The plants were potted in 6 dm³ square pots using a 5:1 mixture of commercial compost and vermiculite and placed in two large walk-in growth rooms with full control of light, temperature, [CO₂] (one chamber maintained ambient atmospheric [CO₂] of 400 ppm and the second an elevated [CO₂] level of 2000 ppm) and humidity for 16-week (technical details of the plant growth chambers are given in Materassi et al., 2005). The plants were watered to pot capacity every 2 days and provided weekly with a commercial liquid plant fertilizer (COMPO Concime Universale, NPK 7-5-7, B, Cu, Fe, Mn, Mo, Zn: COMPO Italia, Cesano Maderno, Italy) to facilitate nutrient availability at free access rates. The growth chambers maintained conditions of 16 h of daylight (14 h at full PAR levels of 1000 μmol m^-2 s^-1 with two 1-h periods of simulated dawn/dusk where light intensity was incrementally increased/decreased), a day and night-time temperature regime of 25/18°C and constant relative humidity of 50%. To avoid potential chamber effects, the growth rooms were alternated every 2 weeks – no significant differences were observed in the measurements conducted under the same conditions in different growth chambers. Timings of day/night programs on the plant growth chambers were staggered to allow the maximum number of plants to be analyzed at the optimal time of the day/night program for photosynthetic activity (07:00–11:00 h), and thus avoid the influence of circadian stomatal behavior; particularly where stomata close at midday or during the early afternoon when leaf water potentials may decrease. Four replicates of each species were grown under ambient and elevated [CO₂], and a minimum of three replicates were used for the measurement of stomatal sensitivity to C_a and closure in response to darkness.

A PP-Systems Ciras-2 attached to a PLC6(U) leaf cuvette and LED light unit (PP-Systems, Amesbury, MA, USA) was used to gauge the physiological response of stomata to darkness and C_a. All physiological measurements were conducted on the same leaf per plant (no more than one measurement was performed on each plant over 48 h) in a well-ventilated air-conditioned room maintained at 25°C. The newest fully expanded leaf was consistently used for analysis (in grasses this was the leaf below the flag leaf) to avoid any age-related effects regarding stomatal functionality. To assess the physiological stomatal behavioral responses of the plants to C_a, the level of [CO₂] within the leaf cuvette was increased in a number of stages (200, 400, 750, 1000, and 2000 ppm CO₂) while temperature (25°C), VPD (1.6–1.8 KPa ± 0.1 KPa), and light intensity (2000 μmol m^-2 s^-1) remained constant. At each [CO₂] step, the stomatal conductance of water vapor (G_{s H2O}) was allowed to stabilize and then remain stable for 5–10 min before being recorded. Maximum stomatal conductance (G_{s max}) was considered to be G_{s H2O} recorded at a [CO₂] of 50 ppm to induce full stomatal opening (Centritto et al., 2003). Stomatal closure was expressed as the percentage of G_{s H2O} values at 2000 ppm CO₂ relative to those recorded at an ambient [CO₂] of 400 ppm (Figure 1). To measure the stomatal response to darkness, a leaf was placed in the leaf cuvette with a temperature of 25°C, PAR of 2000 μmol m^-2 s^-1 and 400 ppm [CO₂]. After G_{s H2O} had remained stable for 15 min the lights in both the cuvette and the room were simultaneously switched off and G_{s H2O} was recorded every 10 s for a minimum of 1 h (Meidner and Mansfield, 1965). Vapor pressure deficit in the cuvette was maintained between 1.6 to 1.8 KPa ± 0.1 KPa throughout the measurement of the stomatal response to darkness. Stomatal closure in response to the cessation of illumination was expressed as the percentage G_{s H2O} after 1-h of darkness. The speed of stomatal closure is not uniform after the onset of darkness (Haworth et al., 2015); the reduction in G_{s H2O} per second over the first 50% reduction in G_{s H2O}, and also over the time to reach the maximum extent of stomatal closure was therefore determined (Figure 1). Measurements of stomatal pore length (SPL) of the plants under ambient and elevated [CO₂] were taken from Haworth et al. (2015).

Response curves of P_N to increasing internal sub-stomatal [CO₂] (C_i) under a saturating light intensity of 2000 μmol m^-2 s^-1 (C_a sequence of 350, 250, 150, 50, 100, 200, 300, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, and 2000 ppm) at a standard cuvette temperature of 25°C were recorded on the same leaves used to assess stomatal control using the same Ciras-2 photosynthesis system. The maximum carboxylation rate of RubisCO (Vc_max), the maximum rate of electron transport for regeneration of ribulose-1,5-bisphosphate (J_max) and mesophyll conductance to CO₂ (G_mCO2) were calculated from the P_N/C_i response curves following Ethier and Livingston (2004). The ‘curve fitting’ method of Ethier and Livingston (2004) estimates G_mCO2 from the response of P_N to increasing partial pressure of CO₂ in the sub-stomatal air-space assuming a constant G_mCO2 across the range of C_i values (cf. Flexas et al., 2007). Total conductance to CO₂ (G_totCO2) was calculated as:

Where G_sCO2 is the stomatal conductance to CO₂ – in the present manuscript to further aid differentiation between measurement of stomatal conductance to H₂O and CO₂, G_{s H2O} is expressed as mmol m^-2 s^-1, while G_{s CO2} is expressed as mol m^-2 s^-1. The maximum rate of photosynthesis (P_{N max}) was considered to be P_N at a saturating light intensity of 2000 μmol m^-2 s^-1 and [CO₂] of 2000 ppm. The maximum (F_v/F_m) and actual (ΦPSII: ΔF/F′m) quantum efficiency of photosystem II was recorded using a Hansatech FMS-2 (saturating pulse of 10,000 μmol m^-2 s^-2) and dark adaptation clips (Hansatech, King’s Lynn, UK) after 30 min of dark adaptation and exposure to actinic light of 1000 μmol m^-2 s^-1 for a minimum of 10 min after the first saturating pulse (Genty et al., 1989; Kalaji et al., 2014).

Statistical analyses were performed using SPSS 20 (IBM, New York, NY, USA). To test [CO₂] treatment effects a one-way ANOVA was used to assess differences in variance between samples. A two-way ANOVA was used to assess species and [CO₂] effects on G_smax (Figure 3). The relative change (Δ) in parameters was expressed as a percentage of the values recorded at 2000 ppm [CO₂] relative to control values measured at 400 ppm [CO₂]. Linear regression was used to investigate potential relationships between stomatal characteristics such as stomatal pore length and speed of stomatal closure and whether relative changes in stomatal behavior (i.e., Δ G_s change to [CO₂] or darkness) were associated with the relative change in photosynthetic physiology.

Photosynthesis of the crop plants grown at ambient and elevated [CO₂] when measured at a common [CO₂] was closely related to stomatal and total conductance to CO₂ (Figures 2A,C). However, G_{m CO2} derived from the curve fitting method (Ethier and Livingston, 2004) did not show any significant relationship to P_N either at a common C_a (Figure 2B) or the respective growth [CO₂] levels of the plants (data not shown). The curve fitting approach calculates a single G_m value along the P_N – C_i curve. The efficacy of this approach is reliant upon G_m remaining constant at a range of C_i values (Tazoe et al., 2009, 2011), however, measurement of G_m using the variable J method of Harley et al. (1992) across a C_i gradient suggests that rates of G_mCO2 may not be uniform and instead vary with the availability of CO₂ in relation to rates of P_N and respiration (Flexas et al., 2007). The curve fitting method has been successfully applied to the determination of G_mCO2 in plants grown at identical [CO₂] but different levels of water availability (Miyazawa et al., 2008). However, no relationship was observed between P_N and G_mCO2 of the crop plants grown at different levels of [CO₂] in the present study (Figure 2B). This may suggest that there is no effect of growth at elevated [CO₂] on G_mCO2 in the crop species analyzed as there were limited reductions in photosynthetic capacity of four of the five species due to the free availability of nutrients (Kitao et al., 2015), or a limitation in the effectiveness of the method due to the size of the cuvette employed (2 cm²; Pons et al., 2009). Movement of CO₂ within the leaf is unlikely to have affected the P_N – C_i curves (Pons et al., 2009), as the species analyzed in this study possess heterobaric leaves (Nikolopoulos et al., 2002).

The speed of stomatal closure showed a positive relationship to P_N._max and G_s._max (Figure 3). This suggests that species with high potential rates of P_N and G_s require a high degree of stomatal control to reduce transpirative water-loss and prevent desiccation when conditions are not conducive to P_N. This relationship may be indicative of natural selective pressures induced by declining [CO₂] during the Cenozoic (65 Ma to present; Haworth et al., 2011) and artificial selective pressures during the domestication of crop species (Evans, 1980; Roche, 2015) favoring highly effective physiological stomatal control alongside high rates of P_N and G_s. The trend of declining [CO₂] during much of the past 65 million years (Berner, 2006) is considered to have favored species with large numbers of small stomata as the most effective arrangement of the epidermal surface to achieve maximum rates of gas exchange (Franks and Beerling, 2009; de Boer et al., 2016) in conjunction with rapid stomatal opening and closing (Hetherington and Woodward, 2003; Raven, 2014). In contrast to previous observations of a negative relationship between stomatal size and the speed of the adjustment in the size of stomatal aperture in closely related species with identical stomatal complex morphology (Drake et al., 2013), this study showed a positive relationship between stomatal size and the speed of the stomatal response to darkness (Figure 4). However, this positive relationship reflects the diversity in stomatal complex morphologies of the five plants studied. The dicots, cotton, and sunflower, both have stomatal complexes composed of ‘kidney-shaped’ guard cells; whereas the monocot grasses, oat, wheat, and barley, have larger ‘dumb-bell’ type guard cells (Haworth et al., 2015). The dumb-bell stomatal complexes of grasses may open and close more rapidly than kidney-shaped stomatal complexes. Stomatal opening relies upon an increase in guard cell turgor as ions are pumped across cell membranes to reduce water potential; however, to effectively open the larger dumb-bell guard cell pairs there needs to be a corresponding loss of turgor in the surrounding cell epidermal subsidiary cells to accommodate an increase in stomatal aperture (Franks and Farquhar, 2007). This rapid control and co-ordination of changes in cell turgor may account for the generally faster rates of stomatal closure observed in the grasses analyzed in this study (Figure 4), and ability of grasses to support larger stomatal pore apertures than species with kidney-shaped stomatal complexes.

Regulation of stomatal aperture size is achieved through a complex hierarchical sensory and signaling network (Hetherington and Woodward, 2003; Kim et al., 2010; Merilo et al., 2014). The results of this study (Figure 6) and others (e.g., Heath and Kerstiens, 1997; Heath, 1998; Zeppel et al., 2012) suggest that growth at elevated [CO₂] may affect either the network of stomatal sensing/signaling or the physical function of stomata. Physiological stomatal control may occur via a signal from the mesophyll to the guard cells (Mott et al., 2008; Fujita et al., 2013), or as a result of metabolic changes within the guard cells (Talbott and Zeiger, 1998). It is not possible from the present dataset to definitively state whether the reduction in photosynthetic physiology and impaired stomatal function at high [CO₂] are causally linked or co-incidental. Quantitative trait loci responsible for P_N and stomatal control occur within the same region of the genome in sunflower (Hervé et al., 2001), indicating the importance of their co-ordination in plant responses to environmental change. The mesophyll is the site where the majority of P_N occurs and mesophyll P_N and G_s are closely linked (Messinger et al., 2006). The results of this study may suggest that the effects on photosynthetic capacity and stomatal control at high [CO₂] are associated. Growth at high [CO₂] can cause damage (Madsen, 1971), inhibition (Van Oosten et al., 1994), and feedback limitations (Sage et al., 1989) to the photosynthetic physiology. However, the species studied in this experiment all exhibit fairly rapid growth that would reduce the impact of sink limitations (Manderscheid et al., 2010), and did not experience any limitations in nutrient availability that might promote any reduction of photosynthetic capacity at elevated [CO₂] (Farage et al., 1998). Three of the five crops showed no effect of growth at elevated [CO₂] on P_N – C_i curves, and none of the five crops exhibited lower F_v/F_m or ΦPSII values at the higher [CO₂] that might indicate some impairment or loss of performance of PSII corresponding to damage to the thylakoid membranes (Shaw et al., 2014; Kalaji et al., 2016) that may account for the patterns observed in P_N and stomatal control reported in this study.

An increase in [CO₂] commonly induces a reduction in stomatal aperture in plants with active physiological stomatal control (Morison and Gifford, 1983; Haworth et al., 2013). Separation of the epidermis and mesophyll layers suggests that this is the result of a signal derived from the mesophyll such as sucrose or malate (Mott et al., 2008; Fujita et al., 2013) regulated by carbonic anhydrase within the mesophyll (Hu et al., 2010). An increase in transport of sucrose to the apoplastic space surrounding the guard cells may act as a signal to induce stomatal closure and limit water-loss during episodes where levels of photosynthetic sugar production exceed rates of transport from the photosynthetic organs (Outlaw, 2003). The plants grown at 2000 ppm [CO₂] in this study exhibited stomatal sensitivity to an increase in C_a from 400 to 2000 ppm; however, the degree of closure in cotton and sunflower was lower, possibly indicating impairment of the mesophyll to guard cell signal, or physical damage to the stomata constraining the ability to close. The leaves of plants grown at high [CO₂] frequently contain greater concentrations of soluble sugars due to CO₂-fertilization and the accumulation of photosynthate if the capacity to transport sugars is exceeded (Van Oosten et al., 1994; Paul and Driscoll, 1997). Such an increase in soluble sugars in the apoplast at high [CO₂] may impair the efficacy of a sucrose mesophyll derived signal, or the sensitivity of guard cells to apoplastic fluctuations in sucrose concentrations. The concentration of sugars such as sucrose derived from guard cell P_N may also play a role in maintaining guard cell turgor after the influx of potassium ions responsible for stomatal opening (Talbott and Zeiger, 1998). Disruption to carbohydrate metabolism may also influence guard and subsidiary cell osmotic balance, thus affecting the mechanics of stomatal opening/closing (e.g., Franks and Farquhar, 2007) and possibly accounting for impaired stomatal control in sunflower and cotton (Figure 5).

Stomatal opening in response to sub-ambient [CO₂] occurs in isolated epidermal strips without the mesophyll, but stomatal closure induced by [CO₂] levels above ambient requires chemical contact between the mesophyll and epidermal layers, suggesting the operation of a diffusible signal (Fujita et al., 2013). No difference was observed in G_smax values recorded at a C_a of 50 ppm [CO₂] (two-way ANOVA: CO₂, F_1,38 = 1.580, P = 0.219; species F_4,38 = 52.511, P = 7.160 × 10^-13) of plants grown at 400 and 2000 ppm [CO₂] (Figure 3C). However, G_s sensitivity to an instantaneous increase in [CO₂] from 400 to 2000 ppm did alter between species; indicative of differential stomatal control mechanisms to sense and signal sub-ambient [CO₂] (Fujita et al., 2013) and the photosynthetic control of [CO₂] at higher levels of [CO₂] (Messinger et al., 2006). The results of this study suggest that control of G_s by mesophyll P_N via C_i is influenced via the effect of growth at elevated [CO₂] on photosynthetic physiology in cotton and sunflower with kidney-shaped stomatal complexes (Figure 6). The similarity in stomatal response to a sub-ambient C_a of 50 ppm, but difference in stomatal sensitivity to super-ambient C_a may indicate that growth at elevated [CO₂] does not affect the physical aspects of stomatal opening/closing but rather disrupts the photosynthetic control of G_s. In essence, the photosynthetic mesophyll to guard cell signaling mechanism appears to be operating in the same manner, but at high [CO₂] the fine control of stomatal behavior is impaired. Stomatal aperture during P_N is linked to C_i (Roelfsema et al., 2002). Stomatal conductance in cocklebur (Xanthium strumarium) showed a gradual change when P_N was CO₂-limited and a more dramatic reduction with C_i when P_N was limited by the regeneration of ribulose-1,5-bisphosphate (Messinger et al., 2006). This may account for the lack of correlation between ΔVc_max and stomatal sensitivity to [CO₂] (Figure 6A) and the negative correlation between ΔJ_max and Δstomatal closure to [CO₂] (Figure 6C) observed in this study; suggesting that alteration of photosynthetic capacity may influence the photosynthetic determination of stomatal aperture in response to fluctuations in C_i.

The pronounced reduction in the effectiveness of stomatal closure to darkness with Vc_max (Figure 6B) may also be suggestive of a link between P_N and physiological stomatal behavior or impaired physical operation of the stomata. A diverse range of stomatal morphologies (Franks and Farquhar, 2007) and physiological behaviors (Haworth et al., 2013, 2015) are observed in evolutionarily diverse plants. The species utilized in this study all exhibit active physiological behavior (Haworth et al., 2015), but possess contrasting stomatal morphologies. The monocot grass species with dumb-bell guard cells generally retained the ability to close stomata, while the kidney-shaped stomata of the two dicots closed less effectively at high [CO₂] (Figure 6C). Dumb-bell guard cells exhibit larger and faster changes in turgor than kidney-shaped guard cells (Franks and Farquhar, 2007). The guard cells of grasses do not possess chloroplasts (Brown and Johnson, 1962) and possibly reply upon the movement of potassium for stomatal opening (Fairley-Grenot and Assmann, 1992). To accommodate the rapid and large change in dumb-bell guard cell turgor, the surrounding subsidiary cells also undergo osmotic adjustment (Franks and Farquhar, 2007). This may suggest that the differential effect of elevated [CO₂] on stomata of grasses and dicots found in this is due to differences in the biochemistry of stomatal opening related to the presence/absence of guard cell chloroplasts, stomatal signaling or photosynthetic control of stomatal aperture.

The results of this study suggest that one unexpected impact of elevated [CO₂] may be a loss of stomatal function in some C3 herbaceous crop species. This impaired stomatal control corresponded to reduced carboxylation capacity, consistent with linkage between G_s and P_N. Growth at elevated [CO₂] generally reduced G_{s H2O} (Table 1). This lower G_{s H2O} was not associated with a decrease in stomatal density, but a reduction in stomatal aperture via physiological stomatal control (Haworth et al., 2015). Under normal growth conditions this lower G_{s H2O} will result in reduced water-loss and correspondingly greater WUE, consistent with observations of enhanced WUE in other experimental studies of elevated [CO₂] (Centritto et al., 2002; Wullschleger et al., 2002; Ainsworth and Rogers, 2007). However, loss of stomatal function at elevated [CO₂] may impair the ability of crop plants with active physiological stomatal behavior to exert stomatal control in the event of a change in growth conditions. Specifically, the reduced ability of stomata to close at elevated [CO₂] may have significant implications for the capacity of crops to tolerate drought and heat stress in the future (e.g., Killi et al., 2016) and warranting further experimental investigation.

The aim of this study was to investigate the effects of elevated [CO₂] on stomatal control by using a very high [CO₂] level of 2000 ppm. This concentration is beyond the range of the IPCC worst case scenario (IPCC, 2007), but not above levels of [CO₂] that have occurred over Earth history since the origination of vascular plants (e.g., Berner, 2009; Haworth et al., 2011). To investigate the loss of stomatal function under more realistic levels of [CO₂] for the next 50–100 years further work should be undertaken in controlled environment chambers and free air CO₂ enrichment (FACE) systems. However, many FACE systems do not increase [CO₂] at night for economic reasons, instead operating enrichment only during daylight hours when P_N occurs. In terms of gauging the effect of elevated [CO₂] on stomatal control it would be necessary for the plants to experience the most realistic simulation of future atmospheric conditions possible via continuous enrichment of [CO₂] levels. The number and duration of heat-waves and drought events are predicted to increase in the future (e.g., Schär et al., 2004; Vautard et al., 2007). The capacity of crop plants to respond to and resist these adverse growth conditions is largely dependent upon effective stomatal control (Killi et al., 2016). At elevated [CO₂], WUE may be higher and delay/mitigate the impact of drought (Wall, 2001; Wullschleger et al., 2002), but the results of our study and that of Heath and Kerstiens (1997) suggests that at severe drought the loss of stomatal function in C3 plants with kidney-shaped stomatal complexes could impair tolerance to drought. Further experimental work at [CO₂] levels equivalent to those predicted in the next 100 years is required to assess whether the loss of stomatal control at high [CO₂] may have negative implications for food security in a water-limited world.