Lignin depletion enhances the digestibility of cellulose in cultured xylem cells

Abstract

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinniaelegans. Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.

Figure 1. Pretreated xylem cells are efficiently degraded by cellulase.

Time-lapse brightfield microscopy was used to image untreated (A) and ASC-pretreated xylem cells (B) incubated with cellulase. (A) Untreated xylem cells have no detectable changes in morphology during a 3-hour incubation. (B) Xylem cells pretreated with ASC are rapidly degraded with noticeable loss of material from the secondary cell wall thickenings within the first hour of incubation with cellulase. Scale bar=20 µm. Also see Video S1.

Figure 2. Polarized light indicates the enzymatic digestion of cellulose in secondary walls.

Time-lapse polarization microscopy was used to image ASC-pretreated xylem cells incubated in the absence (A) or presence (B) of cellulase. (A) Qualitatively, the polarization signal remains stable during a 3-hour incubation without cellulase. (B) The polarization signal rapidly declines during digestion with cellulase. (C) Quantification of the total polarization signal from the cells in (A) and (B) reveals a constant signal during incubation with no cellulase (open symbols) and the rapid loss of signal from the cell digested with cellulase (closed symbols). Scale bar=20µm. Also see Video S2.

Figure 3. CtCBM3-GFP labeling quantitatively reveals the rapid digestion of cellulose in pretreated cells.

(A–D) Fluorescence images show representative xylem cells that were incubated in the absence or presence of cellulase and subsequently labeled with the cellulose-specific CtCBM3-GFP probe. Untreated cells, either undigested (A) or digested with cellulase for 1 hour (B), qualitatively show similar fluorescence levels. Decreased fluorescence levels are evident in cells pretreated with ASC after only 1 hour of digestion with cellulase (D, bottom panel) compared to cells at time 0 (D, top panel) or undigested ASC-pretreated cells (C). (E, F) The total fluorescence values of single xylem cells are plotted as a function of cell area. Four different symbols indicate the time of incubation, as indicated in the inset legends. (E) The fluorescence distributions of untreated cells taken at 1-hour time intervals during a 3-hour incubation without cellulase (top) or with cellulase (bottom) overlap. (F) The fluorescence distributions of ASC-pretreated cells taken at 1-hour intervals during incubation without cellulase (top) are also similar, while the fluorescence levels from cells digested with cellulase (bottom) decreased during the incubation. (G, H) The average of mean fluorescence values of the cells in (E) and (F) are plotted as a function of time. (G) No significant difference is observed between the mean fluorescence of untreated cells incubated without cellulase (open symbols, dashed line) or with cellulase (closed symbols, solid line). (H) The mean fluorescence of ASC-pretreated cells incubated without cellulase (open symbols, dashed line) or with cellulase (closed symbols, solid line) are significantly different (*p<0.001, **p<0.0001). Error bars=SD. Scale bar=20µm.

Figure 4. Inhibitors of lignin deposition significantly reduce secondary wall autofluorescence.

(A–C) Autofluorescence (512-542 nm) from representative xylem cells is shown, either from control cultures (top panels) or from cultures containing 20 µM DPI, 2 mM GSH, or 20 µM DPI combined with 2 mM GSH (bottom panels). For presentation purposes, images were scaled consistently so that the bottom panels had low, yet noticeable signal levels. Scale bar=20µm. (D–F) Mean autofluorescence values from each population of xylem cells were averaged and normalized to the average from the corresponding control sample. The mean autofluorescence is significantly reduced (*p<0.0001) in treated cells compared to controls. Error bars=SD.

Figure 5. Inhibition of lignin biosynthesis significantly improves the enzymatic removal of cellulose.

(A–F) Representative images of xylem cells, either from control cultures (A, C, E), or from cultures containing 20 µM DPI (B), 2 mM GSH (D), or 20 µM DPI combined with 2 mM GSH (F), are shown after incubation without cellulase (top panels) or with cellulase (bottom panels) for 24 hours. All images were corrected for autofluorescence and show fluorescence after labeling cells with CtCBM3-GFP. A reduction in CtCBM3-GFP labeling is evident in cells treated with inhibitors of lignin deposition and then digested with cellulase (B, D, F). Scale bar=20µm. (G–I) The average of mean fluorescence values of populations of xylem cells treated with DPI (G), GSH (H), or DPI combined with GSH (I) and labeled with CtCBM3-GFP are plotted. Mean fluorescence values were corrected for autofluorescence. The fluorescence labeling of undigested xylem cells is significantly increased (*p<0.01) by 73% (G), 40% (H), and 101% (I) in cells treated with inhibitors compared to controls (bracketed blue bars). The mean fluorescence of inhibitor-treated cells is significantly reduced (**p<0.001) by 58% (G), 54% (H), and 74% (I) in digested compared to undigested samples. Error bars=SD.