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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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Fluorescence intensities (561 nm excitation) of click-labeled DHPs.5 mg/ml solutions in DMSO of in vitro-synthesized DHPs incorporated with varying proportions of 3-EPC, 6 and click labeled with 1 μM Alexa 594-azide for 1 h in the dark at room temperature. Blue trace: click labeled 100% 3-EPC DHP; red trace: click labeled 25% 3-EPC + 75% CA DHP; green trace: click labeled 100% CA DHP or G-DHP; black trace: non-labeled G-DHP. The red and blue spectra show significantly higher fluorescence intensities compared to labeled and non-labeled G-DHP. Detectable, but low, fluorescence in click labeled G-DHP is possibly due to physical entanglement of the dye with the polymer.
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pone.0121334.g005: Fluorescence intensities (561 nm excitation) of click-labeled DHPs.5 mg/ml solutions in DMSO of in vitro-synthesized DHPs incorporated with varying proportions of 3-EPC, 6 and click labeled with 1 μM Alexa 594-azide for 1 h in the dark at room temperature. Blue trace: click labeled 100% 3-EPC DHP; red trace: click labeled 25% 3-EPC + 75% CA DHP; green trace: click labeled 100% CA DHP or G-DHP; black trace: non-labeled G-DHP. The red and blue spectra show significantly higher fluorescence intensities compared to labeled and non-labeled G-DHP. Detectable, but low, fluorescence in click labeled G-DHP is possibly due to physical entanglement of the dye with the polymer.

Mentions: To further test the hypothesis that the clickable monolignol analog 3-EPC was in fact incorporated into DHPs in a way that retained some intact alkynyl groups, we performed fluorescent labeling of DHPs prepared as described above using click chemistry. DHPs prepared with 25% or 100% 3-EPC were reacted with 1 μM Alexa 594-azide for 1 h at 25°C in the dark in the presence of Cu(I). Fluorescence measurements of these click-labeled DHP lignins (Fig 5) showed that the DHPs containing 3-EPC had much higher fluorescence intensities than G-DHP subjected to the same click-labeling conditions. G-DHP that was not click labeled was used as a negative control and showed negligible fluorescence at 561 nm excitation. Click labeled G-DHP, however, did show some fluorescence, even though it was much lower than that for 3-EPC-containing DHPs. A possible interpretation of this result is that a small amount of the Alexa 594-azide can become non-specifically entangled within G-DHP.


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)

Fluorescence intensities (561 nm excitation) of click-labeled DHPs.5 mg/ml solutions in DMSO of in vitro-synthesized DHPs incorporated with varying proportions of 3-EPC, 6 and click labeled with 1 μM Alexa 594-azide for 1 h in the dark at room temperature. Blue trace: click labeled 100% 3-EPC DHP; red trace: click labeled 25% 3-EPC + 75% CA DHP; green trace: click labeled 100% CA DHP or G-DHP; black trace: non-labeled G-DHP. The red and blue spectra show significantly higher fluorescence intensities compared to labeled and non-labeled G-DHP. Detectable, but low, fluorescence in click labeled G-DHP is possibly due to physical entanglement of the dye with the polymer.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0121334.g005: Fluorescence intensities (561 nm excitation) of click-labeled DHPs.5 mg/ml solutions in DMSO of in vitro-synthesized DHPs incorporated with varying proportions of 3-EPC, 6 and click labeled with 1 μM Alexa 594-azide for 1 h in the dark at room temperature. Blue trace: click labeled 100% 3-EPC DHP; red trace: click labeled 25% 3-EPC + 75% CA DHP; green trace: click labeled 100% CA DHP or G-DHP; black trace: non-labeled G-DHP. The red and blue spectra show significantly higher fluorescence intensities compared to labeled and non-labeled G-DHP. Detectable, but low, fluorescence in click labeled G-DHP is possibly due to physical entanglement of the dye with the polymer.
Mentions: To further test the hypothesis that the clickable monolignol analog 3-EPC was in fact incorporated into DHPs in a way that retained some intact alkynyl groups, we performed fluorescent labeling of DHPs prepared as described above using click chemistry. DHPs prepared with 25% or 100% 3-EPC were reacted with 1 μM Alexa 594-azide for 1 h at 25°C in the dark in the presence of Cu(I). Fluorescence measurements of these click-labeled DHP lignins (Fig 5) showed that the DHPs containing 3-EPC had much higher fluorescence intensities than G-DHP subjected to the same click-labeling conditions. G-DHP that was not click labeled was used as a negative control and showed negligible fluorescence at 561 nm excitation. Click labeled G-DHP, however, did show some fluorescence, even though it was much lower than that for 3-EPC-containing DHPs. A possible interpretation of this result is that a small amount of the Alexa 594-azide can become non-specifically entangled within G-DHP.

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