Alternative Crassulacean Acid Metabolism Modes Provide Environment-Specific Water-Saving Benefits in a Leaf Metabolic Model

Abstract

Crassulacean acid metabolism (CAM) evolved in arid environments as a water-saving alternative to C₃ photosynthesis. There is great interest in engineering more drought-resistant crops by introducing CAM into C₃ plants. However, it is unknown whether full CAM or alternative water-saving modes would be more productive in the environments typically experienced by C₃ crops. To study the effect of temperature and relative humidity on plant metabolism in the context of water saving, we coupled a time-resolved diel (based on a 24-h day-night cycle) model of leaf metabolism to an environment-dependent gas-exchange model. This combined model allowed us to study the emergence of CAM as a trade-off between leaf productivity and water saving. We show that vacuolar storage capacity in the leaf is a major determinant of the extent of CAM. Moreover, our model identified an alternative CAM cycle involving mitochondrial isocitrate dehydrogenase as a potential contributor to initial carbon fixation at night. Simulations across a range of environmental conditions show that the water-saving potential of CAM strongly depends on the daytime weather conditions and that the additional water-saving effect of carbon fixation by isocitrate dehydrogenase can reach 11% total water saving for the conditions tested.

Figure 1.

Modeling Water-Saving Flux Modes in an Environment-Coupled Model of Leaf Metabolism. (Top left) A diel (24-h) leaf model was constructed by concatenating copies of a core model of plant metabolism (Shameer et al., 2018). The individual models were connected via linker reactions that allowed the transfer of storage compounds in the vacuole and the plastid between successive models. Light uptake was constrained by the diel light curve. The day:night ratios of phloem output and maintenance were set to 3:1 for each hour of the diel cycle, and N uptake was constrained to a ratio of 3:2 based on previous estimates (Cheung et al., 2014). (Top right) The effect of T and RH on stomatal water loss was modeled by a simplified gas-diffusion equation. T and RH data determined the relationship between CO₂ uptake and water loss. The four stomata pores illustrate the water-saving mechanism of nocturnal CO₂ uptake. While respiration occurs in all four scenarios, dominant carbon fixation leads to a net uptake of CO₂ during the day in C₃ plants and at night in CAM plants. (Bottom) Combining metabolic and gas-exchange models allowed us to study the trade-off between productivity and water loss as competing objectives on a Pareto frontier (i.e., the line that denotes combinations of productivity and water-loss values where one objective cannot be improved without compromising the other) and revealed alternative water-saving carbon-fixation mechanisms.

Figure 2.

Metabolic Fluxes and Water Loss for Different Modeling Scenarios. (A) Example of the T and RH data used throughout the simulations. (B) Metabolic flux profiles in a C₃ leaf optimized toward phloem output (100% phloem output). The diel light curve is indicated in yellow and peaks at a maximum intensity of 250 μmol m⁻² s⁻¹. (C) Pareto analysis of phloem output versus water loss in a C₃ leaf (top) and a CAM leaf (bottom). The CAM leaf enabled a better trade-off between the two competing objectives. (D) Metabolic flux profiles for a C₃ leaf (left), a CAM leaf (middle), and a leaf with unlimited vacuolar storage capacity for different Pareto steps (right; 80, 60, 40, and 20% of the maximum phloem output [shown for a C₃ leaf in (B)]). Note the different flux scales on the right plot axis for the C₃ and CAM leaves and the leaf with unlimited storage capacity.

Figure 3.

Different Flux Distributions in a Water-Saving CAM Leaf at 80% Productivity with (Model ICDH_rev) and without (Model ICDH_irrev) Reversible Mitochondrial ICDH. (A) CO₂ budget for the two models reveals different CO₂ turnover fluxes over the course of the day. Shown are all reactions with flux > 0.5 μmol m⁻² s⁻¹ for at least one time point. I to IV indicate the four phases of the CAM cycle. The values for the cumulative contribution are given next to the reaction name for either model ICDH_rev or both models (model ICDH_rev and model ICDH_irrev). c, cytosolic; m, mitochondrial; p, plastidial. (B) Significant linker fluxes for both models. Model ICDH_rev accumulated (iso)citrate as carboxylic acid and additionally Pro and Asp. Model ICDH_irrev accumulated both malate and (iso)citrate but no amino acids. Starch levels in model ICDH_irrev were almost threefold higher than in model ICDH_rev.

Figure 4.

Major Flux Routes Involved in the CAM-Like Temporally Separated Carbon-Fixation Mechanism in a Water-Saving CAM Leaf at 80% Productivity. Analysis with reversible mitochondrial ICDH (model ICDH_rev; [A]) and analysis without reversible mitochondrial ICDH (model ICDH_irrev; [B]) are shown. The two models used different pathways to fix and release CO₂. A-CoA, acetyl-CoA; CS, citrate synthase; (Iso-)Cit, (iso)citrate; Mal, malate; P5C, 1-pyrroline-5-carboxylic acid. The gray area in (B) highlights those reactions that are active in phase II. Roman numerals indicate the sequences of reactions described in the text.

Figure 5.

Water Saving of a Leaf with CAM-Like Nocturnal Carbon Fixation by ICDH at 80% Productivity for Different Environments. (Top) Overview of the environmental conditions analyzed. The T_max-RH_min space was analyzed for different combinations of light intensity and daylength. Conditions A, B, and C are shown below. Conditions with an x are shown in Supplemental Results, Section 2.3. (Bottom) Shown are heat maps for the absolute water saving of model ICDH_rev with respect to the C₃ scenario (orange), the absolute water-saving contribution of ICDH (i.e., the difference in water saving between model ICDH_rev and model ICDH_irrev; blue), and the relative water-saving contribution of ICDH with respect to the C₃ scenario (green; note the different scaling of the color bar) for combinations of T_max and RH_min. Also shown are representative fluxes involved in carbon fixation and the shared nocturnal carbon fixation by PEPC and ICDH for the different environments at T_max = 30°C and RH_min = 0.4 (right column).