Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle

Abstract

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.

Figure 1.

A diagram showing the major factors that determine Δ¹³C during CO₂ exchange in CAM species during the four phases of the CAM cycle according to Equations 5 and 12. The terms p_a, p_i, and p_m are the partial pressures of CO₂ in the atmosphere, the intercellular airspace, and in the mesophyll cells. PCR and PCO refer to the photosynthetic carbon reduction and oxygenation cycles, respectively. The associated fractionation factors are shown by lowercase letters and are described in the “Theory” section.

Figure 2.

Net CO₂ uptake (A) and g_s responses as a function of changing pCO₂ measured from late afternoon (phase IV) and throughout the dark period (phase I) and into the light (phase II; B). Representative data are plotted as individual response curves for one leaf at selected intervals during the dark period. Gas exchange measurements were made at an irradiance of 300 μmol quanta m⁻² s⁻¹ in the light period, with day/night temperatures of 25°C/20°C and a pCO₂ varied against a background gas mixture of 909 mbar nitrogen and 48 mbar of O₂.

Figure 3.

Initial slope of A/p_i response (A) and variation in A_max measured every 2 h from late afternoon (phase IV) and throughout the dark period (phase I) and into the light (phase II; B). Data are plotted as mean ± SEM for three replicate leaves, with inset in A showing the relationship between initial slope and A_max, with experimental details in the Figure 1 legend. Different letters indicate significant differences between plants at P < 0.05.

Figure 4.

A, Net A. B, g_s. C, p_i/p_a measured from late afternoon (phase IV) and throughout the dark period (phase I) and into the light (phase II), with data (±SEM) for three replicate leaves from plants of K. daigremontiana maintained in a controlled-environment chamber on a reverse light/dark cycle. Gas exchange measurements were made at an irradiance of 300 μmol quanta m⁻² s⁻¹ in the light period, with day/night temperatures of 25°C/20°C and an inlet pCO₂ of 531 μbar in 909 mbar of nitrogen and 48 mbar of O₂ gas mixture.

Figure 5.

Δ¹³C measured in association with gas exchange from late afternoon (phase IV) and throughout the dark period (phase I) and into the light period (phase II), with data (white circles, ±SEM) for three replicate leaves from plants of K. daigremontiana maintained in a controlled-environment chamber on a reverse light/dark cycle. Predicted Δ¹³C for the dark period (phase I) determined from the model developed in this article (see “Theory” section for details); dashed line is modeled Δ¹³C including correction for the draw down from substomatal cavity to cytoplasm calculated with g_i = 0.044 mol m⁻² s⁻¹ bar⁻¹ with Equation 8 and 14 b₄ = 6.27‰ at a leaf temperature of 20°C; continuous line is modeled Δ¹³C using infinite g_i (effectively using p_i/p_a) to parameterize the model.

Figure 6.

A, Transpiration rate. B, Leaf to air VPD. C, The ¹⁸O isotopic composition of δ_t and at the δ_e calculated from the Craig-Gordon model (Eq. 18), measured from late afternoon (phase IV) and throughout the dark period (phase I) and into the light (phase II) with data (±SEM) for three replicate leaves from plants of K. daigremontiana that had been maintained in a controlled-environment chamber on a reverse light/dark cycle. The source water used for irrigation of plants had an isotopic composition of δ¹⁸O_VSMOW = −5.5‰.

Figure 7.

Δ¹⁸O measured in CO₂ and in association with gas exchange, from late afternoon (phase IV) and throughout the dark period (phase I) and into the light period (phase II), with data (white circles, ± SEM) for three replicate leaves from plants of K. daigremontiana maintained in a controlled-environment chamber on a reverse light/dark cycle.

Figure 8.

Δ¹⁸O as a function of p_i/p_a (A) and p_m/p_a (B and C). In A, no correction was made for g_i, whereas in B and C, p_m was derived by assuming a g_i of 0.0.85 or 0.044 mol m⁻² s⁻¹ bar, respectively. The lines are not fitted to the data but represent the theoretical relationship of Δ¹⁸O and p_i/p_a or p_m/p_a at full isotopic equilibrium, where a = 7.7‰ and Δ_ea = 45‰ or 51‰ using either the average δ_t or δ_s values, respectively, in Equation 24. The value of g_i = 0.085 in B was estimated from minimizing the variance between measured and theoretical Δ¹⁸O calculated with the average δ_t. The CO₂ supplied to the leaf had a Δ¹⁸O of 24‰ relative to VSMOW. Data points in each section represent the individual samples collected during light (white circles) and dark (black circles) from three replicate leaves of K. daigremontiana (for details, see Figs. 2 and 5, legends above).

Figure 9.

Modeled predictions (Eqs. 8 and 14) of Δ¹³C for two contrasting assimilation rates and a p_i/p_a = 0.45 using b₄ = 6.27‰ at a leaf temperature of 20°C plotted as a function of varying g_i during phase I.