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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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Raman spectra of DHPs.Spectra (1024 nm excitation, 500 scans, data spacing of 1.928 cm-1) of in vitro-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.
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pone.0121334.g004: Raman spectra of DHPs.Spectra (1024 nm excitation, 500 scans, data spacing of 1.928 cm-1) of in vitro-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.

Mentions: Because 3-EPC contains a terminal alkyne group that should impart a characteristic Raman spectroscopic signal, Raman spectra for DHPs prepared with varying proportions of 3-EPC were collected and compared with G-DHP spectra. All of the DHPs showed characteristic lignin peaks at 1600 and 1650 cm-1 [31–33], but DHPs prepared with 3-EPC also showed an additional strong peak at 2100 cm-1, which corresponds to alkynyl stretching vibrations (Fig 4) [56]. This result indicated that at least some intact alkyne groups were present in the DHP and were detectable by Raman spectroscopy, but might have been below the detection threshold of NMR. Because this characteristic Raman peak does not correspond to any of the endogenous molecules present in cells [56,61], this result opens up the future possibility that Raman imaging could be used to detect the incorporation of 3-EPC into plant cell walls without additional labeling by reporter tags.


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)

Raman spectra of DHPs.Spectra (1024 nm excitation, 500 scans, data spacing of 1.928 cm-1) of in vitro-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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0121334.g004: Raman spectra of DHPs.Spectra (1024 nm excitation, 500 scans, data spacing of 1.928 cm-1) of in vitro-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.
Mentions: Because 3-EPC contains a terminal alkyne group that should impart a characteristic Raman spectroscopic signal, Raman spectra for DHPs prepared with varying proportions of 3-EPC were collected and compared with G-DHP spectra. All of the DHPs showed characteristic lignin peaks at 1600 and 1650 cm-1 [31–33], but DHPs prepared with 3-EPC also showed an additional strong peak at 2100 cm-1, which corresponds to alkynyl stretching vibrations (Fig 4) [56]. This result indicated that at least some intact alkyne groups were present in the DHP and were detectable by Raman spectroscopy, but might have been below the detection threshold of NMR. Because this characteristic Raman peak does not correspond to any of the endogenous molecules present in cells [56,61], this result opens up the future possibility that Raman imaging could be used to detect the incorporation of 3-EPC into plant cell walls without additional labeling by reporter tags.

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