Expression of tobacco carbonic anhydrase in the C4 dicot flaveria bidentis leads to increased leakiness of the bundle sheath and a defective CO2-concentrating mechanism

Abstract

Flaveria bidentis (L.) Kuntze, a C4 dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO2 assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O2 concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O2 levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O2 concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C4 CO2-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO2 for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.

Figure 1

Map of the binary construct, pBI-CA-GUS, used in the transformation of F. bidentis. The 660-bp BamHI/SacI fragment encoding the mature tobacco CA polypeptide and the gus gene were flanked by the CaMV 35S promoter and the 3′ end of the nopaline synthase (nos) gene. The nptII gene was flanked by the promoter and the 3′ end of the nos gene. Direction of transcription of the genes is indicated by the arrows. kan^r, Kanamycin resistance; RB and LB, right and left borders of the T-DNA, respectively.

Figure 2

Detection of tobacco CA expression in T₁ leaf extracts using immunoblot analysis. Numbers above the brackets designate F. bidentis primary transformed lines, whereas the numbers directly above the gel lanes represent individual progeny from the primary transformants. Equivalent amounts of tobacco (TOB) and F. bidentis leaf extracts based on equal leaf-area starting material were separated by SDS-PAGE, blotted to nitrocellulose, and labeled with the anti-tobacco CA antiserum. Immunoreactive polypeptides were detected with an HRP-conjugated secondary antibody and the enhanced chemiluminescence labeling method as described in Methods. The position of the 30-kD molecular mass marker is indicated.

Figure 3

Detection of tobacco CA in whole-leaf and bsc extracts of F. bidentis CA transformants. Whole-leaf (WL) and bsc (BSC) extracts from two transformed, negative control F. bidentis plants, 191–2.1 and 191–2.4, and two transformants expressing tobacco CA (TOB CA), 191–7.7 and 191–7.8, were separated by SDS-PAGE and immunolabeled with the anti-tobacco CA antiserum. Equivalent amounts of all of the extracts based on Rubisco content were loaded onto the gels. The position of the 30-kD molecular mass marker is indicated. TOB, Tobacco leaf extract.

Figure 4

Schematic representation of photosynthetic C flow in F. bidentis wild-type plants (A) and CA transformants (B). In F. bidentis, an NADP-ME-type C₄ plant, CO₂ is released from the C₄ acid malate in the bundle-sheath chloroplasts. The two schemes differ in the predicted relative amounts of CO₂ and HCO₃⁻, which diffuse from the bundle sheath into the mesophyll. The dashed arrows in A denote that CO₂ is the major form of inorganic C diffusing back into the mesophyll in wild-type F. bidentis plants. Whereas in the CA transformants (B), inorganic C is lost from the bundle sheath in the form of both CO₂ and HCO₃⁻ (solid arrows) because of the activity of mature tobacco CA (CA_TOB) in this compartment. See text for further details. C₃-P, Triose-P; and RUBP, ribulose-1,5-bisphosphate.

Figure 5

Short-term C isotope discrimination (▵) as a function of the intercellular-to-ambient partial pressure of CO₂ (p_i/p_a) in transformed negative control plants (191–2.1, ▪; 191–2.5, •; and 191–2.4, ▴) and F. bidentis plants expressing tobacco CA (191–7.6, ○; 191–7.8, □; and 191–7.7, ▵). The lines are not a fit to the data but depict the theoretically predicted relationship where: Δ = 4.4 + ([30 − s]Φ − 10.1)p_i/p_a. Slope of line (1) = −3.79; of line (2) = −0.512; and of line (3) = 0.898.

Figure 6

O₂ dependence of A, ΦPSII, and the ratio of ΦPSII to A in F. bidentis wild-type plants (black symbols) and transgenic plants expressing tobacco CA (white symbols; plants were selected from the T₂ progeny of transformant 191–7.8). Measurements were made at an ambient CO₂ concentration of 360 μbar, an irradiance of 1000 μmol quanta m⁻² s⁻¹, and a leaf temperature of 25°C. Values are expressed as a percentage of values at 2% O₂. Mean values at 2% O₂ were: A, 32.1 ± 1.3 and 25.8 ± 1.4; ΦPSII, 0.53 ± 0.02 and 0.502 ± 0.01; and ΦPSII/A, 7.2 ± 0.2 and 8.63 ± 0.2 for F. bidentis wild-type plants and transformants, respectively.

Figure 7

Predicted changes in the CO₂-assimilation rate, bundle-sheath leakiness (Φ) and CO₂ partial pressure (p_s), and C isotope discrimination, with changes in the degree of CO₂ and HCO₃⁻ equilibration in the bundle-sheath cytosol (α = 1 at full equilibration between CO₂ and HCO₃⁻). Calculations were made with the mathematical model of C₄ photosynthesis described by von Caemmerer et al. (1997b) at an intercellular CO₂ partial pressure of 150 μbar, a maximal Rubisco activity of 50 μmol m⁻² s⁻¹, a maximal PEP carboxylase activity of 72 μmol m⁻² s⁻¹, and a bundle-sheath conductance (g_s) of 2 (1 + 5.3 α) mmol m⁻² s⁻¹. C isotope discrimination was calculated from Δ = 4.4 + ([30 − s]Φ − 10.1)p_i/p_a, with p_i/p_a = 0.45 and s = 1.8 − 10.1(5.3α²/[1 + 5.3α]).