Respiratory energy demands and scope for demand expansion and destruction

Nonphotosynthetic plant metabolic processes are powered by respiratory energy, a limited resource that metabolic engineers—like plants themselves—must manage prudently.


Crop Model Description and Parameterization
The model is a simplified representation of crop carbon balance meant to reflect general quantitative relationships between photosynthesis, respiration, and plant carbon accumulation. In particular, the model partitions daily photosynthate carbon between respiration to support maintenance metabolism and a 'remainder' used for growth of new biomass, with the new growth partitioned between plant parts, culminating in growth of seeds only after anthesis. This method of modeling respiration can be considered a growth-and-maintenance respiration approach (Amthor, 2000;Cannell and Thornley, 2000). The model is applied to uniform, unstressed crops. All standing crop mass and flux values are for carbon rather than fresh or dry total phytomass. This study's focus was the maintenance respiration coefficient m (g C [respired] g -1 C [plant] d -1 , or simply d -1 ). The value of m in a crop modified for slower protein turnover was reduced by 6.5% relative to an unmodified crop. The change in protein half-life was assumed to have no effect on plant composition, i.e., the protein content was unchanged and therefore the growth yield YG (carbon appearing in new biomass per unit of photosynthate carbon used in growth processes) was unchanged. If protein turnover were slowed in actual plants, there might be a small reduction in crop nitrogen concentration and corresponding increase in YG due to reduced biosynthesis of enzymatic machinery needed for protein breakdown and re-synthesis.

Model Structure
The (Amthor, 2010) was used to calculate daily PAR incident on the crop (Appendix Figure 1).

Interception of PAR by the canopy (FPAR, fraction [0,1]) is given by:
where the extinction coefficient k is 0.6 in the model and L is crop leaf area index (LAI, m 2 m -2 ). LAI is the product of whole-crop leaf mass (Wleaf, g C m -2 ground) and specific leaf area s (= 0.040 m 2 (g leaf C) -1 ), which is held constant in the model. Photosynthesis (P, g C m -2 d -1 ) is the product ε Pcapacity FPAR fPAR τatm Hh,o, where ε is photosynthetic carbon assimilation (photosynthesis less photorespiration) per unit PAR intercepted (= 1.5 g C (MJ intercepted PAR) -1 in the model; Loomis & Amthor, 1999). To account for loss of canopy photosynthetic capacity due to leaf senescence toward the end of the season, a relative photosynthetic capacity factor (Pcapacity, [0,1]) is included (Appendix Figure 2).
Abscission of senesced leaves is not simulated, leading to slightly larger leaf biomass carbon at the end of the season than would be measured in the field.

Maintenance Respiration (CO2 release)
Maintenance respiration rate (RM, g C m -2 d -1 ) is the product mW, where m is the maintenance coefficient, or specific maintenance respiration rate (g C (g C in biomass) -1 d -1 ; more simply d -1 ), and W is plant carbon content (g C m -2 ground). There are four plant compartments in the model (see below) each with a separate m and W. Experimental data indicates m declines during the season (e.g., McCree and Silsbury, 1978;McCree, 1982McCree, , 1983Stahl and McCree, 1988). To accommodate this, an initial date-of-emergence value of m (m0) is defined for each plant compartment with values for each subsequent day given by: where m* is a time-dependent ontogenetic coefficient (Appendix Figure 2). As implemented for the unmodified crop, i.e., not engineered to reduce protein turnover, m0 values were 0.029, 0.022, 0.015, and 0.006 d -1 for leaf blades, roots, 'other,' and seeds, where 'other' is a mixture of leaf sheath, stem, and spike tissues. Maintenance respiration in seeds is always less than the value associated with m0 = 0.006 d -1 because m* is significantly less than unity by the time of anthesis, which in the model was at day of year 199 when D reached 0.60, the trigger for anthesis. For the reduced-proteinturnover engineered crop, m0 was reduced by 6.5% in all organs.
Whole-plant RM is the sum of mW for all four plant compartments during each day.
The modeled decline in m with crop development might be considered an artificial 'fix' to a weakness of the growth-and-maintenance respiration modeling approach (see Thornley, 2011). Alternativelyand the view taken herein-the decline in m with development reflects a decline in the fraction of biomass undergoing turnover. In leaves, this is reflected in declining nitrogen concentration with age in expanded leaves. In the case of seeds, this is related to the quantitatively significant production and accumulation of long-term storage compounds-be they carbohydrates, proteins and/or lipidsrather than metabolically active cellular fractions.

Growth (Biomass Carbon Accumulation) and Growth Respiration (CO2 Release)
Each daily value of the difference P -RM is used in growth processes (in this model setup, RM was always less than P). The associated growth respiration rate (RG, g C m -2 d -1 ) is given by: where YG is the yield (or carbon-use efficiency) of the growth processes. It is expressed as g C in new biomass per g C in substrate used for growth, with that substrate including the carbon incorporated into new biomass. YG is set to a constant value of 0.72 (McCree and Silsbury, 1978;McCree, 1982;Stahl and McCree, 1988;Cannel and Thornley, 2000;Lötscher et al., 2004) as a simplification, though it can (does) change during the lifespan of actual crops (McCree, 1988;Amthor, 2010) because the composition of biomass being synthesized changes. For example, a change from relatively low-protein, low-lipid vegetative growth early in the season to growth of high-protein and/or high-lipid seeds later in the season can cause YG to decline, whereas a transition to formation of high-carbohydrate seeds or tubers later in the seasonal can cause YG to increase. The whole-plant YG used in this model includes all processes underlying growth such as biosynthesis in growing cells/tissues, source-sink transport of carbon substrate (short-term reserve mobilization and phloem loading), and nitrogen uptake and assimilation. In models simulating direct, local biosynthesis values of YG usually will be (considerably) larger (Penning de Vries et al., 1974;Amthor, 2000;Cannell and Thornley, 2000).
Following from the above, daily growth (ΔW, g C in new biomass m -2 d -1 ) is given by: The carbon in new biomass is a combination of compounds with various longevities ranging from 'permanent' cell wall components to short-term storage compounds.
Nonstructural carbohydrate amount is not tracked in the model. Rather, it is assumed that the crop is in a relatively steady state of carbon acquisition in photosynthesis and use in growth and respiration. As such, the model is applicable to non-stressful environments with gradual and small day-today environmental changes. Further, root death, exudation of carbon from roots, leaf abscission, and herbivory were ignored in the model, as were volatilization and leaching of carbon from the plant. These can be significant amounts in some circumstances.

Partitioning ΔW Among Plant Parts
Whole-crop carbon content at emergence (W0, g C m -2 ground) was set to 0.1 g C m -2 ground and divided between Wleaf (80%) and the root compartment (Wroot, g C m -2 ground) (20%). Partitioning of ΔW among plant fractions during each simulated day after emergence then followed simple partitioning coefficient rules based on D (Appendix Figure 3). In this model application, the magnitude and temporal dynamics of the partitioning coefficients were meant to be typical of cereal crops.

Grain filling and sink strength
Modeled grain carbon (Wgrain, g C m -2 ground) came from two sources, as it does in actual crops: To account for the respiratory cost of mobilization and then biosynthesis of new grain tissue from the mobilized carbon, an amount of carbon equal to 50% of the carbon transferred from Wleaf and Wother to Wgrain is added to daily growth respiration. That respiratory carbon is removed from Wleaf and Wother. This represents the measured 67% efficiency of mobilized carbon retention in new grain biomass during grain filling in wheat (Gebbing et al., 1999). Neither the 15% of grain carbon accumulation arising from mobilization of pre-anthesis carbon accumulation nor the 67% efficiency of converting the mobilized carbon into seed biomass carbon were affected by the reduced maintenance respiration coefficient in the modeled bioengineered crop.
Although the model simulated increased pre-anthesis biomass carbon accumulation in the bioengi- 1 Supplemental Table S1. Experimental estimates for crop leaves or roots of the contribution of protein turnover to maintenance respiration or to dark respiration, depending on the study. These estimates involved a wide range of assumptions and methodologies. As mature leaf respiration supports phloem loading as well as maintenance, the contribution of protein turnover expressed on this basis is necessarily an underestimate of its contribution to maintenance respiration.