Cell Wall Composition and Biomass Recalcitrance Differences Within a Genotypically Diverse Set of Brachypodium distachyon Inbred Lines

Abstract

Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences and recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syringyl:guaiacyl:p-hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p-coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD and biomass accumulation, vernalization was found to affect cell wall composition and free sugars accumulation in some Brachypodium inbred lines, suggesting genetic differences in how vernalization affects carbon flux to polysaccharides. The availability of related RIL populations will allow for the genetic and molecular dissection of this natural variation, the knowledge of which may inform ways to genetically improve bioenergy crop grasses.

Keywords: Pooideae; bioenergy; digestibility; grass; hemicellulose; lignin; polysaccharide; vernalization.

FIGURE 1

Growth habits of Brachypodium distachyon inbred lines. Representative images of non-vernalized plants from lines (A) Bd21, (B) Bd30-1, (C) Bd3-1 (pictures taken 44 days after planting), and (D) Bd2-3 (picture taken 52 days after planting), as well as vernalized plants from lines (E) Bd1-1 and (F) Bd18-1 (pictures are of 115 day-old plants, 37 days after the vernalization period). Bd21-3 plants, which are not shown, looked essentially identical to Bd21 plants. Plants were grown at a density of five plants per 10 cm pot under a 20:4 h light:dark photoperiod.

FIGURE 2

Quantification of growth habits. Means ± SEM for (A) height of each plant’s tallest culm, (B) aboveground dry mass (Log₁₀ backtransformed means ± approximate SEM), (C) culm number per plant (Log₁₀ backtransformed means ± approximate SEM), and (D) heading date (HD). Bd1-1 and Bd18-1 were vernalized 7 weeks to induce flowering (days of vernalization not included in HD). Different letters over the means indicate significant differences, α′ = 0.003, N = 84, n = 12 where n is the number of pots for each line (each pot contained five plants that were measured together to generate a mean).

FIGURE 3

Quantification of cell wall components. Means ± SEM for stem plus leaf sheath cell wall (A) acetyl bromide soluble lignin (ABSL), (B) Syringyl:Guaiacyl lignin units ratio (S:G ratio, Log₁₀ backtransformed means ± approximate SEM), (C) thioacidolysis lignin units composition, and (D) p-coumaric acid (pCA, left) and ferulic acid (FA, right) amounts are shown. Different letters over the means indicate significant differences, for (A–C), α′ = 0.003, N = 84, n = 12 where n is the number of pots for each line (each pot contained five plants whose senesced stem tissue was pooled constituting a sample); for (D), N = 6, n = 3, α′ = 0.02.

FIGURE 4

Principal component analysis (PCA) based upon phenotypic growth and cell wall composition observations. Means included in the PCA were HD, biomass index (BI = height × mass/culm number), and amounts for ABSL, lignin S, G, and H units (from thioacidolysis), xylose, arabinose, galactose, fucose, and crystalline cellulose. N = 84, 7 lines × 12 biological (pot) replicates. Rotated factor pattern and final communality estimates are in Supplementary Table S1. Significant principal components (PC) are in Supplementary Table S2.

FIGURE 5

Effects of vernalization treatment duration on biomass traits. Plants that were vernalized for either 0, 7, 14, 21, or 28 days, transferred to a growth chamber, and then grown to maturity were assessed for (A) height, (B) aboveground dry mass, and (C) heading date. Stem plus leaf sheath biomass was assessed for (D) ABSL, (E) p-coumaric acid (pCA), and (F) ferulic acid (FA) content, and hydrolytic enzyme digestibility following dilute base (6.25 mM NaOH) pretreatment [(G) glucose, (H) pentoses]. Regression was performed to determine the effect vernalization duration on (A–C,G,H), α′ = 0.002. Significant differences indicated for (E,F) were determined by Student’s T-test (^∗ = 0.05 > ρ > 0.01; ^∗∗ = 0.01 > ρ > 0.0005). % dw, percent dry weight. Bars indicate SEM. N = 9, n = 3 where n is the number of pots for each line (each pot contained four plants that were measured together to generate a sample mean).

FIGURE 6

Effects of vernalization treatment duration on free glucose, sucrose, and starch content of senesced biomass. Stem plus leaf sheath biomass from plants that were vernalized for either 0, 7, 14, 21, or 28 days, transferred to a growth chamber, and then grown to maturity was assessed for (A) free glucose, (B) sucrose, and (C) starch content. Regression was performed to determine the effect of increasing vernalization treatment for each compound quantitated, α′ = 0.02. % dw, percent dry weight. Bars indicate SEM. N = 9, n = 3.