Bundle sheath diffusive resistance to CO(2) and effectiveness of C(4) photosynthesis and refixation of photorespired CO(2) in a C(4) cycle mutant and wild-type Amaranthus edulis

Abstract

A mutant of the NAD-malic enzyme-type C(4) plant, Amaranthus edulis, which lacks phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells was studied. Analysis of CO(2) response curves of photosynthesis of the mutant, which has normal Kranz anatomy but lacks a functional C(4) cycle, provided a direct means of determining the liquid phase-diffusive resistance of atmospheric CO(2) to sites of ribulose 1,5-bisphosphate carboxylation inside bundle sheath (BS) chloroplasts (r(bs)) within intact plants. Comparisons were made with excised shoots of wild-type plants fed 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate, an inhibitor of PEPC. Values of r(bs) in A. edulis were 70 to 180 m(2) s(-1) mol(-1), increasing as the leaf matured. This is about 70-fold higher than the liquid phase resistance for diffusion of CO(2) to Rubisco in mesophyll cells of C(3) plants. The values of r(bs) in A. edulis are sufficient for C(4) photosynthesis to elevate CO(2) in BS cells and to minimize photorespiration. The calculated CO(2) concentration in BS cells, which is dependent on input of r(bs), was about 2,000 microbar under maximum rates of CO(2) fixation, which is about six times the ambient level of CO(2). High re-assimilation of photorespired CO(2) was demonstrated in both mutant and wild-type plants at limiting CO(2) concentrations, which can be explained by high r(bs). Increasing O(2) from near zero up to ambient levels under low CO(2), resulted in an increase in the gross rate of O(2) evolution measured by chlorophyll fluorescence analysis in the PEPC mutant; this increase was simulated from a Rubisco kinetic model, which indicates effective refixation of photorespired CO(2) in BS cells.

Figure 1

Schematic representation of the movement of gases and metabolites in the PEPC mutant of A. edulis. CO₂ diffuses into BS cell chloroplasts where it enters the C₃ cycle. Equilibrium between HCO₃⁻ and CO₂ in mesophyll cell is fast, but it is slow in BS because of lack of carbonic anhydrase activity. Glycerate, ammonia, and CO₂ are generated by the photosynthetic carbon oxidation (PCO) cycle. Glycerate metabolism and partial reduction of PGA in mesophyll cells may account for use of some photochemically generated energy in mesophyll chloroplasts.

Figure 2

Example of calculating BS cell resistance from A versus C_i curves measured at low O₂ (0.3 mbar) and PFD of 1,800 μmol m⁻² s⁻¹ in PEPC mutant. The inverse of the initial slope of A/C_i curve is the sum of the diffusive resistance from the cell wall to the sites of Rubisco and of the chemical RuBP carboxylation resistance. A simulated Rubisco CO₂ response curve without diffusive resistance is shown for comparison. Rubisco resistance can be calculated as K_c/V_c. V_c was taken as A_max.

Figure 3

The response of the rates of CO₂ assimilation (A, ○, ●) and gross rate of O₂ evolution from PSII (J_O2, ▴, ▵) on wild type and PEPC mutant with and without feeding DCDP. Measurements were made on leaves of excised plants under 20 mbar O₂. The calculated values of r_bs in wild-type plants in presence of DCDP and in PEPC mutant with and without DCDP are shown. A and B, Wild-type plants grown at 370 μbar CO₂; C and D, wild-type plants grown at 10 mbar CO₂; E and F, PEPC mutant grown at 10 mbar CO₂.

Figure 4

Electron microscopy showing cross sections through interface of mesophyll and BS of leaves of wild type (A) and PEPC mutant (B) with plants grown at 10 mbar CO₂. M, Part of mesophyll cell; BS, part of BS cell; W, cell wall; IS, intercellular air space. Arrows point to plasmodesmata. Scale bar = 0.5 μm. The average thickness of BS cell wall in contact with intercellular air from several sections was 0.34 μm for wild type and 0.32 μm for mutant (n = 3).

Figure 5

Temperature dependence of photosynthetic parameters for wild-type (A) and PEPC mutant (B) A. edulis measured under 0.3 mbar O₂. Shown are internal conductance in the mesophyll for wild type, g_wt (the initial slope of A/C_i curves), the internal conductance in the mutant, g_mut, and the calculated liquid phase-diffusive conductance in the mutant (g_bs) and maximal CO₂ assimilation rate (A_max). C has A_max from A and B plotted in Arrhenius axes (the slope equals −E_a/R). Values of J_O2-net measured under saturating CO₂ (data not shown) were similar to values of A_max.

Figure 6

The response of the rates of CO₂ assimilation (A, ○), net O₂ evolution (J_O2-net, □), and gross O₂ evolution from PSII (J_O2, ▵) in PEPC mutant and wild-type A. edulis to intercellular CO₂ (C_i) at two oxygen partial pressures, 210 and 0.3 mbar. The CO₂ response curves were measured at PFD = 1,800 μmol m⁻² s⁻¹ and at leaf temperature 29°C.

Figure 7

CO₂ response for CO₂ assimilation rate (A, ○), gross O₂ evolution rate (J_O2, ▵), RuBP pool size (●), Rubisco activity (⧫), and Rubisco activation state (□) for PEPC mutant A. edulis at two O₂ pressures, 0.3 and 210 mbar. Leaf temperature was 28°C, PFD = 1,400 μmol m⁻² s⁻¹. Each point is from a different leaf of similar age.

Figure 8

Light response of PEPC mutant and wild-type A. edulis O₂ evolution (J_O2-net, □) at highly saturating levels of CO₂ of 40 mbar CO₂ (O₂ = 0.3 mbar), and CO₂ uptake (A, ○) at limiting CO₂ (2 mbar for PEPC mutant and 0.36 mbar for wild type) and 210 mbar O₂ pressure. Leaf temperature was 28°C. Gross rates of O₂ evolution (J_O2, Δ) were calculated from simultaneous fluorescence measurements as described in “Materials and Methods.”

Figure 9

Oxygen sensitivity of A. edulis photosynthesis at limiting CO₂ concentrations, 30°C, and PFD = 1,800 μmol m⁻² s⁻¹. The rate of PSII O₂ evolution (J_O2) shows an increase with increasing O₂ concentration and continues at CO₂ = 0 because of re-assimilation of CO₂ released from the photorespiration and from the Krebs cycle.

Figure 10

J_O2 for the PEPC mutant of A. edulis from Figure 9 was extrapolated to CO₂ = 0, and the results were plotted against O₂ concentration. The simulated J_O2 shown by the solid line was calculated based on BS O₂ and CO₂ concentration (the latter calculated for each O₂ level considering BS-diffusive resistance) and Rubisco kinetic constants (V_c = 39, K_c = 21 μm, and K_o = 640 μm), where J_O2 equals v_c + v_o (Edwards and Baker, 1993). The rate of CO₂ re-assimilation (at zero external CO₂) is proportional to the ratio of Rubisco conductance and BS-diffusive conductance.

Figure 11

PEPC mutant and wild-type A. edulis leaf RuBP content (●) versus O₂ concentration with C_i of 0.4 mbar for mutant and 0.025 mbar for wild type. Also, CO₂ assimilation rate (A, ○) and O₂ evolution from PSII (J_O2, ▵) are shown. Leaf temperature was 28°C; light PFD = 1,800 μmol m⁻² s⁻¹.

Figure 12

CO₂ response for CO₂ assimilation rate (at leaf temperature of 28°C; PFD = 1,800 μmol m⁻²s⁻¹; ●), calculated CO₂ partial pressure in BS cells (□), and leakiness of CO₂ from BS cells (▵). The CO₂ level in BS cells was calculated according to von Caemmerer (2000), and the leakiness was calculated according to equations 16 and 17 using r_bs value of 113 m² s⁻¹ mol⁻¹. Similar results were obtained from analysis of several experiments on A/C_i responses.