Measuring the Ratio of CO2 Efflux to O2 Influx in Tree Stem Respiration

Tree Physiology 36, 1422–14 31 doi:10.1093/treephys/tpw057

Methods paper

Measuring the ratio of CO2 efflux to 2O influx in tree stem respiration Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021

Boaz Hilman1,2 and Alon Angert1

1The Institute of Earth Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; 2Corresponding author ([email protected])

Received December 21, 2015; accepted June 9, 2016; published online July 14, 2016 ; handling Editor Michael Ryan

In recent studies, the ratio of tree stem CO2 efflux to 2O influx has been defined as the apparent respiratory quotient (ARQ). The metabolism of carbohydrates, the putative respiratory substrate in trees, is expected to yield an ARQ of 1.0. However, previous studies have reported ARQ values ranging between 0.23 and 0.90. These interesting results may indicate internal transport of respired

CO2 within stems; yet no simple field applicable methods for ARQ measurement have been available. Here, we report on the assembly of a closed circulating system called ‘Hampadah’, which uses CO2 and O2 analyzers to measure air samples from stem chambers. We tested the performance of the Hampadah with samples from 36 trees (Tetragastris panamensis (Engl.) Kuntze). Additionally, we showed the feasibility of measuring ARQ directly from stem chambers, using portable CO2 and O2 sensors, in both discrete and con- 2 tinuous modes of operation. The Hampadah measurement proved to be consistent with CO2 gas standards (R = 0.999) and with O2 2 determined by O2/Ar measurements with a mass spectrometer (R = 0.998). The Hampadah gave highly reproducible results for ARQ determination of field samples (±0.01 for duplicates). The portable sensors measurement showed good correlation with the Hampa- 2 dah in measuring CO2, O2 and ARQ (n = 5, R = 0.97, 0.98 and 0.91, respectively). We have demonstrated here that the Hampadah and the sensors’ methods enable accurate ARQ measurements for both laboratory and field research.

Keywords: O2 measurement, oxygen measurement, respiratory quotient, RQ.

Introduction 1918) that metabolize lipids as respiratory substrate and are

In aerobic respiration, CO2 is produced and O2 is consumed. able to accumulate substantial amounts of lipids in woody tis- Such respiration occurs in a tree stem’s living cells (e.g., in the sues, in even higher concentrations than carbohydrates (Hoch phloem, cambium and living parenchyma of the xylem), while et al. 2002). Another factor that affects RQ is assimilation of

CO2 diffuses out and O2 diffuses into the respiration site. The nitrate, which can result in higher than unity values of RQ (Bloom respiratory quotient (RQ) is defined as the ratio of the number et al. 1989, Luo and Zhou 2006). of molecules of CO2 released to that of O2 molecules consumed Earlier work done by our group (Angert and Sherer 2011) during respiration. The RQ is dependent on the oxidation state defined the term ‘apparent RQ’ (ARQ) for the ratio of CO2 efflux of the respiration substrate and when fully oxidized, lipids, car- to O2 influx measured from stem surfaces. Apparent respiratory bohydrates and organic acids are expected to yield RQ values of quotient values of trees in Israel and in a tropical forest in Peru 0.7, 1.0 and 1.5, respectively (Masiello et al. 2008). Given that were well below unity, ranging between 0.23 and 0.90 (Angert carbohydrates are the main compounds for storage in plants and Sherer 2011, Angert et al. 2012a). Apparent respiratory (Chapin et al. 1990) and are apparently the main respiration quotient values lower than 0.7 cannot be the result of substrate substrate (Plaxton and Podestá 2006), an RQ value of 1.0 in metabolism solely, even if the trees were ‘Fat-trees’ and metabo- tree stems is presumed. The exceptions are ‘Fat-trees’ (Sinnott lized lipids. Therefore, it seems that the CO2 in the stem is

© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] CO2 efflux/O2 influx measurement in tree stem 1423 affected by processes other than respiration, and for this reason field measurements are impossible, unless there is a climate- we indicate that at the tree stem surface we measure ‘apparent’ controlled shed on the study site. RQ, and not the actual RQ of the respiring cells. It has been sug- Our main goal in this work was to develop a system (the gested that dissolution of respired CO2 in the xylem sap, as Hampadah , a compound word of the Hebrew words for O2 and reviewed in Teskey et al. (2008), and refixation of CO2 by the CO2) for measuring air samples in the laboratory. The whole enzyme phosphoenolpyruvate carboxylase may result in lower sampling and analysis process was named the ‘Hampadah than 1.0 ARQ values (Angert and Sherer 2011, Angert et al. method’. The Hampadah consists of a fuel-cell analyzer for O2

2012a). The ARQ of tree stems, therefore, may reflect in-stem measurement and an IRGA for CO2 measurement. To estimate processes that are difficult to measure, and further studies on the Hampadah performance in measuring O2 concentration, the factors controlling the ARQ will be of interest. In addition to results of one set of stem chamber samples were compared with stem respiration studies, RQ measurements are also used in soil results of δO2/Ar of a parallel set of samples measured by a Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021 studies (e.g., Dilly 2003, Angert et al. 2014) and in other scien- mass spectrometer. Carbon dioxide accuracy of the Hampadah tific fields. Despite the need for reliable and standard methodol- was estimated by measuring CO2 standards. In addition to the ogy for RQ and ARQ measurements, to our knowledge no simple Hampadah method, we demonstrate the use of a quenching- field operable and cost-effective measurement methods have based O2 optode, and a small IRGA for direct measurement of been developed. ARQ from a stem chamber in the field. The optode and the small

To calculate ARQ values, both CO2 and O2 concentration mea- IRGA were used in two measurement methods: the direct dis- surements are required. Infrared gas analyzers (IRGAs) enable a crete method, which measures the concentrations of gases from relatively easy measurement of CO2, whereas the naturally high the chamber at the same time as flasks sampled in theHampa - background of O2 in air makes detection of small variations of dah method, and the direct continuous method, which produces

O2 technically demanding. Generally, O2 measurements in tree an ARQ value every few hours. Finally, we aimed to validate the physiology studies have focused on in vitro incubation of iso- box model approach for calculating ARQ from CO2 and O2 mea- lated tissues (Goodwin and Goddard 1940, Carrodus and Triffett sured from a static chamber presented in Angert et al. (2012a). 1975, Pruyn et al. 2002, Spicer and Holbrook 2005, 2007a, 2007b) and measurements of within-stem gas concentrations Materials and methods (Eklund 1990, 1993, 1999, Gansert et al. 2001, del Hierro et al. 2002, Pruyn et al. 2002, Mancuso and Marras 2003, Box model Spicer and Holbrook 2005), including continuous measurement Simple modeling is necessary to determine the ARQ value from (Sorz and Hietz 2008). Angert et al. (2012a) evaluated the in closed-static chamber measurements. In previous studies, the vivo O2 influx of stem surface using air sampling in flasks from stem chamber was modeled by analytical and numerical box closed-static stem chambers, and measurements of δO2/Ar models (Angert and Sherer 2011, Angert et al. 2012a). The using a mass spectrometer (Barkan and Luz 2003). In compari- output of the numerical model with a set RQ value of 0.65 is son to standard flow-through stem chambers, in a static cham- presented in Figure 1. ber the O2 decrease during the experiment is markedly higher, which makes the O2 measurement simpler and more accurate. Precise mass spectrometry and gas chromatography methods are laborious and expensive, and using the δO2/Ar method for ARQ calculation requires a second set of samples for separate

CO2 analysis. Recent studies demonstrated the promising use of

Raman spectroscopy in O2 influx and RQ measurements of whole tree branches, studying shifts between respiratory substrates during stress (Fischer et al. 2015, Hanf et al. 2015). Nevertheless, the sensor used in these studies is not yet com- mercially available and the method setup is not adequate for wide field campaigns. Fuel-cell technology provides highly precise and accurate continuous atmospheric O2 measurements. Precision to the order of 1 ppm was achieved by fuel-cell differential analyzers, which measure the difference between atmospheric air and a gas reference continuously (Patecki and Figure 1. The changes in (a) modeled O2 (solid line) and CO2 (dashed Manning 2007, Stephens et al. 2007, Thompson et al. 2007, line) in a tree stem chamber and (b) the ratio between the two gases concentrations change, as predicted by a numerical box model run van der Laan-Luijkx et al. 2010). But fuel-cell analyzers are with an RQ of 0.65, and an arbitrary respiration rate and conductance highly sensitive to air pressure and temperature and therefore values.

Tree Physiology Online at http://www.treephys.oxfordjournals.org 1424 Hilman and Angert

Plant material The initial CO2 concentration in the box is the mean atmospheric air value, and the concentration increases as a result of The measurement methods were applied to: (i) 36 Tetragastris accumulation of stem-emitted CO2. The increase in CO2 in the panamensis (Engl.) Kuntze trees grown in Gigante peninsula box is linear during the first stage, and with time the slope curves (9°06′31″ N, 79°50′37″ W), part of the Barro Colorado Nature and stabilizes (Figure 1a) as CO2 diffuses out from the chamber Monument in the Republic of Panama. Diameter at breast height to the atmosphere. Similar CO2 stabilization in a stem chamber (DBH) of the 36 trees ranged between 12.6 and 58.1 cm, with was observed by Levy et al. (1999) after 24 h. The steady CO2 an average DBH of 30.1 cm. (ii) An oak tree (Quercus calliprinos concentration is a result of a balance between the stem’s CO2 Webb.) with a DBH of 16.2 cm, and an apple tree (Malus domes- efflux into the box and CO2 diffusion out of the box to the atmo- tica Borkh.) with a DBH of 20.1 cm that are located at the Hebrew sphere. The O2 fluxes in the box are opposite in direction to the University campus in Givat Ram, Jerusalem, Israel (31°46′15″ N,

CO2 fluxes. That is, O2 is consumed in the stem and its concentra- 35°11′50″ E). Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021 tion in the box decreases. Oxygen diffusion from the atmosphere into the box increases with time, until steady state is reached. The The Hampadah method diffusion of O2 to stem chamber was shown to be dominantly in Stem chambers and air sampling The stem chambers were the gas phase (Angert et al. 2012b). This assertion is based on constructed out of 10 cm × 12 cm Perspex® plate, cut at the an experiment in which the stem chamber was initially flushed workshop of the Hebrew University Faculty of Science, and a 18 with N2, followed by tracing of the ensuing changes in δ O–O2 double layer of 2-cm thick closed-cell foam frames glued in the chamber air as a result of diffusion and respiration. together (Vidoflex™ manufactured by Anavid, Kibbutz Kiryat

We defineΔ CO2 and ΔO2 as the concentration change of CO2 Anavim, Israel). Four 13.5 mm PVC cable glands with rubber ® and O2 in the box since the beginning of the experiment. The gasket were attached to the Perspex plate and used to connect initial air in the box is defined to have mean atmospheric values, sampling flasks to the chamber. Two holes capped with rubber therefore ΔCO2 and ΔO2 also represent the concentration gradi- septa were used for testing the seal of the chamber, by inserting ent between the box and the atmosphere. The ratio ΔCO2/ΔO2 a syringe needle and pulling the syringe piston to check for equals ARQ in the linear phase of the experiment, i.e., shortly resistance (Figure 2). after sealing the chamber (defined as ‘short incubation’). In Chamber installation in Panama took place 1 day before the steady state, the CO2 and O2 concentrations in the chamber are experiment began. Before attaching a chamber to a tree stem, in equilibrium with the upper layer of the stem directly below the chamber, and the stem CO2 and O2 fluxes are balanced by diffusion fluxes to and from the atmosphere. Apparent respiratory quotient, which is the ratio of the stem fluxes, is given by

g ()∆CO ARQ = CO2 2 (1) g ()∆O O2 2

where gCO2 and gO2 are the CO2 and O2 conductance in the wood and bark between the box and the atmosphere, respectively. The structure of the path along which diffusion occurs is the same for

CO2 and O2 and hence the conductance ratio ggCO22/ O depends solely on the ratio of diffusivities of the gases in air, which is 0.76 (Massman 1998). As a result, in steady state

 ∆CO2  ARQ = 07. 6  (2)  ∆O2 

Diffusivity of gases in air is greater than diffusivity in water by Figure 2. Schematic drawings of a stem chamber installation and a glass flask equipped with Louwers™ O-ring valve (inset). The flask in the inset four orders of magnitude (Marrero and Mason 1972, Jähne et al. is an example of one of the four flasks connected to the Perspex® plate. 1987). Therefore, although diffusion certainly occurs through After sealing, gas fluxes between the chamber, the stem and the atmo- substrates other than air (water, cell membranes, cuticles), the sphere take place. Oxygen diffuses from the chamber to the stem and is presence of a gaseous pathway between the chamber and the consumed there by respiration. Carbon dioxide, produced by respiration within the stem, is emitted to the chamber. In steady state, the above atmosphere justifies the choice of diffusion coefficients in air fluxes are balanced by diffusion of O2 from the atmosphere to the cham- over the coefficients in water. Because the ratio of the diffusivi- ber, and diffusion of CO2 from the chamber to the atmosphere. In a typi- ties of gases in water is 0.79 at 25 °C (Jähne et al. 1987, cal experiment, two flasks are closed after a short incubation (∼30 min) and two flasks are closed after steady state is reached (∼48 h). Incuba- Haynes 2016), diffusion in water would not appreciably change tion times are estimated using a numerical box model and results of estimated ARQ. previous experiments.

Tree Physiology Volume 36, 2016 CO2 efflux/O2 influx measurement in tree stem 1425 lichen and loose bark were removed from it and the surface was filed smooth. The bark of theT. panamensis trees is smooth and thin and no major damage was done to the stem during chamber attachment. After the stem smoothing, the foam frame and the Perspex® plate were fastened by two adjustable nylon straps to the stem, and silicone-based vacuum grease (Silicaid®1010, Aidchim Ltd, Ra’anana, Israel) was applied between the stem, foam frame and the plate. Separate laboratory incubations of the sealant and rubber compartments with gas standard proved that they are inert to CO2 and O2. The chamber headspace was cre- ® ated between the Perspex board, the foam frame and the stem. Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021 After the chamber installation, we blocked the flask connectors Figure 3. A schematic drawing of our closed circulating system, which and tested the seal. When necessary, we added sealant until the measures CO2 and O2 concentrations of air samples (the Hampadah). chamber was sealed. Before we attached the flasks, we ventilated The four-way switching valve has two modes: a purge mode (solid lines), the chambers to ensure initial mean atmospheric air values. After in which CO2 and H2O free atmospheric air flushes the system, and a circulation mode (dashed line), in which air circulates in the system. The samplings, we blocked the flask connectors and filled the cham- sample manifold includes eight connectors, which fit to eight sampling bers with water to estimate their volumes, which ranged between flasks, and servo motors that open and close the flasks. Operation and 75 and 200 cm3. measurement of the Hampadah are entirely automated. The air was sampled in glass flasks with a volume of either 3.7 or 13 cm3, which were connected to O-ring valves (LouwersHa- opening, and calculation of the concentration in the flask that nique, Hapert, The Netherlands) by the glass blowing workshop would yield such change in the system (similar to Levy et al.’s of the Hebrew University Faculty of Science. Sampling took place (1999) method). Detailed description of the Hampadah assem- between 28 September 2013 and 5 October 2013. We sampled bly, calibrations and measurement protocol are presented in air after 30 min of incubation from 11 trees (short incubation, Supplement B and C available as Supplementary Data at Tree some of the samples with duplicates) and after ∼48 h (steady Physiology Online. Briefly, for measuring CO2 we used an IRGA state) in two sets of duplicates in all 36 trees. The incubation (LI 840A, LI-COR, Lincoln, NE, USA), which measures water durations were determined according to our experience. During vapor concentrations as well. For measuring O2 we used a fuel- incubation, the air in the chamber and flasks is mixed by diffusion. cell based analyzer (FC-10 Oxygen analyzer, Sable Systems In contrast to active sampling (Angert and Sherer 2011, Angert International, Las Vegas, NV, USA). The fuel-cell technology is et al. 2012a) in which the chamber air is pushed out to the based on the principle of diffusion of O2 molecules across a flasks, the current sampling method is passive and the flasks are membrane to the cell’s cathode, and the oxidation of the cath- simply closed at the required sampling time. Using the equation ode by the O2 molecules. The reduction induces a current that is of mean-square displacement in diffusion, we calculated the linearly proportional to the partial pressure of the O2. For mole intra-chamber diffusion fluxes and concluded that the air inside fraction calculation, temperature as well as total pressure is mea- the chamber is sufficiently homogeneous during our incubation sured by the O2 analyzer. To reduce temperature and pressure time periods (at least 0.5 h) (see Supplement A available as variations that would change the total pressure, we wrapped the Supplementary Data at Tree Physiology Online). The stem cham- Hampadah’s tubes in insulating material and placed the Hampa- bers and a few centimeters around them were covered with an dah in a closed Perspex box in the laboratory. In addition, to opaque bag to prevent corticular photosynthesis and overheating stabilize the system’s temperature, the first measurement each of the stem. To reduce possible leaks around the flasks’ O-ring day starts only after the system warms up for at least 3 h. during the shipment from Panama to the laboratory in Israel, The analyzers were connected with 1/8″ stainless steel tubes flasks ‘necks’ (the side tube placed between the two O-rings in to a manifold with connectors for eight sampling flasks and to a the inset in Figure 2) were filled with distilled water and blocked small air-tight pump (Micro diaphragm gas pump NMP015L, with a rubber stopper. Samples were analyzed with the Hampa- KNF, Neuberger, Trenton, NJ, USA) set at a flow rate of dah within 2 weeks after field sampling and within 6 weeks after ∼340 cm3 min−1 that runs air in the system. A four-way ball field sampling with the mass spectrometer. valve (Part no. SS-43YFS2, Swagelok, Solon, OH, USA, equipped with an actuator), which is included in the system,

The Hampadah system The Hampadah is a closed-flow sys- enables switching between purge mode, in which CO2 and H2O tem based on two continuous flow analyzers (Figure 3, see free atmospheric air (scrubbed by soda-lime and magnesium Figure S1 available as Supplementary Data at Tree Physiology perchlorate) flushes the system, and circulation mode, in which Online). The principle of operation is measurement of the the sample’s air is mixed in the system. The volume of the 3 change in CO2 and O2 concentrations in the system after flask Hampadah is 88 cm , and while samples are measured, their

Tree Physiology Online at http://www.treephys.oxfordjournals.org 1426 Hilman and Angert

content dilutes within this volume. Consequentially, measure- Box model validation Apparent respiratory quotient evaluation ment of samples with up to 44% CO2 is possible, which is is based on a box model. Therefore, it is essential to validate the much higher than the 2% CO2 limitation of the LI 840A. The box model prediction and its assumption of diffusion in the gas Hampadah runs are controlled by a Matlab (MATLAB R2013a, phase between the chamber and the atmosphere. According to

MathWorks, Inc., Natick, MA, USA) script and are fully automated. the numeric box model, ARQ equals ΔCO2/ΔO2 in the beginning A Hampadah run of eight flasks of 3.7 cm3 takes 4.5 h, and a run of incubation when diffusion fluxes are smallAngert ( et al. 3 of eight flasks of 13 cm takes 6 h. 2012a), and is 0.76(ΔCO2/ΔO2) in steady state (Eq. 2). We

We calibrated the O2 analyzer to 20.95% by streaming CO2 defined the short to steady-state ratio (SSSR): and H O free atmospheric air through the system. The IRGA was 2 ()∆∆CO /O SSSR = 22short (4) calibrated by streaming three gas standards through the system: ()∆∆CO /O 22steady state pure N2 (0% CO2), and span gases of 0.049 and 0.87% CO2. Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021

Due to the samples’ dilution in the Hampadah’s volume, almost The subscripts are the timing when the ratio ΔCO2/ΔO2 is sam- all samples measured in this study fell within the standards pled. Based on the model, we expect SSSR to be 0.76 if diffu- range. We applied corrections to the mass of air in the flasks due sion occurs in the gas phase. We validated the model by to different barometric pressure (BP, hPa) and temperature (T, calculating the SSSR using short incubation and steady-state Kelvin) between the study site and the laboratory, and for water results from Panama, as well as from the experiments in Israel. vapor dilution (vapor saturation in the chambers was assumed). Since ΔCO2 and ΔO2 in steady state are greater than in short The flask’s air volume was corrected based on the ideal gas law: incubation, their ratio is less sensitive to small experimental

noise. Hence, (ΔCO2/ΔO2)steady state was regarded as the more BP T  studysite   lab  accurate value. VVcorrected =      BPlab   Tstudysite  In closed-static chambers, the CO2 build-up and O2 depletion  saaturated vapor pressure increase with time until steady state. The change in the gas com- 1−×  (3)  BPstudy site  position within the chamber most likely alters the stem CO2 efflux and O2 influx since their magnitude depends on the con- where V is the flask’s volume (cm3). The saturated vapor pres- centration gradient between the stem and the air in the chamber. sure (hPa) was calculated using the Clausius–Clapeyron equa- However, SSSR values close to 0.76 would imply that the ratio tion. Barometric pressure and temperature values were obtained between the stem CO2 and O2 fluxes is conservative. from meteorological stations nearby.

For testing the Hampadah CO2 accuracy, we ran flasks with Direct measurement methods Measurements in the direct known concentrations of CO2 in the system. In addition to the methods are in situ, unlike the Hampadah method, and were commercial standards of 0.87 and 4.70% CO2, we used mixes applied on the trees in Israel. The chamber on the apple tree was ® of atmospheric air with different portions of pure CO2. The constructed from a 12 cm × 19 cm Perspex plate with six flask equation [CO2] = 100 − [O2]/0.2095 describes the gas concen- connectors attached to the same base described in Angert and trations in these mixes of air, and by placing the O2 measure- Sherer (2011). The installation and design of the chamber on ment of the Hampadah in the equation we calculated the the oak was similar to that of the chambers installed in Panama, expected CO2 concentration. The Hampadah ΔO2 accuracy was except for the sealant. The oak’s bark had some small cracks, estimated by comparing the measurement of one set of the which required mild filling with hot glue applied with a butane- steady-state samples to ΔO2 as determined by δO2/Ar of the powered hot-glue gun (Portasol G200, Portasol, Carlow, Ire- second set of the steady-state samples. We estimated the Ham- land), which was used also as a sealant. The volume of the 3 padah precision in CO2, O2 and ARQ measurements by the abso- chambers on the apple and the oak trees was 240 and 160 cm , lute difference of duplicates from their average. respectively, as determined by filling the chambers with water.

The CO2 concentration was measured using a small IRGA

δO2/Ar measurement High accuracy O2/Ar (expressed as sensor (COZIR Wide Range 0–20% CO2 Sensor, CO2Meter,

δO2/Ar) measurement is determined by mass spectrometric Inc., Ormond Beach, Florida), which we sealed with epoxy resin analysis. Since Ar is an inert gas, its concentration in the stem to make it water resistant for outdoor operation. In addition, we chamber throughout an experiment is assumed to be constant; fixed the sensor to a 3-cm long stainless steel tube that fits the hence, δO2/Ar depends solely on the O2 concentration within flask connectors on the chamber. To measure 2O , a quenching- the chamber. The sample preparation and mass spectrometry based optode (Fibox 3, PreSens-Precision Sensing, Regensburg, were according to Barkan and Luz (2003). The δO2/Ar was Germany) was used. This sensor measures the chamber’s air calibrated to 20.95% O2 using outside air. In samples with <8% and is similar to the sensor used by del Hierro et al. (2002),

O2 according to the Hampadah, duplicates were pooled together who measured in-stem O2. The optode system includes a trans- to allow enough O2 for the mass spectrometer analysis. mitter, an optical fiber and a fluorescence sensor spot (Figure 4).

Tree Physiology Volume 36, 2016 CO2 efflux/O2 influx measurement in tree stem 1427

sensors attached to the stem chamber installed on the apple tree.

The O2 sensor spot was installed inside the chamber, glued to a transparent Perspex® disk in front of a connector located outside of the chamber, to which the optical fiber was attached. In addition to the sensors, we attached a small KNF pump, and a non- return valve (SMC AKH Non Return Valve, 12 mm tube, RS Components Ltd, Corby, UK) with low cracking pressure (0.005 MPa) (Figure 4) to the chamber (using the flask connectors). During pump operation, the lower pressure in the chamber caused the non-return valve to open, and when the pump was off,

the valve was closed and the chamber was sealed. The pump was Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021 controlled by a timer and was activated for 15 min every 4 h. Water vapor saturation, for simpler vapor dilution correction, was ensured by pouring a small amount of deionized water (∼2 ml)

into the chamber. We assumed CO2 dissolution and release from

the water would not affect the CO2 measurement, since in the

recorded CO2 concentrations in the chamber (up to 4%) the Figure 4. A schematic drawing of a setup of the direct continuous expected pH is ∼5. At such a pH, the total dissolved inorganic method on a stem chamber, in which gas concentrations are measured carbon (DIC) is 1.9 mM, using Henry’s law and the carbonate continuously. When the pump is activated, the one-way valve lets ambi- ent air flush the chamber. After the pump is deactivated, the chamber system constants at 15 °C (the lowest temperature across the encloses. Alternatively, in the direct discrete method, the CO2 sensor and experiment) (McGuire and Teskey 2002). In the 2 ml water the O2 fluoresence sensor spot are left (not connected to a data logger) poured into the chamber, the number of moles in practice is on the chamber with no pump operation. The sensor’s reading is logged at the end of the experiment. 3.8 μmol, which is 4% of the moles in the chamber, while the CO2 concentration is 1% (the chamber volume is 240 cm3). In actuality,

A temperature sensor next to the O2 sensor spot was used for although the boundary layer of the water reacts quickly with the air temperature compensation. above, mixing with deeper layers depends on diffusion in water,

Before measurement, we calibrated the CO2 sensor with pure which is very slow, and thus only a small portion of the total DIC

N2 for 0% CO2 and with gas standards of 0.87 and 4.70% CO2. reacts rapidly with the air above it. Each aeration of the chamber

The O2 sensor was calibrated with pure N2 for 0% O2 and with was used as a calibration point using atmospheric O2 concentra- dry air for 20.95% O2. For water vapor dilution corrections, we tion. Since immediately after aeration the air may not be saturated calculated water vapor pressure in saturation using the Clau- by water, we used relative humidity data as measured in a nearby sius–Clapeyron equation and the temperature from the sensor. meteorological station at the time point of the experiment. The gas sensors were used in their standard utilization, unlike During the experiment, gas concentrations were measured at the Hampadah system where we manipulated open-flow analyz- 30 s intervals. The ratio ΔCO2/ΔO2 of each data point yielded a ers to measure air circulation in a closed system. Nevertheless, somewhat noisy signal; a more robust ΔCO2/ΔO2 signal was we validated the performance of the sensors by measuring RQ achieved by calculating the ratio of the CO2 efflux to the O2 of germinating wheat grains. In wheat grains, carbohydrates are influx. These fluxes were found from the slopes of ΔCO2 and a major storage compound (Lambers et al. 2008), hence the ΔO2 in the first hour after sealing the chamber. The slopes were expected RQ is close to unity. The grains were soaked in water calculated by a linear fit over∼ 120 data points of each gas. The for 15 min and placed into a 250 cm3 beaker for 55 h, while the ARQ was calculated from the ratio of these two slopes. two sensors were connected with a three-way connector (Part No. SS-8-UT-3, Swagelok) to the beaker. The sensor’s reading The direct discrete method In this method, gas concentrations was extracted every 30 s. The experiment took place in the in the stem chamber are measured using portable sensors at the laboratory at a temperature of 22 °C. same time points as the flasks’ sampling times in theHampadah

method. Thus, the CO2 sensor and the O2 sensor spot are left The direct continuous method To obtain an ARQ value every attached to the stem chamber during an experiment, but they few hours, a sequence of short incubations was conducted on the are disconnected from the data logger until sampling time. apple tree between the 13 and 15 of May 2013. During short On 24 April 2014, we connected the CO2 sensor and the O2 incubations, the build-up of CO2 (and decrease of O2) inside the sensor spot to the chamber on the oak. Additionally, two flasks chamber is small, and as a result, diffusion fluxes in and out of the were connected to the same chamber and were analyzed later chamber are small and ARQ can be approximated by ΔCO2/ΔO2. using the Hampadah, for comparison purposes. Sensors and

The concentrations of gases were measured by the CO2 and O2 flasks were attached to the chamber for 46 h. At the end of the

Tree Physiology Online at http://www.treephys.oxfordjournals.org 1428 Hilman and Angert

experiment, the flasks were closed and the readings of the CO2 The calculated root mean-square deviation (RMSD) of the cor- and O2 sensors were taken. On 6 May 2013, we attached four rected CO2 with respect to actual CO2 was 0.09%. The possible flasks in addition to the two sensors to the apple’s chamber. effect of humidity on the system’s adsorption was examined by Data were logged continuously from the sensors, while two running gas standards in flasks with different added water flasks were closed after 2 h (short incubation), and the other amounts. The resulting coefficient of determination of 0.02 two after 48 h (steady state). Apparent respiratory quotient was rejected possible influences. Based on 39 pairs of duplicates, calculated using five practices: the flasks were measured in the the precision of the Hampadah CO2 measurement is 0.06%.

Hampadah (short incubation and steady state), the sensor’s According to the Hampadah, O2 concentrations in the stem reading from the short incubation and steady state (the direct chambers in Panama ranged from 20.56 to 20.79% in the short discrete method), and by the slope ratio of the first hour of incubations (n = 11) and from 4.97 to 19.23% in steady state incubation (the continuous method). A similar experiment was (n = 36). The measured CO2 concentrations ranged from 0.08 Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021 conducted on the apple between 12 and 13 May, just before the to 0.29% in the short incubations (n = 11) and from 1.24 to continuous measurement experiment. Data from the sensors 7.93% in steady state (n = 36). The mean ARQ values (±stan- were logged after 2 and after 23 h of experiment, while two dard deviation (SD)) of samples from Panama were 0.42 ± 0.16 flasks were closed at each time point. Those four ARQs were for short incubations (n = 11), 0.39 ± 0.06 for steady state of compared with the first ARQ measured at the onward direct con- the same chambers and 0.40 ± 0.07 for the steady-state values tinuous measurement experiment. of all the chambers (including ones on which no short incubation was preformed) (n = 36). Apparent respiratory quotient Results precision is 0.01 (n = 39). The mean ± SD SSSR of Panama’s short incubation values and the steady-state values of the same The Hampadah accuracy and precision chambers was 0.83 ± 0.30 (n = 11).

The results of ΔO2 measured by the Hampadah showed good agreement with the results measured by the mass spectrometer Direct measurement methods 2 in samples collected from the trees in Panama (R = 0.998, Carbon dioxide and O2 changes during the wheat germination, as n = 30) (Figure 5). The precision of the Hampadah O2 measure- recorded by the portable sensors, were similar across the exper- ment is 0.08% (n = 39), whereas the mass spectrometer preci- iment (Figure 7a), resulting in a mean RQ of 0.96 in the entire sion is 0.07% (n = 18). experiment (Figure 7b). Carbon dioxide and O2 measurements of

When running flasks with CO2 standards, we observed slightly stem chambers using the portable sensors correlated well with nonlinear CO2 measurement in the Hampadah (Figure 6), which the Hampadah measurement (Figure 8). Close similarity was we suspect is due to the adsorption of CO2 on the system’s sur- found also in comparisons of the different ARQ measurement faces. The relationship between the measured and the actual CO2 methods (Figures 8c and 9). The SSSR values in the Hampadah is best explained with the power equation (R2 = 0.999, n = 53): and the direct discrete methods were 0.79 and 0.82 in the first comparison experiment on the apple, respectively, and 0.62 and CO 0.998 CO10. 8 (5) 2corrected =×2 0.69 in the second comparison experiment, respectively.

Figure 5. Plot of O2 decrease in 30 stem chambers attached to 30 Figure 6. Scatter plot indicating the relationship between CO2 concen- T. panamensis trees as measured by our closed circulating system (the trations in samples contained known gas standard (actual CO2, %) and Hampadah, ΔO2 Hampadah, %) and by δO2/Ar determined by mass our closed circulating system (the Hampadah) CO2 measurement spectrometer (ΔO2 MS, %). (measured CO2, %).

Tree Physiology Volume 36, 2016 CO2 efflux/O2 influx measurement in tree stem 1429

Measurement using the direct continuous method is shown in state results). The RMSD of the Hampadah CO2 measurement with Figure 10. Average ARQ ± SD of this run, based on slope ratios, respect to gas standards was 0.09% after empirical correction. is 0.68 ± 0.04 (n = 10). Changing the ΔCO2 component by a factor of 0.09% in ARQ calculation caused a minor deviation of 0.007 among Panama’s ARQ results. The precision of duplicates measured in the Hampadah for Discussion O2, CO2 and ARQ was 0.08, 0.06 and 0.01, respectively. This The accuracy of tree stem ARQ estimation depends on accurate seems to indicate that most causes of errors in the gas concentra-

O2 and CO2 measurements, and on the validity of our box model. tion measurements do not affect the ratio between the gases, and The linear relationship, with a slope close to 1, of the Hampadah thus resulting ARQ precision is excellent.

O2 against the mass spectrometer O2 (Figure 5) demonstrates As expected, ΔCO2 and ΔO2 in the wheat grains germination the Hampadah’s accuracy in O2 measurements. The slope of 0.96 experiment were very similar across the experiment, resulting in Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021 (Figure 5) is within the range of the instruments’ built-in uncer- RQ close to unity (Figure 7). The wide range of concentrations tainties. Part of the deviation may be attributed to deviation in the (up to 12% CO2) in which ΔCO2 and ΔO2 were similar, and the mass-spectrometer measurements, due to very large δO2/Ar val- comparison of the portable sensor measurement to the Hampa- ues in our samples (>100‰). Nevertheless, the effect of this 4% dah measurement (Figure 8) supports the implementation of variation from a slope of 1 on ARQ is small, and replacing the Ham- the portable sensors also beyond the range of gas standards padah O2 component in ARQ calculation with the mass spectrometer used for calibrations. Further evaluation of the direct sensors

O2 reduces ARQ values by 0.01 on average (for Panama steady- estimate of ARQ is required. Most SSSR values were slightly higher than the expected 0.76 value. It was 0.79 in the Hampadah method and 0.82 in the direct discrete method in the first experiment on the apple tree, and 0.83 ± 0.30 in the Panama results. As shown by the numerical model run (Figure 1b), the SSSR will be higher than 0.76 when the first sampling is delayed and taken when diffusion fluxes from the chamber have already become substantial. Thus, the calculated short incubation ARQ will be higher than the true value. Another concern with the short incubation is demonstrated in the large SD in Panama’s SSSR. Shortly after sealing the cham-

ber, the ΔCO2 and ΔO2 values are still small, and the ARQ is very sensitive to small variations and measurement errors. The continuous method seems to reduce this sensitivity; its ARQ value was very close to steady-state values, closer than that of the short incubations of the Hampadah and the direct discrete methods. This accuracy is probably due to the averaging done during Figure 7. A continuous measurement of wheat grains germination. (a) slope ratio calculation, which sums around 120 data points. In Percentage differences from initial values of O2 (ΔO2, gray solid line, upper curve), and CO2 (ΔCO2, black dashed line). (b) The ratio of ΔCO2 addition to the accuracy of the continuous method, this method to ΔO2 (the respiratory quotient, RQ). showed robustness, with an SD of 0.04 from the average value

Figure 8. A comparison between the measurements of (a) the portable CO2 sensor, (b) the portable O2 sensor and (c) ARQ (ratio CO2 efflux:O2 influx) with measurements of our closed-circulating system (the Hampadah). The gas concentrations were measured in stem chambers on oak and apple trees growing in Jerusalem, Israel using the portable sensors, while the chamber’s air was sampled in flasks. The gas concentrations in the flasks were measured in the laboratory using the Hampadah system. Samplings were conducted during May 2013 and April 2014.

Tree Physiology Online at http://www.treephys.oxfordjournals.org 1430 Hilman and Angert

the numerical model (Figure 1), and confirm the time scale of the steady-state sampling. Based on this similarity of the curves and the proximity of SSSR values to the expected, we conclude that our ARQ measurements agree with the model predictions.

In this work, we demonstrated three methods for coupled CO2

and O2 measurement. The Hampadah method is adequate for wide field campaigns where many chambers are sampled, for studies in remote sites where equipment to be carried is limited and for studies in humid environments where the use of electron- ics is not favorable. Setup and calibration are technically challeng-

ing, although it is more cost effective (∼$16,000 for all the Downloaded from https://academic.oup.com/treephys/article/36/11/1422/2548361 by guest on 30 September 2021 compartments, see cost estimation in Supplement D available as Supplementary Data at Tree Physiology Online) and more acces- Figure 9. A comparison of ARQ (ratio CO2 efflux:O2 influx) assessment sible than mass spectrometry methods. The discrete direct by our different methods, which took place on apple tree in Jerusalem, Israel 2013. method is analytically simpler than the Hampadah method and the results are immediate. It can be applied on several trees

simultaneously, also in remote sites, by leaving CO2 sensors and

O2 spot sensors on a desirable number of stem chambers and returning a few days later to read steady-state values using portable devices. However, it requires in-field calibrations of the sensors and operating electronic equipment in situ, which can be difficult in humid environments. The direct continuous method enables tracking of changes in ARQ over short time periods, and is highly attractive for manipulation experiments. In both the

Hampadah and the discrete methods, respiration rate (O2 uptake)

can be determined from O2 short incubation sampling and from short incubation and steady-state sampling together (Angert et al. 2012a). In the continuous method, respiration rate can be

estimated from the slope of ΔO2. In addition to stem respiration studies, the presented methods can be adapted for ARQ determi- Figure 10. A continuous measurement of gases in a stem chamber nation of detached tissues in incubation experiments. attached to an apple tree during 13–15 May 2013 in Jerusalem, Israel. The chamber was aerated every 4 h, except for two incubations of 8 h each. (a) The percentage differences from initial values of O2 (ΔO2) are Supplementary data represented by the solid line and CO2 (ΔCO2) are represented by the dashed line. The sharp decreases of ΔCO2 and ΔO2 to zero are due to Supplementary data for this article are available at Tree Physiology aeration of the chamber for 15 min. (b) The ratio of the slopes of ΔCO 2 Online. and ΔO2 in the first hour after chamber aeration, which is equal to ARQ (ratio CO2 efflux:O2 influx) (circles). TheΔ CO2/ΔO2 calculated for every data point is represented by the gray line. Acknowledgments for 10 data points over 48 h (part of the deviation may be attrib- We thank S. Joseph Wright and Rufino Gonzales for assistance uted to real variations in ARQ). If the SD in the continuous with field work at Barro Colorado Nature Monument, Eugeni Bar- method indeed reflects actual diurnal variations, it also may cause kan for help with samples analysis, Itsik Simchayov for technical SSSR values to differ from 0.76, and explain the 0.62 and 0.69 support and Aliza Hilman for editing. values calculated in the second comparison on the apple. The resulting SSSR value’s closeness to the expected suggests that Conflict of interest the ratio between the stem CO2 efflux and 2O influx is similar in the beginning of an experiment and at steady state. That is in None declared. contrast to the probable change in the magnitude of fluxes as the experiment progresses, which results from CO build-up and O 2 2 Funding removal in the chamber.

The curves of ΔCO2, ΔO2 and ΔCO2/ΔO2 measured by the This research was funded by the German-Israeli Foundation for Sci- continuous method (Figure 10) conform with those predicted by entific Research and Development (no. 1139/2011), and by a Ring

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