A Versatile Click-Compatible Monolignol Probe to Study Lignin Deposition in Plant Cell Walls

<div><p>Lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into <i>in vitro</i>-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old <i>Arabidopsis</i> seedlings. Incorporation of the analog in stem sections of 6-week-old <i>Arabidopsis thaliana</i> plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly.</p></div

3-EPC incorporation in 40 μm-thick sections of 6 week old <i>Arabidopsis</i> stems.

<p>Autofluorescence (405 nm excitation) and click labeling (561 nm excitation) in (A) control section treated with 20 μM CA for 3 h, labeled with Alexa-594 azide for 1 h, and washed with 96% ethanol for 1 h; (B) section treated with 20 μM 3-EPC and 20 μM CA for 3 h, labeled with Alexa-594 azide for 1 h, and washed with 96% ethanol for 1 h; and (C) section treated with 20 μM 3-EPC for 3 h, labeled with Alexa-594 azide for 1 h, and washed with 96% ethanol for 1 h. (D) Xylem and (E) interfascicular fibers (IFFs) of section treated with 20 μM 3-EPC. (F) Zoomed-in region from image in (E), indicted by yellow box, showing incorporation patterns of 3-EPC in IFFs. Arrowhead in (A) indicates vascular bundle, with interfascicular fibers lying on either side. Images are contrast-enhanced maximum intensity projections of z series recorded with a spinning disk fluorescence confocal microscope. (A-C) were recorded using a 20X objective with a 561 nm laser at 15% power, 100 gain and 400 msec exposure time and a 405 nm laser at 100% power, 100 gain and 400 msec exposure time (scale bar, 100 μm). (D-E) were recorded using a 63X objective with a 561 nm laser at 15% power, 10 gain and 400 msec exposure time and a 405 nm laser at 100% power, 10 gain and 400 msec exposure time (scale bar, 20 μm).</p

Raman spectra of DHPs.

<p>Spectra (1024 nm excitation, 500 scans, data spacing of 1.928 cm^-1) of <i>in vitro</i>-synthesized DHP with 100% CA (black trace); DHP with 25% 3-EPC + 75% CA (red trace); and DHP with 100% 3-EPC (blue trace). The red and blue spectra show a characteristic terminal alkyne peak at 2100 cm^-1.</p

Proposed pathway for the formation of a novel β-ether linkage.

<p>For typical monolignols, external trapping of the β-ether quinone methide occurs via water addition (not experimentally observed with 3-EPC). The alternate pathway illustrates the proposed internal trapping of the quinone methide via the alkyne when 3-EPC is polymerized. The proposed carbocation would undergo additional reactions to yield an as-yet unidentified novel β-ether linkage type.</p

NMR spectra of the DHPs.

<p>The aliphatic regions of the HSQC 2D-NMR spectra of 100% coniferyl alcohol DHP (G-DHP, black), copolymer of 25% 3-EPC, <b>6</b> and coniferyl alcohol, <b>7</b> (red), and 100% 3-EPC, <b>6</b> DHP (blue).</p

<i>Kif3a</i> deletion in osteoblasts and osteocytes has no effect on tibial midshaft geometry.

<p>Imin and Imax are maximum and minimum second moment of inertia, respectively. pMOI is polar moment of inertia. Cortical bone geometry in 16 week old skeletally mature <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice was assessed using as microCT. Data presented as mean±SEM. N.S. is not significant (p>0.15).</p

<i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> were significantly less responsive to mechanical loading than control mice.

<p><i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice responded to mechanical loading with increased mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), however, <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> were significantly less responsive to mechanical loading than control mice. Data presented as mean±SEM.</p>+++<p>p<0.001 for loaded vs. non-loaded values.</p>*<p>p<0.05 for <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> vs. control mice.</p

Axial ulnar loading leads to similar strain at the ulnar midshaft of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> and control mice.

<p>(<b>A</b>) Image of strain gaging and axial ulnar loading experimental set-up. The right forearms of 16 week old skeletal mature mice were axially loaded for 120 cycles per day for 3 consecutive days with a 2 Hz sine wave using an electromagnetic loading system with feedback control. The left forearms were not loaded and used as non-loaded internal controls. (<b>B</b>) Strain in cortical bone at given mechanical loading levels. Open and closed circles indicate <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> (n = 35) and control (n = 27) mice, respectively. Data presented as mean ± SEM. * p<0.05.</p

Kif3a expression in osteoblasts and osteocytes is not critical for embryonic skeletal development.

<p>(<b>A,B</b>) Whole mount Alizarin Red (bone) and Alcian Blue (cartilage) staining of E18.5 <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l (A) and control (B) embryos. The size and limb patterning of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice was similar to that of the control mice. (<b>C,D</b>) Movat's pentachrome staining of cross-sections of the radial/ulnar growth plates (cartilage-blue) in E16.5 <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l (C) and control (D) mice. (<b>E–J</b>) Cross-sections of E16.5 long bones stained with Picrosirius red (E,F-bright field; G,H-polarized light) to illuminate collagen and Safranin O (I,J) to demarcate cartilage. Both control and <i>Colα1(I) 2.3-Cre;Kif3a^fl/f</i>^l mice have similar patterns of osteogenic and chondrogenic differentiation. Scale bar: 100 µm.</p

Skeletally mature <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice exhibit less responsiveness to mechanical loading compared to control mice.

<p>(<b>A</b>) Representative images of non-loaded (left) and loaded (right) ulnae of <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> (top) and control (bottom) mice. Fluorochrome labels (Calcein-green and Alizarin Red-red) given on Days 5 and 12 after the onset of mechanical loading. (<b>B to D</b>) Relative mineralizing surface (rMS/BS, %, B), mineral apposition rate (rMAR, µm per day, C), and bone formation rate (rBFR/BS, µm³/µm² per year, D) of mechanically loaded mice. <i>Colα1(I) 2.3-Cre;Kif3a^fl/fl</i> mice exhibited a decrease of 32% in rMAR and 33% in rBFR/BS when compared to control mice. Data presented as mean ± SEM. * p<0.05.</p