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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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Synthesis of 3-ethynyl p-coumaryl alcohol (3-EPC), 6.(A) Reagents and conditions: a) Iodine monochloride, acetic acid, dichloromethane, 24 hours, 25°C; b) malonic acid, pyridine, piperidine, 90°C, 3.5 hours; c) methanol, sulfuric acid, reflux, overnight; d) trimethylsilyl acetylene, Bis(triphenylphosphine)palladium(II) chloride, cuprous iodide, triethylamine-tetrahydrofuran, 50°C, overnight; e) Diisobutylaluminium hydride (DIBAL-H), dichloromethane, -78°C, 3 hours; f) tetrabutylammonium fluoride, tetrahydrofuran, 25°C, 1 hour. (B) Chemical structures of monolignols and fluorophore. (E)-2-ethynyl-4-(3-hydroxyprop-1-enyl)phenol or 3-ethynyl p-coumaryl alcohol (3-EPC), 6, the monolignol probe developed in this paper; coniferyl alcohol (CA), 7, a natural lignin monomer; and Alexa 594-azide, 8, for fluorescence imaging.
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pone.0121334.g001: Synthesis of 3-ethynyl p-coumaryl alcohol (3-EPC), 6.(A) Reagents and conditions: a) Iodine monochloride, acetic acid, dichloromethane, 24 hours, 25°C; b) malonic acid, pyridine, piperidine, 90°C, 3.5 hours; c) methanol, sulfuric acid, reflux, overnight; d) trimethylsilyl acetylene, Bis(triphenylphosphine)palladium(II) chloride, cuprous iodide, triethylamine-tetrahydrofuran, 50°C, overnight; e) Diisobutylaluminium hydride (DIBAL-H), dichloromethane, -78°C, 3 hours; f) tetrabutylammonium fluoride, tetrahydrofuran, 25°C, 1 hour. (B) Chemical structures of monolignols and fluorophore. (E)-2-ethynyl-4-(3-hydroxyprop-1-enyl)phenol or 3-ethynyl p-coumaryl alcohol (3-EPC), 6, the monolignol probe developed in this paper; coniferyl alcohol (CA), 7, a natural lignin monomer; and Alexa 594-azide, 8, for fluorescence imaging.

Mentions: In the past decade, copper-catalyzed click chemistry has emerged as a useful approach for bioorthogonal metabolic labeling of living cells. In this approach, a metabolite modified with an alkynyl or azido group is added to the cells, which incorporate it into their structure. After incorporation, the modified metabolite can be detected by performing a copper-catalyzed click reaction that covalently links a detection probe, often a fluorophore, to the modified metabolite. This protocol has been used to label carbohydrates and nucleic acids in a variety of organisms, including bacteria, animals, and plants [39–46], allowing for tracing of the dynamics of incorporation and modification of polymers containing the modified metabolite. Due to its chemical mode of polymerization, lignin should be amenable to the incorporation of a click-compatible monolignol analog, allowing for the efficient detection of lignification in vitro and in plant tissues. In this study we designed and synthesized a new coniferyl alcohol analog, (E)-2-ethynyl-4-(3-hydroxyprop-1-enyl) phenol, or 3-ethynyl p-coumaryl alcohol (3-EPC), 6 (Fig 1A), and tested its incorporation in plant tissues. Very recently, incorporation and labeling of click-compatible monolignols into plant tissues was demonstrated by Tobimatsu et al., where the designed monolignols were modified at the γ–O-site instead of the 3’-C position [47]. Another study by Bukowski et al., demonstrated the incorporation and click-labeling of coniferyl alcohol modified at the 3’-C position to include a propargyl group instead of an ethynyl group [48]. In this study, we use nuclear magnetic resonance (NMR) spectroscopy to show that 3-EPC can be incorporated into lignin dehydrogenation polymer (DHP, an in vitro lignin model polymer), and is thus compatible with lignin polymerization. We also demonstrate the incorporation of 3-EPC into specific locations in actively lignifying plant tissues, as detected using fluorescence labeling and imaging. Sites of 3-EPC incorporation can be differentiated from previously existing lignin, which is detectable by its autofluorescence at a different excitation wavelength. Together, these results provide information about the chemical flexibility of lignin polymerization and the process by which plant cell walls become lignified.


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)

Synthesis of 3-ethynyl p-coumaryl alcohol (3-EPC), 6.(A) Reagents and conditions: a) Iodine monochloride, acetic acid, dichloromethane, 24 hours, 25°C; b) malonic acid, pyridine, piperidine, 90°C, 3.5 hours; c) methanol, sulfuric acid, reflux, overnight; d) trimethylsilyl acetylene, Bis(triphenylphosphine)palladium(II) chloride, cuprous iodide, triethylamine-tetrahydrofuran, 50°C, overnight; e) Diisobutylaluminium hydride (DIBAL-H), dichloromethane, -78°C, 3 hours; f) tetrabutylammonium fluoride, tetrahydrofuran, 25°C, 1 hour. (B) Chemical structures of monolignols and fluorophore. (E)-2-ethynyl-4-(3-hydroxyprop-1-enyl)phenol or 3-ethynyl p-coumaryl alcohol (3-EPC), 6, the monolignol probe developed in this paper; coniferyl alcohol (CA), 7, a natural lignin monomer; and Alexa 594-azide, 8, for fluorescence imaging.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401456&req=5

pone.0121334.g001: Synthesis of 3-ethynyl p-coumaryl alcohol (3-EPC), 6.(A) Reagents and conditions: a) Iodine monochloride, acetic acid, dichloromethane, 24 hours, 25°C; b) malonic acid, pyridine, piperidine, 90°C, 3.5 hours; c) methanol, sulfuric acid, reflux, overnight; d) trimethylsilyl acetylene, Bis(triphenylphosphine)palladium(II) chloride, cuprous iodide, triethylamine-tetrahydrofuran, 50°C, overnight; e) Diisobutylaluminium hydride (DIBAL-H), dichloromethane, -78°C, 3 hours; f) tetrabutylammonium fluoride, tetrahydrofuran, 25°C, 1 hour. (B) Chemical structures of monolignols and fluorophore. (E)-2-ethynyl-4-(3-hydroxyprop-1-enyl)phenol or 3-ethynyl p-coumaryl alcohol (3-EPC), 6, the monolignol probe developed in this paper; coniferyl alcohol (CA), 7, a natural lignin monomer; and Alexa 594-azide, 8, for fluorescence imaging.
Mentions: In the past decade, copper-catalyzed click chemistry has emerged as a useful approach for bioorthogonal metabolic labeling of living cells. In this approach, a metabolite modified with an alkynyl or azido group is added to the cells, which incorporate it into their structure. After incorporation, the modified metabolite can be detected by performing a copper-catalyzed click reaction that covalently links a detection probe, often a fluorophore, to the modified metabolite. This protocol has been used to label carbohydrates and nucleic acids in a variety of organisms, including bacteria, animals, and plants [39–46], allowing for tracing of the dynamics of incorporation and modification of polymers containing the modified metabolite. Due to its chemical mode of polymerization, lignin should be amenable to the incorporation of a click-compatible monolignol analog, allowing for the efficient detection of lignification in vitro and in plant tissues. In this study we designed and synthesized a new coniferyl alcohol analog, (E)-2-ethynyl-4-(3-hydroxyprop-1-enyl) phenol, or 3-ethynyl p-coumaryl alcohol (3-EPC), 6 (Fig 1A), and tested its incorporation in plant tissues. Very recently, incorporation and labeling of click-compatible monolignols into plant tissues was demonstrated by Tobimatsu et al., where the designed monolignols were modified at the γ–O-site instead of the 3’-C position [47]. Another study by Bukowski et al., demonstrated the incorporation and click-labeling of coniferyl alcohol modified at the 3’-C position to include a propargyl group instead of an ethynyl group [48]. In this study, we use nuclear magnetic resonance (NMR) spectroscopy to show that 3-EPC can be incorporated into lignin dehydrogenation polymer (DHP, an in vitro lignin model polymer), and is thus compatible with lignin polymerization. We also demonstrate the incorporation of 3-EPC into specific locations in actively lignifying plant tissues, as detected using fluorescence labeling and imaging. Sites of 3-EPC incorporation can be differentiated from previously existing lignin, which is detectable by its autofluorescence at a different excitation wavelength. Together, these results provide information about the chemical flexibility of lignin polymerization and the process by which plant cell walls become lignified.

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