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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 40 μm-thick sections of 6 week old Arabidopsis stems.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).
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pone.0121334.g007: 3-EPC incorporation in 40 μm-thick sections of 6 week old Arabidopsis stems.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).

Mentions: To investigate whether the 3-EPC monolignol analog incorporates into naturally lignifying plant tissue, middle portions of stems from 6-week-old Arabidopsis Col-0 ecotype plants were sectioned using a cryostat and incubated with 3-EPC and/or CA for 3 h, then labeled with Alexa 594-azide for 1 h via a copper catalyzed click reaction. A 405 nm excitation laser was used to detect lignin autofluorescence and 561 nm excitation was used to detect click-associated fluorescence from Alexa 594-azide. Sections that were treated with 3-EPC (Fig 7B and 7C) showed significantly higher fluorescence with 561 nm excitation compared to sections treated with only CA and click labeling solution containing Alexa 594-azide, which showed no visible fluorescence (Fig 7A). Sections that were treated with a mixture of 20 μM 3-EPC and 20 μM CA (Fig 7B) showed lower fluorescence at 561 nm excitation after click labeling as compared to sections treated with 20 μM 3-EPC alone (Fig 7C), suggesting that CA might compete with 3-EPC at incorporation sites in the cell walls of stem sections, resulting in lower overall fluorescence. Imaging after 405 nm excitation of these sections revealed autofluorescence from both previously existing lignin and newly synthesized artificial lignin (Fig 7A–7C). Autofluorescence was higher in the vascular bundles or xylem (Fig 7D) than in interfascicular fibers (Fig 7E), whereas the click-associated 561 nm fluorescence was higher in the interfascicular fibers (Fig 7B–7D). Also, in the interfascicular fibers autofluorescence intensity was highest in the cell corners and middle lamellae and diminished inwards toward cell lumen, whereas click-associated fluorescence was not observed in the cell corners and had low intensity in the middle lamellae (Fig 7F). Lignin autofluorescence did not contribute to fluorescence in the 561 nm channel as observed in stem sections that were not treated with the 3-EPC or Alexa dye (S6 Fig). Quantification of autofluorescence (S8 Fig) revealed that there was no significant increase in autofluorescence after incorporation with 3-EPC and/or CA, which indicates that autofluorescence is mainly contributed by previously existing lignins. Therefore, fluorescence excitation at 405 nm and 561 nm allowed for differentiation between previously existing lignin and newly formed artificial lignin.


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 40 μm-thick sections of 6 week old Arabidopsis stems.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).
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pone.0121334.g007: 3-EPC incorporation in 40 μm-thick sections of 6 week old Arabidopsis stems.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).
Mentions: To investigate whether the 3-EPC monolignol analog incorporates into naturally lignifying plant tissue, middle portions of stems from 6-week-old Arabidopsis Col-0 ecotype plants were sectioned using a cryostat and incubated with 3-EPC and/or CA for 3 h, then labeled with Alexa 594-azide for 1 h via a copper catalyzed click reaction. A 405 nm excitation laser was used to detect lignin autofluorescence and 561 nm excitation was used to detect click-associated fluorescence from Alexa 594-azide. Sections that were treated with 3-EPC (Fig 7B and 7C) showed significantly higher fluorescence with 561 nm excitation compared to sections treated with only CA and click labeling solution containing Alexa 594-azide, which showed no visible fluorescence (Fig 7A). Sections that were treated with a mixture of 20 μM 3-EPC and 20 μM CA (Fig 7B) showed lower fluorescence at 561 nm excitation after click labeling as compared to sections treated with 20 μM 3-EPC alone (Fig 7C), suggesting that CA might compete with 3-EPC at incorporation sites in the cell walls of stem sections, resulting in lower overall fluorescence. Imaging after 405 nm excitation of these sections revealed autofluorescence from both previously existing lignin and newly synthesized artificial lignin (Fig 7A–7C). Autofluorescence was higher in the vascular bundles or xylem (Fig 7D) than in interfascicular fibers (Fig 7E), whereas the click-associated 561 nm fluorescence was higher in the interfascicular fibers (Fig 7B–7D). Also, in the interfascicular fibers autofluorescence intensity was highest in the cell corners and middle lamellae and diminished inwards toward cell lumen, whereas click-associated fluorescence was not observed in the cell corners and had low intensity in the middle lamellae (Fig 7F). Lignin autofluorescence did not contribute to fluorescence in the 561 nm channel as observed in stem sections that were not treated with the 3-EPC or Alexa dye (S6 Fig). Quantification of autofluorescence (S8 Fig) revealed that there was no significant increase in autofluorescence after incorporation with 3-EPC and/or CA, which indicates that autofluorescence is mainly contributed by previously existing lignins. Therefore, fluorescence excitation at 405 nm and 561 nm allowed for differentiation between previously existing lignin and newly formed artificial lignin.

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