Comparative feedstock analysis in Setaria viridis L. as a model for C4 bioenergy grasses and Panicoid crop species

Abstract

Second generation feedstocks for bioethanol will likely include a sizable proportion of perennial C4 grasses, principally in the Panicoideae clade. The Panicoideae contain agronomically important annual grasses including Zea mays L. (maize), Sorghum bicolor (L.) Moench (sorghum), and Saccharum officinarum L. (sugar cane) as well as promising second generation perennial feedstocks including Miscanthus×giganteus and Panicum virgatum L. (switchgrass). The underlying complexity of these polyploid grass genomes is a major limitation for their direct manipulation and thus driving a need for rapidly cycling comparative model. Setaria viridis (green millet) is a rapid cycling C4 panicoid grass with a relatively small and sequenced diploid genome and abundant seed production. Stable, transient, and protoplast transformation technologies have also been developed for Setaria viridis making it a potentially excellent model for other C4 bioenergy grasses. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor response and saccharification dynamics of Setaria viridis are compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A genes was performed identifying eight candidate sequences. Two developmental stages; (a) metabolically active young tissue and (b) metabolically plateaued (mature) material are examined to compare biomass performance metrics.

Keywords: Panicoideae; Setaria; biofuel; cell wall; cellulose synthase; lignocellulose.

FIGURE 1

Representative sample points exemplified during the developmental program of Setaria and sorghum. Plants were sampled at two stages: immature tissue denoted as stage 1 (S1) and mature tissue denoted as stage 2 (S2), exemplified here for Setaria (A) and sorghum (B).

FIGURE 2

Cellulose and lignin determination of acid-insoluble residue (AIR). Cellulose content was determined in aboveground (A) and belowground (B) material for Setaria, maize, sorghum, and switchgrass at both stage S1 and S2. The acid-insoluble lignin portion of aboveground (C) and belowground (D) and acid-soluble lignin (E,F) was quantified. n = 3 biological replicates. Error bars represent the standard error from the mean. One-way ANOVA with a post hoc Tukey test was performed across the four species per stage. Convention adopted is small letters for stage 1 (S1) and capital letters for stage 2 (S2). Same letters indicate non-significance (P > 0.05) different letters indicate significant differences (P < 0.05).

FIGURE 3

Neutral sugar monosaccharide analysis for aboveground material. The relative abundance of each sugar was compared to a true standard and determined Setaria, maize, sorghum, and switchgrass at S1 (A,B) and S2 (C,D). n = 3 biological replicates. Error bars represent the standard error from the mean. One-way ANOVA with a post hoc Tukey test was performed across the four species per stage. Convention adopted is small letters for stage 1 (S1) and capital letters for stage 2 (S2). Same letters indicate non-significance (P > 0.05) different letters indicate significant differences (P < 0.05).

FIGURE 4

Comparing response to cellulose biosynthesis inhibitors (CBIs) in Setaria and sorghum. Response to 2,6-dichlorobenzonitrile (DCB), morlin (7-ethoxy-4-methyl chromen-2-one), and isoxaben (N-[3-(1-ethyl-1-methylpropyl)-1,2-oxazol-5-yl]-2, 6-dimethoxybenzamide) from switchgrass (A) and sorghum (B) was examined during early seedling growth using in plate assays on sterile Murashige and Skoog (MS)-agar media. Average root length was measured (n ≥ 10) and error bars represent standard error from the mean.

FIGURE 5

Phylogenetic trees of CESA proteins (A) and CESA genes (B). Phylogenetic relationships were determined for A. thaliana (AT), Setaria italica (SI), Z. mays (ZM), Sorghum bicolor (SB), and P. vulgare (PV).

FIGURE 6

Pseudo-apparent kinetics and cellulosome biodegradation of total biomasses. Michaelis–Menten enzymatic kinetics were determined for semi-purified cellulose for the aboveground stage 2 (S2) of Setaria viridis (SV); Z. mays (ZM); Sorghum bicolor (SB), and P. vulgare (PV).

FIGURE 7

Total carbon (A) and lactate (B) production obtained through Clostridium thermocellum system during biomass deconstruction. In (A), biological degradation of the four grasses was completed through Clostridium thermocellum system. The saccharification efficiency was determined for stage 1 and 2 (S1, S2) and for aboveground and belowground material for Setaria viridis (SV); Z. mays (ZM); Sorghum bicolor (SB), and P. vulgare (PV). In (B), The graph displays the acetate production for stage 1 and 2 and for three time points (24, 120, and 240 h) representing the initial stage the middle and the final stage of the process. The convention employed in the graph was: name of species plus S (S denotes shoot) for the aboveground and R (R denotes root) for the belowground material.