Volume 24, Issue 3, Pages 326-338 (March 2017) BLISS: A Bioorthogonal Dual-Labeling Strategy to Unravel Lignification Dynamics in Plants  Cedric Lion, Clémence Simon, Brigitte Huss, Anne-Sophie Blervacq, Louis Tirot, Djadidi Toybou, Corentin Spriet, Christian Slomianny, Yann Guerardel, Simon Hawkins, Christophe Biot  Cell Chemical Biology  Volume 24, Issue 3, Pages 326-338 (March 2017) DOI: 10.1016/j.chembiol.2017.02.009 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 326-338DOI: (10. 1016/j. chembiol. 2017 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 The Lignification Process in Plant Cell Walls (A) Phenoloxidase-mediated radical polymerization. (B) BLISS dual-labeling strategy. Cell Chemical Biology 2017 24, 326-338DOI: (10.1016/j.chembiol.2017.02.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Dual Incorporation and Imaging of Monolignol Chemical Reporters in Flax Stems Left: xylem. The arrow indicates the first labeled cell wall from the cambium showing the increased sensitivity of BLISS compared with autofluorescence. Right: close-up on secondary xylem. The arrow shows that BLISS distinguishes lignin produced de novo and can be used to identify cell wall layers in which lignification occurs. (A) Lignin autofluorescence (blue, 405 nm) channel, (B) merged lignin autofluorescence (blue) and HAZ fluorescence (green, 526 nm) channels, (C) merged lignin autofluorescence (blue) and GALK fluorescence (red, 565 nm) channels, (D) merged lignin autofluorescence (blue), HAZ (green) and GALK (red) fluorescence channels. HAZ and GALK co-localization is depicted in yellow. Cell Chemical Biology 2017 24, 326-338DOI: (10.1016/j.chembiol.2017.02.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Flax Xylem Structure and Cell-Specific Monolignol Reporter Incorporation (A) 3D illustration of the structure of secondary xylem in the flax stem and pie chart showing the relative proportions of different major cell wall polymers; V, vessel; FT, fiber tracheid; R, ray parenchyma cell; C, cellulose (40%); H, hemicellulose (30%); L, lignin (25%). (B) View of the secondary xylem; merged lignin autofluorescence (blue), HAZ (green), and GALK (red) fluorescence channels. HAZ and GALK co-localization in yellow. (C–F) Bright-field confocal microscopy view (C) of part of a freehand cross-section from a flax stem used for lignin autofluorescence and monolignol chemical reporter imaging. Pink (ray cells) and yellow (fiber tracheid cells) arrows indicate vectors spanning from the cambial zone toward the pith and scanned for lignin autofluorescence at 405 nm (D), HAZ fluorescence at 526 nm (E) and GALK fluorescence at 565 nm (F). Cell Chemical Biology 2017 24, 326-338DOI: (10.1016/j.chembiol.2017.02.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Lignification Dynamics and Cell Wall Polarization in Differentiating Fiber Tracheids (A and B) Incorporation of HAZ (A) and GALK (B) chemical reporters in cell corners and middle lamella/primary cell wall adjacent to an older xylem cell. Star, cell analyzed in 3D intensity scan; dotted line, limit of analyzed cell wall considered for 3D scan; 1–6, cell corners of analyzed cell. (C–E) Lignin autofluorescence (C), HAZ fluorescence (D), and GALK fluorescence (E) vector scans (yellow arrow) in cell walls of rapidly differentiating fiber tracheids (cells a–d). (F) Merged channels. Note the important polarization (pink arrow) of cell b. Scale bar, 5 μm. Cell Chemical Biology 2017 24, 326-338DOI: (10.1016/j.chembiol.2017.02.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Effect of Incubating Flax Stem Sections with Different Percentage Ratios of HAZ and GALK HAZ:GALK % ratios are given above each column of figures. (A) HAZ channel (green), (B) GALK channel (red), (C) merged HAZ and GALK channels, and (D) histograms indicating average fluorescence intensity of HAZ and GALK for the different % ratios. Values are expressed as mean of the mean fluorescence intensity in gray levels ± SD. Scale bar, 100 μm. Cell Chemical Biology 2017 24, 326-338DOI: (10.1016/j.chembiol.2017.02.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Flax Bast Fiber Structure and Cell-Specific Monolignol Reporter Incorporation (A) 3D illustration of the structure of bast fibers (BF) and adjacent parenchyma cell (P) in the outer tissues of the flax stem and pie chart showing the relative proportions of different major cell wall polymers. M, middle lamella; P, primary cell wall; S1, first layer of secondary cell wall; S2/G, secondary layer/gelatinous layer of secondary cell wall; C, cellulose (75%); H, hemicellulose (15%); L, lignin (4%). (B) High-power merged bright-field, lignin autofluorescence (405 nm), HAZ fluorescence (526 nm), and GALK fluorescence (565 nm) channels of bast fiber with lignified cell corners and middle lamella. (C) Merged HAZ and GALK channels showing that lignification is limited to cell corners (white stars) and middle lamella/primary cell wall of some bast fibers. (D–F) Lignin autofluorescence (D), HAZ fluorescence (E), and GALK fluorescence (F) vector scans (yellow arrow) through the bast fiber cell wall. Scale bars, 10 μm. Cell Chemical Biology 2017 24, 326-338DOI: (10.1016/j.chembiol.2017.02.009) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Application of BLISS In Vivo on Flax Stems (A–C) The roots were removed from the stem of 2-month-old flax plants, the base of which was placed in 1/2 MS containing HAZ and GALK. After incubation, cross-sections were prepared at regular distances from the base and labeled with BLISS. (A) Negative control, (B) cross-section from the region at 0.5–1.0 cm from the base, and (C) cross-section from the region at 1.5–2.0 cm from the base. Tagged HAZ units are detected at 526 nm (green channel), tagged GALK units are detected at 565 nm (red channel), and autofluorescence is detected at 405 nm (blue channel). Cell Chemical Biology 2017 24, 326-338DOI: (10.1016/j.chembiol.2017.02.009) Copyright © 2017 Elsevier Ltd Terms and Conditions