests demonstrated that the system can detect CH 4 fluxes at the scale reported for plant shoots. The system also allows the monitoring water, CO 2 , and VOC fluxes. It is built in a modular way that is easy to customize and/or expand to different chamber types. We have constructed two implementations of this setup that are designed to measure trace gas fluxes from a single plant under controlled environmental conditions in a 440 growth chamber, and from multiple plants in a greenhouse compartment. Future development will aim to adapt the system to allow its deployment under field conditions, :::: e.g., :: at :::: long :::: term :::::::::: monitoring :::: sites.     No CO 2 injections CO 2 injections Figure 6. Mixing ratios of water (a), CO2 (b), and volatile organic compounds (c) during dynamic chamber closures of four shoot chambers in the greenhouse system. Chambers 2 and 3 each contained a shoot of a two year old pine sapling, chambers 1 and 4 were kept empty as blank controls. Black lines represent the measured mixing ratios of water (a) and CO2 (b). In panel (b), the thin black line represents the raw measured CO2 mixing ratio, while the thick black line represents its six minute running average, calculated to compensate for an oscillation in the analyser signal. Shaded areas indicate periods where chamber air was analyzed, with darker colours indicating time periods used to calculate Cout, non-shaded areas periods when the ingoing air was measured bypassing the chamber. The depicted data was measured on  Chamber (j) Figure 8. Observed steady-state mixing ratio of CO2 (a) water (c), and VOCs (e,g,i) in outgoing air (Cout) during dynamic chamber measurements of two empty chambers (grey) and two chambers with pine shoots (black). The mixing ratio of CO2 water in ingoing air (Cin) are indicated by the horizontal lines in each plot. Further, apparent CO2 uptake (b), transpiration (d), and VOC emission (f,h,j) rates calculated from these mixing ratios. Error bars and the shaded area around the horizontal lines indicate one standard deviation. a Diffusive air exchange between chamber and ambient air. Measured by comparing the nighttime CO2 trend at ambient mixing ratios and after injecting CO2 to a mixing ratio of 2000-3000 ppmv. b Flux observed in empty control chambers c Method detection limit for a single measurement, defined as three times the standard deviation of the system blank, and normlized to the foliage dry weight of a typical shoot (3 g). The detection limit for repeated measurements decreases with √ n. Table 3. Shoot fluxes :: and ::::::: stomatal ::::::::: conductance : measured in this study scaled to foliar dry weight and leaf area after after subtracting empty chamber fluxes. All uncertainties include the uncertainties in shoot and blank (empty chamber) measurements. ::::::: Stomatal ::::::::: conductance : (mmol m −2 sec −1 : ; ::: SD) : ::::::: 17.5±1.6 : :::::::