Carbonic anhydrase and its influence on carbon isotope discrimination during C4 photosynthesis. Insights from antisense RNA in Flaveria bidentis

Abstract

In C4 plants, carbonic anhydrase (CA) facilitates both the chemical and isotopic equilibration of atmospheric CO2 and bicarbonate (HCO3-) in the mesophyll cytoplasm. The CA-catalyzed reaction is essential for C4 photosynthesis, and the model of carbon isotope discrimination (Delta13C) in C4 plants predicts that changes in CA activity will influence Delta13C. However, experimentally, the influence of CA on Delta13C has not been demonstrated in C4 plants. Here, we compared measurements of Delta13C during C4 photosynthesis in Flaveria bidentis wild-type plants with F. bidentis plants with reduced levels of CA due to the expression of antisense constructs targeted to a putative mesophyll cytosolic CA. Plants with reduced CA activity had greater Delta13C, which was also evident in the leaf dry matter carbon isotope composition (delta13C). Contrary to the isotope measurements, photosynthetic rates were not affected until CA activity was less than 20% of wild type. Measurements of Delta13C, delta13C of leaf dry matter, and rates of net CO2 assimilation were all dramatically altered when CA activity was less than 5% of wild type. CA activity in wild-type F. bidentis is sufficient to maintain net CO2 assimilation; however, reducing leaf CA activity has a relatively large influence on Delta13C, often without changes in net CO2 assimilation. Our data indicate that the extent of CA activity in C4 leaves needs to be taken into account when using Delta13C and/or delta13C to model the response of C4 photosynthesis to changing environmental conditions.

Figure 1.

Arrangement of the gas flow controllers, the LI-6400 gas exchange system, and the mass spectrometer system used for simultaneous measurements of leaf gas exchange and carbon isotope discrimination. Switching between gas samples was controlled by a manual four-way valve. The zero and reference readings were made before and after each leaf measurement and averaged during the calculations.

Figure 2.

a, Net CO₂ assimilation rate. b, Ratio of intercellular to ambient CO₂ partial pressures (p_i/p_a). c, Carbon isotope discrimination (Δ¹³C). d, Bundle sheath leakiness to CO₂ (φ) as a function of PFD (μmol quanta m⁻² s⁻¹). Measurements were made at a pCO₂ of 52 Pa, a pO₂ of 4.8 kPa, and a leaf temperature of 30°C. Shown are the means ± the se of measurements made on three to five leaves from two F. bidentis wild-type plants. Values for ξ (Eq. 1) were 29.9 ± 0.75, 15.7 ± 0.54, 7.11 ± 0.24, 5.5 ± 0.23, and 4.9 ± 0.17 at PFDs of 150, 300, 800, 1,400, and 2,000 μmol quanta m⁻² s⁻¹. φ was calculated from Equation 5, assuming V_p/V_h = 0.

Figure 3.

Net CO₂ assimilation rate, the ratio of intercellular to ambient pCO₂ (p_i/p_a), and carbon isotope discrimination (Δ¹³C) as a function of the rate constant of leaf CA (k_CA μmol m⁻² s⁻¹ Pa⁻¹). The inset in c shows the expanded scale of Δ¹³C where net CO₂ assimilation is relatively constant. Each point represents a measurement made on a different plant grown in a glasshouse at ambient CO₂ or in a growth cabinet at 1% CO₂: wild-type plants grown at ambient CO₂ (□); anti-CA plants grown at ambient CO₂ (▪); wild-type grown at 1% CO₂ (○, ▵); and anti-CA plants grown at 1% CO₂ (•, ▴). Measurements were made at 2,000 μmol quanta m⁻² s⁻¹, leaf temperature of 30°C, and an inlet CO₂ concentration of either 38 Pa in air (▵, ▴) or 52 Pa of CO₂ in a 90.5 kPa of N₂ and 4.8 kPa of O₂ gas mixture (□○, ▪•). The lines represent the best fit for all measurements (wild-type and anti-CA plants) made at either 38 or 52 Pa of CO₂.

Figure 4.

Carbon isotope discrimination (Δ¹³C) as a function of the ratio of intercellular to ambient pCO₂ (p_i/p_a). The white symbols are wild-type plants and the black symbols are anti-CA plants. Other symbols and measurement conditions are as described in Figure 3. The lines represent the theoretical relationship of Δ¹³C and p_i/p_a, where φ = 0.24, the ratio of the PEPC carboxylation to the CO₂ hydration reaction (V_p/V_h) is either 0 or 1, and the b₄ parameter is calculated with the catalyzed (solid lines, b₄ = −5.7 + 7.9 V_p/V_h) and uncatalyzed (dotted lines, b₄ = −4.5 + 12.5 V_p/V_h) CO₂ and HCO₃⁻ hydration and dehydration fractionation factors. g_w was assumed to be large such that p_i = p_m.

Figure 5.

Leaf dry matter δ¹³C, determined on the entire leaf opposite to the one used for gas exchange and enzyme analysis, plotted against changes in online carbon isotope discrimination (Δ¹³C). The white symbols are wild-type plants and the black symbols are anti-CA plants. All plants were germinated and grown in a glasshouse under ambient atmospheric CO₂ conditions. The plants with extremely low δ¹³C values (•) were transferred to the 1% CO₂ growth cabinets after tissue was collected for Δ¹³C analysis.

Figure 6.

Modeling the response of net CO₂ assimilation (a); the pCO₂ in the BSC and BSC CO₂ leakiness, φ (b); and Δ¹³C (c) in response to changes in PEPC activity (V_p). The C₄ model used a V_cmax of 60 μmol m⁻² s⁻¹, a bundle sheath conductance to CO₂ per leaf area of 0.03 μmol m⁻² s⁻¹ Pa⁻¹, K_m of PEPC for CO₂ (K_p) of 8 Pa, K_m of Rubisco for CO₂ (K_c) and O₂ (K_o) of 65 Pa and 45 kPa, fraction of PSII in the BSC 0.2 and Rubisco specificity of 2,590 Pa/Pa in the gas phase, and mitochondrial respiration was 2 μmol m⁻² s⁻¹, one-half of which was assumed to occur in the mesophyll. The pO₂ in the mesophyll was assumed to be 20 kPa. Carbon isotope discrimination was calculated using the C₄ photosynthetic model output and a constant p_i/p_a of 0.4. V_h was set at 2,000 μmol m⁻² s⁻¹ and V_p/V_h varied between 0.001 and 0.1, causing only a 0.3‰ shift in Δ¹³C at a constant φ. The lines for Δ¹³C represent models determined with Equation 2 by substituting the b₄ and b₃ factors with Equations 3 and 4, respectively. The lines for Δ¹³C represent models using different fractionation factors for respiration (3‰ dotted line and −6‰ solid and dashed lines) and photorespiration (10‰ dashed line and −6.8‰ dotted and solid lines).