Eight elite wheat varieties adapted to the United Kingdom were selected: Alchemy, Brompton, Claire, Hereward, Rialto, Robigus, Soissons, and Xi19. These are the founder lines of the ‘NIAB Elite MAGIC’ multi-parent advanced generation inter-cross (MAGIC) population (Mackay et al., 2014). Seeds were sown in plastic trays containing compost and germinated in a growth cabinet (Reftech BV, Sassenheim, Netherlands) at ∼200 μmol m^-2 s^-1 PPFD, 14 h/10 h photoperiod (light/dark), ∼15°C on average and ∼60% relative humidity (RH). The compost material (Levington F2S) contained fertilizer (144 mg L^-1 N, 73 mg L^-1 P, 239 mg L^-1 K, adjusted to pH 5.3–6.0 with dolomitic lime) and incorporated coir and sand. Plants were watered every 2 days. At BBCH (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) growth stage (GS) 12 (GS12, two seedling leaves unfolded; Lancashire et al., 1991) seedlings were moved into a cold room for vernalization: 4°C, ∼50 μmol m^-2 s^-1 PPFD at 10 h/14 h photoperiod (light/dark) for 8 weeks. After vernalization, seedlings (one per pot) were transplanted into 1.5 L (15 cm diameter; 12 cm deep) or 4 L (16.5 cm diameter; 21 cm deep) pots (depending on the experiment) containing Levington F2S compost (Everris, Ipswich, United Kingdom). After which plants where transferred to either the glasshouse or controlled growth environment depending on experimental design (see below).

Statistical analyses were conducted using SPSS (v.16; SPSS, Inc., Chicago, IL, United States) and R¹. A two-way analysis of variance (ANOVA) was used for gas exchange data when two factors (genotype × growth stage) were present (i.e., for the variables A, g_s, K_i, K_d, T_95%A, g_slimitA, T_iWUE for Experiment 1). Single factor analyses were carried out using one-way ANOVA (i.e., for A, g_s, K_i, K_d in Experiments 2 and 3). Shapiro–Wilk and Levene’s tests were used to test data for normality and homogeneity of variance, respectively. Duncan’s test was used for multiple comparisons. When present, linear curves were fitted with major axis regression thus minimizing the variability for the traits of interest in both the axes. The strength of trait associations at GS41-45 (between steady-state and dynamic gas exchange, anatomical and photosynthetic capacity traits) and for all developmental stages (between steady-state and dynamic gas exchange traits) were measured using Pearson’s correlation coefficient.

Significant genotypic variation (P < 0.001) in steady-state A and g_s at 1500 μmol m^-2 s^-1 was recorded for the eight wheat cultivars investigated (Figure 2A–F). Soissons and Xi19 showed the highest A and g_s values, whereas Hereward showed consistently lower values. There was a significant effect of growth stage on A and g_s (P < 0.001 for both), with most of the cultivars showing higher values at GS41. Significant variation in the time constants for stomatal opening (K_i) was recorded between cultivars (P = 0.038) (Figure 2G–I) and developmental stage significant impact K_i (P < 0.001) in the majority of cultivars, with a lower time constant (thus faster g_s responses) at GS31 and GS41 compared with GS71. However, in cultivars Claire, Rialto and Robigus, there was no significant effect of growth stage on K_i. Similarly, K_d varied significantly between the different growth stages (P < 0.001), although no significant genotypic differences were found (P = 0.343) (Figure 2J–L). Most of the cultivars achieved 95% A between 7 and 15 min following a step increase in light intensity when analyzed at GS31 and GS41 and significant variation (P = 0.045) existed between cultivars (Figure 2M–O). At GS71, T_95%A was significantly longer than GS31 and GS41 (P < 0.001), between 14 and 25 min.

When plants were subjected to a step increase in light intensity, photosynthesis was limited by the slow increase in g_s, with an average limitation (g_slimitA) between 7 and 15% across genotypes (P = 0.019) and growth stages (Figure 3A–C). Soissons and Alchemy showed the greatest limitation of A by g_s (∼12% on average) while, Rialto, Hereward and Claire were less limited at ∼8% on average. Generally, g_slimitA was exacerbated at GS71 (P < 0.001), although some genotypes (Claire, Rialto) did not show any significant increases in g_slimitA at GS71 compared to GS41.

The time to restore _iWUE (T_iWUE) was generally faster at GS31 compared to GS41 and GS71 (P < 0.001) (Figure 4A–C). Hereward was the quickest to restore _iWUE due to fast stomatal closure (low K_d) at GS31 and GS41, whilst the slowest responses were observed in Alchemy at GS31 and Soissons at both GS41 and GS71 (P = 0.014). g_slimitiWUE was significantly different between cultivars (P = 0.030) and growth stages (P < 0.001) (Figure 4D–F). Across all of the growth stages measured, the temporal response of g_s for opening and closing were significantly correlated with T_95%A and g_slimitA (Figure 5). At the same time, significant correlations were found between T_iWUE and the time constant for stomatal closing.

Significant genotypic variation in V_cmax (P < 0.024) was observed within the eight cultivars analyzed (Figure 6A,B). Rialto, Soissons, and Xi19 showed the highest values for both V_cmax and J_max (∼160 and 260 μmol m^-2s^-1 on average, respectively) whereas Robigus and Hereward displayed the lowest values. Significant positive correlations were observed between photosynthetic capacity traits (A, V_cmax, J_max), g_s, speed of stomatal responses and stomatal density (Figure 7). A significant positive relationships was observed between g_s and A whilst a negative relationship between g_s and _iWUE was recorded. In addition, A was significantly and positively correlated with most of the stomatal kinetics related traits (K_i, K_d, T_95%A, g_slimitA). Interestingly, _iWUE positively correlated with the g_slimitA. Significant and positive correlations were found between the g_slimitA, T_95%A, and K_i.

Stomatal density and pore length were significantly different between the cultivars (P = 0.002 for abaxial and P < 0.001 for adaxial stomatal density while P = 0.013 for abaxial and P = 0.001 for adaxial pore length) (Table 1). The abaxial density ranged from 63.7 to 81.6 stomata mm^-2 while the adaxial density was between 61.0 and 90.4 mm^-2. Stomatal density was correlated with K_i (adaxial, positive) and T_95%A (abaxial, negative) (Figure 7) while abaxial pore length was negatively correlated with abaxial stomatal density (Figure 7).

Using the variety Soissons, reduced water availability significantly reduced A and g_s at 1500 μmol m^-2s^-1 PPFD by 45 and 63% respectively (P < 0.001) (Figure 8A,B). The time constant K_i was increased (P = 0.036) in plants grown under water stress (WS) conditions compared to the well-watered controls (WW) (Figure 8C). In contrast, a significantly lower K_d (P = 0.022) was recorded under WS compared with WW (Figure 8D).

The cv. Soissons grown under 700 μmol mol^-1 [CO₂] showed a 25% increase in A compared to the rate in control plants grown at 400 μmol mol^-1 [CO₂] (Figure 8E). In contrast, a small reduction in g_s (6%) was recorded under elevated [CO₂], although this was not significantly different from g_s at 400 μmol mol^-1 [CO₂] (Figure 8F). Elevated [CO₂] significantly reduced K_i (P = 0.047), while no differences were found for K_d (Figure 8G,H).

Previous work has demonstrated the presence of significant interspecific (Vico et al., 2011; McAusland et al., 2016) and intraspecific (e.g., rice, Qu et al., 2016) variation in the rapidity of stomatal responses or photosynthetic induction (Salter et al., 2019) in crops. Here, we show that significant genotypic variation in the rapidity of g_s is present in wheat in response to step changes in irradiance. Consistent with the conclusions of previous work (e.g., Vico et al., 2011; Lawson et al., 2012; McAusland et al., 2016), the time to reach maximum steady state g_s ranged from 7 to 27 min between cultivars. Cultivars with faster g_s opening responses (lower K_i) (e.g., Hereward, Claire) achieved 95% A more rapidly than those cultivars with slower g_s kinetics (e.g., Xi19, Soissons) supported by the positive correlation between K_i and T_95%A. At the same time, cultivars with faster stomatal closing (lower K_d, e.g., Hereward and Claire at GS41) following a high to low light transition achieve a higher _iWUE, more rapidly. These findings support previous reports in which the ‘speedy stomata’ trait has been considered a potential target for maximizing CO₂ diffusion and A, as well as _iWUE, particularly under dynamic light regimes (Lawson and Vialet-Chabrand, 2018). Significant differences in stomatal density and pore length were also observed between cultivars; interestingly, the variation in stomatal density was greater on the adaxial than the abaxial surface. However, only a weak correlation between stomatal density (adaxial) and K_i was apparent, indicating minimal anatomical influence on the speeds of g_s response in the panel of wheat cultivars analyzed. Additionally, our findings are contrary to previous research on the relationship between stomatal speed and density in the non-domesticated species of the dicot genus Banksia that have reported higher stomatal density results in faster responses (Drake et al., 2013).

In rice (Qu et al., 2016) and other species (McAusland et al., 2016), asymmetric stomatal responses (e.g., faster stomatal closure that opening) have been suggested as a strategy of prioritizing water conservation over CO₂ uptake. In our work, the relatively conserved ratio of K_i:K_d (at all growth stages) in all the cultivars studied indicates a balance between carbon gain and water conservation. However, although the time constant for opening and closure were comparable, the fact that T_iWUE was significantly higher than T_95%A indicates that slow g_s responses had a greater impact on _iWUE than A. While the varieties studied in this work are adapted to a north–west European environment (Mackay et al., 2014), and therefore likely optimized for carbon gain rather than _iWUE, wheat cultivars adapted to lower rainfall regimes may provide a more extensive natural diversity for water conservation (i.e., faster stomatal closure rather than opening). To our knowledge, this is the first report demonstrating natural variation in the speed of stomatal responses in wheat at the leaf level. However, new cutting-edge technologies, for example whole plant gas-exchange (Jauregui et al., 2018), would enable the impact of the speed of stomatal responses on whole plant net carbon assimilation and water use to be evaluated. Cultivars with fast g_s responses (e.g., Claire, Robigus, and Hereward) were followed by lower A and g_s values thus showing potential elevated adaptation to dynamic light environment and potentially water deficit conditions. In contrast, Soissons and Xi19 demonstrated high overall g_s values, but slow g_s responses, traits that may be useful for environments in which light is high and constant with higher temperatures but with sufficient water to support high g_s and evaporative cooling. These data suggest that phenotyping wheat lines for stomatal rapidity has the potential to identify novel targets for improving wheat productivity for exploitation in breeding programs.

In our study, a significant variation for V_cmax and steady-state A and g_s was found between cultivars, consistent with previous studies in wheat (e.g., Driever et al., 2014; Gaju et al., 2016). However, cultivars with greater g_s rapidity displayed lower photosynthetic capacity demonstrated by the positive relationship between A and g_s with T_95%A, and the time constants for stomatal opening and closing (K_i and K_d), respectively. This suggests a compromise between the rapidity of stomatal behavior and the values of steady state A and g_s achieved. Stomatal movement involves a series of hierarchical processes based on the transport, accumulation, and release of osmotically active solutes (Lawson and Blatt, 2014) as well as subsidiary cell physiology (Raissig et al., 2017), and any variation in these processes could result in differences in stomatal behavior. For example, variation in vascular connectivity (e.g., vein density) could explain the positive relationship between steady state A and g_s and the speed of g_s. Feldman et al. (2017) recently showed stronger photosynthetic performance in rice with increased leaf vein densities via mutagenesis. It is therefore conceivable that concurrent improvements for stomatal rapidity, photosynthetic capacity and for maximum A and g_s could be attained if vein density and hydraulic efficiency were improved.

A novel finding of this work is the significant effect of growth stage on stomatal responses. The rapidity of g_s was reduced at post-anthesis stage (GS71) compared to the earlier developmental stages (GS31 and GS41) and this corresponded with a significant decrease in both steady-state A and g_s. The decrease in post-anthesis photosynthetic capacity, and therefore reduction in radiation use-efficiency in cereals, has been reported previously (Bingham et al., 2007; Carmo-Silva et al., 2017), and mainly attributed to the onset of leaf senescence (Gaju et al., 2016). The activation of the senescence signaling pathway, thought to be triggered by sink feedback (e.g., Bingham et al., 2007), leads to degradation of chlorophyll and Rubisco and subsequent re-allocation of nutrients from the senescing parts (i.e., leaves) to the growing sink (i.e., grain), thus leading to reduction in the efficiency of the source (Camargo et al., 2016). However, to our knowledge, this is the first report showing developmental effects on stomatal responses to changes in light intensity. In particular, the data highlight growth stage- and genotype-dependent variation in stomatal rapidity, and the importance of taking into account these variables when quantifying dynamic stomatal traits. Additionally, periods of low precipitation and/or high temperature are more common during the post-anthesis stage, often leading to significant yield reductions. Faster stomatal opening could facilitate greater utilization of sudden increases in irradiance, and thus not only provide more assimilates for grain filling, but avoid any potential damage from excess excitation pressure (Yamasaki et al., 2002). Increased A is particularly important in view of the potential source-limitation (or at least source-sink co-limitation), which has been reported during grain filling in several wheat cultivars (Álvaro et al., 2008; Acreche and Slafer, 2009). Additionally, as wheat is extremely sensitive to temperature (Yamasaki et al., 2002) rapid g_s responses to increasing irradiance will facilitate maintenance of nearer optimal leaf temperatures to support maximum photosynthetic performance (Lawson and Vialet-Chabrand, 2018).

On the other hand, water conservation strategies would be enhanced by faster stomatal closure when carbon gain is reduced (e.g., during high to low light transition), thus improving the water-use budget and helping to reduce early soil water exhaustion (Bodner et al., 2015). For example, Hereward, Claire, and Robigus showed very quick g_s responses overall with minimal developmental effects (apart from Hereward at GS71), thus being good candidates for breeding exploitation for stomatal rapidity. The fact that a significant variation was observed for K_i, as well as a stage × genotype interaction for K_d and T_iWUE, suggests that the targeted exploitation of existing natural variation could be used to facilitate carbon gain for photosynthesis and optimize water-use under dynamic field environments and at different stages of wheat development.

The effect of elevated [CO₂] and water stress on stomatal rapidity has received little attention to date. A recent report on Arundo donax (Haworth et al., 2018), showed that water stress increases the rapidity of stomatal closure and reduced the speed of opening, consistent with our data in wheat. Similarly, in Lawson and Blatt (2014), Vicia faba plants subjected to water stress showed a faster g_s reduction during a shade fleck whilst a slower g_s increase was recorded for a sun fleck. However, recent work by Gerardin et al. (2018) reported an increase in rapidity for both the opening and the closing phase in Nicotiana tabacum under reduced water availability. It should be noted that in N. tabacum a strong asymmetry between the opening and closing phase (due to a faster closing phase) under control conditions was also reported. Under optimal soil water availability, asymmetric stomatal responses have not been previously described in wheat (e.g., McAusland et al., 2016), thus suggesting that the opening/closing ratio under optimal growth conditions might be species-specific and strongly influenced by water status. Thus, the presence of asymmetric stomatal responses under stress conditions could be considered as: (1) an adaptation to reduce water loss (stronger coordination between A and g_s) and (2) a mechanism to limit increasing g_s after steady state A has been achieved (McAusland et al., 2016). Our data suggests that both possibilities are conceivable under reduced water availability, with both high K_i and low K_d values observed in wheat. Water stress therefore exacerbates conservative responses under dynamic light in wheat allowing further opportunities for adaptation to reduced water availability conditions.

Only a handful of studies have examined the effect of atmospheric [CO₂] on stomatal kinetics, with most research focusing on the effects for steady stage g_s or changes in stomatal anatomy (Ainsworth and Rogers, 2007). Leakey et al. (2002) reported that in Shorea leprosula, the relative enhancement of biomass driven by elevated [CO₂] was greater under dynamic irradiance compared to uniform irradiance. Consistent with our findings in wheat, Leakey et al. (2002) suggested that faster stomatal opening under dynamic conditions reduced the time to reach maximum g_s and reduced CO₂ limitation of A. Therefore, a faster stomatal opening phase (in response to an increase in irradiance) might be a leaf trait that has an additional positive effect under elevated [CO₂] that deserves further investigation at the field level. Further efforts should focus on understanding and quantifying the effects of these major environmental factors on stomatal dynamics under fluctuating light environments.