The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s election without traverse of group I, claims 1-6 and 8-9 in the reply filed on December 11, 2024 is acknowledged. Claim 7 is withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected invention.
Examiner makes the following observations regarding the currently examined claims and the originally examined claims in the parent application. Each of the currently examined independent claims requires solving a chamber mass balance equation as part of a determining step. The originally examined claims did not include that language but require determining assimilation rates on an empty chamber as well as determining an apparent assimilation rate on the same chamber containing a photosynthesis capable sample. This is followed by determining a net assimilation rate by determining the net assimilation range through subtracting the empty chamber assimilation rate value from the apparent assimilation rate value. The currently examined independent claims do not require the measurements on an empty chamber, but are written in open language so that they cover the possibility of extra steps that could be needed to perform the determining step with a mass balance equation. Since the currently examined claims require a mass balance equation in the determining step, examiner attempted to determine the scope of what the instant disclosure describes/teaches as a mass balance equation.
The first description of note is found in instant paragraph [0022] of the originally filed specification. It teaches that the “embodiments disclosed herein provide novel analytical systems and methods for measuring plant leaf gas exchange based upon instantaneous mass balance in the leaf sample chamber, due to the close physical proximity of the gas analyzer(s) to the points (i) where the incoming airflow is divided into sample and reference air flows, (ii) where the sample flow rate is measured and enters the leaf chamber, and/or (iii) where the sample flow leaves the leaf chamber.” The paragraph further explains that the “close physical proximity of the gas analyzers to the points (i) where the incoming airflow is divided into sample and reference air flows, (ii) where the sample flow rate is measured, and (iii) where the sample flow leaves the leaf chamber, makes it possible to perform a near instantaneous mass balance on gases entering and leaving the leaf chamber.” The paragraph clearly teaches the “close proximity as “an important characteristic (1) allowing near instantaneous measurement of gas concentrations entering and leaving the leaf chamber and (2) for reducing diffusive sources and sinks.” The paragraph teaches that the measurements can be made just outside or inside the chamber as the air flow enters and leaves the chamber. In other words, the mass balance is based on measuring the gas flow entering and leaving the chamber. It is noted that for an “instantaneous mass balance” the relative proximity to the chamber of the measurement points of the gas flow into and out of the chamber is important.
The next description of note is found in instant paragraphs [0024]-[0026] of the originally filed specification. In particular instant paragraph teaches that in the new approach includes applying analyses that exploit the ability to measure instantaneous mass balance in the leaf chamber due to the close proximity of components as mentioned above. The paragraph is also clear that two examples will illustrate the principles. Thus each of these examples would constitute use of mass balance or a mass balance equation in the process of performing the described embodiments.
The first example is given in instant paragraph [0025] which describes a method in which measurements are made on an empty chamber and a chamber containing a photosynthesis capable sample and resulting measurements are combined together to determine the desired answer. While the "mass balance” language is not found in the paragraph, the described process is a mass balance process because the gas flowing into and from the chamber is measured and used to determine the assimilation of carbon dioxide by the photosynthesis capable sample. Thus, although not explicitly using the mass balance language, this paragraph describes an example that one of ordinary skill in the art would have considered to be using a mass balance equation. It is noted that the described method appears to be within the scope or the claims previously examined in application 15/811210.
The second example is given in instant paragraph [0026] which describes the "Integration Method" to produce an average assimilation from the mass balance of the gas flowing into and out from the chamber. This appears to be describing at least what is found in instant claims 8-9.
The next description of note is found in instant paragraph [0035] instant paragraph teaches equation 1 as representing the chamber mass balance. It is noted that equation 1 is required in instant claims 3 and 6. It is further noted that paragraph [0035] teaches that preliminary tests were performed with an empty chamber although it “does not require an empty chamber test to be paired with each sample measurement”. In other words, although the paired empty chamber test is not required it is not prohibited by the instant disclosure. Another way of looking at this language is that a preliminary empty chamber test may be performed and the correction determined therefrom can be used repeatedly rather than performing an empty chamber test with each sample measurement.
Relative to the currently examined claims, examiner notes that the mass balance equation language of the independent claims does not require any specific form. Thus, any equation that can be considered a mass balance equation is within the scope of these claims. As a result of the above analysis examiner will treat claims currently examined independent claims as having a scope that covers any mass balance equation and also covers additional steps such as making measurements on an empty chamber and combining the two sets of measurements to perform the required determination of assimilation. Claims 2 and 5 will also be treated at a similar scope since they do not modify the scope of claims 1 or 4 relative to the specific mass balance equation used or by excluding an empty chamber test as part of the process being performed. Claims 3 and 6 will be treated as having a scope that requires the specific mass balance equation being claimed but will be treated as including the possibility of performing tests on an empty chamber to provide the needed corrections even though the empty chamber test is not paired/performed with each sample measurement.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-6 and 11-15 are rejected under 35 U.S.C. 103 as being unpatentable over Kositsup (Photosynthetica 2010) in view of Hari (Plant, Cell and Environment 1999), Johnson (US 2012/0073355), Nugawela (Journal of Natural Rubber Research 1995) and McDermitt (Ann 1989), Andrews (Physiology and management of mangroves. Tasks for vegetation science 1984, newly cited and applied), Thorpe (Journal of Experimental Botany 1991, newly cited and applied), Lawton (Philosophical Transactions: Biological Sciences 1993, newly cited and applied), Jost (Geophysical Research Abstracts 2014, newly cited and applied) or Marynick (Plant Physiology 1975) and further in view of van Iersel (Journal of the American Society for Horticultural Science 1999) or Long (Journal of Experimental Botany 2003, 54, 2393-2401). In the paper Kositsup investigated the effect of leaf age and position on light-saturated CO2 assimilation rate, photosynthetic capacity, and stomatal conductance in rubber trees. Shoots of the tropical latex-producing tree Hevea brasiliensis (rubber tree) grow according to a periodic pattern, producing four to five whorls of leaves per year. All leaves in the same whorl were considered to be in the same leaf-age class, in order to assess the evolution of photosynthesis with leaf age in three clones of rubber trees, in a plantation in eastern Thailand. Light-saturated CO2 assimilation rate (Amax) decreased more with leaf age than did photosynthetic capacity (maximal rate of carboxylation, Vcmax, and maximum rate of electron transport, Jmax), which was estimated by fitting a biochemical photosynthesis model to the CO2-response curves. Nitrogen-use efficiency (Amax/Na, Na is nitrogen content per leaf area) decreased also with leaf age, whereas Jmax and Vcmax did not correlate with Na. Although measurements were performed during the rainy season, the leaf gas exchange parameter that showed the best correlation with Amax was stomatal conductance (gs). An asymptotic function was fitted to the Amax-gs relationship, with R² = 0.85. Amax, Vcmax, Jmax and gs varied more among different whorls in the same clone than among different clones in the same whorl. They concluded that leaf whorl was an appropriate parameter to characterize leaves for the purpose of modelling canopy photosynthesis in field-grown rubber trees, and that stomatal conductance was the most important variable explaining changes in Amax with leaf age in rubber trees. Of particular relevance to the instant claims are the second full paragraph of page 68 teaching that all gas exchange measurements were made on attached leaves with a portable photosynthesis system (LI-6400). Light was supplied with red-blue light emitting diodes (6400-02B LED light source). The CO2 concentration of the reference air (360 μmol mol–1) entering the leaf chamber was controlled with a CO2 mixer. Air relative humidity and leaf temperature were maintained constant at ambient level during the measurement. Also relevant toward the claims is the paragraph bridging pages 69-70 teaching that CO2-response curves were fit according to the Farquhar model in which net assimilation (A) is limited either by the activity of Rubisco at saturating RuBP (Ac) or by RuBP concentration (Aj). Photosynthetic capacity (Vcmax and Jmax) was estimated from the response of A to intercellular leaf CO2 concentration (Ci, CO2-response curve or A/Ci curve). The recording of an A/Ci curve was started at an ambient CO2 concentration of 360 µmol mol–1 and a saturating PPF of 1600 µmol m–2 s–1. The leaf chamber was equilibrated for at least 15 min in order to reach a steady state. The CO2 concentration was then decreased stepwise to 250, 200, 150, 100, and 50 µmol mol–1 and then increased stepwise from 360 to 600, 800, 1,000, 1,100, 1,200, 1,400 and 1,600 µmol mol–1 to obtain Ac and Aj parts of each full curve (RuBP carboxylation and regeneration limited parts of A/Ci curve, respectively). The value of A at each concentration was recorded only once A and gs had stabilized. Leaf temperature was maintained at a constant ambient level during the measurement cycle. A/Ci curves were fit by nonlinear least squares regression using commercially available software, assuming where P25 is the value of Vcmax (or Jmax) at 25 °C, P is the value of Vcmax (or Jmax) at leaf temperature, Tref is the reference temperature of 25 °C (298.15 K), Ea is the activation energy [J mol–1], R the gas constant (8.314 J K–1 mol–1) and T is leaf temperature [K]. Also relevant is figure 1 showing an example of one A/Ci curve fitting of a 2-year-old potted RRIM 600 clone. Farquhar’s model was fit to the data of the response of light-saturated CO2 assimilation rate (Amax) to intercellular CO2 concentration (Ci) in order to estimate Vcmax and Jmax. Kositsup does not teach the specifics of the LI-6400 portable photosynthesis system structure, using a continuously varied concentration over the CO2 concentration ranges measured or measuring the A/Ci values in an empty leaf chamber.
In the paper Hari teaches an improvement in a method of calibrating photosynthetic carbon dioxide flux. Measurements of rapid changes in concentrations and fluxes of gaseous compounds relating to photosynthetic gas exchange are commonly performed using flow-through cuvettes in connection with infrared gas analyzers. The accuracy and repeatability of these measurements relies ultimately upon the design of the system as a whole, rather than upon each of its components, and therefore the calibration and testing of the system should be performed keeping this in mind. Presented is a simple and efficient method for the calibration of such a measurement system using a precisely determined CO2 flow. This method provides the opportunity to take into account any disturbing effects caused by undesired properties of the chamber or tubing materials. With the proposed calibration method, the accuracy of the CO2 flux measurement is improved from 8% up to the level of 2%, determined mainly by the accuracy of the control gas used for calibration of the CO2 analyzer. The paragraph bridging the columns of page 1297 teaches that although straightforward in principle, chamber studies are subject to various uncertainties: modifications of flow and turbulence, variations in pressure and temperature, adsorption and reactions with wall materials, uncertainties in calibration procedures, influences of biological processes and the overall representativity of the measured object. CO2 and H2O are rather inert molecules compared with many reactive pollutants, but their interactions with all the construction materials should not be ignored in accurate measurements of their net exchange by leaves, shoots, branches or a soil surface. Starting on page 1298 the calibration principle based on mass balance in the chamber (cuvette) is explained. The CO2 concentration inside the cuvette depends on the sum of different sources and sinks. Generally, the sources consist of the ambient air pumped or leaking into the cuvette, possible emissions from the fragment of the plant and cuvette surface, and also the atmospheric chemical reactions that produce the substance considered. Sinks are the sampled air, deposition into the plant fragment and cuvette surface, and chemical conversion. Figure 1 presents a flow chart of the CO2 fluxes in the measurement system. P denotes the flux due to the presence of a sink (photosynthetic CO2 flux), Cc the CO2 concentration in the cuvette, qin the compensating air flow and Cin the CO2 concentration in it, qs the air flow rate into the gas analyzer, qout the leakage from the cuvette caused by the difference between qc and qs, and qk the extra gas flow and Ck the CO2 concentration in it. During calibration, p is replaced with qk Ck and qk Cc. Equation (1) is related to the photosynthetic CO2 flux and is obtained from the mass-balance equation. Equation (2) is a rearrangement of equation (1) that results from adding a known sink through adding an extra flow. In equation (2) the left-hand side gives the CO2 flux generated into the cuvette, and the right-hand side is identical with the photosynthetic CO2 flux measured within the cuvette. Thus, a calibration coefficient was derived for the measurement of CO2 flux as a ratio between the generated and measured fluxes. Subsequently the method was applied to a chamber/cuvette made of a material known to have a substantial diffusivity toward CO2.
In the Johnson patent publication, figure 1a illustrates the flow path in a prior photosynthesis measurement system where the flow is split at the console, remote from the sensor head. Paragraph [0038] in describing experiments using the LI-COR Biosciences LI-6400 teaches that diffusion through flexible tubing, gaskets, and pneumatic connectors is a significant and quantifiable issue. In one experiment, to eliminate diffusion sources/sinks from the leaf chamber, the chamber was removed from the LI-6400 head (see, e.g., figure 1a) and was replaced with an aluminum block and a vinyl gasket as shown in FIG. 2. In other words, figure 1a shows a structure of an LI-6400 instrument. Paragraph [0009] references figure 1a and teaches that in open photosynthesis systems, a conditioned air stream is typically split into two streams at the console, remote from the sensor head, and flows to the sensor head via two separate paths as illustrated in figure 1a. The first flow path (known as reference) passes through a gas analyzer (e.g., Infra-Red Gas Analyzer or IRGA) which measures constituent gas concentrations (CO2 and/or H2O). The second flow path (known as sample) passes through a sample chamber (leaf chamber) in which gas exchange occurs. This second sample flow path exits the chamber and enters a second gas analyzer (e.g., IRGA). The difference between the sample and reference gas concentrations is used to calculate photosynthesis (CO2) and transpiration (H2O). As photosynthesis and transpiration measurements are based on concentration differences in these two gas streams, the accuracy in measuring the difference is more critical than measuring the absolute concentration of either. Diffusive parasitic sources and/or sinks present in the tubing, connectors, and fittings that supply the head with the sample and reference gas streams can compromise measurement accuracy. Paragraph [0012] teaches that embodiments provide system flow path designs that help minimize the impact of diffusion. By reducing the magnitude of parasitic source and sinks, lower rates of photosynthesis and transpiration can be more accurately measured, e.g., without the need for extensive empirical compensation. Paragraphs [0034]-[0035] teach that figure 1b illustrates an embodiment of a flow path in a gas exchange measurement system (10) . The gas exchange measurement system includes a console (15) and a sensor head (20) remote from the console. The console typically includes, or is connected with, one or more gas sources and gas conditioning equipment. For example, in the context of photosynthesis and transpiration measurements, gas sources would include reservoirs of CO2 and H2O, and conditioning equipment for conditioning each gas concentration. A flow path (17) connecting the console with the sensor head typically includes flexible tubing and connectors. The flow path provides a single stream or gas flow path to a flow splitting mechanism (25) in the sensor head. The flow splitting mechanism receives a stream of gas from the console and splits the flow into two separate flow paths. One stream is provided to the chamber (30) (e.g., sample stream) and the other stream (e.g., reference stream) is provided to a reference gas analyzer (50). A second gas analyzer (40) receives and analyzes gas from the chamber. Both gas analyzers might each include an Infra-Red Gas Analyzer (IRGA), as is known in the art, or other gas analyzer. It is desirable that flow path lengths and the number of connections downstream of the flow split device be minimized to reduce parasitic sources and sinks which differentially affect concentrations in the two flow paths. Hence, the flow path is split in the sensor head proximal to the sample chamber. The majority of parasitic sources and sinks, which are located upstream of the sensor head in figure 1b, affect only a single air stream (flow path 17) when the flow is split at the sensor head. Parasitic sources and sinks which impact the sample and reference streams independently are advantageously minimized. Paragraph [0047] teaches an open-path gas exchange analysis system that includes a first gas analyzer (e.g., IRGA) configured to measure a first concentration of a gas entering the gas inlet port of the sample chamber at a plurality of times and a second gas analyzer (e.g., IRGA) configured to measure a second concentration of the gas exiting the gas outlet port of the sample chamber at the plurality of times. The enclosed sample chamber defines a measurement volume for analysis of a sample, where the gas inlet port of the sample chamber is coupled with a gas source. The system also includes a processing module, communicably coupled with the first and second gas analyzers, configured to determine at each of the plurality of times a concentration differential between the first measured concentration and the second measured concentration. The results (data) can be output, displayed or otherwise provided to another system or device for further manipulation. A method of measuring a concentration of a gas in such a gas exchange analysis system includes measuring a first concentration of a gas at the input port of the sample chamber at each of a plurality of times, measuring a second concentration of the gas at the output port of the sample chamber at each of said plurality of times, and thereafter determining at each of the plurality of times a concentration differential between the first measured concentration and the second measured concentration. In certain aspects, the gas includes CO2 and/or H2O.
In the paper Nugawela studied genotypic variation in non-steady state photosynthetic carbon dioxide assimilation of Hevea brasiliensis plants. These genotypes are described in the second paragraph on page 267. The second full paragraph on page 267 describes the gas exchange system as an open gas exchange system. The leaf section chamber is described in the paragraph bridging the columns of page 267. The last full paragraph on page 267 teaches that measurements were made using attached leaflets, under controlled conditions, using the open gas exchange system. Particularly relevant to the instant claims is the paragraph bridging pages 268-269. This paragraph teaches that an apparent lag in the response of the leaf to a step change in light could be caused by the time taken to replace the air in the I.R.G.A. analysis cell in addition to the time required for physiological adjustment by the leaf. To correct for this system error, a step change in CO2 differential was produced by a gas diluter. The time taken for the system to respond fully to a 1 p.p.m. increase or decrease in CO2 was 1.5 s and 2.5 s, respectively. The computations of CO2 uptake loss due to the actual lag in response to light fluctuations and the undershoot in CO2 assimilation rates when lowering light levels were made as described in Figure 1. These are rapid responses to small changes in this open system. The first full paragraph on page 274 discusses changes that occur in the non-steady state and teaches that this emphasizes the importance of the non-steady state responses and genotypic differences.
In the paper McDermitt teaches that CO2 response curves can be measured with a field-portable closed-loop photosynthesis system. Assimilation rate versus internal CO2 response curves provide an important tool for assessing the efficiency and capacity of the photosynthetic system. Prior to the paper, measurement of CO2 response curves was mostly limited to laboratory studies, where elaborate gas exchange systems were available, or to mobile field laboratories. The paper reports the use of a portable photosynthesis system (LI-6200) for measurement of response curves. The LI-6200 uses a closed-loop design in which continuously varying CO2 concentrations are provided as the leaf removes CO2 from the system. A typical measurement requires 10-25 minutes, depending upon chamber volume, leaf area and assimilation rate. Response curves measured on well-watered soybean and cotton were compared to those measured with a fully controlled steady state system (see figure 1. The last two paragraphs of page 417s teach that a baseline CO2 response curve was measured by placing a single soybean leaflet in the assimilation chamber of the LI-6200 and allowing the leaflet to remove CO2 until the compensation point was reached. Assimilation rate, conductance and internal CO2 concentration were computed every 5 ppm or so as the chamber CO2 mole fraction declined. This was repeated 2 more times and all curves were coincident. A 4th curve was prepared in which the CO2 mole fraction was held constant (± 5 µmol•mol-l) for 5 minutes at 7 different levels using a CO2 injector. Assimilation, conductance and Ci were then measured in transient mode by allowing the CO2 mole fraction to decline a few ppm from each of the preset levels. Since the curve measured by continuous drawdown is coincident with that measured after a 5 minute equilibration at each CO2 level, they concluded that the 2 methods are equivalent. Soybean leaflets are evidently able to maintain a quasi-steady state with a slowly declining (0.01-1 ppm•s-1) external CO2 concentration. Three other experiments gave the same result. To further evaluate results obtained with the LI-6200, response curves were measured on soybeans with a steady state system and side-by-side measurements were made on the same leaves under similar conditions with the LI-6200 (see figure 2). Correspondence between the 2 methods is generally excellent except that the CO2 compensation point is slightly overestimated by the LI-6200. They taught that at low chamber CO2 mole fractions, a large CO2 gradient exists between chamber air and ambient air exaggerating chamber leaks that are normally small. Leaks would cause an underestimation of the assimilation rate, and consequently, an overestimation of the compensation point. The effects of system leaks and control of leaf temperature were tested and discussed. The first two paragraphs of page 418s teach that chamber leaks can be modeled by the following expression: (dCchamber/dt) = (Cambient – Cchamber/) in which dCchamber/dt is the CO2 change rate due to chamber leaks (s-1), Cambient is the CO2 mole fraction of ambient air surrounding the chamber (µmol•mol-l or ppm), Cchamber is the chamber CO2 mole fraction, and is the leak rate time constant (s). A simple leak test was performed by first reducing the chamber CO2 mole fraction to 50-100 ppm using the system CO2 scrubber, and then measuring the rate of CO2 increase (dCchamber/dt) with a filter paper leaf replica in the chamber. Since the chamber CO2 mole fraction is always known, and the ambient CO2 mole fraction is constant and easily measured, can be computed. They found that t was constant and independent of the CO2 gradient for a given set of conditions. Once , Cchamber and Cambient are known, the leak rate can be computed and subtracted from the measured CO2 change rate. The LI-6200 can be programmed to calculate the leak rate and correct each assimilation measurement as the chamber CO2 mole fraction declines. Additionally, both corrected and uncorrected data can be stored. As the experiments reported in figures 2-5 progressed, declined from about 15,000 s to about 7,000 s, presumably due to chamber gasket deterioration. The effects of leaks on the LI-6200 data from figure 2 are shown in figure 3 for 2 values of . Chamber leaks have important effects at low chamber CO2 mole fractions, but negligible effects at ambient levels. In ordinary photosynthesis measurements where CO2 concentrations are near ambient, only small gradients exist to drive CO2 diffusion into the chamber, so chamber leaks are not a problem. However, when CO2 response curves are being measured, leak tests should be performed regularly, and the data corrected accordingly. Figure 4 shows the LI-6200 data from figure 2 after the leak correction was applied. The correspondence between the steady state and LI-6200 results is excellent. Similar results were obtained in a 2nd experiment. The paragraph bridging the columns of page 419s teaches that these and other experiments support the conclusion that well-watered C-3 plant leaves are able to maintain a quasi-steady state with respect to CO2 mole fractions which change at the rates observed in typical experiments (e.g., 0.01-1 ppm•s-1). Under these conditions, the transient approach provides a valid method for measuring CO2 response curves. It is rapid and convenient inasmuch as it does not require a series of mixed gasses or long equilibration times, and it can be performed with a compact and portable instrument.
In the paper Andrews examined the gas exchange properties of fully expanded leaves on mature trees of Rhizophora stylosa and R. apiculata using a measurement system based on infrared gas analyzers and dew point hygrometers. In some cases, CO2 and water vapor exchange were followed in diurnal ‘tracking’ experiments under natural illumination. Leaf temperature and the vapor pressure deficit inside the leaf chamber were maintained under changing conditions which simulated those experienced by other leaves in the canopy at the time. These experiments revealed that leaf temperature was a key factor controlling assimilation under natural conditions. In other experiments, the response of gas exchange parameters to variation in CO2 partial pressure, light flux and leaf temperature under otherwise constant conditions were studied using artificial illumination. The stomata were found to be very sensitive to changes in environmental conditions, irrespective of whether these changed when simulating canopy conditions or in response to experimental manipulation. In both cases there appeared to be parallel changes in stomatal conductance and assimilation rate so that the calculated CO2 partial pressure in the intercellular spaces was maintained more or less constant. The only case when this nexus between conductance and assimilation rate was not complete occurred when CO2 partial pressures were manipulated. The paragraph bridging the columns of page 16 teaches that gas exchange by a single leaf enclosed in a well-ventilated chamber, constructed of aluminum and glass, was measured with infrared gas analyzers (CO2), dew point hydrometers (water vapor) and a mass flow meter (air flow). For some experiments the gas exchange system was used in an open-ended mode and for others in a closed configuration. Leaf temperature was measured with two fine-gauge copper-constantin thermocouples fixed with transparent tape to the upper surface of the leaf close to either end. The photon flux density of photosynthetically active radiation ( 400-700 nm) inside the chamber was measured with a Lambda quantum sensor. Leaf temperature and the partial pressures of CO2 and water vapor inside the chamber were controlled using the appropriate transducers and a small computer. Under steady state conditions it was possible to control leaf temperature to ± 0.2 °C, pH2O to ± 0.2 mbar and pCO2 to ± 2 μbar (approx. ± 2 ppm). Under non-steady state conditions (i.e. when forcing conditions inside the chamber to track those prevailing outside) the degree of control was in some cases slightly worse (see figure 1). The computer was also used for data acquisition, providing an on-the-spot output of both primary and derived data. This allowed experimental procedures and conditions to be varied immediately. The following paragraph on page 16 teaches that in some experiments, CO2 and water vapor exchange were followed in 'diurnal tracking experiments' under natural illumination, in which leaf temperature and water vapor pressure inside the chamber were controlled to simulate conditions measured with an independent set of sensors placed on or near other leaves in the canopy outside the leaf chamber. In other experiments, the response of photosynthesis and transpiration to variation in the CO2 partial pressure, light flux and leaf temperature, under otherwise constant conditions, were studied using artificial illumination provided by a multi-metal halide discharge lamp. The first full paragraph on page 18 teaches that an example of the kind of tracking results obtained are shown in figure 1. These data were obtained using a reference leaf artificially held horizontal outside the chamber and a CO2 partial pressure of 400 μbars. The paragraph bridging the columns of page 19 teaches that it was evident from the 'tracking' experiments that the stomata were remarkably responsive to small increases in leaf temperature when the latter was in the supra-optimal range. This was also found in the course of the other described experiments. By contrast, the response of the stomata to a stepwise reduction or increase in light was slower, and periods of up to 30 minutes at a given photon flux density were often required before the stomatal conductance settled to a relatively constant value.
In the paper Thorpe described a technique is for continuous, simultaneous and in vivo monitoring of the photosynthetic uptake of carbon, the transport of carbon from the leaf, and its transport throughout the entire plant. Continuous but varying amounts of carbon-11 dioxide are supplied to a leaf while observing the amount of labelled photoassimilate that appears in different parts of the plant. Appropriate analysis of these tracer profiles gives physiologically meaningful parameters such as the fraction of recent assimilate which is exported from the leaf. Variation in these parameters can be followed over an indefinite time, to follow the effects of natural rhythms or treatments. The method does not require any assumptions about mechanism, nor the assumption that transport processes within the leaf are constant, unlike compartmental analysis of efflux data sometimes used to infer fluxes in leaves. The second full paragraph on page 462 teaches that the method is another application of the technique of systems identification to analyze temporal non-steady-state profiles of isotope activity, a technique which gives a continuous measure of system properties. The experimental section on page 462 teaches that a leaf chamber that is part of a closed loop around which air circulated with a controlled dew point (see at least figure 1). CO2 was measured with a URAS 2 infra-red gas analyzer, and an aliquot of CO2 admitted whenever CO2 fell to a preset value. CO2 in the loop varied by about ± 3% about ambient. By injecting 0.5 cm3 of CO2 at known pressure and temperature, they deduced the sensitivity of the gas analyzer to changes in mass of CO2 in the loop. The slope of a chart recorder trace of CO2 concentration gave the photosynthetic rate. The closed loop extended to another room where 11CO2 could be admitted through a reed valve from a reservoir which was replenished at intervals of about 100 min with about 2 GBq of 11CO2 in 120 cm3 of air. The duty cycle of the valve was computer controlled according to a requested rate of supply of radioactive label, while allowing for the initial activity of isotope in the reservoir and for the decay of isotope within it. The last full paragraph on page 462 teaches that the data were analyzed by an 'input-output' method for the analysis of carbon-11 tracer profiles. The technique estimates the best parameter values of an input-output model to describe the data. Because of the recursive nature of the method it is also possible to detect time-variation in the model's parameter values, by giving greater weight to the most recent observations when calculating the parameters for each sampling time. The method involves continuous, not constant labelling. The photosynthesis section of pages 462-463 derives/explains the mathematical equations used to determine photosynthesis from the data. The paragraph bridging pages 463-464 teaches that the technique can be applied to any system where tracer inflow and outflow can be measured, provided the system is linear in tracer, i.e. the amount of tracer transported is proportional to the amount entering the system. Linearity is assured since the actual amount of carbon tracer is minuscule (around 10-12 moles).
In the paper Lawton described a controlled environmental facility for the investigation of population and ecosystem processes. Section 5(h) on page 190 describes the various micro-environment sensors used in the controlled environment including sensors to measure air temperature and photosynthetically active radiation (PAR) irradiance at different heights above the ground surface. Figure 4 presents temperature, humidity, and light-intensity conditions in the Ecotron during an early experiment. This figure shows how physical factors can be smoothly regulated by computer control. Relative to temperature, the relevant paragraph on page 188 teaches that air enters each chamber at a controlled temperature, regulated by computer control with sensor input from two sensors per bank of chambers. Individual chambers have sensors that monitor temperature for information only. Temperature can be held constant or cycled smoothly over daily or longer time intervals. A typical cycle used in preliminary experiments is shown in figure 4. The system can deliver air temperatures in the range of 5 °C to 30 °C dry bulb (± 0.5 °C) and be continuously varied as required (see figure 5). Regarding the lighting, the paragraph bridging pages 188-189 teaches that gradual brightening and dimming of dawn and dusk is achieved by electronic control of the power supply voltage. This produces red to far-red radiation (R: FR) ratio shift resembling that which occurs during dawn and dusk. The paragraph bridging pages 189-190 teaches that light intensity is computer controlled and can be smoothly varied between maximum output and darkness to mimic natural diurnal cycles (see figure 4).
In the abstract Jost described the performance of a laser based CO2 isotope infrared spectrometer used to study biosphere-atmosphere exchange processes. the Isotope Ratio Infrared Spectrometers (IRIS) is capable of simultaneously determining both d18O and d13C isotope ratios of carbon dioxide utilizing a simple, direct absorption approach with a robust multi pass cell and a cryogen free setup. In a plant chamber simulation, the concentration ramp speed was increased up to 40 ppm per min. For 1 minute averaged samples, the precision was d13C = 0.097 ‰ and d18O = 0.121 ‰. The IRIS analyzer was also integrated into a large plant chamber experiment involving multiple instruments to study CO2 fluxes using d18O-CO2. Plant chamber in and out was alternatingly monitored for 5 minutes.
In the paper Marynick presents a mathematical treatment of rate data obtained in biological flow systems under nonsteady state conditions. The problem of determining gas exchange rates from flow system data under nonsteady state conditions was analyzed. A correction factor is presented for obtaining constant rates under nonsteady state conditions. A general formula for obtaining any rate under nonsteady state conditions is also given. Turnover time is defined and discussed in terms of the mathematics presented. The origins of nonsteady states and steady states in flow systems are discussed, as are some of the experimental advantages of working under nonsteady state conditions. The second paragraph on page 680 teaches that in a flow system, tissue is enclosed in a container and air or a modified atmosphere is passed through the container at a known rate. One of the principal advantages of a flow system over the older manometric and volumetric techniques is the ability to closely control the composition of the atmosphere surrounding the tissue. Volume, pressure, flow rate, and usually temperature are constant, and changes in the composition of the gas stream with time are related to the activities of the living tissue. The paragraph bridging the columns of page 680 teaches that the paper is concerned with three basic questions: (a) When does equation 1 (R = %C•F/100) apply? (b) When equation 1 does not apply what is the appropriate mathematical framework to deal with the experimental data? (c) What are the potential experimental advantages in using this general solution? The experimental section on page 680 describes the flow system and how samples were obtained for measurement of carbon dioxide. The derivation of the nonsteady state equation is found on pages 680-681. The last paragraph of page 681 in describing a feature of figure 2 notes that the feature is characteristic of al open flow systems. The first full paragraph of the right column of page 682 teaches that the mathematics presented are generally valid for all open flow systems for which instantaneous mixing is a good approximation. It is important to note that the mathematical treatment deals with gas concentrations in the chamber. If there were any significant length of tubing connecting the chamber to the site of sampling (as is common when IRGA instruments are used) the approximation of instantaneous mixing would not be valid and the mathematical treatment would not necessarily apply. The sampling, as well as the mathematics, must reflect the instantaneous situation in the chamber. The paragraph bridging pages 682-683, teaches that depending on the accuracy of the measuring equipment, the non-steady state techniques will possibly allow accurate measurements of rates from two to twenty times faster than the usual steady state procedures. The first full paragraph on page 683 teaches that nonsteady state techniques are useful whenever a large free volume or a slow flow rate, or both, is either unavoidable or desirable, or when speed in obtaining data is critical. Whenever a rate to be measured in a flow system is rapidly changing relative to the time needed to obtain steady state, nonsteady state techniques are essential. In other cases where work under steady state is practical, the elimination of the wait for steady state with use of nonsteady state rate calculations could result in a significant saving of time.
In the paper van Iersel investigated temperature-response curves for photosynthesis and respiration which are useful in predicting the ability of plants to perform under different environmental conditions. Whole crop CO2 exchange rates of three magnolia (Magnolia grandiflora L.) cultivars (‘MGTIG’, ‘Little Gem’, and ‘Claudia Wannamaker’) were measured over a 25 °C temperature range. Plants were exposed to cool temperatures (13 °C day, 3 °C night) temperatures before the measurements. Net photosynthesis (Pnet) of all three cultivars increased from 3 to 15 °C and decreased again at higher temperatures. ‘MGTIG’ had the highest and ‘Little Gem’ the lowest Pnet, irrespective of temperature. The Q10 (relative increase in the rate of a process with a 10 °C increase in temperature) for Pnet of all three cultivars decreased over the entire temperature range. ‘MGTIG’ had the lowest Q10 at low temperatures (1.4 at 8 °C), while ‘Little Gem’ had the lowest Q10 for Pnet at temperatures >17 °C and a negative Q10 > 23 °C. This indicates a rapid decline in Pnet of ‘Little Gem’ at high temperatures. All three cultivars had the same optimal temperature (11 °C) for net assimilation rate (NAR), and NAR was not very sensitive to temperature changes from 3 to 17 °C. This indicates that the plants were well-adapted to their environmental conditions. The results suggest that respiration rate may limit magnolia growth when temperatures get high in winter time. The first two full paragraphs of page 278 teaches that CO2 exchange data of groups of whole magnolias was collected using a multichamber, semicontinuous CO2 exchange system. Ambient air was blown into acrylic gas-exchange chambers and air flow into the chambers was measured with mass flow meters. The CO2 concentration of the incoming air was measured with an infrared gas analyzer (IRGA). The difference in the CO2 concentration of the air entering and exiting the chamber was measured with an IRGA in differential mode (LI-6251). Air flow to the differential IRGA was controlled by opening and closing solenoid valves. Whole-chamber CO2 exchange (mmol·s–1) was calculated as the product of mass flow (mol·s–1) and the difference in CO2 concentration of the air entering and exiting the chamber (mmol·mol–1). Every chamber was measured for 30 seconds, once every 10 minutes. There was a 30 second delay in data collection after solenoids were switched to measure the next chamber, to assure that all air from the previous gas exchange chamber was purged from the tubing. The data from the 30 second measuring period was automatically collected, averaged and stored by a datalogger. Four plants of a cultivar were enclosed in each acrylic gas exchange chamber and eight chambers were placed in larger growth chambers. Errors in the measurements due to zero drift of the differential IRGA were corrected by subtracting the CO2 exchange rates of empty gas exchange chambers from the measured CO2 exchange rate of the plants.
In the paper Long discusses gas exchange measurements and what they can tell us about the underlying limitations to photosynthesis? There is also a discussion of procedures and sources of error. Leaf CO2 uptake (A) versus intercellular CO2 concentration (Ci) curves may now be routinely obtained from commercial gas exchange systems. The potential pitfalls, and means to avoid these, are examined. The paragraph bridging the columns of page 2394 is directed to off‐the‐shelf gas exchange systems and some pitfalls. In particular the first full paragraph of the right column of page 2394 deals with leaks. Some CO2 can escape through the gasket, this may not be a constant and will vary with the leaf. It is worse among leaves with prominent veins where small air channels may form between the gasket and the sides of the vein. This is particularly significant at low fluxes when errors due to artefactual apparent fluxes will have their greatest effect and in the measurement of A/Ci responses, when differences between the air outside and that within the chamber are greatest. A partial solution, recommended commonly by manufacturers is the measurement of flux in the absence of a leaf. Here, when the chamber is closed, a perfect seal should give a zero flux, regardless of the difference in [CO2] between the inside and outside of the chamber. However, gaskets have some permeability and may release or absorb some CO2. These leaks may be measured and used to correct fluxes. However, when the leaf is placed in the chamber additional leaks may be introduced. There are two partial solutions: 1) use a dead leaf, formed by rapidly drying a live specimen and establish the rate of leakage at each [CO2] that will be used in constructing an A/Ci response and 2) enclose the chamber in a container filled with the gas mixture that is being introduced into the chamber.
It would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the instrument used by Kositsup to control it to perform the A/Ci measurements of Kositsup using a continuously varying change in a variable known to affect the A/Ci measurements of Kositsup such as light intensity, or temperature as taught by at least Lawton or pressure (a nonsteady state technique such as taught by Marynick) from a first value to a second value such as pressure (ambient) to a second pressure (both lower and greater than ambient) in which the CO2 exchange rate is measured a plurality of times by measuring the concentration of CO2 in the gas entering the leaf chamber simultaneously with the concentration of CO2 in the gas leaving the chamber and determining a change/differential due to the photosynthesis capable sample in the chamber because of the expected rapid response to small changes in CO2 amount as shown by Nugawela, the expectation that as taught by McDermitt plant leaves are able to maintain a quasi-steady state with respect to CO2 mole fractions which change at the rates observed in typical experiments (e.g., 0.01-1 ppm•s-1), under these conditions, the transient (continuously varying) approach provides a valid method for measuring CO2 response curves which is rapid and convenient inasmuch as it does not require a series of mixed gasses or long equilibration times, the rapid response of leaves to small increases in leaf temperature compared to larger step increases as taught by Andrews