Impaired Stomatal Control Is Associated with Reduced Photosynthetic Physiology in Crop Species Grown at Elevated [CO2]

Abstract

Physiological control of stomatal conductance (G_s) permits plants to balance CO₂-uptake for photosynthesis (P_N) against water-loss, so optimizing water use efficiency (WUE). An increase in the atmospheric concentration of carbon dioxide ([CO₂]) will result in a stimulation of P_N and reduction of G_s in many plants, enhancing carbon gain while reducing water-loss. It has also been hypothesized that the increase in WUE associated with lower G_s at elevated [CO₂] would reduce the negative impacts of drought on many crops. Despite the large number of CO₂-enrichment studies to date, there is relatively little information regarding the effect of elevated [CO₂] on stomatal control. Five crop species with active physiological stomatal behavior were grown at ambient (400 ppm) and elevated (2000 ppm) [CO₂]. We investigated the relationship between stomatal function, stomatal size, and photosynthetic capacity in the five species, and then assessed the mechanistic effect of elevated [CO₂] on photosynthetic physiology, stomatal sensitivity to [CO₂] and the effectiveness of stomatal closure to darkness. We observed positive relationships between the speed of stomatal response and the maximum rates of P_N and G_s sustained by the plants; indicative of close co-ordination of stomatal behavior and P_N. In contrast to previous studies we did not observe a negative relationship between speed of stomatal response and stomatal size. The sensitivity of stomata to [CO₂] declined with the ribulose-1,5-bisphosphate limited rate of P_N at elevated [CO₂]. The effectiveness of stomatal closure was also impaired at high [CO₂]. Growth at elevated [CO₂] did not affect the performance of photosystem II indicating that high [CO₂] had not induced damage to the photosynthetic physiology, and suggesting that photosynthetic control of G_s is either directly impaired at high [CO₂], sensing/signaling of environmental change is disrupted or elevated [CO₂] causes some physical effect that constrains stomatal opening/closing. This study indicates that while elevated [CO₂] may improve the WUE of crops under normal growth conditions, impaired stomatal control may increase the vulnerability of plants to water deficit and high temperatures.

Keywords: drought; food security; photosynthetic down-regulation; stomatal behavior; stomatal evolution; stomatal sensitivity.

FIGURE 1

Illustration of gas exchange measurements in sunflower to determine: (A) Stomatal sensitivity to C_a – plants grown in atmospheres of 400 ppm (open symbols, black line) and 2000 ppm (closed symbols, gray line) were exposed to instantaneous increases in C_a. Stomatal conductance was allowed to stabilize at each C_a before data was logged. The difference between G_s values at C_a values of 400 and 2000 ppm [CO₂] is used to infer stomatal closure. (B) Stomatal closure to darkness – the leaves of plants grown in atmospheres of 400 ppm (black line) and 2000 ppm (gray line) were placed in a cuvette, after G_s had remained stable for 10 min the light in the cuvette and room were simultaneously switched off (represented by the vertical black line). Fifty percent of the maximum and maximum stomatal closure are marked by dashed horizontal lines. The speed of stomatal closure at 50% and maximum stomatal closure are marked by dashed vertical lines labeled a and b, respectively. The lack of stability in G_s values immediately after the cessation of illumination may be an artifact related to changes in the temperature of the leaf cuvette affecting the measurement of relative humidity of the air within the cuvette. For clarity, the points taken to illustrate the determination of stomatal closure are only showed in relation to the plants grown at 400 ppm [CO₂] (black lines). Plants grown at 2000 ppm (gray lines) are included to illustrate the impact of [CO₂] on stomatal control, with the black arrow indicating the loss of stomatal control incurred by growth at elevated [CO₂].

FIGURE 2

The relationship between photosynthesis (P_N) and (A) stomatal conductance to CO₂ (G_sCO2; linear regression F_1,37 = 107.922; P = 1.607 × 10^-12; R² = 0.863), (B) mesophyll conductance to CO₂ (G_mCO2; linear regression F_1,37 = 0.052; P = 0.821), and (C) total conductance to CO₂ (G_totCO2; linear regression F_1,37 = 46.763; P = 4.659 × 10^-8; R² = 0.747). Solid black line indicates best fit, gray lines either side indicate 95% confidence intervals of the mean.

FIGURE 3

The relationship between maximum rates of photosynthesis (P_{N max}) and speed of stomatal closure during the initial 50% closure (A) (linear regression F_1,37 = 6.814; P = 0.0130; R² = 0.394) and to the maximum extent of stomatal closure, (B) (linear regression F_1,37 = 17.654; P = 0.00016; R² = 0.568); and the relationship between maximum rates of stomatal conductance (G_smax) and speed of stomatal closure during the initial 50% closure, (C) (linear regression F_1,37 = 7.235; P = 0.0107; R² = 0.404) and to the maximum extent of stomatal closure, and (D) (linear regression F_1,37 = 0.058; P = 0.811). Solid black line indicates best fit, gray lines either side indicate 95% confidence intervals of the mean. Symbols as in Figure 2.

FIGURE 4

The relationship between the stomatal pore length (SPL) and speed of stomatal closure to darkness: (A) speed of stomatal closure during the initial 50% of closure versus SPL of plants grown at 400 ppm [CO₂] (linear regression F_1,37 = 1.358; P = 0.259); (B) speed of stomatal closure during the initial 50% of closure versus SPL of plants grown at 2000 ppm [CO₂] (linear regression F_1,37 = 0.0331; P = 5.378; R² = 0.490); (C) speed of stomatal closure during the time taken to achieve maximum closure versus SPL of plants grown at 400 ppm [CO₂] (linear regression F_1,37 = 16.304; P = 0.0008; R² = 0.689), and (D) speed of stomatal closure during the time taken to achieve maximum closure versus SPL of plants grown at 2000 ppm [CO₂] (linear regression F_1,37 = 24.190; P = 0.0001; R² = 0.766). Solid black line indicates best fit, gray lines either side indicate 95% confidence intervals of the mean. Symbols as in Figure 2.

FIGURE 5

The impact of growth at an elevated [CO₂] of 2000 ppm on photosynthetic physiology and stomatal sensitivity to C_a and closure to darkness. Linear regression was used to calculate F and P values are as follows: (A) F_1,6 = 2.889, P = 0.140; (B) F_1,7 = 0.079, P = 0.787; (C) F_1,6 = 29.981, P = 0.00155, R² = 0.913; (D) F_1,4 = 28.643, P = 0.00587, R² = 0.877; (E) F_1,6 = 0.312, P = 0.597; (F) F_1,6 = 0.726, P = 0.427; (G) F_1,7 = 0.0252, P = 0.878; (H) F_1,6 = 1.430, P = 0.277; (I) F_1,4 = 81.006, P = 0.0008, R² = 0.953; (J) F_1,6 = 5.604, P = 0.0557; (K) F_1,6 = 0.0325, P = 0.863; (L) F_1,7 = 1.549, P = 0.253; (M) F_1,6 = 1.600, P = 0.253; (N) F_1,4 = 9.705, P = 0.0357, R² = 0.708; (O) F_1,6 = 0.312, P = 0.597; (P) F_1,6 = 0.108, P = 0.754; (Q) F_1,7 = 0.378, P = 0.558; (R) F_1,6 = 1.309, P = 0.296; (S) F_1,4 = 46.374, P = 0.00243, R² = 0.921; (T) F_1,6 = 0.184, P = 0.682. Symbols as in Figure 2.

FIGURE 6

The relationship between the proportional change in the maximum rate of carboxylation of ribulose-1,5-bisphosphate carboxylase/oxygenase (ΔVc_max) with (A) the change in stomatal closure to a change in C_a from 400 to 2000 ppm [CO₂] (Δstomatal closure to C_a; linear regression F_1,17 = 0.819; P = 0.378), and (B) stomatal closure to darkness in plants grown at an elevated [CO₂] of 2000 ppm (Δstomatal closure to darkness; linear regression F_1,17 = 44.454; P = 3.966 × 10^-6; R² = 0.851), and the relationship between the proportional change in the maximum rate of electron transport required for ribulose-1,5-bisphosphate regeneration (ΔJ_max) with (C) Δstomatal closure to C_a (linear regression F_1,17 = 7.244; P = 0.0154; R² = 0.547), and (D) Δstomatal closure to darkness (linear regression F_1,17 = 4.506; P = 0.0488; R² = 0.458). Solid black line indicates best fit, gray lines either side indicate 95% confidence intervals of the mean. Symbols as in Figure 2.