A mass and charge balanced metabolic model of Setaria viridis revealed mechanisms of proton balancing in C4 plants

Abstract

Background: C4 photosynthesis is a key domain of plant research with outcomes ranging from crop quality improvement, biofuel production and efficient use of water and nutrients. A metabolic network model of C4 "lab organism" Setaria viridis with extensive gene-reaction associations can accelerate target identification for desired metabolic manipulations and thereafter in vivo validation. Moreover, metabolic reconstructions have also been shown to be a significant tool to investigate fundamental metabolic traits.

Results: A mass and charge balance genome-scale metabolic model of Setaria viridis was constructed, which was tested to be able to produce all major biomass components in phototrophic and heterotrophic conditions. Our model predicted an important role of the utilization of NH[Formula: see text] and NO[Formula: see text] ratio in balancing charges in plants. A multi-tissue extension of the model representing C4 photosynthesis was able to utilize NADP-ME subtype of C4 carbon fixation for the production of lignocellulosic biomass in stem, providing a tool for identifying gene associations for cellulose, hemi-cellulose and lignin biosynthesis that could be potential target for improved lignocellulosic biomass production. Besides metabolic engineering, our modeling results uncovered a previously unrecognized role of the 3-PGA/triosephosphate shuttle in proton balancing.

Conclusions: A mass and charge balance model of Setaria viridis, a model C4 plant, provides the possibility of system-level investigation to identify metabolic characteristics based on stoichiometric constraints. This study demonstrated the use of metabolic modeling in identifying genes associated with the synthesis of particular biomass components, and elucidating new role of previously known metabolic processes.

Keywords: Ammonium and nitrate usage; Bioenergy grasses; C4 photosynthesis; Gene association; Genome-scale metabolic network model; Lignocellulosic biomass; Mass and charge balance; Millet; Setaria viridis.

Fig. 1

a Flowchart of model construction process from PlantCyc dataset; b Construction of multi-tissue model from GSM. Input/output (_tx) transporters were used to transport minerals, water, CO₂, O₂, etc., to/from external (Ext) environment. Biomass synthesis reactions (_biomass) were included to allow the model to produce biomass components which can be used to explore quantitative biomass production. Abbreviations: Chl chloroplast, Mit mitochondria, Per peroxisome, Vac vacuole, Cyto cytosol

Fig. 2

Schematic description of constraints used with the multi-tissue model to simulate the production of cellulose, hemi-cellulose (XLFG-Xyloglucan) and lignin (Sinap, sinapyl alcohol; Conol, coniferyl alcohol and Coumol, coumaryl alcohol) in stem. Gaseous exchanges of CO₂ and O₂ from environment were allowed from M and S cell types. All other mineral nutrients and water uptake were only allowed through S that can be distributed to BS and M (dashed linked arrow). Photon influx was allowed into both M and BS. Under two different case scenarios, model was either restricted (P _B) or allowed to exchange H ⁺ from environment in the three cell types. This figure only illustrates P _B condition. Four carbon compounds, Pyr, 3-PGA, DHAP and Mal were allowed to transport between M and BS cells. O₂ evolution to environment directly from BS was restricted but its exchange with M was allowed. Sucrose was allowed to transport from BS to S. Input of mineral nutrients from the external environment are shown (rectangle box), however only water was utilized under this simulation

Fig. 3

Sets of genes (a) and reactions (b) in stem involved in the production of cellulose, hemi-cellulose and lignin under P _B. One gram of each biomass were fixed to produced (one at a time) and the genes/reactions expressed in stem for the biomass synthesis were used to construct the Venn diagrams

Fig. 4

Main biosynthetic pathway for the biosynthesis of cellulose, XLFG-xylogucan and monolignols i.e., coumaryl, coniferyl and sinapyl alcohols under P _B. The flux map shown here for M and BS is common to all the three biomass synthesis scenarios. Main routes for lignin, cellulose and XLFG-xylogucan biosynthesis are shown in the stem. Abbreviations: CA, carbonic anhydrase; PEPC, phosphoenolpyruvate carboxylase; MDH, malate dehydrogenase, PPDK, pyruvateorthophosphate-dikinase, RBC, ribulose-bisphosphate-carboxylase