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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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3-EPC incorporation in roots of 4-day-old Arabidopsis seedlings.(A, D) control seedlings treated with 20 μM CA for 4 h and labeled with Alexa-594 azide for 1 h. (B, E) seedlings treated with 5 μM 3-EPC and 15 μM CA for 4 h and labeled with Alexa-594 azide for 1 h. (C, F) seedlings treated with 20 μM 3-EPC for 4 h and labeled with Alexa-594 azide for 1 h. Images were collected with a spinning disk confocal microscope using a 561 nm laser at 10% power and 500 gain with an exposure time of 400 msec. (A-C) contrast-enhanced mosaics of contiguous images, starting at the root tip and going through the late differentiation zone, recorded using a 20X objective (Scale bar, 100 μm). (D-F) contrast-enhanced maximum intensity projections of z series recorded at the indicated root zones with a 100X oil-immersion objective (Scale bar, 20 μm).
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pone.0121334.g006: 3-EPC incorporation in roots of 4-day-old Arabidopsis seedlings.(A, D) control seedlings treated with 20 μM CA for 4 h and labeled with Alexa-594 azide for 1 h. (B, E) seedlings treated with 5 μM 3-EPC and 15 μM CA for 4 h and labeled with Alexa-594 azide for 1 h. (C, F) seedlings treated with 20 μM 3-EPC for 4 h and labeled with Alexa-594 azide for 1 h. Images were collected with a spinning disk confocal microscope using a 561 nm laser at 10% power and 500 gain with an exposure time of 400 msec. (A-C) contrast-enhanced mosaics of contiguous images, starting at the root tip and going through the late differentiation zone, recorded using a 20X objective (Scale bar, 100 μm). (D-F) contrast-enhanced maximum intensity projections of z series recorded at the indicated root zones with a 100X oil-immersion objective (Scale bar, 20 μm).

Mentions: After demonstrating that 3-EPC could be polymerized into a lignin-like polymer in vitro, we next investigated whether this monolignol analog could undergo polymerization in the cell walls of living plants. Arabidopsis Col-0 seedlings were incubated with 3-EPC in order to study its incorporation into root cell walls. Four-day-old seedlings were incubated with 3-EPC and/or CA in liquid MS medium for 4 h, reacted with Alexa 594-azide via a copper-catalyzed click reaction to fluorescently label any incorporated alkynyl groups, and imaged using fluorescence microscopy. Seedlings treated with 3-EPC showed significantly higher fluorescence in the root epidermis (Fig 6B and 6C, 6E and 6F) as compared to control seedlings that were treated with only coniferyl alcohol and reacted with Alexa 594-azide, which showed negligible fluorescence (Fig 6A and 6D). In seedlings treated with 3-EPC, fluorescence intensity increased from the root tip to the differentiation zone, with the highest intensities observed in the late differentiation zone (Fig 6E and 6F). Seedlings treated with a mixture of 5 μM 3-EPC and 15 μM CA to test whether the presence of a natural lignin precursor affects the incorporation of the synthetic monolignol showed significantly higher fluorescence (Fig 6B and 6E) as compared to seedlings treated with 20 μM 3-EPC (Fig 6C and 6F). Fluorescence quantification data for each of these treatments is shown in S7 Fig. The finding that the addition of CA resulted in higher incorporation of 3-EPC in the root epidermis suggests that either synergistic monolignol uptake might be occurring, or that 3-EPC self-reactivity in the absence of CA before or during its incorporation might result in the loss of free alkynyl groups.


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)

3-EPC incorporation in roots of 4-day-old Arabidopsis seedlings.(A, D) control seedlings treated with 20 μM CA for 4 h and labeled with Alexa-594 azide for 1 h. (B, E) seedlings treated with 5 μM 3-EPC and 15 μM CA for 4 h and labeled with Alexa-594 azide for 1 h. (C, F) seedlings treated with 20 μM 3-EPC for 4 h and labeled with Alexa-594 azide for 1 h. Images were collected with a spinning disk confocal microscope using a 561 nm laser at 10% power and 500 gain with an exposure time of 400 msec. (A-C) contrast-enhanced mosaics of contiguous images, starting at the root tip and going through the late differentiation zone, recorded using a 20X objective (Scale bar, 100 μm). (D-F) contrast-enhanced maximum intensity projections of z series recorded at the indicated root zones with a 100X oil-immersion objective (Scale bar, 20 μm).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0121334.g006: 3-EPC incorporation in roots of 4-day-old Arabidopsis seedlings.(A, D) control seedlings treated with 20 μM CA for 4 h and labeled with Alexa-594 azide for 1 h. (B, E) seedlings treated with 5 μM 3-EPC and 15 μM CA for 4 h and labeled with Alexa-594 azide for 1 h. (C, F) seedlings treated with 20 μM 3-EPC for 4 h and labeled with Alexa-594 azide for 1 h. Images were collected with a spinning disk confocal microscope using a 561 nm laser at 10% power and 500 gain with an exposure time of 400 msec. (A-C) contrast-enhanced mosaics of contiguous images, starting at the root tip and going through the late differentiation zone, recorded using a 20X objective (Scale bar, 100 μm). (D-F) contrast-enhanced maximum intensity projections of z series recorded at the indicated root zones with a 100X oil-immersion objective (Scale bar, 20 μm).
Mentions: After demonstrating that 3-EPC could be polymerized into a lignin-like polymer in vitro, we next investigated whether this monolignol analog could undergo polymerization in the cell walls of living plants. Arabidopsis Col-0 seedlings were incubated with 3-EPC in order to study its incorporation into root cell walls. Four-day-old seedlings were incubated with 3-EPC and/or CA in liquid MS medium for 4 h, reacted with Alexa 594-azide via a copper-catalyzed click reaction to fluorescently label any incorporated alkynyl groups, and imaged using fluorescence microscopy. Seedlings treated with 3-EPC showed significantly higher fluorescence in the root epidermis (Fig 6B and 6C, 6E and 6F) as compared to control seedlings that were treated with only coniferyl alcohol and reacted with Alexa 594-azide, which showed negligible fluorescence (Fig 6A and 6D). In seedlings treated with 3-EPC, fluorescence intensity increased from the root tip to the differentiation zone, with the highest intensities observed in the late differentiation zone (Fig 6E and 6F). Seedlings treated with a mixture of 5 μM 3-EPC and 15 μM CA to test whether the presence of a natural lignin precursor affects the incorporation of the synthetic monolignol showed significantly higher fluorescence (Fig 6B and 6E) as compared to seedlings treated with 20 μM 3-EPC (Fig 6C and 6F). Fluorescence quantification data for each of these treatments is shown in S7 Fig. The finding that the addition of CA resulted in higher incorporation of 3-EPC in the root epidermis suggests that either synergistic monolignol uptake might be occurring, or that 3-EPC self-reactivity in the absence of CA before or during its incorporation might result in the loss of free alkynyl groups.

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