Engineering Strategies to Boost Crop Productivity by Cutting Respiratory Carbon Loss

Abstract

Roughly half the carbon that crop plants fix by photosynthesis is subsequently lost by respiration. Nonessential respiratory activity leading to unnecessary CO₂ release is unlikely to have been minimized by natural selection or crop breeding, and cutting this large loss could complement and reinforce the currently dominant yield-enhancement strategy of increasing carbon fixation. Until now, however, respiratory carbon losses have generally been overlooked by metabolic engineers and synthetic biologists because specific target genes have been elusive. We argue that recent advances are at last pinpointing individual enzyme and transporter genes that can be engineered to (1) slow unnecessary protein turnover, (2) replace, relocate, or reschedule metabolic activities, (3) suppress futile cycles, and (4) make ion transport more efficient, all of which can reduce respiratory costs. We identify a set of engineering strategies to reduce respiratory carbon loss that are now feasible and model how implementing these strategies singly or in tandem could lead to substantial gains in crop productivity.

Figure 1.

Plant Respiration and Its Relation to Biomass Yield. (A) The plurality of diverse metabolic processes that underlie respiratory CO₂ losses (red) contrasted with the unitary process of photosynthetic carbon gain (blue). (B) The main current strategy to enhance crop productivity through modification of central metabolism is to increase net photosynthetic carbon gain. A now-viable alternative is to decrease respiratory carbon loss. These two strategies can be pursued in parallel and then combined.

Figure 2.

Growth and Maintenance Respiration. (A) The conceptual split between respiration that drives the stoichiometric synthetic and transport processes required for growth and respiration that fuels cyclic maintenance processes. The bottom part of the figure shows processes that can to some degree be assigned to growth or maintenance. (B) Experimental measurement of growth and maintenance components of respiration. This relationship is derived by dividing both sides of Equation 1 by biomass amount, giving specific (i.e., per unit dry mass) rates of respiration and growth as follows: R/W = g(dW/dt)/W + m. Respiration rates are measured at various growth rates; the specific respiration rate at zero growth (derived by extrapolation) is taken as the maintenance respiration coefficient m. The maintenance coefficient can be assumed, as a first approximation, not to depend on growth rate. The slope of the relationship is an estimate of the growth respiration coefficient g. This relationship, and Equation 1, is referred to as a two-component coupled respiration model (Hunt and Loomis, 1979) because respiration rate R is coupled to growth rate dW/dt through g.

Figure 3.

Reducing THI4 Turnover, Reconfiguring Lignin Synthesis, and Suppressing Futile Cycles. (A) Plants synthesize the thiazole phosphate precursor of thiamin from NAD and Gly via a suicidal THI4 enzyme that takes the sulfur atom needed for the thiazole ring from an active-site Cys residue. This converts the Cys to dehydroalanine (DHAla), which inactivates THI4 (Chatterjee et al., 2011). Plant THI4 proteins must consequently be completely degraded and resynthesized after mediating a single reaction. Certain prokaryotes such as Methanococcus jannaschii have THI4s with a His residue in place of the active-site Cys (shown in sequence alignment with Arabidopsis THI4) that use free sulfide as sulfur donor (Eser et al., 2016). These prokaryotic THI4s are true catalysts, i.e., they perform multiple reaction cycles and so make thiazole synthesis far less energetically expensive than when a suicidal THI4 is used. (B) Alternative plant pathways for conversion of arogenate to 4-coumarate, a precursor to lignin. The reaction set involving Phe, which oxidizes one NADPH per 4-coumarate formed, is found in all plants, but the alternative pathway via Tyr, which reduces one NADP per 4-coumarate formed, is found predominantly in monocots. The overall efficiency of lignin biosynthesis via Tyr is expected to be significantly greater than that of the more common pathway via Phe because of a net reduction in NADPH use. (C) Futile cycling between Suc synthesis and degradation, highlighting ATP-consuming processes. Suc is synthesized via Suc phosphate synthase (SPS, blue) and sucrose 6-phosphate phosphatase (SPP) and degraded and metabolised either via sucrose synthase (SuSy, green), UDP Glc pyrophosphorylase (UGPase), and fructokinase (FK) or via invertase (INV, purple), hexokinase (HK), and FK. Suc synthesis from two hexose phosphates requires one UTP per Suc, with one PPi being salvaged (not shown). Suc degradation via SuSy requires one PPi per Suc (UTP is internally recycled; not shown). Suc degradation via INV requires two ATP per Suc, plus additional energy for active transport steps (not shown). In the futile cycles, net energy loss is one ATP per Suc in the cycle with SuSY, UGPase and INV, and two ATP per Suc in the cycle with INV, HK, and FK. It is assumed that FK utilizes UTP and/or that the uridine and adenine nucleotide systems are equilibrated via nucleoside diphosphate dikinase (Geigenberger et al., 1993). For simplicity, transport steps needed when INV is located in the vacuole or apoplast are omitted. The rate of cycling can be measured by analyzing labeling patterns after supplying isotopically labeled Suc, Fru, and Glc. (D) Futile cycling between fructose-6P and fructose-1,6BP. This cycle can occur due to simultaneous activity of two of these three enzymes: ATP-phosphofructokinase (PFK), fructosebisphosphatase (FBPase), and pyrophosphate fructose 6-phosphate phosphotransferase (PFP). PFP (blue) catalyzes a readily reversible reaction. Net energy loss per cycle per unit fructose-6P is one ATP for the cycle between PFK and FBPase, the difference in free energy between one ATP and one PPi for the cycle between PFK and PFP, and one PPi for the cycle between PPi and FBPase. Cycling between fructose-6P and fructose-1,6BP can be measured as randomization of ¹⁴C or ¹³C label between the carbon-1 and carbon-6 positions.

Figure 4.

Strategies to Reduce the Costs of NO₃⁻ Acquisition. In the schemes above the line, the native state is on the left and the reduced-cost engineered state is on the right. Red arrows are proton fluxes or malate (Mal²⁻) fluxes as indicated, solid black arrows are NO₃⁻ transport fluxes, and dashed black arrows are NO₃⁻ leaks. Blue ovals are NO₃⁻ transporters, i.e., NRT family members and the NAXT NO₃⁻ excretion transporter. The red oval represents aluminum-activated malate transporters (ALMTs). The green oval is the ATP-driven H⁺ pump that generates the electrochemical proton gradient for NO₃⁻ uptake. Cost reduction strategies are as follows. (A) Identifying and eliminating NO₃⁻ leaks, e.g., via the NAXT1 transporter. (B) Modifying selectivity of NO₃⁻ transporters and anion channels. Anion channels such as the ALMTs that can be permeable to NO₃⁻ and that would normally allow leakage out of the cytoplasm to the external medium (negative membrane potential and low external NO₃⁻), potentially enable a futile cycle. Altering the selectivity of NRT transporters is possible to reduce competing Cl⁻ transport. (C) Increasing the density on specific membranes (e.g., in root hair cells) of an optimized NO₃⁻ transporter would effectively increase the flux density (V_max) that may more efficiently capture NO₃⁻ when and where it becomes available in soil and possibly linked to high water flows to the root. (D) Location, timing, and flux capacity are critical for any strategy. For instance, eliminating an efflux transporter from the xylem parenchyma as well as from the root cortex would compromise transport to the shoot. Similarly, expression of an influx transporter in the xylem parenchyma could result in a futile cycle where a net efflux is required to the xylem. Heterogeneous concentrations of NO₃⁻ in the soil require control of lateral root foraging matched with transport capacity and demand by the shoot.

Figure 5.

Potential Biomass Gains Accruing from Reduced Maintenance Respiration. A simple integrative model to quantify potential effects of a fractional reduction in the maintenance respiration coefficient (m) on the potential proportional gain in whole-plant growth written as αβ/(1 − β), where α is the fraction of m that is “engineered away” and β is the whole-plant mW:P (maintenance respiration [mW = R_M]/photosynthesis) ratio. Potential biomass gains are plotted for discrete values of α (different lines) over the range 0.10 to 0.40 for β. Over a growing season, we expect β to be in the range 0.20 to 0.25 for non-stressed crops.