DOI: 10.5061/dryad.pk0p2ngst
title: The Dynamic Assimilation Technique measures photosynthetic CO2 response curves with similar fidelity as steady-state approaches in half the time

description: The net CO2 assimilation (A) response to intercellular CO2 concentration
 (Ci) is a fundamental measurement in photosynthesis and plant physiology
 research. The conventional A/Ci protocols rely on steady-state
 measurements and take 15-40 minute per measurement, limiting data
 resolution or biological replication. Additionally, there are several CO2
 protocols employed across the literature, without clear consensus as to
 the optimal protocol or systematic biases in their estimations. We
 compared the non-steady state Dynamic Assimilation Technique (DAT)
 protocol and the three most used CO2 protocols in steady-state
 measurements, and tested whether different CO2 protocols lead to
 systematic differences in estimations of the biochemical limitations to
 photosynthesis. The DAT protocol reduced the measurement time by almost
 half without compromising estimations accuracy or precision. The monotonic
 protocol was the fastest steady-state method. Estimations of biochemical
 limitations to photosynthesis were very consistent across all CO2
 protocols, with slight differences in ribulose 1·5- bisphosphate
 carboxylase/oxygenase carboxylation limitation. The A/Ci curves were not
 affected by the direction of the change of CO2 concentration but rather
 the time spent under TPU-limited conditions. Our results suggest that
 maximum rate of ribulose 1·5- bisphosphate carboxylase/oxygenase
 carboxylation (Vcmax), linear electron flow for NADPH supply (J) and
 triose phosphate utilization (TPU) measured using different protocols
 within the literature are comparable, or at least not systematically
 different based on the measurement protocol used.
description: Photosynthetic gas exchange was
 measured with portable infrared gas analyzer (LI-6800, LI-COR, Lincoln,
 NE, USA) on fully developed leaves. Chamber conditions were set to mimic
 species growing conditions (Specific conditions outlined in Table S2). To
 increase battery life in these field measurements, temperature was
 controlled at the block level. Across all species, our measurements
 spanned from 20 – 30 °C and 1000 – 1500 μmol photons
 m<sup>-2</sup> s<sup>-1</sup>. While we understand
 that these light intensities are likely slightly sub-saturating, we
 selected them to minimize the confounding effects of long-term build-up of
 non-photochemical quenching resulting from the longer total measuring
 times required to test all the regimes on the same leaf. Leaves were
 acclimated to this saturating light intensity for at least 5 minutes to
 ensure rubisco activation before the measurement commenced.
 We used 10 biological reps in tobacco
 to fine-tune the different protocols. We then used 6 biological replicates
 for all other species. In tobacco, the 10 biological replicates were
 measured using multiple Li-6800 over multiple days. For all other species,
 all biological replicates were measured on the same day using three
 Li-6800 that were used to measure two separate leaves per day. The order
 in which all CO<sub>2</sub> protocols were implemented on the
 leaf was randomized separately for each replicate using the random
 variation in atmospheric data (Haahr, 2023), which outperforms computer
 algorithms that generate random numbers. An example random protocol for a
 single measurement is shown in figure S1. All steady-state
 CO<sub>2</sub> protocols controlled the
 CO<sub>2</sub> concentration in the reference channel. We
 tested the following protocols and CO<sub>2</sub> sequences;
 monotonic (400, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800,
 1000, 1250, 1500, 1750<sub> </sub>μmol
 CO<sub>2</sub> mol<sup>-1</sup>), split up-down
 (400, 500, 700, 1000, 1500, 1750, 1250, 800, 600, 400, 350, 250, 150, 50,
 100, 200, 300 400 μmol CO<sub>2
 </sub>mol<sup>-1</sup>), split down- up, (400, 300, 200,
 100, 50, 150, 250, 350, 400, 600, 800, 1250, 1750, 1500, 1000, 700, 500,
 400 μmol CO<sub>2 </sub>mol<sup>-1</sup>), random
 (with a different CO<sub>2</sub> sequence for each replicate)
 and, the non-steady state DAT-monotonic protocol (Fig. S1).
 In tobacco measurements, we also
 implemented a random protocol and an abrupt CO<sub>2</sub>
 increase of CO<sub>2</sub> concentration. These random
 measurements were implemented to minimize the possibility that a previous
 measurement CO<sub>2</sub> concentration affected the subsequent measurement. These measurements included a period in between each concentration to return the leaf back to steady-state photosynthesis at 400 μmol CO<sub>2</sub> mol<sup>-1 </sup>between each random CO<sub>2</sub> concentration. For the random protocol, the CO<sub>2</sub> levels were randomly ordered for each of the 10 plants CO<sub>2 </sub>with two 400 μmol CO<sub>2</sub> mol<sup>-1 </sup>steps in between each concentration to return the leaf to initial ambient CO<sub>2</sub>. An abrupt increase of CO<sub>2</sub> concentration from 400 to 1500 μmol CO<sub>2 </sub>mol<sup>-1</sup> was also performed to measure oscillations associated with CO<sub>2</sub> entry into TPU-limiting CO<sub>2</sub> concentrations (Oscillation test). The oscillation test ended once the leaves reach a new steady state at 1500 μmol CO<sub>2</sub><sub> </sub>mol<sup>-1</sup>. The DAT-monotonic protocol ranged from 50 – 2000 μmol CO<sub>2</sub><sub> </sub>mol<sup>-1</sup> at CO<sub>2</sub> concentration ramp speed of 200 μmol CO<sub>2</sub><sub> </sub>mol<sup>-1</sup> min<sup>-1</sup>. Leaves were left to acclimate at 50 μmol CO<sub>2</sub> mol<sup>-1 </sup>for 5 minutes before starting the CO<sub>2</sub> ramp to allow the CO<sub>2</sub> concentrations in the chamber to reach a steady-state before the DAT curve is initiated. This pre-ramp wait time is included in the total time of the A/Ci measurement. Before clamping on the first leaf in the morning and afternoon a CO<sub>2</sub> and H<sub>2</sub>O range matching, and a CO<sub>2</sub> dynamic tunning all with an empty chamber were performed (Saathoff and Welles, 2021; LI-6800 Operating Instructions, 2023). This time was not included in the total time of the A/Ci measurement. A complete set of four or five (for tobacco only) CO<sub>2</sub> protocols was implemented on the same leaf without unclamping the leaf. A minimum of five minutes was allowed for leaves upon clamping to acclimate to chamber conditions and reach steady state before starting the A-C<sub>i</sub> curves. A minimum of five minutes was also allowed in between A/Ci curves for the leaf to reach steady state again. At each step of the A/Ci measurements, CO<sub>2</sub> concentration in the reference channel remained constant, and gas exchange variables were recorded upon reaching steady state or the elapsed time reaching 3 min. The stability criteria for auto logging were for A slope &lt; 1 and standard deviation &lt; 0.5, and for slope &lt; 0.2 and standard deviation &lt; 0.01 over a 20 second period. For field measurements the period was extended to 30 seconds. In all steady-state protocols, gas analyzers in the reference and sample channel were matched after every measurement. For the DAT protocols, gas exchange variables were recorded every 5 seconds for tobacco and 1 second for all other species. The parameters underlying the biochemical limitations to photosynthesis on each A/Ci measurement were estimated by fitting the Farquhar-von Caemmerer-Berry model of C3 photosynthesis (FvCB model; Farquhar <em>et al.</em>, 1980) including TPU limitation (Harley and Sharkey, 1991) to the experimental data using the non-linear fitting procedure developed by Sharkey (2016) and implemented on the msuRACiFit R package (Gregory <em>et al.</em>, 2021; McClain <em>et al.</em>, 2023). The parameters for the three main photosynthesis limitations, Vcmax, J, and TPU, along with respiration in the light (RL), mesophyll conductance (gm) and carbon flow out of photorespiration as glycine (alpha<sub>g</sub>) or serine (alpha<sub>s</sub>) were estimated simultaneously. FvCB estimations also considered the leaf temperature at which the A/Ci was measured. Vcmax was also estimated as the initial slope of the A/Ci response curve for Ci &lt; 300 μmol CO<sub>2</sub><sub> </sub>mol<sup>-1</sup><sub> </sub>(Vcmax). The A oscillations in the Oscillation test were quantified using the following indices (Fig. S2); <em>Amplitude </em>(μmol CO<sub>2</sub> m<sup>-2</sup> s<sup>-1</sup>), A difference between first A maximum and the projected A value of the linear trend of 1500-CO<sub>2</sub> steady state at the time of first maximum, <em>Wavelength </em>(min), average time between two consecutive maximum or minimum, <em>Oscillation length</em> (min), time elapsed between the first A minimum and the first observation at the new steady state at 1500 μmol CO<sub>2</sub><sub> </sub>mol<sup>-1</sup><sub> </sub>(1500-CO<sub>2</sub> steady state), <em>Number of minimums </em>(count), number of times the oscillation reached a local minimum or valley, <em>Overshot CO<sub>2</sub> </em>(μmol CO<sub>2</sub> m<sup>-2</sup>), total area of CO<sub>2</sub> assimilated above the linear trend of the 1500-CO<sub>2</sub> steady state, <em>Forgone CO<sub>2</sub> </em>(μmol CO<sub>2</sub> m<sup>-2</sup>), area between the linear trend of the 1500-CO<sub>2</sub> steady state and the assimilated CO<sub>2</sub> below the trend, and <em>Total </em>or <em>Net CO<sub>2</sub> </em>(μmol CO<sub>2</sub> m<sup>-2</sup>)<em>, </em>sum or difference between <em>Overshot </em>CO<sub>2</sub> <em>and</em> <em>Forgone </em>CO<sub>2</sub>, respectively. For consistency, the first maximum of the oscillation was not included in the estimations, given its large variability across replicates. The shapiro.test function was used to test the normality assumption, and plots of the residual variance across the fitted values to asses the homoscedasticity assumption. The emmeans function in the emmeans package (Lenth et al., 2018) was used for mean comparison, and emtrends in the same package to test for significant differences between slopes. For the linear regression we tested whether the slope of the regression was different from zero, and the intercept was different from zero. To investigate the correlation between A/Ci curves and A oscillations, we correlated the A/Ci parameters with the A oscillation indices. We used spearman correlation coefficient (r) to find significant correlations (P &lt; 0.1).
description: We used R Statistical Software for all statistical analyses based
 on code developed by Mauricio Tejera-Nieves available at https://github.com/PerennialDr/ACi Methods Please direct correspondence to Berkley Walker (berkely@msu)<em><br></em>
description: # The Dynamic Assimilation Technique measures photosynthetic CO2 response
 curves with similar fidelity as steady-state approaches in half the time
 Dataset compiles net CO2 assimilation (A) measurements measured at
 different internal CO2 concentrations (Ci) using different CO2 protocols
 (i.e., different sequences of CO2). The aim of the study was to evaluate
 whether the different CO2 protocols had any impact on the A – Ci response
 curve. Each CO2 regime was measured in 5 species: Apple, Arabidopsis,
 Potato, Soybean and Tobacco. For each plant, all 5 protocols were
 performed on the same leaf in a randomized order on 10 biological reps for
 tobacco and 6 for all other species. ## Description of the data and file
 structure Data are presented in a long format. Each row corresponds to a
 net CO2 assimilation observation at a given internal CO2 concentration
 (Ci), for a given CO2 regime and a given replicate. Columns represent:
 Species: Name of the species used in the experiment (one of Apple,
 Arabidopsis, Potato, Soybean, Tobacco) Protocol: CO2 regime used in the
 measurement (one of Monotonic, split UpDown, split DownUp, Random,
 DAT-monotonic) RunID: Unique combination of species, treatment and
 replicate. Elapsed: Time in seconds since the beginning of the measurement
 A: net CO2 assimilation (μmol CO2 m-2 s-1) Ci: Internal CO2 concentration
 (μmol CO2) Tleaf: Leaf temperature measured with a thermocouple (oC)
 CO2_r: CO2 concentration (μmol CO2) of the air stream before reaching the
 leaf PAR: Photosynthetically active radiation (μmol photon m-2 s-1)
 VPDleaf : Vapor pressure deficit (kPa) ## Sharing/Access information
 Companion manuscript is submitted to the Journal of Experimental Botany
 for publicacion. Please direct correspondence to Berkley Walker
 (berkely@msu) ## Code/Software We used R Statistical Software for all
 statistical analyses based on code developed by Mauricio Tejera-Nieves
 available at [https://github.com/PerennialDr/ACi
 Methods](https://github.com/PerennialDr/SinkDepletion). The biochemical
 limitations to photosynthesis on each A – Ci measurement were estimated by
 fitting the Farquhar-von Caemmerer-Berry model of C3 photosynthesis
 including TPU limitation to the experimental data using the non-linear
 fitting procedure developed by Sharkey (2016) and implemented on the
 msuRACiFit R package. The emmeans function in the emmeans package (Lenth
 et al., 2018) was used for mean comparison.