Carboxylation Capacity Can Limit C3 Photosynthesis at Elevated CO2 throughout Diurnal Cycles

The response of carbon fixation in C3 plants to elevated CO2 is relatively larger when photosynthesis is limited by carboxylation capacity (VC) than when limited by electron transport (J). Recent experiments under controlled, steady-state conditions have shown that photosynthesis at elevated CO2 may be limited by VC even at limiting PPFD. These experiments were designed to test whether this also occurs in dynamic field environments. Leaf gas exchange was recorded every 5 min using two identical instruments both attached to the same leaf. The CO2 concentration in one instrument was controlled at 400 μmol mol−1 and one at 600 μmol mol−1. Leaves were exposed to ambient sunlight outdoors, and cuvette air temperatures tracked ambient outside air temperature. The water content of air in the leaf cuvettes was kept close to that of the ambient air. These measurements were conducted on multiple, mostly clear days for each of three species, Glycine max, Lablab purpureus, and Hemerocallis fulva. The results indicated that in all species, photosynthesis was limited by VC rather than J at both ambient and elevated CO2 both at high midday PPFDs and also at limiting PPFDs in the early morning and late afternoon. During brief reductions in PPFD due to midday clouds, photosynthesis became limited by J. The net result of the apparent deactivation of Rubisco at low PPFD was that the relative stimulation of diurnal carbon fixation at elevated CO2 was larger than would be predicted when assuming limitation of photosynthesis by J at low PPFD.


Introduction
Photosynthesis by terrestrial vegetation is a large component of the annual global carbon balance, and predicting how the photosynthetic CO 2 assimilation (A) of terrestrial vegetation will respond to increased concentrations of CO 2 in the atmosphere is vital to understanding the future global carbon cycle [1][2][3]. Plants with C 3 photosynthetic metabolism are of predominant importance in terms of numbers of species, total CO 2 fixation, use as food for humans, and the responsiveness of photosynthesis to projected changes in atmospheric CO 2 . Predictions of photosynthesis of C 3 plants at elevated CO 2 concentrations often use the Farquhar-von Caemmerer-Berry biochemical model of photosynthesis [4] and its recent modifications [5].
In the Farquhar-von Caemmerer-Berry model of C 3 photosynthesis [4], A at high photosynthetic photon flux density (PPFD) is limited by the maximum carboxylation capacity of Rubisco (V Cmax ) at low CO 2 concentrations, by maximum electron transport capacity (J max ) at higher CO 2 , and sometimes by the rate of utilization of triose phosphates (TPU) at the highest CO 2 concentrations [5], with all of these rate-limiting parameters having different temperature dependencies. Because TPU limitation did not occur in these experiments, the focus here will be on V Cmax and J max at saturating PPFD, and V C and J at limiting PPFD. The external CO 2 at the transition between limitation by V Cmax and J max at high PPFD varies among species, and with implementations of the FvCB model, from about 450 to 700 µmol mol −1 [3], so the issue is highly relevant to global change issues. Experiments exposing plants to elevated CO 2 have sometimes found photosynthesis at elevated CO 2 at high PPFD to be limited by V Cmax and sometimes by J max , depending upon species and level of elevated CO 2 (reviewed in [6]).
The relative increase in A caused by increased CO 2 concentration inside the leaf (C i ) is less at a given temperature when A is limited by J than when limited by V C [3,4]. This is illustrated in Figure 1, which shows that for an increase in C i from 250 to 375 µmol mol −1 (i.e., approximate C i for ambient and 1.5 × ambient CO 2 ), A is stimulated about 20% more when A is limited by V C than when limited by J, over a wide range of temperatures. Experiments exposing plants to elevated CO2 have sometimes found photosynthesis at elevated CO2 at high PPFD to be limited by VCmax and sometimes by Jmax, depending upon species and level of elevated CO2 (reviewed in [6]). The relative increase in A caused by increased CO2 concentration inside the leaf (Ci) is less at a given temperature when A is limited by J than when limited by VC [3,4]. This is illustrated in Figure 1, which shows that for an increase in Ci from 250 to 375 μmol mol −1 (i.e., approximate Ci for ambient and 1.5 × ambient CO2), A is stimulated about 20% more when A is limited by VC than when limited by J, over a wide range of temperatures. Hypothetical light-saturated rates of CO2 assimilation (A) at internal CO2 concentration (Ci) = 375 μmol mol −1 relative to those measured at Ci = 250 μmol mol −1 as a function of temperature, for leaves where A is limited by carboxylation capactity (Vc) or electron transport (J), and the ratio of these two values of A, using the Farquhar-von Caemmerer-Berry model [4].
At lower PPFD, it is often assumed that carboxylation capacity remains constant while electron transport rate decreases. This primarily lowers A at higher Ci ( Figure 2) and would result in a smaller relative stimulation of A between a Ci of 250 and 375 μmol mol −1 , for example [7]. However, the assumption of constant carboxylation capacity at lower PPFD is incorrect, in the steady-state [8]. Bunce [8] found that the initial slope of A vs. Ci curves, an in situ measure of Rubisco carboxylation capacity, decreased as PPFD decreased in the steady-state, in a range of C3 species measured, over a range of temperatures. The result was that the relative stimulation of A at elevated vs. ambient CO2 did not decrease as PPFD decreased, at any temperature [8]. Hypothetical light-saturated rates of CO 2 assimilation (A) at internal CO 2 concentration (C i ) = 375 µmol mol −1 relative to those measured at C i = 250 µmol mol −1 as a function of temperature, for leaves where A is limited by carboxylation capactity (V c ) or electron transport (J), and the ratio of these two values of A, using the Farquhar-von Caemmerer-Berry model [4].
At lower PPFD, it is often assumed that carboxylation capacity remains constant while electron transport rate decreases. This primarily lowers A at higher C i ( Figure 2) and would result in a smaller relative stimulation of A between a C i of 250 and 375 µmol mol −1 , for example [7]. However, the assumption of constant carboxylation capacity at lower PPFD is incorrect, in the steady-state [8]. Bunce [8] found that the initial slope of A vs. C i curves, an in situ measure of Rubisco carboxylation capacity, decreased as PPFD decreased in the steady-state, in a range of C 3 species measured, over a range of temperatures. The result was that the relative stimulation of A at elevated vs. ambient CO 2 did not decrease as PPFD decreased, at any temperature [8]. Experiments exposing plants to elevated CO2 have sometimes found photosynthesis at elevated CO2 at high PPFD to be limited by VCmax and sometimes by Jmax, depending upon species and level of elevated CO2 (reviewed in [6]). The relative increase in A caused by increased CO2 concentration inside the leaf (Ci) is less at a given temperature when A is limited by J than when limited by VC [3,4]. This is illustrated in Figure 1, which shows that for an increase in Ci from 250 to 375 μmol mol −1 (i.e., approximate Ci for ambient and 1.5 × ambient CO2), A is stimulated about 20% more when A is limited by VC than when limited by J, over a wide range of temperatures. Hypothetical light-saturated rates of CO2 assimilation (A) at internal CO2 concentration (Ci) = 375 μmol mol −1 relative to those measured at Ci = 250 μmol mol −1 as a function of temperature, for leaves where A is limited by carboxylation capactity (Vc) or electron transport (J), and the ratio of these two values of A, using the Farquhar-von Caemmerer-Berry model [4].
At lower PPFD, it is often assumed that carboxylation capacity remains constant while electron transport rate decreases. This primarily lowers A at higher Ci ( Figure 2) and would result in a smaller relative stimulation of A between a Ci of 250 and 375 μmol mol −1 , for example [7]. However, the assumption of constant carboxylation capacity at lower PPFD is incorrect, in the steady-state [8]. Bunce [8] found that the initial slope of A vs. Ci curves, an in situ measure of Rubisco carboxylation capacity, decreased as PPFD decreased in the steady-state, in a range of C3 species measured, over a range of temperatures. The result was that the relative stimulation of A at elevated vs. ambient CO2 did not decrease as PPFD decreased, at any temperature [8]. Hypothetical examples of assimilation rates (A) vs. internal CO 2 concentrations (C i ) at saturating and low PPFD, as predicted by the FvCB photosynthesis model [4]. Rates of A based on limitation by V Cmax and by J at high and low PPFD are given. In all cases, actual A is the minimum of the A (V Cmax ) and A (J) curves. The purpose of these experiments was to determine whether the same apparent decrease in V C (deactivation of Rubisco) at low PPFD observed in the steady-state also occurred under naturally varying PPFD in dynamic field environments in C 3 species. This was tested by determining whether A at elevated CO 2 during different parts of diurnal cycles was better predicted from A at ambient CO 2 using the assumption of limitation of A at elevated CO 2 by Vc or by J.

Results
A representative diurnal time course of environment and leaf gas exchange for leaves of G. max at 400 and 600 µmol mol −1 CO 2 is presented in Figure 3 of the A (VCmax) and A (J) curves.
The purpose of these experiments was to determine whether the same apparent decrease in VC (deactivation of Rubisco) at low PPFD observed in the steady-state also occurred under naturally varying PPFD in dynamic field environments in C3 species. This was tested by determining whether A at elevated CO2 during different parts of diurnal cycles was better predicted from A at ambient CO2 using the assumption of limitation of A at elevated CO2 by Vc or by J.

Results
A representative diurnal time course of environment and leaf gas exchange for leaves of G. max at 400 and 600μmol mol −1 CO2 is presented in Figure 3    Because no differences occurred between morning and afternoon time periods in the outcome of the modeling of assimilation, either at the stable low or stable high PPFD periods, or after sudden decreases in PPFD due to clouds, values averaged over mornings and afternoons are presented in Table 1. The data reported in Table 1 for stable PPFD conditions represent means for two measurements (morning and afternoon) per day for three days in the cases of G. max and H. fulva, and four days in L. purpurea. The data for the sudden decrease in PPFD are means for two time points per day for each species, with three days in the case of G. max and H. fulva, and four days in L. purpurea. The results indicate that for all three species, when PPFD was stable, A at 600 µmol mol −1 CO 2 was accurately modeled by assuming limitation of A at both 400 and 600 µmol mol −1 CO 2 by Vc rather than J (Table 1). This was true for both high and low PPFD measurements, despite the rates of A being much lower at the low PPFD. In contrast, after sudden decreases in PPFD, A at 600 µmol mol −1 CO 2 was more accurately modeled by assuming limitation of A at both 400 and 600 µmol mol −1 CO 2 by J rather than by Vc (Table 1).     Because no differences occurred between morning and afternoon time periods in the outcome of the modeling of assimilation, either at the stable low or stable high PPFD periods, or after sudden decreases in PPFD due to clouds, values averaged over mornings and afternoons are presented in Table 1. The data reported in Table 1 for stable PPFD conditions represent means for two measurements (morning and afternoon) per day for three days in the cases of G. max and H. fulva, and four days in L. purpurea. The data for the sudden decrease in PPFD are means for two time points per day for each species, The Vc of all three species, measured at low PPFDs at 400 µmol mol −1 CO 2 , had similar approximately linear increases in Vc with PPFD ( Figure 6). Leaf temperatures ranged from 30 to 35 • C in this comparison. These similar values among species at low PPFD levels occurred despite large differences among species in V Cmax , which ranged from about 160 µmol m −2 s −1 in H. fulva to 320 µmol m −2 s −1 in G. max, at 35 • C. Table 1. CO 2 assimilation rates (A) of leaves measured at ambient (400 µmol mol −1 ) and elevated (600 µmol mol −1 ) CO 2 at stable, high PPFD (>1200 µmol m −2 s −1 ), stable low PPFD (<400 µmol m −2 s −1 ), or within 10 min after PPFD abruptly decreased by at least 700 µmol m −2 s −1 due to clouds. Also presented are assimilation rates at elevated CO 2 modeled using the model parameters fitted to the measured rates at ambient CO 2 , assuming limitation of rates at elevated CO 2 either by V C or by J at elevated CO 2 . See text for details. creases in PPFD, A at 600 μmol mol −1 CO2 was more accurately modeled by assuming limitation of A at both 400 and 600 μmol mol −1 CO2 by J rather than by Vc (Table 1). Table 1. CO2 assimilation rates (A) of leaves measured at ambient (400 μmol mol −1 ) and elevated (600 μmol mol −1 ) CO2 at stable, high PPFD (>1200 μmol m −2 s −1 ), stable low PPFD (<400 μmol m −2 s −1 ), or within 10 min after PPFD abruptly decreased by at least 700 μmol m −2 s −1 due to clouds. Also presented are assimilation rates at elevated CO2 modeled using the model parameters fitted to the measured rates at ambient CO2, assuming limitation of rates at elevated CO2 either by VC or by J at elevated CO2. See text for details. The Vc of all three species, measured at low PPFDs at 400 μmol mol −1 CO2, had similar approximately linear increases in Vc with PPFD ( Figure 6). Leaf temperatures ranged from 30 to 35 °C in this comparison. These similar values among species at low PPFD levels occurred despite large differences among species in VCmax, which ranged from about 160 μmol m −2 s −1 in H. fulva to 320 μmol m −2 s −1 in G. max, at 35 °C.

Discussion
The results of this study indicated that for gradual changes in PPFD over the course of a day, caused by changes in the solar angle, leaf photosynthesis was always limited by Vc rather than by J in all three of these C 3 species, both at the approximate current ambient CO 2 concentration and at 1.5 times the current concentration. Models which assume limitation of C 3 photosynthesis by J at less than saturating PPFDs would underestimate the stimulation of daily CO 2 fixation at 1.5 times the current CO 2 concentration by approximately 50%.
The observed contrasting limitation of A by J after sudden decreases in PPFD caused by clouds obstructing direct solar radiation indicates that the assay of the type of limitation to photosynthesis used here was able to distinguish limitation by Vc from limitation by J. The change in type of limitation at low PPFD caused by the rate of decrease in PPFD is consistent with deactivation of Rubisco at low PPFD requiring a few to several minutes.
Conducting rapid A vs. C i curves [9,10] under field conditions may be a new alternative method of determining the biochemical limitations to photosynthesis throughout a day, although to date these seem to only have been conducted at saturating PPFD. However, field-based rapid A vs. C i measurements would be much more labor intensive than the method used here, and would also require information on the operational C i throughout the day.
Several field experiments have indicated that V Cmax corrected to a constant temperature may change during the course of a day [11][12][13], although the cause of the changes is not clear, and can occur even in shade [13]. In those cases, V Cmax was assayed using steady-state A vs. C i curves at high PPFD, although it was not clear whether the assays allowed sufficient time at high PPFD to fully re-activate Rubisco. Clearly, models of C 3 photosynthesis which assume constant V Cmax throughout a day may be substantially in error. Furthermore, there is a notable lack of information on daily patterns of A vs. C i curves at less than saturating PPFD, even at ambient CO 2 . This is particularly important in predicting responses of photosynthesis to past and future changes in atmospheric CO 2 , and may be one explanation for the observed insensitivity of biochemically based global photosynthesis models to past increases in atmospheric CO 2 [6,14].

Materials and Methods
Three C 3 species, Glycine max L. Merr., cultivar Clark, Hemerocalus fulva L., and Lablab purpureus L. Sweet were grown at the South Farm of the USDA Beltsville Agricultural Research Center, Beltsville, Maryland. The G. max and L. purpureus plants were grown from seed planted in the spring of 2019 in rows 70 cm apart, with plants thinned to about 7 cm between plants after emergence. The H. fulva plants were grown from tubers in rows 70 cm apart, with about 20 cm between tubers. The soil was a silt loam soil with a water table at about 1.5 m depth. Weeds were removed by hand. The prior crop was soybean, and no fertilizer was added to the soil after the well-fertilized soybean crop. Frequent precipitation prevented significant water deficits.
Two Ciras-3 portable photosynthesis systems (PP Systems, Amesbury, MA, USA) were used simultaneously each measurement day, with leaf chambers installed on opposite side leaflets of the same leaf in the case of G. max and L. purpureus, and both leaf chambers were on the same leaf in H. fulva, with about 5 cm between the chambers. Fully expanded, upper leaves were selected for measurement. Leaf chambers had circular windows 1.8 cm in diameter and were held horizontal. Leaf chambers were positioned so as to not be shaded by other leaves throughout the day. Leaf chambers were programmed to track ambient air temperature, and the external air temperature sensors were shaded at all times. Each chamber had internal PPFD sensors. The inlet air humidity setting was adjusted so that the chamber air had approximately the same water vapor content as that of the outside air. The CO 2 concentrations of air streams entering the two chambers were controlled at 400 and 600 µmol mol −1 . Leaf gas exchange data and environmental data were automatically recorded every 5 min from each leaf chamber throughout a 24 h period. Recording times of the two leaf chambers were not precisely synchronized, and the timing changed in each due to periodic automatic instrument self-tests, but recordings were synchronous within about 2 min. Measurements were begun soon after dew had evaporated from the leaves in the morning, and continued for 24 h. Measurements were continued for 24 h in order that rates in morning could be obtained without problems caused by dew on the leaves. Rates of respiration in darkness were not analyzed, for two reasons. First, flow rates were chosen based on obtaining accurate photosynthesis measurements, and created this very low, about 1 µmol mol −1 , differentials in darkness, so respiration measurements would be quite imprecise. Secondly, measuring small CO 2 differentials using clamp-on cuvettes has long been noted to produce artefactual apparent responses of respiration to CO 2 concentration because of leakage [15].
Measurements were made on three days each for G. max and H. fulva, and four days for L. purpureus, with days randomly assigned to the species, and with random assignment of the two gas exchange systems to the two CO 2 concentrations. All measurements were made in July and early August of 2019, on days selected as forecast to be mostly clear and without precipitation. All plants had flowered prior to the gas exchange measurements, but were plants were measured at least a month prior to reproductive maturity.
For each measurement day, detailed gas exchange analysis and modeling was applied to measurements during four periods: (1) periods of high (>1200 µmol m −2 s −1 ), stable PPFD before mid-day, (2) periods of high, stable PPFD after mid-day, (3) periods of low (100-300 µmol m −2 s −1 ), stable PPFD in early morning, and (4) periods of low, stable PPFD in late afternoon. Additionally, detailed analysis was made of periods of low (<500 µmol m −2 s −1 ) PPFD which occurred after PPFD had decreased by at least 700 µmol m −2 s −1 within the last 10 min, because of clouds. This later type of data was available each measurement day, which is typical for this climate, where intermittent afternoon clouds are very common in mid-summer.
Assimilation rates for the 400 µmol mol −1 treatment under each of the above conditions were used to estimate values of Vc and J which would be consistent with the measured values of A, C i , and temperature, using the FvCB model ( [16], Equations (1) and (2)), with the temperature dependencies from Bernacchi et al. [17]. where Ci is intercellular (CO 2 ), O is the oxygen concentration, γ* is the photosynthetic CO 2 compensation point without dark respiration, Kc and Ko are the Michaelis-Menton constants for CO 2 and O 2 , Vc is the carboxylation capacity of Rubisco, and J is the photosynthetic electron transport rate. Mesophyll conductance was assumed to be infinite, so that the results would not be influenced by assumed finite values, given the lack of information about values of mesophyll conductance for H. fulva and L. purpureus, or its CO 2 dependence [10]. It was then tested whether values of A at 600 µmol mol −1 taken at the approximately the same time points were better fit by assuming limitation of A by the values of Vc or J estimated from the leaves at 400 µmol mol −1 . It could hypothetically occur that A at 400 µmol mol −1 would be limited by Vc while rates at 600 µmol mol −1 would be limited by J. That would lead to rates at elevated CO 2 in between those predicted by limitation by Vc or by J [6], but that did not occur in these experiments.

Conclusions
Field leaf gas exchange measurements on three herbaceous species indicated that, under clear sky conditions, photosynthesis was limited by the carboxylation capacity of Rubisco rather than by electron transport rate throughout diurnal cycles of solar radiation both at approximately the current atmospheric CO 2 concentration and at 1.5 times the current concentration. Abrupt decreases in radiation due to clouds temporarily resulted in limitation by electron transport capacity.