The role of phosphoenolpyruvate carboxylase during C4 photosynthetic isotope exchange and stomatal conductance

Abstract

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) plays a key role during C(4) photosynthesis and is involved in anaplerotic metabolism, pH regulation, and stomatal opening. Heterozygous (Pp) and homozygous (pp) forms of a PEPC-deficient mutant of the C(4) dicot Amaranthus edulis were used to study the effect of reduced PEPC activity on CO(2) assimilation rates, stomatal conductance, and (13)CO(2) (Delta(13)C) and C(18)OO (Delta(18)O) isotope discrimination during leaf gas exchange. PEPC activity was reduced to 42% and 3% and the rates of CO(2) assimilation in air dropped to 78% and 10% of the wild-type values in the Pp and pp mutants, respectively. Stomatal conductance in air (531 mubar CO(2)) was similar in the wild-type and Pp mutant but the pp mutant had only 41% of the wild-type steady-state conductance under white light and the stomata opened more slowly in response to increased light or reduced CO(2) partial pressure, suggesting that the C(4) PEPC isoform plays an essential role in stomatal opening. There was little difference in Delta(13)C between the Pp mutant (3.0 per thousand +/- 0.4 per thousand) and wild type (3.3 per thousand +/- 0.4 per thousand), indicating that leakiness (), the ratio of CO(2) leak rate out of the bundle sheath to the rate of CO(2) supply by the C(4) cycle, a measure of the coordination of C(4) photosynthesis, was not affected by a 60% reduction in PEPC activity. In the pp mutant Delta(13)C was 16 per thousand +/- 3.2 per thousand, indicative of direct CO(2) fixation by Rubisco in the bundle sheath at ambient CO(2) partial pressure. Delta(18)O measurements indicated that the extent of isotopic equilibrium between leaf water and the CO(2) at the site of oxygen exchange () was low (0.6) in the wild-type and Pp mutant but increased to 0.9 in the pp mutant. We conclude that in vitro carbonic anhydrase activity overestimated as compared to values determined from Delta(18)O in wild-type plants.

Figure 1.

Carbon isotope discrimination (Δ¹³C) as a function of the ratio of intercellular to ambient pCO₂ (p_i/p_a) in wild-type and mutant A. edulis plants. The dashed line represents the theoretical relationship of Δ¹³C₄ and p_i/p_a during C₄ photosynthesis where φ = 0.24, using Equation 6. The solid lines represent the theoretical relationship of Δ¹³C₃ and p_i/p_a using the C₃ model (Eq. 3). Gas-exchange conditions are as in Table I. Each point represents the means ± se of measurements made on three to five leaves from separate plants from wild type (▪), Pp mutant (▴), and pp mutant (•).

Figure 2.

Oxygen isotope discrimination (Δ¹⁸O) as a function of the ratio of mesophyll cytosolic to ambient CO₂ partial pressure (p_m/p_a). p_m was calculated with g_m = 1 mol m⁻² s⁻¹ bar⁻¹. 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. 8) and the CO₂ supplied to the leaf had a δ¹⁸O of 24‰ relative to VSMOW. Symbols are as in Figure 1 and measurement conditions are as in Table I. The inset shows the expanded scale of Δ¹⁸O for the wild-type and Pp plants.

Figure 3.

Stomatal conductance for wild-type and pp mutant plants under growth conditions (indicated by arrow) at 400 μmol quanta m⁻² s⁻¹, leaf temperature of 30°C, and the leaf chamber humidity was 29.6 ± 0.6 and 32.4 ± 0.4 mmol mol⁻¹ for the wild-type and pp mutant, respectively. Plants were subsequently transferred from the growth cabinets at time zero and a leaf was immediately placed into the gas-exchange chamber under growth conditions except the CO₂ concentration was 360 μbar instead of 9.8 mbar. The leaf chamber humidity was maintained at 30.01 ± 0.01 for both the wild-type and pp mutant. Data are the means of measurements of four different wild-type and five different pp mutants, error bars represent se; wild type (○) and pp mutant (•).

Figure 4.

Light induction of stomatal conductance (A) and net CO₂ assimilation (B) in wild-type and the pp mutant. Plants were grown under 9.8 mbar CO₂ and then transferred to ambient CO₂ in the dark overnight. Leaf gas exchange was measured for several minutes prior to the start of illumination at 2,000 μmol quanta m⁻² s⁻¹ (indicated by the arrow). The rate of stomatal opening for the wild-type and pp mutant was 7.8 ± 1.9 and 2.4 ± 0.5 (mmol water m⁻² s⁻²), respectively. The ambient CO₂ partial pressure was maintained at 364 μbar for the duration of the measurements. Data are the means of measurements of six different plants, error bars represent se; wild type (○) and pp mutant (•).

Figure 5.

A, The net change in stomatal conductance over time after a step change from 364 μbar to 48 μbar CO₂, normalized by the initial conductance in 364 μbar CO₂. Steady-state conductance at low CO₂ was 0.83 ± 0.08 (wild type) and 0.41 ± 0.05 (pp mutant) mol water m⁻² s⁻¹ and the rate of stomatal opening for the wild-type and pp mutant was 20.2 ± 4.5 and 11.9 ± 2.3 (mmol water m⁻² s⁻²), respectively. Illumination was kept at 2,000 μmol quanta m⁻² s⁻¹ for the duration of the experiment. B, The change in CO₂ assimilation rate over time at low CO₂ partial pressure is shown for comparison. Data are the means of measurements of four and five different plants, respectively, for the wild-type (○) and homozygous pp mutant (•). Error bars represent se.

Figure 6.

Soluble protein profile of leaf discs and epidermis of A. edulis wild-type (WT) and pp mutant. A, Coomassie Blue-stained SDS-PAGE gel of soluble leaf protein from leaf and epidermal (epi) fraction. B, Immunoblot of PEPC and the large subunit of Rubisco (RbcL), shown as a loading control. Thirty micrograms of total protein were loaded per lane. The epidermal PEPC protein content of the pp mutant was 53% ± 4% of wild type. The relative abundance of epidermal PEPC protein in the pp mutant compared to wild type was determined from immunoblot labeling of three wild-type and four pp mutant epidermal extractions. Shown here is one representative blot.