The Dynamic Assimilation Technique measures photosynthetic CO2 response curves with similar fidelity as steadystate approaches in half the time
Data files
Jan 12, 2024 version files 1.94 MB

ACiMethod_4.0_RepositoryData.csv

README.md
Abstract
The net CO_{2} assimilation (A) response to intercellular CO_{2} concentration (C_{i}) is a fundamental measurement in photosynthesis and plant physiology research. The conventional A/Ci protocols rely on steadystate measurements and take 1540 minute per measurement, limiting data resolution or biological replication. Additionally, there are several CO_{2} protocols employed across the literature, without clear consensus as to the optimal protocol or systematic biases in their estimations. We compared the nonsteady state Dynamic Assimilation Technique (DAT) protocol and the three most used CO_{2} protocols in steadystate measurements, and tested whether different CO_{2} 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 steadystate method. Estimations of biochemical limitations to photosynthesis were very consistent across all CO_{2} 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 CO_{2} concentration but rather the time spent under TPUlimited conditions. Our results suggest that maximum rate of ribulose 1·5 bisphosphate carboxylase/oxygenase carboxylation (V_{cmax}), 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.
README: The Dynamic Assimilation Technique measures photosynthetic CO2 response curves with similar fidelity as steadystate 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, DATmonotonic)
RunID: Unique combination of species, treatment and replicate.
Elapsed: Time in seconds since the beginning of the measurement
A: net CO2 assimilation (μmol CO2 m2 s1)
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 m2 s1)
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 TejeraNieves available at https://github.com/PerennialDr/ACi Methods.
The biochemical limitations to photosynthesis on each A – Ci measurement were estimated by fitting the Farquharvon CaemmererBerry model of C3 photosynthesis including TPU limitation to the experimental data using the nonlinear 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.
Methods
Photosynthetic gas exchange was measured with portable infrared gas analyzer (LI6800, LICOR, 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^{2} s^{1}. While we understand that these light intensities are likely slightly subsaturating, we selected them to minimize the confounding effects of longterm buildup of nonphotochemical 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 finetune the different protocols. We then used 6 biological replicates for all other species. In tobacco, the 10 biological replicates were measured using multiple Li6800 over multiple days. For all other species, all biological replicates were measured on the same day using three Li6800 that were used to measure two separate leaves per day. The order in which all CO_{2} 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 steadystate CO_{2} protocols controlled the CO_{2} concentration in the reference channel. We tested the following protocols and CO_{2} sequences; monotonic (400, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 1000, 1250, 1500, 1750_{ }μmol CO_{2} mol^{1}), split updown (400, 500, 700, 1000, 1500, 1750, 1250, 800, 600, 400, 350, 250, 150, 50, 100, 200, 300 400 μmol CO_{2 }mol^{1}), split down up, (400, 300, 200, 100, 50, 150, 250, 350, 400, 600, 800, 1250, 1750, 1500, 1000, 700, 500, 400 μmol CO_{2 }mol^{1}), random (with a different CO_{2} sequence for each replicate) and, the nonsteady state DATmonotonic protocol (Fig. S1).
In tobacco measurements, we also implemented a random protocol and an abrupt CO_{2} increase of CO_{2} concentration. These random measurements were implemented to minimize the possibility that a previous measurement CO_{2} concentration affected the subsequent measurement. These measurements included a period in between each concentration to return the leaf back to steadystate photosynthesis at 400 μmol CO_{2} mol^{1 }between each random CO_{2} concentration. For the random protocol, the CO_{2} levels were randomly ordered for each of the 10 plants CO_{2 }with two 400 μmol CO_{2} mol^{1 }steps in between each concentration to return the leaf to initial ambient CO_{2}. An abrupt increase of CO_{2} concentration from 400 to 1500 μmol CO_{2 }mol^{1} was also performed to measure oscillations associated with CO_{2} entry into TPUlimiting CO_{2} concentrations (Oscillation test). The oscillation test ended once the leaves reach a new steady state at 1500 μmol CO_{2}_{ }mol^{1}.
The DATmonotonic protocol ranged from 50 – 2000 μmol CO_{2}_{ }mol^{1} at CO_{2} concentration ramp speed of 200 μmol CO_{2}_{ }mol^{1} min^{1}. Leaves were left to acclimate at 50 μmol CO_{2} mol^{1 }for 5 minutes before starting the CO_{2} ramp to allow the CO_{2} concentrations in the chamber to reach a steadystate before the DAT curve is initiated. This preramp 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_{2} and H_{2}O range matching, and a CO_{2} dynamic tunning all with an empty chamber were performed (Saathoff and Welles, 2021; LI6800 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_{2} 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 AC_{i} 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_{2} 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 < 1 and standard deviation < 0.5, and for slope < 0.2 and standard deviation < 0.01 over a 20 second period. For field measurements the period was extended to 30 seconds. In all steadystate 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 Farquharvon CaemmererBerry model of C3 photosynthesis (FvCB model; Farquhar et al., 1980) including TPU limitation (Harley and Sharkey, 1991) to the experimental data using the nonlinear fitting procedure developed by Sharkey (2016) and implemented on the msuRACiFit R package (Gregory et al., 2021; McClain et al., 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_{g}) or serine (alpha_{s}) 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 < 300 μmol CO_{2}_{ }mol^{1}_{ }(Vcmax).
The A oscillations in the Oscillation test were quantified using the following indices (Fig. S2); Amplitude (μmol CO_{2} m^{2} s^{1}), A difference between first A maximum and the projected A value of the linear trend of 1500CO_{2} steady state at the time of first maximum, Wavelength (min), average time between two consecutive maximum or minimum, Oscillation length (min), time elapsed between the first A minimum and the first observation at the new steady state at 1500 μmol CO_{2}_{ }mol^{1}_{ }(1500CO_{2} steady state), Number of minimums (count), number of times the oscillation reached a local minimum or valley, Overshot CO_{2} (μmol CO_{2} m^{2}), total area of CO_{2} assimilated above the linear trend of the 1500CO_{2} steady state, Forgone CO_{2} (μmol CO_{2} m^{2}), area between the linear trend of the 1500CO_{2} steady state and the assimilated CO_{2} below the trend, and Total or Net CO_{2} (μmol CO_{2} m^{2}), sum or difference between Overshot CO_{2} and Forgone CO_{2}, 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 < 0.1).
Usage notes
We used R Statistical Software for all statistical analyses based on code developed by Mauricio TejeraNieves available at https://github.com/PerennialDr/ACi Methods
Please direct correspondence to Berkley Walker (berkely@msu)