A transgenic approach to understanding the influence of carbonic anhydrase on C18OO discrimination during C4 photosynthesis

Abstract

The oxygen isotope composition of atmospheric CO(2) is an important signal that helps distinguish between ecosystem photosynthetic and respiratory processes. In C(4) plants the carbonic anhydrase (CA)-catalyzed interconversion of CO(2) and bicarbonate (HCO(3)(-)) is an essential first reaction for C(4) photosynthesis but also plays an important role in the CO(2)-H(2)O exchange of oxygen as it enhances the rate of isotopic equilibrium between CO(2) and water. The C(4) dicot Flaveria bidentis containing genetically reduced levels of leaf CA (CA(leaf)) has been used to test whether changing leaf CA activity influences online measurements of C(18)OO discrimination (Delta(18)O) and the proportion of CO(2) in isotopic equilibrium with leaf water at the site of oxygen exchange (theta). The Delta(18)O in wild-type F. bidentis, which contains high levels of CA relative to the rates of net CO(2) assimilation, was less than predicted by models of Delta(18)O. Additionally, Delta(18)O was sensitive to small decreases in CA(leaf). However, reduced CA activity in F. bidentis had little effect on net CO(2) assimilation, transpiration rates (E), and stomatal conductance (g(s)) until CA levels were less than 20% of wild type. The values of theta determined from measurements of Delta(18)O and the (18)O isotopic composition of leaf water at the site of evaporation (delta(e)) were low in the wild-type F. bidentis and decreased in transgenic plants with reduced levels of CA activity. Measured values of theta were always significantly lower than the values of theta predicted from in vitro CA activity and gas exchange. The data presented here indicates that CA content in a C(4) leaf may not represent the CA activity associated with the CO(2)-H(2)O oxygen exchange and therefore may not be a good predictor of theta during C(4) photosynthesis. Furthermore, uncertainties in the isotopic composition of water at the site of exchange may also limit the ability to accurately predict theta in C(4) plants.

Figure 1.

Net CO₂ assimilation rate, transpiration rate (E), and stomatal conductance (g_s) as a function of the rate constant of leaf CA (k_CA mol m⁻² s⁻¹ Pa⁻¹). Each point represents a measurement made on a different plant grown in a glass house at ambient CO₂ or in a growth cabinet at 0.96 kPa CO₂: wild-type plants grown at ambient CO₂ (□); anti-CA plants grown at ambient CO₂ (▪); wild type grown at 0.96 kPa CO₂ (○); and anti-CA plants grown at 0.96 kPa CO₂ (•). Gas exchange measurements were made at an irradiance of 2,000 μmol quanta m⁻² s⁻¹, leaf temperature of 30°C and an inlet CO₂ concentration of 52 Pa in 90.5 kPa of N₂ and 4.8 kPa of O₂ gas mixture.

Figure 2.

The isotopic composition (‰) of the water at the site of evaporation (δ_e) and the oxygen isotope discrimination (Δ¹⁸O) as a function of the rate constant of leaf CA (k_CA mol m⁻² s⁻¹ Pa⁻¹). Values of δ_e were calculated as described in the text using Equation 1 and source water (δ_s) was −5.3 ± 0.3‰. The line represents a linear regression (R² = 0.87) for all data except the plants with high Δ¹⁸O values. Symbols and gas exchange conditions are as in Figure 1 and δ values are expressed in reference to VSMOW.

Figure 3.

Oxygen isotope discrimination (Δ¹⁸O) as a function of the ratio of mesophyll cytosolic to ambient CO₂ partial pressure (p_m/p_a). Where p_m was calculated with g_w = 10 mol m⁻² s⁻¹ Pa⁻¹ for the C₄ plants and 5 mol m⁻² s⁻¹ Pa⁻¹ for tobacco. The line represents the theoretical relationship of Δ¹⁸O and p_m/p_a at full isotopic equilibrium where a = 7.7‰ and Δ_ea = 33.7‰ (Eq. 4) and the CO₂ supplied to the leaf had a δ¹⁸O of 24‰ relative to VSMOW. Each point represents a measurement made on a different plant grown under ambient conditions in a glass house or at 0.96 kPa CO₂ in growth chambers. Measurement conditions are as in Table II and Figure 1. Each point represents a measurement made on a different plant or different light level: wild-type F. bidentis (□); anti-CA F. bidentis (▪); anti-SSU F. bidentis (▴); wild-type Z. mays (⋄); and wild-type tobacco (▵). Plants grown in ambient and 0.96 kPa CO₂ were grouped together in this figure.

Figure 4.

The extent of isotopic equilibrium (θ) as a function of the rate constant of leaf CA (k_CA mol m⁻² s⁻¹ Pa⁻¹) in wild-type and anti-CA F. bidentis plants. The predicted values of θ were determined from in vitro CA assays using Equation 9 (A) and the measured values of θ were determined from Δ¹⁸O using Equation 7 (B). Symbols and gas exchange conditions are as in Figure 1. Calculations were made with g_w = 10 mol m⁻² s⁻¹ Pa⁻¹.

Figure 5.

The predicted isotopic equilibrium (θ) determined with various isotopic compositions of water at the site of CO₂-H₂O oxygen exchange (δ_ex). Calculations were made with g_w values ranging from 10 to 5 mol m⁻² s⁻¹ Pa⁻¹. θ was calculated using Equation 7 where Δ_ea was determined using Equation 5 and substituting δ_e with various values of δ_ex. Δ_ca was calculated from parameters taken from the high light wild-type F. bidentis measurements in Table II. δ_ex values are presented relative to VSMOW.