Radiometric Method for Determining Canopy Stomatal Conductance in Controlled Environments
Radiometric Method for Determining Canopy Stomatal Conductance in Controlled Environments
Monje, Oscar;Bugbee, Bruce
2019-02-27 00:00:00
agronomy Article Radiometric Method for Determining Canopy Stomatal Conductance in Controlled Environments 1 , 2 Oscar Monje * and Bruce Bugbee AECOM, Air Revitalization Lab, Mail code: LASSO-008, Kennedy Space Center, Merritt Island, FL 32899, USA Plants, Soils and Biometeorology Depatment, Utah State University, Logan, UT 84322, USA; bruce.bugbee@usu.edu * Correspondence: oscar.a.monje@nasa.gov; Tel.: +1-321-861-2935 Received: 1 January 2019; Accepted: 20 February 2019; Published: 27 February 2019 Abstract: Canopy stomatal conductance is a key physiological factor controlling transpiration from plant canopies, but it is extremely difficult to determine in field environments. The objective of this study was to develop a radiometric method for calculating canopy stomatal conductance for two plant species—wheat and soybean from direct measurements of bulk surface conductance to water vapor and the canopy aerodynamic conductance in controlled-environment chambers. The chamber provides constant net radiation, temperature, humidity, and ventilation rate to the plant canopy. In this method, stepwise changes in chamber CO alter canopy temperature, latent heat, and sensible heat fluxes simultaneously. Sensible heat and the radiometric canopy-to-air temperature difference are computed from direct measurements of net radiation, canopy transpiration, photosynthesis, radiometric temperature, and air temperature. The canopy aerodynamic conductance to the transfer of water vapor is then determined from a plot of sensible heat versus radiometric canopy-to-air temperature difference. Finally, canopy stomatal conductance is calculated from canopy surface and 2 1 aerodynamic conductances. The canopy aerodynamic conductance was 5.5 mol m s in wheat and 2 1 1 2.5 mol m s in soybean canopies. At 400 umol mol of CO and 86 kPa atmospheric pressure, 2 1 2 1 canopy stomatal conductances were 2.1 mol m s for wheat and 1.1 mol m s for soybean, comparable to canopy stomatal conductances reported in field studies. This method measures canopy aerodynamic conductance in controlled-environment chambers where the log-wind profile approximation does not apply and provides an improved technique for measuring canopy-level responses of canopy stomatal conductance and the decoupling coefficient. The method was used to determine the response of canopy stomatal conductance to increased CO concentration and to determine the sensitivity of canopy transpiration to changes in canopy stomatal conductance. These responses are useful for improving the prediction of ecosystem-level water fluxes in response to climatic variables. Keywords: canopy stomatal conductance; aerodynamic conductance; elevated CO ; climate change 1. Introduction Understanding boundary layer and land surface feedbacks on canopy transpiration is essential for developing simpler and realistic climate change models and for improving the prediction of ecosystem-level water fluxes in response to climatic variables [1,2]. Canopy stomatal conductance (G ), a key physiological factor controlling transpiration from plant stands, is an important component of land surface feedbacks because it regulates evapotranspiration and surface temperature changes in response to incident radiation, CO concentration, and vapor pressure deficit (VPD). This regulatory function is reflected in canopy temperature, which in turn, determines the magnitude and direction Agronomy 2019, 9, 114; doi:10.3390/agronomy9030114 www.mdpi.com/journal/agronomy Agronomy 2019, 9, 114 2 of 23 of sensible heat exchange between the vegetation and its environment. At regional scales, stomata exert little control, and daily transpiration of well-watered vegetation is predominantly controlled by radiation and temperature [3–5], in part due to feedbacks that cannot be predicted from single leaf measurements alone [3]. Since canopy-scale transpiration is determined by the ratio between canopy aerodynamic conductance (g ) and G [4,6,7], improved methods for measuring g , as well A S A as measuring responses of G to environmental variables (e.g., light, CO , VPD, soil moisture, and S 2 temperature), are needed for studying the processes controlling feedback and stomatal control of evaporation from regional land surfaces. In the field, g is often approximated by the conductance to momentum transfer determined using the log-wind profile approximation, which requires at least 100 m of fetch and thus cannot be used in controlled-environment chambers [8]. In controlled environments, leaf boundary conductance has been estimated from measurements with wet filter paper analogs [9], from cooling curves of metal models of leaves [10], or from combined energy balance and temperature measurements using metal leaf models [11,12]. However, Jarvis and McNaughton [3] argue that leaf level measurements of stomatal control of transpiration may not be applicable to plant canopies in the field because the amount of ventilation in leaf cuvettes and plant chambers typically prevents feedback between transpiration and VPD observed in the field. G can be derived using energy balance approaches from canopy surface conductance to water vapor (G ), latent heat flux (LE), and the VPD at the leaf surface (Ds). Similarly, the single-layer SFC or “big leaf” G may be computed from G , provided the boundary layer conductance to water S SFC vapor (i.e., g ) and the mean aerodynamic canopy temperature (T ) are known [13,14]. In the field, A Aero G is calculated from canopy-level LE obtained using lysimeters, Bowen ratio, and eddy correlation SFC systems [15], or by inverting the Penman–Monteith equation [13]. However, these approaches for measuring canopy-level LE do not permit partitioning of transpiration among individual species and often cannot distinguish between transpiration and evaporation from the soil or from wet leaf surfaces. Thus, G is not always related to estimates of canopy G derived from single leaf measurements SFC S because it often includes significant contributions from soil evaporation [13]. Smith et al. [16] used canopy-level energy balance measurements to estimate sensible heat flux (H) of a wheat field from radiometric canopy temperature when canopy g and LE were known. Their approach produced accurate estimates of hourly LE, which suggests that g could be estimated if H and the canopy-to-air temperature difference are measured accurately. However, canopy g determined from changes in radiometric canopy temperature differs from g determined using the log-wind profile approximation because it includes the conductance to heat and water vapor across leaf boundary layers, as well as the turbulent conductance caused by the movement of air eddies between the canopy and the atmosphere [17,18]. Canopy G obtained from energy balance approaches may contain considerable errors because Ds and LE are estimated using measurements of canopy radiometric temperature (T ) to approximate the aerodynamic canopy temperature [19–21]. In the field, estimating T from infrared measurements Aero is complicated because radiometric measurements depend on the view angle of the sensor, sun angle, degree of crop cover, spatial variability of canopy emissivity, and atmospheric attenuation, and they often include significant temperature contributions from soil surfaces [20,22–25]. A systematic difference of 1 C was measured between radiometric and aerodynamic temperatures by Huband and Monteith [26], although differences ranging from 2 to 6 C have also been observed [13]. The difference between T and T can be very small in dense canopies, but it can exceed 10 C in sparse vegetation Aero R because of contributions from soil temperature ([18,27]. These differences are significant because small errors of 1 C in the surface-to-air temperature difference can represent an uncertainty in latent heat fluxes of ~40 W m [13]. Many complicating factors that affect infrared measurements of canopy temperature in field settings can be minimized in controlled environments by using high planting density canopies grown under constant lighting. In dense canopies, canopy brightness temperature measured with infrared sensors approximates canopy radiometric temperature [28], but errors due Agronomy 2019, 9, 114 3 of 23 to radiation reflected into the sensor and artifacts caused by fluctuating sensor body temperatures remain [29]. Agronomy 2018, 8, x FOR PEER REVIEW 3 of 23 The purpose of this study was to develop a radiometric method for measuring canopy G of well-watered plant canopies in controlled environments. The hypothesis tested was that a radiometric 97 radiometric method utilizing canopy-level energy balance measurements provides more accurate method utilizing canopy-level energy balance measurements provides more accurate estimates of 98 estimates of canopy stomatal conductance than bottom-up methods scaling leaf-level to canopy-level canopy stomatal conductance than bottom-up methods scaling leaf-level to canopy-level conductance 99 conductance or top-down methods that estimate canopy surface conductance from field data. or top-down methods that estimate canopy surface conductance from field data. Bottom-up methods 100 Bottom-up methods require that leaf area index be known and must integrate the responses of leaf require that leaf area index be known and must integrate the responses of leaf stomatal conductance to 101 stomatal conductance to vertical gradients in radiation, temperature, and humidity. Conductances vertical gradients in radiation, temperature, and humidity. Conductances from top-down methods 102 from top-down methods using field data typically include significant contributions of soil using field data typically include significant contributions of soil evaporation, and field radiometric 103 evaporation, and field radiometric data include soil surface temperatures that cause significant data include soil surface temperatures that cause significant differences between radiometric and 104 differences between radiometric and aerodynamic temperatures [13]. aerodynamic temperatures [13]. 105 Simultaneous measurements of energy balance, gas fluxes, and canopy temperature at constant Simultaneous measurements of energy balance, gas fluxes, and canopy temperature at constant 106 environmental condit environmental conditions ions were were used used to to compute compute can canopyoG py G from S from surface suG rfaceand GSFC canopy and ca g nop (Figur y gA e 1 (Fi ). gure S SFC A The relation between radiometric and aerodynamic temperatures was studied by varying incident 107 1). The relation between radiometric and aerodynamic temperatures was studied by varying incident radiation and wind speed. Canopy G and g of high planting density wheat (Triticum aestivum L. cv. S A 108 radiation and wind speed. Canopy GS and gA of high planting density wheat (Triticum aestivum L. cv. USU Apogee) and soybean (Glycine max L. cv. Hoyt) canopies were measured at 400 umol mol CO . 2−1 109 USU Apogee) and soybean (Glycine max L. cv. Hoyt) canopies were measured at 400 umol mol CO2. The radiometric method was used to explore the effects of rising CO concentration on canopy G and 2 S 110 The radiometric method was used to explore the effects of rising CO2 concentration on canopy GS to describe stomatal feedbacks to transpiration using the canopy-scale decoupling coefficient. 111 and to describe stomatal feedbacks to transpiration using the canopy-scale decoupling coefficient. Figure 1. A two chamber, open gas exchange system capable of determining canopy aerodynamic 113 Figure 1. A two chamber, open gas exchange system capable of determining canopy aerodynamic conductance from measures of sensible heat flux and canopy-to-air temperature difference was used to 114 conductance from measures of sensible heat flux and canopy-to-air temperature difference was used calculate canopy stomatal conductances of wheat and soybean canopies. 115 to calculate canopy stomatal conductances of wheat and soybean canopies. 2. Materials and Methods 116 2. Materials and Methods 2.1. Chamber System In this study, 18–35-day-old, closed wheat and soybean canopies were used to examine various 117 2.1. Chamber System aspects of the method—energy balance responses to changes in radiation forcing, responses of 118 In this study, 18–35-day-old, closed wheat and soybean canopies were used to examine various vertical gradients in canopy-to-air temperature to fan speed or light level, responses in canopy-to-air 119 aspects of the method—energy balance responses to changes in radiation forcing, responses of temperature and sensible heat flux to CO concentration, etc. Each test took several days to conduct, 120 vertical gradients in canopy-to-air temperature to fan speed or light level, responses in canopy-to-air and plant canopies of different ages were used because the logistics of growing canopies to the same age for each test was impractical. Thus, conductances observed in a vegetative 20-day-old wheat 121 temperature and sensible heat flux to CO2 concentration, etc. Each test took several days to conduct, 122 and plant canopies of different ages were used because the logistics of growing canopies to the same 123 age for each test was impractical. Thus, conductances observed in a vegetative 20-day-old wheat 124 canopy may not be the same as in a reproductive 35-day-old canopy due to ontogenetic changes in 125 canopy structure (i.e. the presence of heads). However, overall the method is robust as long as energy 126 balance components and canopy-to-air temperature differences are measured accurately and 127 simultaneously. 128 2.2. Cultural and Environmental Conditions Agronomy 2019, 9, 114 4 of 23 canopy may not be the same as in a reproductive 35-day-old canopy due to ontogenetic changes in canopy structure (i.e., the presence of heads). However, overall the method is robust as long as energy balance components and canopy-to-air temperature differences are measured accurately and simultaneously. 2.2. Cultural and Environmental Conditions Wheat and soybean canopies were grown in sealed, water-cooled, controlled-environment chambers (Model EGC-13, Environmental Growth Chambers, Chagrin Falls, OH, USA). Two canopies of the same species were grown simultaneously in adjacent chambers. Wheat was seeded into lids containing a 10 mm layer of inert media (Isolite, size CG-2, Sumitomo Corp., Denver, CO, USA) at a density of 1100 plants m . Soybean seedlings were transplanted into closed cell foam plugs in a Styrofoam lid at a planting density of 60 plants m . The seedling roots grew into a recirculating hydroponic solution after germination. The hydroponic system is described in Monje and Bugbee [30]. Inside each chamber, a polished aluminum, reflective side-wall was built around the perimeter of the ~1 m canopy to minimize edge effects and side lighting. The incident photosynthetic photon flux 2 1 2 1 (PPF ) was 1600 mol m s for wheat and 750 mol m s for soybean. Lighting was provided by four, 1000 W high-pressure sodium (HPS) lamps, which were adjusted with neutral density filters to achieve 5% PPF uniformity over the crop surface. PPF was measured at the top of the canopy with o o a quantum sensor (Model LI-190SB, LI-COR, Lincoln, NE, USA), and was adjusted daily throughout the life cycle by lowering the canopy platform as the plants grew taller. Longwave radiation emitted by the lamps was removed by a 10 cm deep water filter. The filter consisted of a glass box filled with recirculating, chilled water located below the lamps. The water filter under the lamps was removed over the course of several days during tests that change surface radiation forcing by increasing incident PPF and longwave radiation impinging on the canopy. Advective conditions existed in the chamber because the temperature control system heated the air to maintain the chamber temperature setpoint, and the canopy was exposed to a continuous flow of warm air. Air temperature was 21.0 0.3 C, the barometric pressure was 86 0.1 kPa, and chamber CO varied between 400 and 1400 mol mol to manipulate canopy temperature, LE and H. Relative humidity at night was 50% 5%. During the day, transpiration humidified the 1300 L of chamber air and daytime relative humidity was 70 5%. Wheat was grown under a 20 h light/4 h dark photoperiod and soybean under a 12 h light/12 h dark photoperiod. The canopies grew in a chamber supplied with a constant temperature (T ) and VPD of bulk air surrounding the vegetation (D ) Air Bulk as well as a constant wind speed. Thus, boundary layer forcing (T and D ) and surface layer Air Bulk feedbacks (chamber wind speed) were held constant, but in nature they are dictated by diurnal changes in local climate (T , D , and wind speed). Air Bulk 2.3. Gas Exchange System Each chamber used an open gas exchange system to measure canopy photosynthesis [30,31]. The open flow system ensured that humid air (~50% relative humidity) of a constant CO concentration 1 1 (setpoint10 mol mol ) fed the chambers at flow rates between 500 and 1100 L min . Air mass flow (MF; mol s ) into the chambers was measured with mass flow meters (Model 730, Sierra Instruments, Monterey, CA, USA). The gas exchange systems were modified to use a dew point hygrometer to measure the water vapor concentration of pre- and post-chamber air from which evapotranspiration was calculated. Two solid-state multiplexers (Model AM-25T, Campbell Scientific, Logan, UT, USA), each referenced to a 100 Ohm platinum resistance thermometer, were used for precision thermocouple measurements. Data acquisition and control were performed with a datalogger (Model CR-10T, Campbell Scientific, Logan, UT, USA). Gas exchange fluxes in each chamber were measured continuously and averaged for 2 min every 8 min. Net photosynthesis, P , and dark respiration rates were calculated from the difference between net pre- and post-chamber CO concentrations (DCO ), multiplied by MF of air into the chambers. DCO 2 2 2 Agronomy 2019, 9, 114 5 of 23 was measured with a differential infrared gas analyzer (Model LI-6251, LI-COR, Lincoln, NE, USA). The temperature, water vapor band broadening, and dilution corrections used for the C fluxes are described in Monje and Bugbee [30]. Chamber evapotranspiration (ET) was determined from the difference in mole fraction of water vapor between pre- and post-chamber air (DX ), multiplied h20 by mass flow rate entering the chamber (ET = DX MF). DX was determined from sequential h20 h20 measurements of pre- and post-chamber air dewpoint made with a dewpoint hygrometer (Model Dew-10, General Eastern, Watertown, MA, USA). Air flow was changed to increase DCO and DX h20 in the chamber. The flow rate of air entering the chamber was not corrected for the amount of water vapor added by canopy transpiration (a maximum of ~15 L/day) because this correction was negligible, which would not be the case in smaller leaf gas exchange systems [32]. Water use efficiency (mol mmol ) was calculated from the ratio of P to ET. net 2.4. Chamber Wind Speed Wind speed above and within the canopies was measured with heat transfer needle anemometers (Model AN-27, Soiltronics, Burlington, WA, USA). These anemometers were well-suited for making wind measurements within canopies because they are small, have fast response times (half-life t = 1 1/2 s), and are omnidirectional. The anemometers were calibrated in a wind tunnel for windspeeds between 0.05 and 5 m s [33]. Vertical gradients in mean wind speed above and within the canopies were measured with anemometers spaced between 4 and 6 cm apart. Each chamber was modified to include variable speed centrifugal blowers so that wind speed above the vegetation could be controlled 1 1 1 over a wide range. Three wind speed settings (high: 2.3 m s ; medium: 1.7 m s , and low: 0.8 m s ) were used in the chambers, but the majority of the measurements were made at the medium setting. 2.5. Temperature Measurements The temperature sensor used to control chamber air temperature was situated 20 cm above the canopy and 10 cm below the lamps. This reference location was chosen because the lamps were found to heat the air in the top 5 cm of the chamber near the water filter. Mean air temperature (T ) at the reference location was measured using a shielded and aspirated thermocouple (Type-E, air 30 gauge). Vertical profiles of air temperature within the canopies were measured with an aspirated thermocouple manifold. The thermocouples were arranged in parallel within a manifold that held the thermocouples evenly spaced (10 cm apart) and were ventilated at about 1–2 m s by a single aspirator (a vacuum cleaner). The aspirated thermocouples were shielded from incident radiation by plastic tubing wrapped in aluminum foil. The vertical profiles in temperature were expressed as an air temperature difference from the reference air temperature above the canopy. Canopy temperature measurements made using infrared temperature sensors are described using the nomenclature and definitions of Norman and Becker [28]. Two nadir-viewing (e.g., perpendicular to the canopy) infrared sensors in each chamber (Model IRTS-P, Apogee Instruments, Logan, UT, USA) were used to measure canopy brightness temperature (T ), which is a directional temperature canopy,IR that depends on the angle of observation, the wavelength band of the infrared sensor, the sensor body temperature, and sensor position above the top of the canopy. The IRTS-P infrared sensors have a 90 field of view and an accuracy of 0.2 C. The 8–14 m wavelength band was viewed. They were placed in the center of the canopy at a height of 10 cm above the foliage, where the chamber walls could not be seen. The calibration procedures, the field of view considerations, and the functions used to correct for sensor body temperature for these sensors are described in Bugbee et al. [29]. Canopy T , formally defined as the extrapolation of air temperature profile down to an effective Aero height within the canopy at which the vegetation components of sensible and latent heat flux arise [18], is the mean canopy temperature felt by the air that solves the energy balance equation exactly. T Aero cannot be measured directly. It can be obtained from H when T and g are known, but is typically air A approximated by T , the canopy radiometric temperature [13,27]. Canopy T was derived from R R T after correcting for the sky irradiance (e.g., proportional to sky temperature, T ) that is canopy,IR Sky Agronomy 2019, 9, 114 6 of 23 reflected by the canopy and by the chamber walls into the field of view of the infrared sensor [28]. In the controlled-environment chambers, sky irradiance is emitted by the warm chamber surface areas above the canopy, which were proportionally divided into a 20% chamber wall and an 80% glass water filter. The difference between T and T depends on the canopy emissivity, " (Equation (1)): R canopy,IR 4 4 4 T = " **T + (1 " )**T (1) c c canopy,IR R Sky 2 4 where = Stefan–Boltzman constant (W m K ), and T = temperature of the chamber surfaces Sky above the canopy (K). In this paper, it was assumed that T T because the correction for R canopy,IR canopy emissivity is small ( 0.2 C). For example, if T = 30 C, T = T = 23 C, T = 24 C air canopy,IR glass wall (e.g., 20% wall and 80% glass temperature), and " = 0.97, then the difference between T and T c R canopy,IR is only 0.14 C. If the water filter under the lamps is removed, T increases by ~0.5 C, T = 45 C, glass and the difference between T and T rises to ~0.5 C (Equation (1)). These conditions are R canopy,IR unique to controlled-environment conditions because such high T temperatures are never observed Sky in the field. 2.6. Absorbed Radiation Energy exchange and photosynthesis are proportional to the amount of radiation absorbed by plant canopies, which is determined by the direct beam fraction of incident radiation, the canopy structure, and the optical properties of the plant elements [34]. Incident PPF and shortwave radiation within the growth chamber were measured at canopy height. Shortwave radiation between 0.285 and 2.8 m was measured with a precision spectral pyranometer (The Eppley Laboratory, Model PSP, Newport, RI, USA). Incident non-photosynthetic, shortwave radiation (NPSW ) was determined by 2 1 2 subtracting PPF (converted to energy units assuming 5 mol m s per W m for HPS lamps) from the total shortwave radiation. The fraction of PPF absorbed by the canopy (PPF ) was calculated abs from the product of radiation capture and PPF , as described by Monje and Bugbee [30]. A diffuse light fraction of 0.7 was measured in the chamber using a shadow band to shield the quantum sensor from direct radiation. The fraction of non-photosynthetic, shortwave radiation absorbed by the canopy (NPSW = (1 $ ) NPSW ) depends on the canopy reflection coefficient (or surface albedo), $ , in abs c o c the near-infrared (NIR). $ was estimated using Equation (2) from the single leaf scattering coefficient ( ) [35]: 1/2 1/2 $ = [1 (1 ) ]/[1 + (1 ) ]. (2) S S varies with the wavelength of the radiation and equals the sum of the fractions of reflected and transmitted light. In the visible spectrum, the $ of the high planting density wheat canopies was 0.055 during vegetative growth [36], which corresponds to a of 0.2. Since the NIR $ was not S c measured directly, it was derived from Equation (2) assuming an NIR of 0.8. For comparison, the single leaf reflectance (0.43) and transmittance of winter wheat (0.33) in the NIR combine to give an NIR of 0.76 [37]. Thus, NPSW was 0.62 NPSW for a $ of 0.38 in the NIR. Although this o c S abs approximation overestimates $ in sunny conditions (e.g., high direct beam radiation), it predicts it accurately under overcast conditions [38], similar to the highly diffuse radiation found in these controlled-environment chambers. 2.7. Net Radiation, Evapotranspiration, and Photosynthesis The net radiation above the canopy, R , was assumed proportional to net input of shortwave net radiation and incoming longwave radiation (Equation (3)). R = PPF + NPSW + #L "L (3) net abs abs g c where PPF = absorbed photosynthetic radiation (W m ), NPSW = absorbed non-photosynthetic abs abs shortwave radiation (W m ), #L = longwave radiation emitted by the glass from the water filter and g Agronomy 2019, 9, 114 7 of 23 2 2 the chamber walls (W m ), and"L = longwave radiation emitted by the canopy (W m ). Assuming 4 4 4 that the longwave radiation components (#L "L = " #L " T = " T " T ) g c c g c c c R Sky R nearly canceled each other was acceptable as long as the differences between T and T were also Sky small. For example, if T = 30 C, T = T = 23 C, and T = 24 C, then T = 28.6 C, and #L glass wall air R sky "L = 27 W m . Although Equation (3) ignores changes in longwave radiation within the canopy caused by vertical gradients in temperature, it was a better estimate than direct measurements with a net radiometer. Most net radiometers are calibrated for field operation, where the fraction of longwave radiation is much smaller than in these chambers, and the dimensions of the chambers placed the net radiometer close to the top of the foliage, where self-shading led to significant overestimates of the net radiation flux. Net radiometers are preferred in chambers illuminated by solar radiation, but they are affected during cloudy days with highly diffuse radiation. Net radiation in the chamber could be varied by either changing PPF with neutral density filters (window screen filters) or by draining the water filter under the lamps. Shading with neutral density filters does not alter the spectral composition of the incident radiation. In contrast, the water filter under the lamps reduces the amount of longwave radiation impinging on the canopy, thereby increasing the ratio of PPF to R [39]. Removing the water filter increased #L compared to "L abs net g c and added ~100 W m to R , as the glass temperature measured with a thermocouple reached 45 C. net The PAR to R ratio was 83% of R in a chamber with a water filter below the lamps, but was abs net net only 64% of R when the water filter was removed. These changes in surface radiation forcing (R ) net net were used to change canopy temperature and H for studying the relation between T and T . Aero R 2 1 Chamber ET (mmol m s ) consisted of canopy transpiration (E ) and evaporation (E) from can the hydroponic solution through the porous media sustaining the plants (Equation (4)). ET = E + E. (4) can Chamber latent heat flux (LE; W m ) was determined from the product of ET and the heat of vaporization of water (44 kJ mol ). Evaporation from the hydroponic tubs, covered with lids but without a canopy, was small (~2% of R when expressed in W m ). This made ET essentially equal net to E in this study and ensured that T , calculated from the energy balance measurements, was can Aero mostly due to the flux of sensible heat between the foliage and the air flowing above the canopy. In controlled environments, P should be included in the energy balance equation at high light intensities because it becomes a large fraction of R . Photosynthesis (P; W m ), the conversion net of energy in radiation into stored chemical energy, was derived from the product of canopy photosynthesis, P [30], and the enthalpy of combustion for CHO (479 KJ mol ) [40]. net 2.8. Canopy Sensible Heat Flux In the steady state, H is the energy exchanged by conduction and convection between the canopy and the chamber air. The canopy energy balance equation was rearranged for calculating H by residual (Equation (5)), where R = net radiation, LE = latent heat flux, G = soil heat flux, and P = energy net storage in photosynthesis. H = R LE G P. (5) net LE includes water vapor fluxes mostly due to canopy E because evaporation was only 2% of can R . The soil heat flux, G, is a component of land surface feedbacks that depends on the amount of net energy available below the canopy. G was assumed to be zero due to a poor transfer of heat through the dense canopies (high planting densities and leaf area indices > 15) used in this study, but this may not be a valid assumption during early development when the plants are seedlings. P was determined from canopy photosynthesis, which can be as much as 10% of R at high light intensities. For example, net 2 1 2 1 2 if P = 60 umol m s at a PPF of 1400 mol m s , then P = 29 W m . Equation (5) allows for net o a comparison of energy fluxes in common energy units (W m ) and allows H to be determined by residuals. However, Equation (5) ignores the thermal storage within the canopy, which is small for Agronomy 2019, 9, 114 8 of 23 the short vegetation used in this study, but this storage can be as high as 5–10% of the net radiation in forest canopies [13]. 2.9. Canopy Aerodynamic Conductance In field settings, the log-wind profile approximation allows canopy g to be determined from H provided DT , the aerodynamic canopy-to-air temperature difference (DT = T T ), the A A Aero air displacement height, and the roughness length are known [41]. However, the short fetch (1 m) of the canopies used in this study precludes the use of the log-wind profile approximation for calculating g in controlled-environment chambers. Instead, an analog of Ohm’s law (Equation (6)) that relates the surface-to-air temperature difference to the sensible heat loss from the surface was used to describe the energy transfer between the canopy and the chamber air [8]: H = $*Cp*g *(T T ) (6) A R air 3 3 1 where $ = density of air (kg m ), Cp = heat capacity of air at constant pressure (kJ m C ), 2 1 g = canopy aerodynamic conductance (mol m s ), and T ( C) was approximated by T . A R canopy,IR T was measured at the reference height above the canopy, and used to determine the radiometric air canopy-to-air temperature difference (DT = T T ). Equation (6) assumes that the slope between IR R air H and DT equals the slope between H and DT , when T = T . This assumption is valid for fully IR A Aero R covered canopies, whereby the contribution to DT from the temperature of the surface below the IR vegetation (e.g., soil or hydroponic tray) is negligible. The canopy leaf boundary layer conductance component depends on leaf shape and size, and the turbulent conductance component depends on wind speed and canopy aerodynamic roughness [8]. Canopy aerodynamic conductances of dense wheat and soybean canopies with distinct canopy architectures were calculated from the slopes of plots of H vs. measured DT following Equation (6). IR Radiometric DT and H were varied simultaneously by manipulating chamber CO concentration at IR 2 constant environmental conditions (wind speed and VPD) over the course of several days. The g measured for each species results from the amount of drag generated by the interaction between canopy architecture and the chamber air recirculating at constant wind speed. Although changes in CO affect H and DT through changes in stomatal conductance, g remains 2 IR A constant at a fixed chamber wind speed. The highly turbulent conditions in the chamber ensure that free convection effects are negligible compared to forced convection, so changes in light level should not significantly affect canopy g . Estimates of g obtained from the slope of a plot of H vs. DT A A IR are also insensitive to systematic errors in H (e.g., offset errors in R ) because these do not affect the net slope. In this context, the canopy g obtained by this radiometric method represents the canopy leaf boundary layer conductance, as well as the conductance for turbulent heat transfer between the leaves at T and T measured at the reference height above the canopy. Aero air The H vs. DT plot is also useful for exploring differences between T and T . The offset, IR Aero R defined as the value of DT when H and DT are zero (Equation (7)), quantifies this difference because IR A DT and DT are referenced to a common T . IR A air DT = DT + Offset. (7) A IR The behavior of Offset was studied by varying the intensity of the radiation incident on the canopy using neutral density filters and by changing the chamber wind speed. These changes effectively alter surface radiation forcing (PPF ) and surface layer feedbacks (wind speed). 2.10. Canopy G and G SFC S The measurement of canopy ET in controlled environments makes it possible for calculating a “big-leaf” surface canopy conductance (G ) with a corresponding effective VPD at the “big-leaf” SFC surface (D ). Surface G was calculated from the ratio of E to D (Equation (8)): S SFC can S Agronomy 2019, 9, 114 9 of 23 G = E P /D (8) SFC can atm S where G = canopy surface conductance, E = canopy transpiration measured using the gas SFC can 2 1 exchange system (mmol m s ), and P = atmospheric pressure. D was calculated using T Atm Bulk Air measured at the reference location above the canopy. D is the VPD of the air within the canopy at Aero T . When D = D in Equation (8), each leaf surface is at the mean aerodynamic temperature and Aero S Aero sees the same saturation deficit at its surface, which treats the canopy as a giant single leaf where the average canopy leaf temperature equals T . Aero Canopy G calculated from Equation (8) includes canopy G and g [10] because these SFC S A conductances are additive in series. Canopy G was calculated from surface G and g using S SFC A Equation (9), the resistance subtraction method [7]. G is metabolically controlled canopy stomatal conductance that influences land atmosphere interactions via land surface feedbacks. Gs = G g /(g G ) = ((1/G ) (1/g )) . (9) SFC A A SFC SFC A 2.11. Canopy Decoupling Coefficient At the canopy level, relative magnitudes of G and g determine the effect of changes in stomatal S A conductance on the transport of heat and water vapor from an average leaf surface, through leaf and canopy boundary layers to an effective sink for heat and water vapor above the canopy [3]. The boundary layer surrounding vegetation allows transpired water vapor to humidify air near the leaf surface (e.g., it lowers D compared to D ), altering the driving force for transpiration; thus, S Bulk E becomes less sensitive to changes in stomatal conductance. This feedback between E and D is can can important for diminishing the sensitivity of E to proportional changes in G [1,3,4]. can S The dimensionless decoupling coefficient, W, quantifies the sensitivity of E to changes in can stomatal aperture and depends on the influence that G and g exert on how closely conditions at S A the leaf surface (e.g., D ) are linked to D of the free air stream. Equation (10) calculates W from g , S Bulk A G , and " = s/
, where s = the slope of the saturation vapor pressure versus temperature, and
= the psychrometric constant [10]. W = (" + 1)/[" + 1 + (g /G )]. (10) A S Equation (10) assumes that the available energy is independent of surface temperature and neglects changes in leaf temperature due to changes in stomatal conductance [4]. In spite of this simplification, W is useful for (1) exploring how differences in canopy architecture (e.g., wheat and soybean) affect canopy transpiration and (2) quantifying the sensitivity of E to changes in can stomatal conductance. Typical values for g , G , and W for crops and forests are depicted in Table 1. A S The magnitude of W effectively determines whether Ecan is primarily controlled by stomata or by the supply of energy. Generally, forests are well coupled, and their transpiration rate is accurately predicted by the Priestley–Taylor equation [3,4]. The sensitivity of transpiration to stomatal control, dE , is determined by the degree of coupling (1 W) between D and D (Equation (11); [3,6,7]). can S Bulk dE = (1 W) (E /G ) dG . (11) can can S S Table 1. Typical land surface properties that influence the control of transpiration rate from conifers or crops. Species Coupling T T g W Relative Magnitude Transpiration Control Aero air A Conifer coupled small low ~ 0.1 g >> G Radiation DR net A S Crop decoupled large high ~ 0.8 g << G Stomatal DG A S S 2.12. Responses of Transpiration to Elevated CO Responses of transpiration to CO concentration were measured at a constant PPF using the same vegetative wheat canopy over a span of 8 days. During this time, chamber CO was increased in 2 Agronomy 2019, 9, 114 10 of 23 a stepwise fashion from 400, to 700, to 950, and to 1200 umol mol . Canopy gas exchange fluxes and energy balance components were held at each CO concentration for 48 h to allow the incremental buildup of sugar pools in tissues throughout the canopy. These data were used to measure canopy aerodynamic conductance and to determine the response of canopy transpiration to increased CO concentration. Daily average values of E , P , LE, H, G , W, and WUE were calculated because G can net S S and E did not remain constant throughout the day due to diurnal changes in stomatal conductance. can 3. Results 3.1. Wind and Temperature Profiles Average wind speed and air temperature profiles were measured at different heights above and within wheat and soybean canopies in a ventilated chamber. The mean wind speed at any given plane above the canopy was highly spatially and temporally variable, typically ranging from 0.5 to 2.4 m s in wheat (Figure 2A), and from 0.4 to 1.4 m s in soybean (Figure 2B). The average wind speed at Agronomy 2018, 8, x FOR PEER REVIEW 10 of 23 the canopy surface was attenuated rapidly within the first few centimeters of foliage. Wind speed 402 the canopy surface was attenuated rapidly within the first few centimeters of foliage. Wind speed within the canopies was more uniform than above and was often below 0.4 m s , reaching as low as 1 −1 403 within the canopies was more uniform than above and was often below 0.4 m s , reaching as low as 0.1 m s at the bottom of the wheat canopy. −1 404 0.1 m s at the bottom of the wheat canopy. Figure 2. Vertical wind profiles of wheat and soybean canopies measured with needle anemometers. 406 Figure 2. Vertical wind profiles of (a) wheat and (b) soybean canopies measured with needle Y-axis units are fraction of canopy height. 407 anemometers. Y-axis units are fraction of canopy height. Vertical air temperature profiles within the growth chamber were homogeneous in an empty, dark 408 chamber Vertic since al air te theremperature pr was no foliage ofiles w to trap ithin pockets the gr ofowth chamber were air, and because the homogeneous in surfaces within the an empt chamber y, 409 (glass dark ch and ambe chamber r since there walls,was no fo and the liag surface e to trap po of the gr ckets of air, owth media) and because the surface equilibrated at nearly s wit the hin the same 410 temperatur chamber (ge. lass When and ch theam lights ber wa wer ll es, turned and thon, e sur the face vertical of the growt air temperatur h media e)pr eq ofiles uilibrat within ed atthe nea canopy rly the 411 wer same temper e spatiallyature. When variable; air the lights wer near the plants e turned could be on1–5 , the vertic C higher al air than temperature the reference profiles w air temperatur ithin the e 412 measur canopyed we above re spat them ially v depending ariable; aion r ne the ar t rh elative e plant magnitudes s could be 1– of5 incide °C high nt radiation er than thor e rwind eference a speed ir 413 within temperature the chamber measured (Figur above them depending on the e 3). These large air temperatur rel e dif atifer ve m ences agnit within udes o the f incident canopies rr aesult diation or from 414 vertical wind spe dife fer d ences within the c in light h intensity amber ,(Fig leafure temperatur 3). Thes e,e la and rgleaf e aitranspirat r tempera ion turrates. e diffe Tre ranspiration nces within cools the 415 cooled canopie the s re lower sult from layers vert ofic the al di canopy fferences to temperatur in light intes ensit below y, lethe af te refer mperat ence ure air , a temperatur nd leaf trans e, and piratthe ion 416 uppermost rates. Transpiration leaf layers cools c remain ooled warmer the lo because wer laythey ers of the ca are heated nopy to tempera by the absorption turesof below the incident reference radiation. 417 air te Incident mperature, PPF af and t fected he up the p air erm temperatur ost leaf la e yers differ re ence main above warm and erwithin becaus ae t wheat hey canopy are heat (Figur ed by e t 3h A;e 1 1 418 20-day absorpt old; ion o [CO f inci ] dent r = 400 adiation. mol mol ; T = 21 C; RH = 68%; medium wind speed: 1.7 m s ). In the 2 air 419 dark,Incident PP the top layers F affected the of foliage rair temperat emained warmer ure diffe than rence the lower above and w layers because ithin a whe they at canopy were heated (Figur by e −1 −1 420 sensible 3A; 20-day o heat lflux d; [CO from 2] = 40 the0 warm µmol chamber mol ; Taiair r = 21 flowing °C; RH = 68 above%; medi the canopy um wi . During nd speed: the phot 1.7 m operiod, s ). In the the 421 top dark of , the the top canopy layerrs of fo emained liage rem hotter than ained the warmer lower th leaf an layers the lower as the lay top ers bec layers ause t of foliage hey wer absorbed e heated by most 422 of sensib the incident le heat flux radiation. from the The warm ch air within ambe the r aitop r flow 5 cm ingof above the c the canopy anbecame opy. During hotter ththan e photoperiod, the the reference 2 1 423 air top of temperatur the canopy remai e as incident ned hotter tha light levels nincr the l eased ower to lea 1050 f layer and s as the top lay 1850 mol m ers o sf foliage (Figur absorb e 3A).ed most 424 of the incident radiation. The air within the top 5 cm of the canopy became hotter than the reference −2 −1 425 air temperature as incident light levels increased to 1050 and 1850 µmol m s (Figure 3A). −1 −1 −1 426 At constant PPFo, the fan speed setting (high: 2.3 m s ; medium: 1.7 m s ; low: 0.8 m s ) changed 427 the amount of forced convection in the chamber and affected the vertical air temperature profiles −2 −1 428 above and within the wheat canopy (Figure 3B; 35-day old; PPF = 1800 µmol m s ; [CO2] = 1200 −1 −1 429 µmol mol ; Tair = 21 °C; RH = 68%). At the low chamber wind speed setting (0.8 m s ), the upper 7 −1 −1 430 cm of the canopy was 1.5 °C warmer than at medium (1.7 m s ) and high (2.3 m s ) settings (Figure 431 3B). Air temperature up to 12 cm above the canopy was also heated by 0.5–1.2 °C by the warm foliage 432 at the low wind speed. This suggests a threshold in turbulence in the chamber, above which an 433 increase in wind speed does not continue to affect canopy-air heat exchange. Agronomy 2019, 9, 114 11 of 23 Agronomy 2018, 8, x FOR PEER REVIEW 11 of 23 Agronomy 2018, 8, x FOR PEER REVIEW 11 of 23 Figure 3. Vertical canopy-to-air temperature difference profiles of wheat canopies were affected by 435 Figure 3. Vertical canopy-to-air temperature difference profiles of wheat canopies were affected by (A) (A) light intensity and (B) chamber wind speed. 436 light intensity and (B) chamber wind speed. 1 1 1 At constant PPFo, the fan speed setting (high: 2.3 m s ; medium: 1.7 m s ; low: 0.8 m s ) 437 3.2. Diurnal Changes in Energy Balance Components changed the amount of forced convection in the chamber and affected the vertical air temperature 2 1 profiles above and within the wheat canopy (Figure 3B; 35-day old; PPF = 1800 mol m s ; 438 Sensible heat flux (Figure 4; pink line) was calculated from direct measurements of canopy 1 1 [CO ] = 1200 mol mol ; T = 21 C; RH = 68%). At the low chamber wind speed setting (0.8 m s ), 439 energy balance components (net radiation—red line; latent heat—blue line; photosynthesis—green 2 air 1 1 −1 −2 −1 the upper 7 cm of the canopy was 1.5 C warmer than at medium (1.7 m s ) and high (2.3 m s ) 440 line) in wheat (18-day-old; [CO2] = 680 µmol mol ; PPF = 1600 µmol m s ; Tair = 21 °C; RH = 68%) −1 −2 −1 settings (Figure 3B). Air temperature up to 12 cm above the canopy was also heated by 0.5–1.2 C by 441 and soybean (25-day-old; [CO2] = 1200 µmol mol ; PPF = 750 µmol m s ; Tair = 21 °C; RH = 64%) 435 Figure 3. Vertical canopy-to-air temperature difference profiles of wheat canopies were affected by (A) the warm foliage at the low wind speed. This suggests a threshold in turbulence in the chamber, above 442 using Equation (5). In the dark, net radiation was negligible and the canopies were always cooler than 436 light intensity and (B) chamber wind speed. which an increase in wind speed does not continue to affect canopy-air heat exchange. 443 air temperature because the transpiration rate and latent heat flux of hydroponic plants remains high 444 [42]. However, the topmost leaf layers remained warm compared to the lower layers of the canopy 437 3.2. Diurnal Changes in Energy Balance Components 3.2. Diurnal Changes in Energy Balance Components 445 (Figure 3A), as advection of warm air from the chamber temperature control system supplies 438 Sensible heat flux (Figure 4; pink line) was calculated from direct measurements of canopy 446 additional energy for transpiration. Latent heat increased and sensible heat decreased in the hours Sensible heat flux (Figure 4; pink line) was calculated from direct measurements of canopy energy 439 energy balance components (net radiation—red line; latent heat—blue line; photosynthesis—green 447 preceding the photoperiod (Figure 4), probably due to circadian increases in predawn stomatal balance components (net radiation—red line; latent heat—blue line; photosynthesis—green line) in −1 −2 −1 440 line) in wheat (18-day-old; [CO2] = 680 µm 1 ol mol ; PPF = 1600 µm 2 ol 1 m s ; Tair = 21 °C; RH = 68%) 448 conductance [43]. wheat (18-day-old; [CO ] = 680 mol mol ; PPF = 1600 mol m s ; T = 21 C; RH = 68%) and 2 air −1 −2 −1 441 and soybean (25-day-old; [CO2] = 1200 µmol mol 1 ; PPF = 750 µmol m 2 s 1 ; Tair = 21 °C; RH = 64%) soybean (25-day-old; [CO ] = 1200 mol mol ; PPF = 750 mol m s ; T = 21 C; RH = 64%) 2 air 442 using Equation (5). In the dark, net radiation was negligible and the canopies were always cooler than using Equation (5). In the dark, net radiation was negligible and the canopies were always cooler 443 air temperature because the transpiration rate and latent heat flux of hydroponic plants remains high than air temperature because the transpiration rate and latent heat flux of hydroponic plants remains 444 [42]. However, the topmost leaf layers remained warm compared to the lower layers of the canopy high [42]. However, the topmost leaf layers remained warm compared to the lower layers of the canopy 445 (Figure 3A), as advection of warm air from the chamber temperature control system supplies (Figure 3A), as advection of warm air from the chamber temperature control system supplies additional 446 additional energy for transpiration. Latent heat increased and sensible heat decreased in the hours energy for transpiration. Latent heat increased and sensible heat decreased in the hours preceding the 447 preceding the photoperiod (Figure 4), probably due to circadian increases in predawn stomatal photoperiod (Figure 4), probably due to circadian increases in predawn stomatal conductance [43]. 448 conductance [43]. 450 Figure 4. Diurnal course of canopy energy balance components: net radiation, latent heat flux, sensible 451 heat flux, and photosynthesis in (A) wheat and (B) soybean canopies. 452 Generally, sensible heat increased during the photoperiod as the canopy became warmer 453 because evaporative cooling from latent heat diminished during the course of the day, even though 454 incident PPF was constant. This decrease in latent heat is probably due to diurnal changes in stomatal 455 conductance [42]. In wheat, sensible heat was negative whenever latent heat plus photosynthesis 456 exceeded net radiation, but the canopy became hotter than air temperature and sensible heat was Figure 4. Diurnal course of canopy energy balance components: net radiation, latent heat flux, sensible 457 450 positive Figure 4. at the end of th Diurnal course of e day canopy energy balance componen (Figure 4A). The soybean ca ts: no net radiation, py remained latent cooler than heat flux, sensible the air heat flux, and photosynthesis in (A) wheat and (B) soybean canopies. 458 451 temperat hure, eat flu and x, an lad te ph ntotosy heatn rem thesis ai in ned (Agrea ) whter tha eat andn ( net B) soy radi bean ation canopie in spit s. e of decreasing latent heat at 459 the end of the day (Figure 4B). 452 Generally, sensible heat increased during the photoperiod as the canopy became warmer 453 because evaporative cooling from latent heat diminished during the course of the day, even though 454 incident PPF was constant. This decrease in latent heat is probably due to diurnal changes in stomatal 455 conductance [42]. In wheat, sensible heat was negative whenever latent heat plus photosynthesis 456 exceeded net radiation, but the canopy became hotter than air temperature and sensible heat was 457 positive at the end of the day (Figure 4A). The soybean canopy remained cooler than the air 458 temperature, and latent heat remained greater than net radiation in spite of decreasing latent heat at 459 the end of the day (Figure 4B). Agronomy 2019, 9, 114 12 of 23 Agronomy 2018, 8, x FOR PEER REVIEW 12 of 23 Generally, sensible heat increased during the photoperiod as the canopy became warmer because 460 3.3. Canopy-to-Air Teperature Difference evaporative cooling from latent heat diminished during the course of the day, even though incident 461 The difference between aerodynamic ΔTA and radiometric ΔTIR is affected by two physical PPF was constant. This decrease in latent heat is probably due to diurnal changes in stomatal 462 factors: the field of view of the IR transducers and the chamber wind speed. The field of view of the conductance [42]. In wheat, sensible heat was negative whenever latent heat plus photosynthesis 463 sensor with respect to the canopy surface influenced the magnitude of the radiometric TR measured exceeded net radiation, but the canopy became hotter than air temperature and sensible heat was 464 by the infrared transducers. Differences in TIR and TAero are probably due to differences in how well positive at the end of the day (Figure 4A). The soybean canopy remained cooler than the air temperature, 465 radiometric measurements truly represent the average canopy temperature profile. With constant Tair and latent heat remained greater than net radiation in spite of decreasing latent heat at the end of the 466 and PPFo provided by the chamber, radiometric TR was affected by the vertical positioning of the day (Figure 4B). 467 infrared transducers above or within the canopy. Generally, TR was higher in the surface layers of 3.3. Canopy-to-Air Teperature Difference 468 foliage and became lower as the IR transducer was inserted into the canopy foliage. Once the IR 469 transducers were positioned, the canopy-to-air temperature difference was compared to the canopy- The difference between aerodynamic DT and radiometric DT is affected by two physical factors: A IR 470 to-air temperature difference obtained from H. the field of view of the IR transducers and the chamber wind speed. The field of view of the sensor with 471 The relation between H, ΔTIR, and ΔTA was explored in soybean by changing the amount and respect to the canopy surface influenced the magnitude of the radiometric T measured by the infrared −1 −2 −1 472 quality of incident radiation (Figure 5; 45-day-old; [CO2] = 400 µmol mol ; PPF = 1050 µmol m s ; transducers. Differences in T and T are probably due to differences in how well radiometric IR Aero 473 Tair = 22 °C; RH = 62%). In the dark, the energy balance components under each water filter were measurements truly represent the average canopy temperature profile. With constant T and PPF air −2 474 similar, yielding H ~ −75 W m (Figure 5, top), but radiometric ΔTIR and aerodynamic ΔTA differed provided by the chamber, radiometric T was affected by the vertical positioning of the infrared 475 by a nearly constant offset (Figure 5, bottom). The spikes in H observed at the beginning and at the transducers above or within the canopy. Generally, T was higher in the surface layers of foliage and 476 end of the photoperiod are artifacts that occur when H is obtained by subtraction and chamber energy became lower as the IR transducer was inserted into the canopy foliage. Once the IR transducers were 477 fluxes and temperatures equilibrate. positioned, the canopy-to-air temperature difference was compared to the canopy-to-air temperature 478 During the photoperiod, removing the water filter under the HPS lamps increased Rnet by ~50 W difference obtained from H. −2 479 m due to a 30% greater PPFo transmission and due to increased longwave radiation as the lamps The relation between H, DT , and DT was explored in soybean by changing the amount and IR A 480 heated the glass of the water filter. Without the water filter, the rati o of photosyntheti 1 c to non 2 1- quality of incident radiation (Figure 5; 45-day-old; [CO ] = 400 mol mol ; PPF = 1050 mol m s ; 481 photosynthetic shortwave radiation dropped from 83:17 to 66:34, and sensible heat flux increased up T = 22 C; RH = 62%). In the dark, the energy balance components under each water filter were air −2 −2 482 to approximately −95 W m (Fi g2ure 5A, top), from approximately −120 W m (Figure 5B, top) as the similar, yielding H ~ 75 W m (Figure 5, top), but radiometric DT and aerodynamic DT differed IR A 483 additional radiation from the lamps warmed the canopy. Radiometric ΔTIR was consistently higher by a nearly constant offset (Figure 5, bottom). The spikes in H observed at the beginning and at the 484 than aerodynamic ΔTA, and the offset was between 0.8 and 1.0 °C higher than it was in the dark. In end of the photoperiod are artifacts that occur when H is obtained by subtraction and chamber energy 485 fact, the sensible heat flux calculated from ΔTIR using Equation (6) often had an opposite sign to values fluxes and temperatures equilibrate. 486 of sensible heat flux calculated from the energy balance equation (Figure 5, middle). Figure 5. Diurnal changes in canopy sensible heat, radiative, and aerodynamic canopy-to-air 488 Figure 5. Diurnal changes in canopy sensible heat, radiative, and aerodynamic canopy-to-air temperature differences, and the offset of soybean illuminated by lamps (A) without and (B) with a 489 temperature differences, and the offset of soybean illuminated by lamps (A) without and (B) with a water filter to remove excess longwave radiation. 490 water filter to remove excess longwave radiation. 491 However, relative changes in the magnitude of ΔTIR as a function of time paralleled the relative 492 changes in H and ΔTA (Figure 5, bottom), and the difference between the measured ΔTIR and ΔTA 493 remained constant throughout the photoperiod. Agronomy 2019, 9, 114 13 of 23 During the photoperiod, removing the water filter under the HPS lamps increased R by net ~50 W m due to a 30% greater PPF transmission and due to increased longwave radiation as the lamps heated the glass of the water filter. Without the water filter, the ratio of photosynthetic to non-photosynthetic shortwave radiation dropped from 83:17 to 66:34, and sensible heat flux increased 2 2 up to approximately 95 W m (Figure 5A, top), from approximately 120 W m (Figure 5B, top) as the additional radiation from the lamps warmed the canopy. Radiometric DT was consistently IR higher than aerodynamic DT , and the offset was between 0.8 and 1.0 C higher than it was in the dark. In fact, the sensible heat flux calculated from DT using Equation (6) often had an opposite sign IR to values of sensible heat flux calculated from the energy balance equation (Figure 5, middle). However, relative changes in the magnitude of DT as a function of time paralleled the relative IR Agronomy 2018, 8, x FOR PEER REVIEW 13 of 23 changes in H and DT (Figure 5, bottom), and the difference between the measured DT and DT A IR A remained constant throughout the photoperiod. 494 Wind speed determines canopy gA and affects how the foliage warms as PPFo is increased. In a Wind speed determines canopy g and affects how the foliage warms as PPF is increased. In a A −1 o 495 wheat canopy (25-day-old; [CO2] = 1200 µmol mol ; Tair = 22 °C; RH = 68%; no water filter), changes wheat canopy (25-day-old; [CO ] = 1200 mol mol ; T = 22 C; RH = 68%; no water filter), changes 2 air 496 in PPFo at two chamber wind speeds were used to explore the offset between ΔTA and ΔTIR (Figure in PPF at two chamber wind speeds were used to explore the offset between DT and DT (Figure 6). o A IR 497 6). At each wind speed, ΔTA calculated from H by inverting Equation (6) was compared with values At each wind speed, DT calculated from H by inverting Equation (6) was compared with values of 498 of ΔTIR measured by the IR sensors. DT measured by the IR sensors. IR Figure 6. The radiometric (DT ) and aerodynamic (DT ) temperatures and the offset were measured IR A 501 Figure 6. The radiometric (ΔTIR) and aerodynamic (ΔTA) temperatures and the offset were measured at (A) low and (B) high chamber wind speed settings. 502 at (A) low and (B) high chamber wind speed settings. 2 1 As incident PPF increased from 0 to 1700 mol m s , DT increased linearly from 0 C o IR −2 −1 503 As incident PPFo increased from 0 to 1700 µmol m s , ΔTIR increased linearly from 0 °C to +4 to +4 C at low wind speed (Figure 6A, top; 1.7 m s ) and increased from 1 C to +2 C at high 1 −1 504 wind °C at low speed win (Figur d speed e 6B,(F top; igure 6A, top; 2.3 m s ).1.7 m s Although ) and inc the aer reased odynamic from D−T 1 °C to also+2 °C increased at high linearly wind speed with −1 505 (Figure 6B, top; 2.3 m s ). Although the aerodynamic ΔTA also increased linearly with increasing PPFo increasing PPF (Figure 6A,B), its sign was negative at low to moderate light levels and it had a steeper 506 response (Figures 6A to PPF ,B), it than s signD was nega T (e.g., changing tive at low to mode from 3 C rato te light levels +4 C at theand low it wind had a steeper setting; Figur respeonse to 6A). o IR 507 PPFo than ΔTIR (e.g., changing from −3 °C to +4 °C at the low wind sett ing; Figure 6A). The radiometric DT never equaled zero when DT was zero (Figure 6) and was often opposite IR A 508 in sign The radiomet to the aerodynamic ric ΔTIRD neve T (Figur r equaled e 6A,B, zero top). when TheΔof TA fset wacorr s zeection ro (Figbetween ure 6) and the wa radiometric s often oppDo Tsite A IR 509 in sign to the aerodynamic ΔTA (Figures 6A,B, top). The offset correction between the radiometric and the aerodynamic DT increased linearly with increasing PPF , but did not vary with chamber 510 wind ΔTIR speed and the (Figur aerodyn e 6A,B, amic bΔ ottom TA incr graphs; eased lthe inearly dashed withlines incre ar as eing the P95% PFo, but di confidence d not va interval). ry with cha Smaller mber 511 wind speed (Figures 6A,B, bottom graphs; the dashed lines are the 95% confidence interval). Smaller values of the offset at high light intensities suggest that the warmer leaves at the top of the canopy 512 play valu aes o greater f the roffs ole et in at H hi and ghr li educe ght int the ens dif ities ferences sugges between t that the wa T and rmer l T ea . ves at the top of the canopy R Aero 513 play a greater role in H and reduce the differences between TR and TAero. This analysis suggests that DT cannot be used to determine H directly, that is, without correcting IR 514 for the This an offset. Thus, alysis su thegge offset sts that in partΔT corr IR ca ects nnot be used to determi estimates of H for differ nences e H di between rectly, tha DTt isand , wiD thout T IR A 515 correcting for the offset. Thus, the offset in part corrects estimates of H for differences between ΔTIR and allows Equations (6) and (7) to accurately describe the energy balance of dense canopies. 516 and ΔTA and allows Equations (6) and (7) to accurately describe the energy balance of dense canopies. 517 3.4. Canopy Aerodynamic Conductance 518 In controlled-environment chambers, gA is determined by an interaction between canopy 519 architecture and air circulation in the chamber. Typically, fan speed is constant and canopy 520 architecture remains constant over several days once the canopy is closed. In these conditions, 521 Equation (6) permits canopy gA to be calculated from the slope of a plot of H versus ΔTIR. Stepwise −1 522 increases in chamber CO2 concentration from 400 to 1200 µmol mol were used to simultaneously 523 alter H and ΔTIR via physiological changes in canopy GS at a constant canopy gA. H and ΔTIR increase 524 simultaneously when chamber ambient CO2 increases because elevated CO2 reduces stomatal 525 conductance, and the canopy is warmed due to less evaporative cooling. 526 A plot of H and radiometric ΔTIR was used to calculate the gA of a wheat canopy (Figure 7; 25- −2 −1 527 day-old; PPF = 1200 µmol m s ; Tair = 22 °C; RH = 70%). The slopes of H versus ΔTIR at each CO2 Agronomy 2019, 9, 114 14 of 23 3.4. Canopy Aerodynamic Conductance In controlled-environment chambers, g is determined by an interaction between canopy architecture and air circulation in the chamber. Typically, fan speed is constant and canopy architecture remains constant over several days once the canopy is closed. In these conditions, Equation (6) permits canopy g to be calculated from the slope of a plot of H versus DT . Stepwise increases in chamber A IR CO concentration from 400 to 1200 mol mol were used to simultaneously alter H and DT via 2 IR physiological changes in canopy G at a constant canopy g . H and DT increase simultaneously S A IR when chamber ambient CO increases because elevated CO reduces stomatal conductance, and the 2 2 canopy is warmed due to less evaporative cooling. A plot of H and radiometric DT was used to calculate the g of a wheat canopy (Figure 7; IR A Agronomy 2018, 8, x FOR PEER REVIEW 14 of 23 2 1 25-day-old; PPF = 1200 mol m s ; T = 22 C; RH = 70%). The slopes of H versus DT at each air IR CO concentration were similar (separate regressions not shown in Figure 7), which suggests that g 2 A 528 concentration were similar (separate regressions not shown in Figure 7), which suggests that gA did did not respond to changes in ambient CO . 529 not respond to changes in ambient CO2. Figure 7. Plot of H versus DT (red line) and the offset from a 25-day-old wheat canopy exposed to IR 531 Figure 7. Plot of H versus ΔTIR (red line) and the offset from a 25-day-old wheat canopy exposed to changing CO concentration. 532 changing CO2 concentration. The variability in H (~25 W m ) corresponds to an uncertainty in DT of ~0.4 C, which is IR −2 533 The variability in H (~±25 W m ) corresponds to an uncertainty in ΔTIR of ~±0.4 °C, which is close to the error in determining T from T . The dashed line in Figure 7 represents the plot of R canopy,IR 534 close to the error in determining TR from Tcanopy,IR. The dashed line in Figure 7 represents the plot of H versus DT , determined by subtracting a constant offset to DT (Equation (7)). This offset equals A IR 535 H versus ΔTA, determined by subtracting a constant offset to ΔTIR (Equation (7)). This offset equals the value of the difference between DT and DT when H is zero. In wheat, this offset was +0.75 C at IR A 2 1 2 1 536 the value of the difference between ΔTIR and ΔTA when H is zero. In wheat, this offset was +0.75 °C 1600 mol m s and was +1.0 C in soybean at 750 mol m s . −2 −1 −2 −1 2 1 537 at 1600 µmol m s and was +1.0 °C in soybean at 750 µmol m s . The g of the 25-day-old wheat canopy was 5.5 mol m s (Figure 7). The g of a 45-day-old A A 2 1 −2 −1 538 The gA of the 25-day-old wheat canopy was 5.5 mol m s (Figure 7). The gA of a 45-day-old soybean canopy was 2.5 mol m s . These conductances correspond to aerodynamic resistances of −2 −1 539 soybean canopy was 2.5 mol m s . These conductances correspond to aerodynamic resistances of 7.5 and 16.5 s m , respectively. Soybean has a smaller g compared to wheat because soybean leaves −1 540 7.5 and 16.5 s m , respectively. Soybean has a smaller gA compared to wheat because soybean leaves are wider than wheat leaves and have a smaller leaf boundary layer conductance. 541 are wider than wheat leaves and have a smaller leaf boundary layer conductance. 3.5. Canopy Surface and Stomatal Conductances 542 3.5. Canopy Surface and Stomatal Conductances Canopy surface G of wheat was calculated from E and D using Equation (8) (green line; SFC can S 1 2 1 Figure 8A; 26-day-old; [CO ] = 400 mol mol ; PPF = 1600 mol m s ; T = 21 C; RH = 68%). 543 Canopy surface GSFC of wheat was calculated from Ecan and DS using Equation (8) (green line; 2 air −1 −2 −1 Once g was determined, G was used for estimating canopy G using Equation (9) (green line; 544 Figure 8A; 26-day-old; [CO2] = 400 µmol mol ; PPF = 1600 µmol m s ; Tair = 21 °C; RH = 68%). Once A SFC S Figure 8B). Assuming that canopy G equals G , that is, without taking g into account (green lines 545 gA was determined, GSFC was used for estimating canopy GS using Equation (9) (green line; Figure S SFC A in Figure 8A,B) underestimates G by 40% in wheat. 546 8B). Assuming that canopy GS equals GSFC, that is, without taking gA into account (green lines in 547 Figures 8A,B) underestimates GS by 40% in wheat. 548 The sensitivity of GSFC (Equation (8)) to errors from using ΔTIR instead of ΔTA was also explored 549 (Figure 8). Surface GSFC of wheat (Figure 8A) was only slightly greater when calculated from 550 radiometric TR instead of aerodynamic TAero. The average surface GSFC at the radiometric TR was 1.6 −2 −1 −2 −1 551 mol m s (red line; Figure 8A) and 1.5 mol m s (green line; Figure 8A) at the aerodynamic TAero. 552 Therefore, neglecting the offset correction between ΔTIR and ΔTA in wheat resulted in only a –6% −2 −1 −1 553 error in surface GSFC. The difference between the average radiometric GS (2.3 mol m s or 18.0 s m ) −2 −1 −1 554 and aerodynamic GS (2.1 mol m s or 19.7 s m ) was also small (red vs. green line; Figure 8B). Thus, 555 canopy GS of wheat computed using the observed TR instead of TAero was only 8% higher. −1 −2 −1 556 In soybean at 400 µmol mol of CO2, the surface radiometric GSFC was 0.56 mol m s and −2 −1 557 aerodynamic GSFC was 0.75 mol m s , thus using TR instead of TAero to estimate surface GSFC of 558 soybean resulted in a larger (−34%) error. The corresponding radiometric and aerodynamic values of −2 −1 −2 −1 559 canopy GS were 0.7 mol m s and 1.1 mol m s , a difference of 49%. Agronomy 2019, 9, 114 15 of 23 Agronomy 2018, 8, x FOR PEER REVIEW 15 of 23 Figure 8. Daily courses of (A) canopy surface stomatal conductance (G ; Equation (8)), and (B) canopy SFC 561 Figure 8. Daily courses of (A) canopy surface stomatal conductance (GSFC; Equation (8)), and (B) stomatal conductance (G ; Equation (9)) of wheat. 562 canopy stomatal conductance (GS; Equation (9)) of wheat. The sensitivity of G (Equation (8)) to errors from using DT instead of DT was also explored SFC IR A 563 In wheat, the sensitivity of GSFC to errors in gA was examined by comparing the measured GSFC (Figure 8). Surface G of wheat (Figure 8A) was only slightly greater when calculated from radiometric SFC 564 with the GSFC obtained from the observed GS and a typical value of field gA reported in the literature 2 1 T instead of aerodynamic T . The average surface G at the radiometric T was 1.6 mol m s R Aero SFC R −2 −1 565 (gA = 2 mol m s ; [44]). This field value of gA corresponds to less turbulent conditions (a smaller gA) 2 1 (red line; Figure 8A) and 1.5 mol m s (green line; Figure 8A) at the aerodynamic T . Therefore, Aero 566 than were observed for wheat in the chamber, and it is closer to the gA of soybean. The GSFC calculated neglecting the offset correction between DT and DT in wheat resulted in only a –6% error in surface IR A 567 by inverting Equation (9) using measured GS and field gA (Figure 8A; dashed line) underestimates 2 1 1 G . The difference between the average radiometric G (2.3 mol m s or 18.0 s m ) and aerodynamic SFC S 568 the measured GSFC (Figure 8A; green line) by 33%. This analysis shows that large differences in surface 2 1 1 G (2.1 mol m s or 19.7 s m ) was also small (red vs. green line; Figure 8B). Thus, canopy G of S S 569 GSFC exist between field settings and controlled-environment chambers and that these occur because wheat computed using the observed T instead of T was only 8% higher. R Aero 570 of differences in turbulence that can be accounted for only when gA is known. 1 2 1 In soybean at 400 mol mol of CO , the surface radiometric G was 0.56 mol m s and 2 SFC −1 −2 −1 571 The canopy gA and GS for wheat (18-day-old; [CO2] = 400 µmol mol ; PPF = 1600 µmol m s ; 2 1 aerodynamic G was 0.75 mol m s , thus using T instead of T to estimate surface G of SFC R Aero SFC −1 −2 −1 572 Tair = 21 °C; RH = 68%) and soybean (25-day-old; [CO2] = 400 µmol mol ; PPF = 750 µmol m s ; Tair soybean resulted in a larger ( 34%) error. The corresponding radiometric and aerodynamic values of 573 = 21 °C; RH = 64%) canopies are reported in Table 2. 2 1 2 1 canopy G were 0.7 mol m s and 1.1 mol m s , a difference of 49%. In wheat, the sensitivity of G to errors in g was examined by comparing the measured G SFC A SFC −1 574 Table 2. Canopy gA, GS, and Ω from two crop architectures at 400 umol mol CO2 and 86 kPa. with the G obtained from the observed G and a typical value of field g reported in the literature SFC S A 1 2 2 1 Species Architecture gA Gs Ω Ds-DBulk DBulk (g = 2 mol m s ; [44]). This field value of g corresponds to less turbulent conditions (a smaller g ) A A A Wheat Erectophile 5.5 (7.5) 2.3 (18) 0.67 0.38 0.74 than were observed for wheat in the chamber, and it is closer to the g of soybean. The G calculated A SFC Soybean Planophile 2.5 (16.5) 1.1 ( 37) 0.39 0.56 1.15 by inverting Equation (9) using measured G and field g (Figure 8A; dashed line) underestimates the S A 1 −2 −1 −1 2 575 µmol m s (s m ). kPa. measured G (Figure 8A; green line) by 33%. This analysis shows that large differences in surface SFC G exist between field settings and controlled-environment chambers and that these occur because SFC 576 3.6. Diurnal Changes in GS of differences in turbulence that can be accounted for only when g is known. 1 2 1 The canopy g and G for wheat (18-day-old; [CO ] = 400 mol mol ; PPF = 1600 mol m s ; A S 2 577 Diurnal changes in Ω reflect changes in GS because chamber gA and DBulk are constant (Equation 1 2 1 T = 21 C; RH = 68%) and soybean (25-day-old; [CO ] = 400 mol mol ; PPF = 750 mol m s ; air 2 578 (10)). The effect of CO2 concentration on the diurnal course of GS was examined in wheat (Figure 9A; T = 21 C; RH = 64%) canopies are reported in Table 2. −2 −1 air 579 (18-day-old; PPF = 1600 µmol m s ; Tair = 21 °C; RH = 68%)) and soybean (Figure 9B; (27-day-old; −2 −1 580 PPF = 750 µmol m s ; Tair = 21 °C; RH = 64%)) canopies. Table 2. Canopy g , G , and W from two crop architectures at 400 umol mol CO and 86 kPa. A S 2 581 In the dark, Ω of both canopies was below 0.2, Ds was coupled to DBulk, and canopy transpiration 1 2 582 was small. Indeed, the nighttime VPDs within the wheat canopy (DAero 1.12 kPa, and DS 1.15 kPa) Species Architecture g Gs W Ds-D D A Bulk Bulk 583 were near the VPD (DBulk 1.27 kPa) of the chamber. When the lights came on, the stomata opened, the Wheat Erectophile 5.5 (7.5) 2.3 (18) 0.67 0.38 0.74 584 chamber humidity and Gs increased, the boundary layer within the canopy became humidified by Soybean Planophile 2.5 (16.5) 1.1 ( 37) 0.39 0.56 1.15 585 transpiration, and DBulk decreased du 1 ring the pho 2 1 top e1rio 2d. In wheat, VPDs within the canopy (DAero mol m s (s m ). kPa. −1 586 0.57 kPa and DS 0.36 kPa) became decoupled from DBulk (0.74 kPa). At 400 µmol mol CO2, the mean 587 daily Ω of wheat was 0.67 and 0.55 in soybean (Equation (10); Figure 9) due to the differences in their 588 corresponding GS and gA. Ω during the early part of the day rose to ~0.8 in wheat and to ~0.75 in 589 soybean. As the photoperiod progressed, Ω gradually declined to ~0.6 in wheat and to ~0.6 in soybean −1 590 as a consequence of a diurnal decrease in GS. At 1200 µmol mol CO2, Ω of wheat and soybean Agronomy 2019, 9, 114 16 of 23 3.6. Diurnal Changes in G Diurnal changes in W reflect changes in G because chamber g and D are constant (Equation (10)). S A Bulk Agronomy 2018, 8, x FOR PEER REVIEW 16 of 23 The effect of CO concentration on the diurnal course of G was examined in wheat (Figure 9A; 2 S 2 1 (18-day-old; PPF = 1600 mol m s ; T = 21 C; RH = 68%)) and soybean (Figure 9B; (27-day-old; air 591 reached a maximum o 2 f 0. 14 due to reduce d stomatal conductance and declined to near 0.2 at the end PPF = 750 mol m s ; T = 21 C; RH = 64%)) canopies. air 592 of the photoperiod (Figure 9). Figure 9. Diurnal changes in W of (A) wheat and (B) soybean at two chamber CO concentrations. 594 Figure 9. Diurnal changes in Ω of (A) wheat and (B) soybean at two chamber CO2 concentrations. In the dark, W of both canopies was below 0.2, Ds was coupled to D , and canopy transpiration Bulk 595 3.7. Control of Canopy Transpiration by CO2 Concentration was small. Indeed, the nighttime VPDs within the wheat canopy (D 1.12 kPa, and D 1.15 kPa) Aero S 596 The effect of CO2 concentration on canopy transpiration and Ω was explored using a wheat were near the VPD (D 1.27 kPa) of the chamber. When the lights came on, the stomata opened, Bulk −2 −1 597 canopy (27 to 34 day old; PPF = 1600 µmol m s ; Tair = 21⁰C; RH = 68%) exposed to varying chamber the chamber humidity and Gs increased, the boundary layer within the canopy became humidified −1 598 CO2 concentrations ranging between 400 and 1200 µmol mol (Figure 10; Table 3). by transpiration, and D decreased during the photoperiod. In wheat, VPDs within the canopy Bulk (D 0.57 kPa and D 0.36 kPa) became decoupled from D (0.74 kPa). At 400 mol mol CO , Aero S 2 Bulk the mean daily W of wheat was 0.67 and 0.55 in soybean (Equation (10); Figure 9) due to the differences in their corresponding G and g . W during the early part of the day rose to ~0.8 in wheat and to S A ~0.75 in soybean. As the photoperiod progressed, W gradually declined to ~0.6 in wheat and to ~0.6 in soybean as a consequence of a diurnal decrease in G . At 1200 mol mol CO , W of wheat and S 2 soybean reached a maximum of 0.4 due to reduced stomatal conductance and declined to near 0.2 at the end of the photoperiod (Figure 9). 3.7. Control of Canopy Transpiration by CO Concentration The effect of CO concentration on canopy transpiration and W was explored using a wheat 2 1 canopy (27 to 34 day old; PPF = 1600 mol m s ; T = 21 C; RH = 68%) exposed to varying air chamber CO concentrations ranging between 400 and 1200 mol mol (Figure 10; Table 3). The CO concentration was raised in steps from 400, to 700, to 950, and to 1200 mol mol and allowing a 48 h acclimation period at each CO concentration. Simulated decoupling coefficients 2 1 2 1 were calculated for increasing values of g (Figure 10; 2 mol m s (dotted line), 4 mol m s 2 1 (dashed line), and 8 mol m s (solid line)). The decoupling coefficient increased as canopy G 2 1 1 increased, reaching an average W of 0.65 with a canopy G of 2.1 mol m s at 400 mol mol CO S 2 (Figure 10; Table 3). Increasing CO concentration from 390 to 690 mol mol led to 1.77 X CO or 600 Figure 10. The decoupling coefficient, Ω, expressed as a function of canopy GS in wheat measured at 2 2 −1 601 nearly CO2 concentrations ranging between a doubling in ambient CO . This 400 and 1200 µmol mol change decreased mean . daily G by 35%, resulting in a 23% 2 S lower transpiration rate and a -23% reduction in latent heat (Table 3). Since g remained constant, −1 602 The CO2 concentration was raised in steps from 400, to 700, to 950, and to 1200 μmol mol and this decrease in G caused a 26% decrease in W. These results indicate that E is less sensitive to can 603 allowing a 48 h acclimation period at each CO2 concentration. Simulated decoupling coefficients were changes in stomatal conductance due to the decoupling of D from D . In addition, an increase in Bulk −2 −1 −2 −1 604 calculated for increasing values of gA (Figure 10; 2 mol m s (dotted line), 4 mol m s (dashed line), P of 15% and a 23% decrease in E resulted in a 150% increase in WUE (Table 3). net can −2 −1 605 and 8 mol m s (solid line)). The decoupling coefficient increased as canopy GS increased, reaching −2 −1 −1 606 an average Ω of 0.65 with a canopy GS of 2.1 mol m s at 400 µmol mol CO2 (Figure 10; Table 3). −1 607 Increasing CO2 concentration from 390 to 690 μmol mol led to 1.77 X CO2 or nearly a doubling in 608 ambient CO2. This change decreased mean daily GS by −35%, resulting in a −23% lower transpiration 609 rate and a -23% reduction in latent heat (Table 3). Since gA remained constant, this decrease in GS 610 caused a −26% decrease in Ω. These results indicate that Ecan is less sensitive to changes in stomatal Agronomy 2018, 8, x FOR PEER REVIEW 16 of 23 591 reached a maximum of 0.4 due to reduced stomatal conductance and declined to near 0.2 at the end 592 of the photoperiod (Figure 9). 594 Figure 9. Diurnal changes in Ω of (A) wheat and (B) soybean at two chamber CO2 concentrations. 595 3.7. Control of Canopy Transpiration by CO2 Concentration 596 The effect of CO2 concentration on canopy transpiration and Ω was explored using a wheat −2 −1 597 canopy (27 to 34 day old; PPF = 1600 µmol m s ; Tair = 21⁰C; RH = 68%) exposed to varying chamber Agronomy 2019, 9, 114 17 of 23 −1 598 CO2 concentrations ranging between 400 and 1200 µmol mol (Figure 10; Table 3). Figure 10. The decoupling coefficient, W, expressed as a function of canopy G in wheat measured at 600 Figure 10. The decoupling coefficient, Ω, expressed as a function of canopy GS in wheat measured at CO concentrations ranging between 400 and 1200 mol mol . −1 601 CO2 concentrations ranging between 400 and 1200 µmol mol . Table 3. Canopy level responses to elevated CO at constant PPFo and g . 2 A −1 602 The CO2 concentration was raised in steps from 400, to 700, to 950, and to 1200 μmol mol and CO Concentration (mol mol ) 603 allowing a 48 h acclimation period at each CO2 concentration. Simulated decoupling coefficients were Parameter Symbol Units 390 690 930 1230 −2 −1 −2 −1 604 calculated for increasing values of gA (Figure 10; 2 mol m s (dotted line), 4 mol m s (dashed line), 2 1 Transpiration E mmol m s 11.2 8.7 7.9 7.0 can −2 −1 605 and 8 mol m s (solid line)). The decoupling coefficient increased as canopy GS increased, reaching % 100 77 71 63 −2 −1 −1 606 an average Ω of 0.65 with a canopy GS of 2.1 mol m s at 400 µmol mol CO2 (Figure 10; Table 3). 2 1 Photosynthesis A mol m s 63 73 81 79 can −1 607 Increasing CO2 concentration from 390 to 690 μmol mol led to 1.77 X CO2 or nearly a doubling in % 100 115 128 125 Latent Heat LE 460 356 324 288 608 ambient CO2. This change decreased mean daily W G m S by −35%, resulting in a −23% lower transpiration Sensible Heat H W m 175 70 42 3 609 rate and a -23% reduction in latent heat (Table 3). Since gA remained constant, this decrease in GS 2 1 Stomatal Conductance G mmol m s 2145 1394 1152 1031 610 caused a −26% decrease in Ω. These results indicate that Ecan is less sensitive to changes in stomatal % 100 65 54 48 W dim. 0.65 0.48 0.45 0.39 Decoupling coefficient % 100 74 69 60 Water Use Efficiency WUE mol mmol 5.6 8.4 10.2 11.2 % 100 150 183 199 2 1 dEcan mmol m s - 2.5 3.3 4.2 Changes in E , Gs, LE can dLE - 104 136 172 W m as CO increased from 2 1 dG mol m s - 0.75 0.99 1.11 1 S 400 mol mol dE /dG mmol mol - 3.33 3.32 3.75 can The relative changes in G , E , and W of wheat as CO concentration increased, from Table 3, S can 2 are shown in Figure 11. Canopy G decreased by 52%, but E only decreased by 37% when CO S can 2 concentration was raised from 390 to 1230 mol mol because of the feedback between E and D . can S In these chamber settings, E is much less sensitive to a proportional change in G and the reduction can S in E is largely explained by W. can Agronomy 2018, 8, x FOR PEER REVIEW 17 of 23 611 conductance due to the decoupling of Ds from DBulk. In addition, an increase in Pnet of 15% and a −23% 612 decrease in Ecan resulted in a 150% increase in WUE (Table 3). 613 Table 3. Canopy level responses to elevated CO2 at constant PPFo and gA. −1 CO2 Concentration (µmol mol ) 390 690 930 1230 Units Parameter Symbol −2 −1 Transpiration Ecan mmol m s 11.2 8.7 7.9 7.0 % 100 77 71 63 −2 −1 Photosynthesis Acan µmol m s 63 73 81 79 % 100 115 128 125 −2 Latent Heat LE W m 460 356 324 288 −2 Sensible Heat H W m −175 −70 −42 −3 −2 −1 Stomatal Conductance GS mmol m s 2145 1394 1152 1031 % 100 65 54 48 Decoupling coefficient Ω dim. 0.65 0.48 0.45 0.39 % 100 74 69 60 −1 Water Use Efficiency WUE µmol mmol 5.6 8.4 10.2 11.2 % 100 150 183 199 −2 −1 dEcan mmol m s - 2.5 3.3 4.2 Changes in Ecan, Gs, LE −2 dLE W m - 104 136 172 as CO2 increased from −2 −1 dGS mol m s - 0.75 0.99 1.11 −1 400 µmol mol −1 dEcan/dGS mmol mol - 3.33 3.32 3.75 615 The relative changes in GS, Ecan, and Ω of wheat as CO2 concentration increased, from Table 3, 616 are shown in Figure 11. Canopy GS decreased by 52%, but Ecan only decreased by 37% when CO2 −1 617 concentration was raised from 390 to 1230 μmol mol because of the feedback between Ecan and DS. 618 In these chamber settings, Ecan is much less sensitive to a proportional change in GS and the reduction Agronomy 2019, 9, 114 18 of 23 619 in Ecan is largely explained by Ω. Figure 11. Relative changes in G , E , and W as chamber CO concentration increases. S can 2 621 Figure 11. Relative changes in GS, Ecan, and Ω as chamber CO2 concentration increases. 4. Discussion 622 4. Discussion 4.1. Canopy Stomatal Conductance Land components of climate and carbon models require accurate descriptions of the stomatal 623 4.1. Canopy Stomatal Conductance control of canopy energy exchange, evapotranspiration, and carbon exchange because land surfaces 624 Land components of climate and carbon models require accurate descriptions of the stomatal provide a continuous feedback of latent and sensible heat fluxes to the atmosphere, which drives 625 control of canopy energy exchange, evapotranspiration, and carbon exchange because land surfaces weather and climate [45]. The method developed in this study expands the usefulness of controlled environments for improving land surface models because it allows the measurement of responses of canopy-level G , an essential control of canopy gas exchange, to environmental variables. In this study, G was measured when surface radiation forcing (R ), boundary layer forcing S net (T & D ), surface layer feedbacks (g ), and soil moisture were held constant during the air Bulk A photoperiod. Furthermore, the use of well-watered plant stands grown at constant light reduced much of the environmental variability that confounds estimates of G in natural ecosystems, such as periodic drought, the diurnal change in solar radiation, or short, temporal fluctuations in radiation due to cloud cover. Moreover, the carbon and water vapor fluxes measured in this study do not include significant contributions from soil respiration and evaporation as compared to field measurements. Canopy G was derived from direct measurements of surface G , g , and energy balance (R , S SFC A net LE, and P) in controlled environments. Once the IR transducers were positioned above the canopy, chamber CO concentration was manipulated to alter stomatal conductance, which in turn resulted in corresponding changes in sensible heat flux and the canopy–air temperature difference. Canopy g was obtained radiometrically from the slope of a plot of H vs. DT (Figure 7), and the offset correcting IR for differences between T and T was determined. R aero The radiometric method presented here differs from other methods for calculating G because canopy g E , and canopy-to-air temperature differences are measured directly. This avoids A, can the complexity of methods for scaling leaf level observations to the canopy scale because these must integrate responses of leaf stomatal conductance to vertical profiles in radiation, temperature, humidity, and wind speed within a canopy. Separating g from the measured canopy G permits the A SFC determination of the physiologically controlled canopy-scale G , which is equivalent to the “big-leaf” stomatal conductance, where the stomatal conductances of individual leaves of the canopy act in parallel, and the vertical gradients in temperature and humidity are averaged by the aerodynamic T and D . The strength of this approach is that canopy G responses are measured at the correct Aero Aero S scale for predicting ET in future elevated CO and climate change scenarios. Furthermore, estimates of canopy ET made using the measured G also account for the feedback of Ds on ET, as shown by the decoupling coefficient. Agronomy 2019, 9, 114 19 of 23 In the chamber, g was set constant by the air flow rate provided by its recirculation fans. However, the g established in the chamber was different for each species when measured at the same 2 1 1 turbulent field provided by the chamber fans. The g for the wheat (5.5 mol m s or 7.5 s m ) and 2 1 1 for the soybean (2.5 mol m s or 16.5 s m ) canopies were within the range of typical aerodynamic 2 1 1 conductances of field crops (ranging from 3.2–10 mol m s or 4–13 s m ; [46]). A smaller g for soybean compared to wheat reflects a larger canopy boundary layer associated with the broader soybean leaves. In this study, G values of wheat and soybean were measured at an elevation of 1460 m (4800 ft), a 1 2 1 barometric pressure of 86 kPa, and 400 mol mol CO (Table 2). For wheat, G was 2.3 mol m s 2 S 1 2 1 1 or 18 s m , which is slightly higher than field G values (1.8 mol m s or 22.7 s m ) reported by Hatfield [47] at sea level, under optimal available soil water. The G of soybean was also slightly higher than typical conductances measured in field crops [46]. Soybean G at 400 mol mol CO was S 2 2 1 1 1.1 mol m s or 37 s m , nearly one-half the value found in wheat probably due to less leaf area and because it was measured at a lower PPF . The G values of this study are expected to be higher than those o S measured at sea level because, at lower atmospheric pressures, the diffusion coefficients of water vapor and CO in air increase, so G also increases [48,49]. 2 S 4.2. The Control of Transpiration by CO Concentration The radiometric method developed in this study was used to determine the response curve of G to CO concentration in wheat (Figure 11, Table 3). As CO concentration increased, the measured 2 2 decrease in E was lower than the measured decrease in G because feedback between E and can can D operating at the canopy scale effectively reduces the sensitivity of E to changes in G . Thus, a S can S smaller change in E was observed as CO increased, and the reduction in E is largely explained can can by changes in W, which are determined by the relative magnitudes of g and G . A S In this study, an increase of 1.77 X CO (that is, an increase from 390 to 690 mol mol ; Table 3) caused a 23% drop in E and a +15% increase in photosynthesis. These changes are comparable can to the results of Friend and Cox [50], who used a combined climate-vegetation model to predict a similar 25% drop in ET and a +19.4% increase in GPP for a doubling ambient CO (2 X CO ). In a 2 2 four-year SoyFACE study, Bernacchi et al. [51] found a 9–16% reduction in canopy ET and reported that meta-analyses across FACE experiments indicate a 17–22% drop in leaf level stomatal conductance 1 1 when daytime CO was raised by 175 umol mol from 375 to 550 umol mol . In this study, the two regression equations in Figure 11 (E and G as a function of CO concentration) predict a 13% can S 2 decrease in E and a 22% decrease in canopy G for an increase of 175 umol mol of CO . These can S 2 comparisons suggest that the responses of G and E to CO reported in this study are similar to S can 2 responses in canopy G and ET observed in CO -enriched plant canopies in field settings. S 2 5. Conclusions The controlled-environment experiments conducted in this study provide a new methodology for measuring canopy stomatal and aerodynamic conductances. A radiometric method for determining canopy aerodynamic conductance from changes in vegetation temperature and energy balance was developed in controlled environments using a canopy-level gas exchange system. The gas exchange system measured canopy gas fluxes (CO and water vapor), energy balance (net radiation, latent and sensible heat fluxes), and canopy temperatures as CO concentration was varied. Two key assumptions of this method are that radiative canopy temperature is approximated by canopy brightness temperature and that the difference between aerodynamic and radiative canopy-to-air temperature differences is constant during the photoperiod. Once canopy aerodynamic conductance was determined from a plot of sensible heat flux versus the radiative canopy-to-air temperature difference, canopy stomatal conductance was calculated from measurements of canopy transpiration. The method was used to determine the curves of the response of canopy stomatal conductance and canopy ET to increased CO concentration in wheat (Table 3; Figure 11). Predictions of canopy ET 2 Agronomy 2019, 9, 114 20 of 23 made from the measured response of G to elevated CO are comparable to land surface model S 2 predictions and to observed changes in ET found in FACE studies [50,51]. Future work should focus on studying how canopy stomatal conductance measured using this methodology responds to drought, vapor pressure deficit, and temperature to provide data sets for calibrating global climate models. The method should also be used to characterize how canopy aerodynamic conductance changes during the growth cycle of different crop species. Author Contributions: O.M. and B.B. conceived the study. O.M. wrote the original manuscript. O.M. and B.B. reviewed and revised the manuscript. Project administration and funding acquisition: B.B. All authors read and approved the manuscript. Funding: This research was supported by the Advanced Life Support Program of the National Aeronautics and Space Administration and by the Utah State Agricultural Experiment Station, Utah State University. This manuscript has been approved as journal paper number 9178. Acknowledgments: The authors would like to thank John Norman, Marc Van Iersel, and Larry Hipps for critically reviewing the manuscript. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Abbreviations Symbol Description Units 3 1 C Heat capacity of air at constant pressure kJ m C [CO ] CO concentration umol mol 2 2 DAE Days after emergence d 2 1 E Chamber evaporation rate mmol m s 2 1 Ecan Canopy transpiration rate mmol m s 2 1 ET = DX *MF Chamber evapotranspiration mmol m s h20 2 1 g Single leaf stomatal conductance mol m s 2 1 g Canopy aerodynamic conductance mol m s G Soil heat flux W m 2 1 G Canopy surface conductance mol m s SFC 2 1 G Canopy stomatal conductance mol m s H Sensible heat flux W m LE Latent heat flux W m "Lc Longwave radiation emitted by the canopy W m #Lg Longwave radiation emitted by the glass of the water filter W m MF Mass flow rate of air mol s NPSW Absorbed non-photosynthetic shortwave radiation W m abs Offset Difference between DT and DT C A IR P Energy storage in photosynthesis W m 2 1 P Canopy net photosynthetic rate mol m s net 2 1 PPF Incident photosynthetic photon flux mol m s 2 1 PPF Fraction of incident PPF absorbed by the canopy mol m s abs R Net radiation W m net Slope of the relation between saturation vapor pressure and temperature T Mean air temperature measured above the canopy C air T Canopy aerodynamic temperature C Aero T Canopy brightness temperature measured by IR transducers C canopy,IR T T Water filter glass and chamber wall temperatures C glass, wall T Canopy radiometric temperature C T Composed of 20% chamber T and 80% T C Sky wall glass Agronomy 2019, 9, 114 21 of 23 Psychrometric constant kPa K DT Aerodynamic canopy-to-air temperature difference (T T ) C A Aero air DT Radiometric canopy-to-air temperature difference (T T ) C IR R air DX Mole fraction difference between pre- and post-chamber water vapor h20 ratio of the increase of latent heat content to the increase of sensible " = s/ heat content of saturated air " Canopy emissivity $ Density of air kg m $ Canopy reflection coefficient 2 4 Stefan–Boltzman constant W m K Scattering coefficient W Decoupling coefficient X (T ) Mol fraction of water vapor at T above the canopy H2O air air X (T ) Mol fraction of water vapor at T H2O Aero Aero References 1. 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