Antisense reduction of NADP-malic enzyme in Flaveria bidentis reduces flow of CO2 through the C4 cycle

Abstract

An antisense construct targeting the C(4) isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO(2) to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (α-NADP-ME) exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ). In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO(2) assimilation rates at high intercellular CO(2) were significantly reduced, whereas the in vitro activities of both phosphoenolpyruvate carboxylase and Rubisco were increased. Δ measured concurrently with gas exchange in these plants showed a lower Δ and thus a lower calculated leakiness of CO(2) (the ratio of CO(2) leak rate from the bundle sheath to the rate of CO(2) supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of increased Δ and leakiness. We use these measurements to estimate the C(4) cycle rate, bundle sheath leak rate, and bundle sheath CO(2) concentration. The comparison of α-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of the C(3) and C(4) cycles that exist during environmental perturbations by light and CO(2) can be disrupted through transgenic manipulations. Furthermore, our results suggest that the efficiency of the C(4) pathway could potentially be improved through a reduction in C(4) cycle activity or increased C(3) cycle activity.

Figure 1.

CO₂ assimilation rate (A), PEPC activity (B), Rubisco activity (C), dry matter carbon isotope composition (D), and nitrogen (E) as a function of NADP-ME activity in wild-type (open circles) and α-NADP-ME (closed circles) F. bidentis plants. Error bars show three technical replicates of individual plants. Linear correlations were fitted to B, C, D, and E, yielding r² values of 0.17, 0.33, less than 0.01, and 0.46 respectively.

Figure 2.

Gas exchange of wild-type (n = 4) and α-NADP-ME (n = 8) F. bidentis plants. CO₂ assimilation rates are given over a complete (A) and low (B) range of C_i. Lines depicted are four wild-type plants (open circles), four α-NADP-ME plants with NADP-ME activity from 55% to 95% of the wild type (closed triangles), and four α-NADP-ME plants with NADP-ME activity below 40% of the wild type (closed circles). Error bars show three technical replicates of individual plants. Measurements were made in the glasshouse at 1,500 µmol quanta m⁻² s⁻¹ and a leaf temperature of 25°C.

Figure 3.

Concurrent measurement of CO₂ assimilation rate (A), stomatal conductance (B), C_i/C_a (C), Δ (D), and ϕ (E) as a function of C_i. Lines and error bars represent averages and se of measurements on three individual wild-type (open circles) and α-NADP-ME (closed circles) F. bidentis plants, respectively. Measurements were made at 1,500 µmol quanta m⁻² s⁻¹ and a leaf temperature of 25°C.

Figure 4.

Concurrent measurement of CO₂ assimilation rate (A), stomatal conductance (B), C_i/C_a (C), Δ (D), and ϕ (E) as a function of irradiance. Lines and error bars represent averages and se of measurements on three individual wild-type (open circles) and α-NADP-ME (closed circles) F. bidentis plants, respectively. Measurements were made at a CO₂ concentration of 380 µmol mol⁻¹ and a leaf temperature of 25°C.

Figure 5.

A, Δ of wild-type (open circles, open squares), α-NADP-ME (closed circles), and α-SSu (closed squares) F. bidentis plants as a function of C_i/C_a. Lines represent the theoretical relationship between Δ and C_i/C_a during C₄ photosynthesis (Eq. 1) at infinite g_m with ϕ of 0.184, 0.25, and 0.34 [(Δ = 4.64 + (−5.9 − 4.64 + 29.2 × ϕ) × (C_i/C_a)]. Measurements were made as described in Figure 3. B, ϕ of wild-type (open circles, open squares), α-NADP-ME (closed circles), and α-SSu (closed squares) plants as a function of C_i. Measurements made as described in Figure 3.

Figure 6.

CO₂ assimilation rate (A and D), C₄ cycle rate (B and E), and bundle sheath leak rate (C and F) as a function of C_i (A–C) and irradiance (D–F) in wild-type (open circles, open squares), α-NADP-ME (closed circles), and α-SSu (closed squares) F. bidentis plants. The C₄ cycle and bundle sheath leak rate were calculated from Equations 6 and 7.

Figure 7.

Estimated C_s as a function of C_i (A), irradiance (B), and bundle sheath resistance (C). Symbols represent estimated bundle sheath CO₂ levels at the bundle sheath resistance assumed in this study for ϕ measurements (approximately 333 m² s⁻¹ bar⁻¹ mol⁻¹).