Leaf Gas Exchange and Photosystem II Fluorescence Responses to CO₂ Cycling

Abstract

Experimental systems to simulate future elevated CO₂ conditions in the field often have large, rapid fluctuations in CO₂. To examine possible impacts of such fluctuations on photosynthesis, the intact leaves of the field-grown plants of five species were exposed to two-minute cycles of CO₂ between 400 and 800 μmol mol⁻¹, lasting a total of 10 min, with photosynthesis, stomatal conductance and PSII fluorescence measured at the end of each half-cycle and also 10 min after the end of the cycling. Prior to the cyclic CO₂ treatments, the steady-state responses of leaf gas exchange and fluorescence to CO₂ were determined. In four of the five species, in which stomatal conductance decreased with increasing CO₂, the cyclic CO₂ treatments reduced stomatal conductance. In those species, both photosynthesis and the photochemical efficiency of PSII were reduced at limiting internal CO₂ levels, but not at saturating CO₂. In the fifth species, there was no change in stomatal conductance with CO₂ and no change in either photosynthesis or PSII efficiency at any CO₂ level with CO₂ cycling. It is concluded that in many, but not all, species, fluctuations in CO₂ may reduce photosynthesis at low CO₂, partly by decreasing the photochemical efficiency of photosystem II as well as by decreasing stomatal conductance.

1. Introduction

With the continuing increase in CO₂ concentrations in the atmosphere [1], there has been considerable research examining the impacts of changes in CO₂ concentration on plant functions and growth [2,3,4,5]. As a substrate for photosynthesis, CO₂ is still currently a growth-limiting resource for plants that have C₃ metabolism. Experiments imposing different CO₂ concentrations on growing plants generally use CO₂ sensors to dynamically regulate the supply of CO₂ to the experimental system, while any removal of CO₂ required during daylight is usually accomplished by plant photosynthesis and/or wind. Concerns over the impacts of short-term variations in CO₂ concentration on plant function resulted primarily from the recognition of large-magnitude CO₂ fluctuations in free-air-carbon dioxide-enrichment facilities. Free-air-CO₂-enrichment (FACE) facilities were developed to provide elevated CO₂ treatments to plant ecosystems outdoors with a minimal disturbance from other environmental factors, such as wind, light, air temperature and humidity, and soil conditions [6]. However, in most FACE systems, CO₂ release is at the perimeter of the plot, while the CO₂ concentration sampled to control the CO₂ release is near the center of the plot, often many meters from the release points. Because air movement is needed to distribute CO₂ across the plot, there is a variable time lag between CO₂ release and the detection of the achieved concentration as well as the disturbance by air turbulence. A few papers documented large fluctuations in CO₂ concentrations over time within a given plot with FACE systems, using sampling systems that averaged CO₂ concentrations over about 5 s periods [7,8]. Surprisingly, despite the existence of rapid-response open-path CO₂ analyzers for about the last 25 years, rapid (seconds) CO₂ concentration measurements in FACE plots have only recently been published [9,10]. Based on measurements in a FACE system of the Brookhaven National Laboratory design, Allen et al. [10] concluded that “due to the difficulty of controlling elevated CO₂ concentrations in turbulent air, the range of fluctuations of CO₂ in FACE experiments are more than 10-fold greater than plants experience in natural conditions”. After reviewing experiments comparing plant responses to elevated CO₂ with different degrees of fluctuation, it was concluded that plant growth was suppressed by the larger CO₂ fluctuations in FACE systems, probably by reducing photosynthesis [10].

Because of the difficulty of reproducing fluctuations observed in FACE plots in controlled experiments, most experiments to assess the impacts of fluctuating CO₂ have used either regular cycles of CO₂ or brief pulses of high CO₂ [11,12,13,14,15]. Hendrey et al. [11] measured chlorophyll fluorescence responses to the short-term cyclic variation in CO₂ concentration of several frequencies. Holtum and Winter [12] measured responses of CO₂ uptake to the short-term cyclic variation in CO₂ concentration but did not measure stomatal conductance, and found that variations in CO₂ reduced photosynthesis in two tree species. Bunce [13] provided long-term cyclic CO₂ treatments compared with constant elevated CO₂ treatments at the same mean elevated CO₂ in open top chambers, and found that the cyclic CO₂ treatments reduced photosynthesis, stomatal conductance and plant growth in wheat and cotton. Short-term series of pulses of elevated CO₂ mimicking those observed in FACE plots reduced photosynthesis and stomatal conductance in wheat and rice leaves [14]. In indoor chambers, a larger magnitude of continuously applied fluctuations of CO₂ reduced photosynthesis, stomatal conductance and the growth of four herbaceous species compared with a smaller amplitude of CO₂ variation [15]. Although reduced stomatal conductance often occurs in response to CO₂ fluctuations, it is not the sole cause of reductions in photosynthesis, even if the stomatal closure is entirely “patchy” in nature [15,16].

This work examined whether a reduced photochemical efficiency of photosystem II occurred in response to CO₂ fluctuations and might cause some of the suspected reductions in photosynthesis in field-grown plants in FACE systems, in addition to reductions in stomatal conductance.

2. Results

Throughout the cycling of CO₂, four of the five species studied, G. max, L. purpureus, L. tulipifera and S. lycopersicum, had a reduced assimilation rate (A) and PSII efficiency (ΦPSII) at rate-limiting sub-stomatal CO₂ (C_i) values of about 250 to 300 μmol mol⁻¹ occurring at 400 μmol mol⁻¹ external CO₂ (Figure 1). At a higher C_i, occurring at 800 μmol mol⁻¹ external CO₂, A was actually slightly increased in all of these species, except L. tulipifera, and the ΦPSII was the same as before the cycling of CO₂ in all four of these species (Figure 1). The reduction in ΦPSII and A to below steady-state values was evident at the end of the first 400 μmol mol⁻¹ half-cycle and continued throughout the cycling of CO₂ in all of these four species. In G. max, the stomatal conductance decrease caused by cycling was nearly complete in the first half-cycle, while the other species had slower decreases in stomatal conductance, but stomatal conductance had stabilized before the end of the 10 min of cycling. All species were the same as G. max in terms of the speed of the ΦPSII decrease, i.e., it decreased by the end of the first half-cycle. The decrease in ΦPSII during CO₂ cycling, observed at the lower C_i, was accompanied by increased non-photochemical quenching. Ten minutes after the end of CO₂ cycling, a lower stomatal conductance remained at each CO₂ level in all four of these species (

Table 1. Mean values of stomatal conductance measured at 400 and 800 μmol mol⁻¹ CO₂ before, during, and 10 min after cycling of CO₂ between 400 and 800 μmol mol⁻¹, with a full cycle length of 2 min, for a total of 10 min, in five species. Within rows, numbers followed by different letters are different at p = 0.05, using repeated measures ANOVA.

Species	Stomatal Conductance (mmol mol−1)
		Before Cycling		During Cycling	After Cycling
	CO2 (μmol mol−1):	400	800	Both CO2s	400	800
G. max		1643 a	1465 b	1168 c	956 d	808 e
L. purpureus		652 a	437 b	269 c	280 c	240 d
L. tulipifera		205 a	183 b	159 c	152 c	144 c
S. lycopersicum		797 a	638 b	493 c	537 c	531 c
P. crispum		313 a	310 a	315 a	322 a	316 a

). Additionally, at ten minutes after the end of CO₂ cycling, ΦPSII and photosynthesis measured at 400 μmol mol⁻¹ both remained lower than before the CO₂ cycling. However, the values of A and ΦPSII measured at 600 μmol mol⁻¹ did not differ significantly from control values when measured at 600 μmol mol⁻¹ (

Table 2. Means values of A and ΦPSII at 600 μmol mol⁻¹ CO₂ before and 10 min after the end of cycling of CO₂ between 400 and 800 μmol mol⁻¹ CO₂ for 10 min. Within rows, numbers followed by different letters are different at p = 0.05, using repeated measures ANOVA.

Species	A (μmol m−2 s−1)		ΦPSII
	Before	After	Before	After
G. max	37.1 a	36.5 a	0.333 a	0.313 a
L. purpureus	22.9 a	21.7 a	0.175 a	0.165 a
L. tulipifera	16.3 a	15.2 a	0.095 a	0.094 a
S. lycopersicum	34.5 a	33.7 a	0.310 a	0.308 a
P. crispum	35.1 a	35.3 a	0.255 a	0.257 a

) in any species, despite a lower stomatal conductance in all species except P. crispum.

The P. crispum, in contrast to the other four species, had no reduction in the A vs. C_i curve, or in ΦPSII after the cycling of CO₂ (Figure 2), and also showed no change in stomatal conductance with CO₂ (Table 1).

Stomatal conductance before the cycling of CO₂ was lower at 800 than at 400 μmol mol⁻¹ CO₂ in all species except P. crispum (Table 1). Stomatal conductance during CO₂ cycling was reduced in all species, except P. crispum (Table 1). Ten minutes after cycling ended, the stomatal conductance remained lower than before cycling in all species, except P. crispum, in which the stomatal conductance was unchanged by all treatments (Table 1).

3. Discussion

All of these species had fairly typical A vs. C_i curves for C₃ species, with no decreases in A at the highest C_i values, which would be clear evidence of a limitation by triose phosphate utilization (TPU) [17]. However, all species had some decrease in ΦPSII at the highest C_i values, which McClain et al. [18] suggest is indicative of TPU limitation. A premature leveling off of A vs. C_i curves is more difficult to discern than reductions in ΦPSII as an indication of TPU limitation, except possibly by the fitting of a photosynthesis model that includes a TPU limitation to the observed data.

The reductions in the photochemical efficiency of PSII (ΦPSII) at 400 μmol mol⁻¹ external CO₂ levels caused by the cycling of CO₂ concentration, which occurred in four of the five species examined, provide a new explanation of reduced photosynthesis rates for a given sub-stomatal CO₂ concentration, which has frequently been reported in CO₂ fluctuation experiments [12,13,14,15]. Prior suggestions that reduced photosynthesis might be the result of “patchy” stomatal closure [13,15] admittedly could not account for the lack of reduction in photosynthesis at elevated measurement CO₂ [15]. In the current experiments, the reduction in ΦPSII that occurred at the lower measurement CO₂ did not occur at the higher measurement CO₂. At the higher measurement CO₂, photosynthesis was also not inhibited by the cycling of CO₂ in these experiments, despite the continued lower stomatal conductance. The lack of decrease in A despite a lower stomatal conductance is to be expected at nearly saturating values of CO₂. Similar to the results presented here, in long-term cyclic CO₂ exposures in open top chambers, the relative reductions in photosynthesis in cotton were much larger for measurements made at the lower (near-ambient CO₂) than at the higher external CO₂ of the cycles [13].

McClain et al. [18] also reported reductions in ΦPSII in response to a large step increase in CO₂, which they proposed was related to a triose-phosphate limitation of photosynthesis at high CO₂. They provided no information on the stomatal conductance response to their treatments. However, in the fluctuating CO₂ experiments reported here, reduced ΦPSII only occurred at limiting CO₂ concentrations, not at elevated CO₂. This difference in plant response might be related to the much shorter duration of exposure to high CO₂ and lower elevated CO₂ concentrations in the present experiment (800 μmol mol⁻¹) compared with those of McClain et al. (1500 μmol mol⁻¹). In these experiments, leaves were actually at 800 μmol mol⁻¹ during the cycling of CO₂ for less than five minutes.

I speculate that P. crispum had a qualitatively different photosynthetic response to the cyclic CO₂ treatment than the other four species studied here, because it had no response at all of stomatal conductance to CO₂ in the range of 400 to 800 μmol mol⁻¹, in contrast to all of the other species. Similar results for more species with stomates unresponsive to changes in CO₂ would be required to confirm this correlation. L. tulipifera was chosen for these experiments, based on the generally smaller response of stomatal conductance to CO₂ in tree species [19,20]. It did have a smaller relative response than the other three herbaceous species, but not a zero response, as occurred in P. crispum. It remains unclear how the presence or absence of changes in stomatal conductance during fluctuations in CO₂ could influence photochemical limitations on photosynthesis at low CO₂. However, the decrease in photosynthesis and ΦPSII observed in this tree species at the lower measurement CO₂ is consistent with the decreases in photosynthesis found by Holtum and Winter in two tropical tree species [12]. This suggests that FACE experiments may also not give the most accurate indication of tree responses to climate change.

Allen et al. [10] reviewed yield data in FACE and open top chambers (OTC) for several major C₃ crop species, and they concluded that the yield stimulation caused by the same elevated CO₂ treatments was, in FACE, on average, only about 0.66× of that occurring in OTC. A smaller yield stimulation by elevated CO₂ in FACE than in OTC was documented for wheat and soybeans in the only side-by-side simultaneous FACE and OTC comparisons of crop yield [21] that exist to date. Allen et al. [10] tentatively attributed this smaller yield stimulation to a reduced stimulation of photosynthesis by elevated CO₂ in FACE than in OTC. The smaller stimulation of photosynthesis was thought to be caused by the much larger fluctuations in CO₂ in elevated CO₂ treatments in FACE than in OTC. Allen et al. [10] carefully documented larger CO₂ fluctuations in FACE with all of the available rapid CO₂ measurement data, and I am not aware of any more recent published data on CO₂ fluctuations in FACE. However, at the time that paper was written [10], reasons why rapid fluctuations in CO₂ would cause reduced photosynthesis were unclear, despite some documented cases of high-CO₂ pulses or the cycling of CO₂ reducing photosynthesis [12,13,14]. Deceases in photosynthesis caused by the pulses of elevated CO₂ or by the cycling of CO₂ have now been documented in many of the most important C₃ crop species, wheat [14], rice [14], soybean ([15] and this paper), and cotton [14], in two minor crop species, tomato and lablab [this paper], and also in three tree species ([12] and this paper). Up until the current work, the only clue about the reasons why fluctuations in CO₂ would inhibit photosynthesis were observations of a reduced stomatal conductance to water vapor [13,14,15].

The results presented here provide a new mechanism by which fluctuations in CO₂ around leaves can inhibit photosynthesis, a decrease in the photochemical efficiency of photosystem II. Of course, these results beg the question of why ΦPSII was decreased by the cycling of CO₂. Furthermore, the extent to which this decrease in ΦPSII at low CO₂ occurs in experiments exposing plants to a long-term elevation of CO₂, for example in FACE experiments, has not been determined. It is interesting to consider that reduced photosynthesis in FACE systems may primarily occur during those periods in which CO₂ fluctuations bring CO₂ levels down to near-ambient CO₂ levels, based on the results presented here. Most measurements of photosynthesis in FACE systems have been conducted at the targeted elevated CO₂ concentration, not at lower CO₂ concentrations. The only experiment to date that directly compared photosynthesis in plants grown simultaneously at elevated CO₂ in open top chambers and in FACE systems only measured leaf gas exchange at the elevated CO₂ [21], in the plants grown at elevated CO₂, and thus would have missed photosynthetic responses resembling those presented here.

4. Materials and Methods

Leaf gas exchange and chlorophyll fluorescence measurements were conducted on four species of herbaceous plants and one tree species grown outdoors at ambient CO₂. The species studied were Glycine max L. Merr. cv. Clark, Lablab purpureus L. Sweet, Petroselinum crispum Mill. Fuss var. neopolitanum, Solanum lycopersicum L. cv. Better Boy, and Lireodendron tulipifera L. The four herbaceous species were grown in Annapolis, Maryland in an unshaded plot with a sandy loam soil. Plants were grown from seed and planted in late April 2020. The plot was fertilized with a complete fertilizer containing 12% N, 4% P, and 8% K at 200 g of fertilizer per m², and it did not experience soil water stress. The L. tulipifera trees sampled were saplings, about 6 years old, growing at a south-facing forest edge in Annapolis, on a sandy loam soil. Leaf gas exchange and chlorophyll fluorescence measurements were conducted from mid-June through to the end of June 2020. The mean temperature in Annapolis in May 2020 was 16.0 °C, slightly below the long-term mean of 17.7 °C, and in June 2020 it was 23.3 °C, which equals the long-term mean temperature.

All leaf gas exchange and chlorophyll fluorescence measurements were conducted at 27 °C leaf temperature, 1500 μmol m⁻² s⁻¹ PPFD, with a leaf-to-air water vapor pressure difference of 1 to 1.5 kPa, using a Ciras-3 portable photosynthesis system with a PLC3 leaf chamber/fluorometer, with an air flow rate of 400 cm³ min⁻¹. The “stored differential balance” function of the instrument was used to correct measurements for changes in calibration with background CO₂. The values of sub-stomatal CO₂ (C_i) were calculated from photosynthesis, stomatal and boundary layer conductances, and external CO₂ by the system software. During the mornings of sunny days, a fully expanded upper-canopy leaf was selected for measurement. Steady-state responses of stomatal conductance, photosynthesis, and PS II chlorophyll fluorescence at CO₂ concentrations of 400, 600, and 800 μmol mol⁻¹ were determined on a leaf, allowing sufficient time for the stomatal conductance to adjust to each CO₂ level, as observed on the graphical display of incoming data. Steady-state values were used to ensure that C_i values were accurate. The efficiency of PSII was assessed using multipulse fluorescence measurements at each CO₂ level. The CO₂ concentration was then returned to 400 μmol mol⁻¹, and cycles of CO₂ from 400 to 800 μmol mol⁻¹ with a total cycle length of 2 min were then applied for 10 min, that is, one minute at 400 μmol mol⁻¹, one minute at 800 μmol mol⁻¹, one minute at 400 μmol mol⁻¹, etc., for a total of 10 min. Photosynthesis, stomatal conductance, and PSII efficiency were recorded at the end of each half-cycle. At the end of the cyclic CO₂ treatment, CO₂ was returned to 400 μmol mol⁻¹, and beginning ten minutes after the end of the CO₂ cycling, photosynthesis, stomatal conductance, and PSII efficiency were measured at 400, 600, and 800 μmol mol⁻¹ CO₂. These measurements were made on at least four different plants of each species. On a few different leaves of each species, the responses of stomatal conductance, photosynthesis, and PS II chlorophyll fluorescence to CO₂ concentrations from 100 to 1200 μmol mol⁻¹ were determined. There were nine steps of CO₂ (400, 300, 200, 100, 400, 600, 800, 1000, 1200 μmol mol⁻¹). Leaves were kept at each step of CO₂ for three to four minutes, waiting for the leaf gas exchange to stabilize, before measuring the photochemical efficiency of PSII using a multipulse measurement at each step in CO₂. The leaf-to-air water vapor pressure difference changed by less than 10% of its initial value of 1 to 1.5 kPa during the cycling of CO₂, which would have a minimal impact on the stomatal conductance.