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A versatile click-compatible monolignol probe to study lignin deposition in plant cell walls.

Pandey JL, Wang B, Diehl BG, Richard TL, Chen G, Anderson CT - PLoS ONE (2015)

Bottom Line: We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings.Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural and Biological Engineering, The Pennsylvania State University, University Park, Pennsylvania, United States of America; Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

ABSTRACT
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 in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana 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.

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NMR spectra of the DHPs.The aliphatic regions of the HSQC 2D-NMR spectra of 100% coniferyl alcohol DHP (G-DHP, black), copolymer of 25% 3-EPC, 6 and coniferyl alcohol, 7 (red), and 100% 3-EPC, 6 DHP (blue).
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pone.0121334.g002: NMR spectra of the DHPs.The aliphatic regions of the HSQC 2D-NMR spectra of 100% coniferyl alcohol DHP (G-DHP, black), copolymer of 25% 3-EPC, 6 and coniferyl alcohol, 7 (red), and 100% 3-EPC, 6 DHP (blue).

Mentions: Heteronuclear single quantum coherence (HSQC) NMR spectra were collected for 100% coniferyl alcohol DHP (G-DHP, black), 25% 3-EPC DHP (red), and 100% 3-EPC DHP (blue) and are shown in Fig 2. The pure G-DHP contained shifts typical of G-DHPs. Interestingly, increasing the 3-EPC content dramatically decreased the shift near 5.0/72.0 ppm (Aα), which was attributed to β-ether/α-hydroxyl linkages (i.e., “typical” β-O-4 linkages whose quinone methide intermediates were inter-molecularly quenched via reaction with water) [60]. In fact, when 3-EPC is the sole monolignol, β-ether/α-hydroxyl signals are essentially absent. Our interpretation of this result is that the alkyne group might react with the quinone methide intermediate in an intra-molecular fashion, as shown in Fig 3, to form a carbocation that undergoes additional transformation to yield an as-yet unidentified novel linkage type that has not been observed in natural lignins. This might happen so efficiently that β-ether/α-hydroxyl linkages do not form. Novel shift 1 (4.3/85.0 ppm) appears in the 25% 3-EPC sample and is prominent in the 100% 3-EPC sample at the expense of the shift at 4.6/85.0 ppm (Aβ, the β-O-4/α-OH β-position). This provides further evidence that β-ether/α-hydroxyl structures do not form, and novel shift 1 is likely attributable to a novel β-position. Novel shifts 2 (4.9/83.0 ppm) and 3 (5.8/78.0 ppm) remain unidentified, but one of these shifts is likely attributable to the α-position of the novel β-ether linkage type. Although the NMR data strongly suggest the formation of a new lignin inter-unit linkage type, most likely arising from participation of the alkyne in quinone methide trapping as in Fig 3, further work involving additional model lignin compounds will be necessary to determine the exact structure of this new linkage. Surprisingly, we did not detect unreacted alkyne functionality in the HSQC NMR spectra (Fig 3); additional proton and carbon NMR experiments (not shown) similarly failed to show alkyne shifts. As discussed above, some of the alkyne group may have been consumed via quinone methide trapping, or potentially via other side reactions.


A versatile click-compatible monolignol probe to study lignin deposition in plant cell walls.

Pandey JL, Wang B, Diehl BG, Richard TL, Chen G, Anderson CT - PLoS ONE (2015)

NMR spectra of the DHPs.The aliphatic regions of the HSQC 2D-NMR spectra of 100% coniferyl alcohol DHP (G-DHP, black), copolymer of 25% 3-EPC, 6 and coniferyl alcohol, 7 (red), and 100% 3-EPC, 6 DHP (blue).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401456&req=5

pone.0121334.g002: NMR spectra of the DHPs.The aliphatic regions of the HSQC 2D-NMR spectra of 100% coniferyl alcohol DHP (G-DHP, black), copolymer of 25% 3-EPC, 6 and coniferyl alcohol, 7 (red), and 100% 3-EPC, 6 DHP (blue).
Mentions: Heteronuclear single quantum coherence (HSQC) NMR spectra were collected for 100% coniferyl alcohol DHP (G-DHP, black), 25% 3-EPC DHP (red), and 100% 3-EPC DHP (blue) and are shown in Fig 2. The pure G-DHP contained shifts typical of G-DHPs. Interestingly, increasing the 3-EPC content dramatically decreased the shift near 5.0/72.0 ppm (Aα), which was attributed to β-ether/α-hydroxyl linkages (i.e., “typical” β-O-4 linkages whose quinone methide intermediates were inter-molecularly quenched via reaction with water) [60]. In fact, when 3-EPC is the sole monolignol, β-ether/α-hydroxyl signals are essentially absent. Our interpretation of this result is that the alkyne group might react with the quinone methide intermediate in an intra-molecular fashion, as shown in Fig 3, to form a carbocation that undergoes additional transformation to yield an as-yet unidentified novel linkage type that has not been observed in natural lignins. This might happen so efficiently that β-ether/α-hydroxyl linkages do not form. Novel shift 1 (4.3/85.0 ppm) appears in the 25% 3-EPC sample and is prominent in the 100% 3-EPC sample at the expense of the shift at 4.6/85.0 ppm (Aβ, the β-O-4/α-OH β-position). This provides further evidence that β-ether/α-hydroxyl structures do not form, and novel shift 1 is likely attributable to a novel β-position. Novel shifts 2 (4.9/83.0 ppm) and 3 (5.8/78.0 ppm) remain unidentified, but one of these shifts is likely attributable to the α-position of the novel β-ether linkage type. Although the NMR data strongly suggest the formation of a new lignin inter-unit linkage type, most likely arising from participation of the alkyne in quinone methide trapping as in Fig 3, further work involving additional model lignin compounds will be necessary to determine the exact structure of this new linkage. Surprisingly, we did not detect unreacted alkyne functionality in the HSQC NMR spectra (Fig 3); additional proton and carbon NMR experiments (not shown) similarly failed to show alkyne shifts. As discussed above, some of the alkyne group may have been consumed via quinone methide trapping, or potentially via other side reactions.

Bottom Line: We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings.Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural and Biological Engineering, The Pennsylvania State University, University Park, Pennsylvania, United States of America; Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

ABSTRACT
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 in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana 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.

Show MeSH
Related in: MedlinePlus