Monolignol export by diffusion down a polymerization-induced concentration gradient

Abstract

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export.

Figure 1

Multiphoton autofluorescence microscopy simultaneously reveals Arabidopsis root protoxylem lignin and soluble phenolics. A, Conventional epifluorescence imaging of Arabidopsis root stained with basic fuchsin (magenta) (white arrow) and calcofluor (green). Scale bar 500 µm. Inset represents the equivalent portion of the root imaged using multiphoton autofluorescence microscopy shown in (B)–(D). XY and YZ planes represent the 3D multiphoton data captured in a z stack. B, Cross-section of a representative Col-0 Arabidopsis root with cell types labeled. C, Control and 24 h 5-µM piperonylic acid (PA)-treated dark-grown roots, lignin imaged by two-photon excitation at 730 nm and emission filter 520–560 nm (green) and propidium iodide (PI) imaged by two-photon excitation at 1,000 nm and emission filter 630 nm (magenta). Images are maximum intensity Z-projections. D, Control and 24 h 5-µM PA-treated roots grown in continuous light or continuous dark conditions imaged by two-photon excitation at 730 nm and emission filter 520–560 nm. Images are maximum intensity Z-projections. Scale bars in (B)–(D) are 25 µm.

Figure 2

Intrinsic fluorescence shows that lac4 lac17 mutant roots have increased intracellular phenolics. A, Schematic of an Arabidopsis root with the region of cell wall lignification highlighted by the black box. B, pLAC4:LAC4-mCherry and pLAC17:LAC17-mCherry in young root xylem tracheary elements imaged for mCherry fluorescence and lignin autofluorescence. Two-photon excitation at 730 nm and emission filter 520–560 nm for the lignin/phenolic channel. C, Coniferin levels in Col WT roots compared to lac4 lac17 measured by HPLC/QTOF. n = 5 experimental replicates of two to three plates of seedlings containing approximately 200 seedlings each, two-sample t test, P = 0.000045. D, Representative images of lignin/phenolic autofluorescence of Col-0 WT controls and lac4 lac17 double mutants. White rectangles indicate representative portion of tracheary element sampled for quantification. Gray rectangles indicate region of neighbor cells sampled for vacuolar fluorescence quantification. Scale bar 25 µm. E(i), Quantification of lignin autofluorescence intensity in WT Col-0 compared to lac4 lac17 double mutants. n = 17 roots from a total of four experimental replicates consisting of two to three plates of seedlings grown and images on separate occasions. Two-sample t test, P<0.01. E(ii), Quantification of lignin autofluorescence intensity in WT Col-0 compared to lac4 lac17 double mutant, lac4 lac17 LAC4-mCherry, and lac4 lac17 LAC17-mCherry lines. ANOVA and Tukey’s post hoc test, P <0.05. n = 7 roots from three experimental replicates. Letters indicate statistical significance. F(i), Quantification of neighboring cell autofluorescence intensity in WT Col-0 compared to lac4 lac17 double mutants. n = 17 roots from a total of four experimental replicates. Two-sample t test, P<0.01. F(ii), Quantification of neighboring cell autofluorescence intensity in WT Col-0 compared to lac4 lac17 double mutant, lac4 lac17 LAC4-mCherry, and lac4 lac17 LAC17-mCherry lines. ANOVA and Tukey’s post hoc test, P <0.05. n = 7 roots from three experimental replicates consisting of two to three plates of seedlings grown and images on separate occasions. Letters indicate statistical significance. Box and whiskers in C, E, and F represent median values and quartiles.

Figure 3

Vanadate treatment does not change lignification in root protoxylem. A, Representative lignin signals from control and 24 h 50-µM vanadate-treated roots. White rectangles indicate representative portion of tracheary element sampled for quantification. Two-photon excitation at 730 nm and emission filter 520–560 nm. Scale bar 25 µm. B, Quantification of lignin signal from two-photon microscopy in control and 24 h 50-µM vanadate-treated roots. n = 14 roots from a total of three experimental replicates consisting of independently grown plates of plants. Two-sample t test indicated no significant difference (n.d.). C, Comparison of representative two-photon autofluorescent signals Col-0 WT roots, lac4lac17 mutant roots, and vanadate-treated roots. te indicates xylem tracheary elements. en indicates endodermal cells neighboring xylem vessels. co indicates cortical cells. ep indicates epidermal cells. Scale bar indicates 25μm.

Figure 4

Monolignols cross pure lipid bilayers to encounter laccase inside. A, Schematic representation of the lignifying thickened secondary wall of a root protoxylem vessel and the in vitro laccase-containing liposome system. Top row: laccase (black bean) has been exported to the cell wall in advance of monolignol production; in liposomes, lipid bilayer (magenta) encapsulates laccase proteins. Middle: CA monomers (blue) cross the lipid bilayer. Bottom: Monolignols are oxidized by laccase to form monolignol radicals (blue with highlight and dot) that form dimers of CA. B, Cryo-transmission electron microscopy of liposomes before and after incubation with CA. Scale bar 100 nm. C, CA levels over time with liposomes containing laccase, BSA, or buffer. n = 3 replicated independent reactions. Bars indicate standard deviation. D, CA incubated with liposomes containing laccase generate β-β-, β-O-4-, and β-5-linked dimers over time. Bars indicate standard deviation. CA and dimer analysis by HPLC was replicated three times. Bars indicate standard deviation.

Figure 5

Stable laccase-containing liposomes accumulate phenolic polymer. A(i), Gel permeation chromatographic analysis of products formed by laccase-containing liposomes fed CA. A(ii), GPC standards of laccase DHP, pure CA, and blank solvent control. GPC analysis was replicated four times by preparing liposomes in four separate tubes. B, Super-resolution total internal reflection micrographs of immobilized laccase-containing liposomes (arrows) following 24 h 1-mM CA incubation. Autofluorescence of phenolic material (405 nm) green in merged image, signal from the DiI (DiIC18(3)) lipid dye (561 nm) magenta in merged images. Representative images of laccase-containing liposomes with CA (LAC+CA) and without CA (LAC−CA), and DHP formed by T. versicolor laccase incubated directly with CA (no liposomes). ≥20 fields of view from three replicated experiments, consisting of independent reactions with liposomes, analyzed per treatment, a representative image is shown for each treatment. C, Quantification of (i) density and (ii) size of autofluorescent puncta in laccase-contain liposomes with (LAC + CA) or without CA (LAC − CA). Control liposomes contained BSA with (BSA + CA) or without CA (BSA − CA). More than 20 fields of view from three replicated experiments per treatment. ***Indicates Dunn’s pairwise comparison, P < 0.001. D, Schematic representation of the end point of the liposome reaction in Figure 5A (96 h). Laccase-containing liposomes with polymer in the interior and no monolignols remaining in the surrounding solution.