Plant Physiology Preview. Published on April 1, 2016, as DOI:10.1104/pp.16.02012
Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
Revision following review, highlighting removed, March 31, 2016
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Maize Tricin-Oligolignol Metabolites and their Implications for Monocot Lignification
Wu Lan,1,2,§ Kris Morreel,3,4§ Fachuang Lu,1,5,6 Jorge Rencoret,7 José Carlos del Río,7 Wannes
Voorend,3,4 Wilfred Vermerris,8 Wout Boerjan,3,4,* and John Ralph.1,2,5,*
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DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of
Wisconsin, Madison, WI, USA
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Department of Biological System Engineering, University of Wisconsin, Madison, WI, USA
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Department of Plant Systems Biology, VIB, B-9052 Gent, Belgium
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Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent,
Belgium
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Department of Biochemistry, University of Wisconsin, Madison, WI, USA
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State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,
Guangzhou, People’s Republic of China
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Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS), CSIC, Seville, Spain
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Department of Microbiology and Cell Science - IFAS, and UF Genetics Institute, University
of Florida, Gainesville, FL, USA
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Co-first authors [Contributed equally to the the work performed and the writing of this paper]
Correspondence to (co-corresponding authors):
Prof. John Ralph
Prof. Wout Boerjan
Dept. of Biochemistry, GLBRC
Department of Plant Systems Biology, VIB
Wisconsin Energy Institute
Dept. Plant Biotechnology & Bioinformatics, UGent
U. Wisconsin–Madison
Technologiepark 927
1552 University Ave
9052 Ghent
Madison, WI 53726
BELGIUM
USA
Tel: 001-608-890-2429
Tel: +32 (0)9 331 38 92
Email: [email protected]
Email: [email protected]
Research Area: Biochemistry and Metabolism
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Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
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Maize Tricin-Oligolignol Metabolites and their Implications for Monocot Lignification
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Wu Lan,1,2,§ Kris Morreel,3,4§ Fachuang Lu,1,5,6 Jorge Rencoret,7 José Carlos del Río,7 Wannes
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Voorend,3,4 Wilfred Vermerris,8 Wout Boerjan,3,4,* and John Ralph.1,2,5,*
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1
DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of
Wisconsin, Madison, WI, USA
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Department of Biological System Engineering, University of Wisconsin, Madison, WI, USA
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Department of Plant Systems Biology, VIB, B-9052 Gent, Belgium
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Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent,
Belgium
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Department of Biochemistry, University of Wisconsin, Madison, WI, USA
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State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,
Guangzhou, People’s Republic of China
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Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS), CSIC, Seville, Spain
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Department of Microbiology and Cell Science - IFAS, and UF Genetics Institute, University
of Florida, Gainesville, FL, USA
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Co-first authors [Contributed equally to the the work performed and the writing of this paper]
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Summary
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Tricin-oligolignol maize metabolites, including variously acylated derivatives, validate
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combinatorial lignification and the incorporation of tricin into monocot lignins.
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Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
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Maize Tricin-Oligolignol Metabolites and their Implications for Monocot Lignification
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Financial Sources
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• WL, FL and JR were funded by the DOE Great Lakes Bioenergy Research Center (DOE BER
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Office of Science DE-FC02-07ER64494).
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• JR, KM, and WB were funded by Stanford’s Global Climate and Energy Program (GCEP).
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• WB and KM received additional support from the Institute for Promotion of Innovation
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through Science and Technology in Flanders (IWT), via the SBO project ‘Bioleum’.
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• WL is supported by the China Scholarship Council, State Education Department, for his PhD
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Program in the Department of Biological System Engineering, University of Wisconsin,
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Madison.
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Author Contributions
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Wu Lan performed all of the synthesis and NMR analysis, optical activity determination, wrote
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the paper and drew Figures 1, 2 and 5. [Equal first author]
Kris Morreel performed all the MS and CSPP analysis and provided the raw figures used for
Figures 3 and 4, and wrote the CSPP section. [Equal first author]
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Fachuang Lu co-supervised Wu Lan, advised on organic synthesis, and edited the paper.
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Jorge Rencoret first discovered the tricin on lignin, aided with NMR interpretation, and edited
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the paper.
José Carlos del Río supervised Jorge Rencoret, co-discovered the tricin on lignin, and edited the
paper.
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Wannes Voorend prepared samples for the MS analysis and aided Kris Moreel.
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Wilfred Vermerris initially discovered tricin in corn samples (but it remained unidentified at the
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time), helped with the design of the project, and edited the paper.
Wout Boerjan supervised Kris Morreel and Wannes Voorend, is project leader for the CSPP, MS
and general metabolomics work, and edited the paper.
John Ralph supervised Wu Lan and Fachuang Lu, devised the study, significantly edited the
paper, oversaw the research.
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Combinatorial Tricin-Oligolignols in Maize
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Maize Tricin-Oligolignol Metabolites and their Implications for Monocot Lignification
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ABSTRACT
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Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in
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which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-
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hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true
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monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the
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metabolites of maize were extracted from lignifying tissues and profiled using the recently
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developed Candidate Substrate Product Pair (CSPP) algorithm applied to Ultra-High-
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Performance Liquid Chromatography and Fourier Transform-Ion Cyclotron Resonance-Mass
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Spectrometry (UHPLC-FT-ICR-MS). Twelve tricin-containing products (each with up to eight
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isomers), including those derived from the various monolignol acetate and p-coumarate
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conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-
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oligolignol dimeric and trimeric compounds. The identification of such compounds helps
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establish that tricin is an important monomer in the lignification of monocots, acting as a
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nucleation site for starting lignin chains. The array of tricin-containing products provides further
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evidence for the combinatorial coupling model of general lignification and supports evolving
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paradigms for the unique nature of lignification in monocots.
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Keywords: Lignin, flavonoid, flavone, flavonolignan, flavonolignin, NMR, LC-MS, Candidate
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Substrate Product Pair (CSPP) analysis
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INTRODUCTION
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Lignin is one of the major components in plant cell walls and is predominantly deposited in the
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walls of secondary-thickened cells. It is a complex phenylpropanoid polymer composed
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primarily of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units derived from the
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monolignols p-coumaryl 2H, coniferyl 2G, and sinapyl 2S alcohols, respectively (Figure 1)
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(Freudenberg and Neish, 1968). These monolignols are biosynthesized in the cytoplasm and
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translocated to the cell wall where they are oxidized by laccases and peroxidases to monolignol
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radicals (Boerjan et al., 2003; Dixon and Reddy, 2003; Ralph et al., 2004b; Vanholme et al.,
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2008; Bonawitz and Chapple, 2010; Vanholme et al., 2010; Mottiar et al., 2016). The polymer
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can be started by radical coupling between two monolignol radicals to form a dehydrodimer from
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which the chain extends by endwise polymerization with additional monolignols, producing β–
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O–4-, β–5-, β–1-, and β–β-linked units in the lignin. Two growing oligomers may also radically
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couple to increase the polymer size producing 4–O–5- and 5–5-linked units. During such radical
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coupling reactions, the monomer-derived units are therefore linked together via various C–C and
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C–O bonds with different frequencies depending primarily on the monomer distribution and
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supply (Ralph et al., 2004b).
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A rather remarkable discovery regarding monocot lignins was recently made during a
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characterization study on wheat (del Río et al., 2012b). Previously unassigned correlation peaks
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in short-range 2D 1H–13C correlation (HSQC) NMR spectra of wheat (and other monocot)
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lignins were ultimately attributed to tricin 1 (Figure 1), a flavone, that was shown by long-range
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correlation (HMBC) experiments to be etherified by putative 4'–O–β-coupling with coniferyl
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alcohol 2G. Although tricin itself, various glycosides, and the flavonolignan tricin 4'–O–(β-
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guaiacylglyceryl) ether 3G are known (Bouaziz et al., 2002), as recently reviewed (Li et al.,
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2016), more profound implications arose from the demonstrated presence of this structure in
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polymeric lignins (del Río et al., 2012a; del Río et al., 2012b; Rencoret et al., 2013; del Río et
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al., 2015), as recently fully established by biomimetic coupling reactions and product
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authentication (Lan et al., 2015). It is the first time a phenolic derived from a pathway
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independent of the canonical monolignol biosynthetic pathway has been shown to polymerize
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into lignin in wild-type plants. Second, tricin’s structure, and its inability to undergo radical
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dehydrodimerization (below), implies that it can only start a lignin chain and can not be
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incorporated into an existing one. Tricin therefore provides a nucleation site for lignin chain
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growth, in a manner analogous to that proposed for arabinoxylan-bound ferulates (Ralph et al.,
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1995; Ralph et al., 2004a; Ralph et al., 2004b; Ralph, 2010). [We prefer not to use the term
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‘initiation site’ as this implies some kind of active role (Ralph, 2010)]. Given the facile detection
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of tricin in monocot lignins analyzed to date, a modest fraction of lignin chains must be
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covalently linked with tricin (at their starting ends).
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We have recently supported the involvement of tricin in lignification in the first disclosure
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(del Río et al., 2012b) by synthesizing a variety of authentic compounds 3 to confirm the veracity
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of the NMR assignments, and have shown that tricin indeed cross-couples with all three
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monolignols 2 via the radical coupling reactions that typify lignification (Lan et al., 2015). Tricin
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was shown to not undergo dehydrodimerization, which meant that it is restricted to cross-
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coupling reactions (with monolignols) during lignification, and was found even in the highest
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molecular weight fractions of the lignin isolated from maize (Zea mays) (Lan et al., 2015).
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In this study, we aimed to elucidate the incorporation pathways of tricin into maize lignins by
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applying liquid chromatography-mass spectrometry (LC-MS)-based tools developed for
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oligolignol profiling (Morreel et al., 2004a; Morreel et al., 2004b; Morreel et al., 2006; Morreel
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et al., 2010a; Morreel et al., 2010b; Morreel et al., 2014). We sought to provide evidence that
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tricin undergoes coupling with monolignols 2, and that endwise chain-extension polymerization
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continues in planta. What was not fully anticipated was the array of tricin-oligolignols derived
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from not only tricin’s coupling with monolignols but also with acylated monolignols (both
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acetates and p-coumarates) known to be involved in maize lignification (Ralph, 2010). The
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variety of structures extracted from maize and implicated by mass spectrometric analysis, and
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then in many cases authenticated via synthesis of the genuine compounds, is not only evidence
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for tricin’s role in lignification but additionally provides compelling support for the
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combinatorial nature of the lignification process itself.
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RESULTS
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Metabolite profiling by CSPP analysis
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The phenolics from the (lignifying) internode bearing the maize cob were extracted with
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methanol and profiled via Ultra-High-Performance Liquid Chromatography coupled to Fourier
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Transform-Ion Cyclotron Resonance-Mass Spectrometry (UHPLC-FT-ICR-MS) (Morreel et al.,
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2004a; Morreel et al., 2004b; Morreel et al., 2014; Niculaes et al., 2014; Dima et al., 2015) to
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reveal the presence of tricin-oligolignols. Using the recently developed Candidate Substrate
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Product Pair (CSPP) algorithm (Morreel et al., 2014), a network was constructed in which m/z
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features that might be derived from each other via well-known enzymatic or chemical
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conversions, are connected. This approach facilitates the tracking of m/z features representing
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similar compounds. Subsequently, network nodes of the tricin-oligolignol sub-network were
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further characterized via MSn-based oligolignol sequencing (Morreel et al., 2010a; Morreel et al.,
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2010b). This approach revealed a rather expansive set of tricin-oligolignol compounds 3, 4, and
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5 (Figure 1). The compounds include the products of coupling of all three monolignols 2 (at their
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usual β-positions) with tricin 1 (at its 4'–O-position), resulting in the tricin-4'–O–(β-arylglyceryl)
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or arylglycerol-β–O–4'-tricin ethers 3, along with the products 3G'/S' and 3G"/S" from the
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coupling of tricin with the acylated monolignols, the coniferyl and sinapyl acetate and p-
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coumarate conjugates 2' and 2" (Figure 1 and Table 1). Even more striking was the suggested
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presence of the trimers 4 and 5 resulting from the tricin-(4'–O–β)-monolignol with a further 4–
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O–β or 5–β linkage to another (acylated) monolignol (Figure 1 and Table 1). All of the observed
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compounds, along with their retention times, m/z values, and formulae are listed in Table 1.
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Syntheses of authentic compounds
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Over the past few years, an understanding of the gas-phase fragmentation patterns that result
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when oligolignol anions are subjected to collision-induced dissociation (CID), has allowed us to
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become more confident with the structural assignments of the peaks revealed by LC-MS.
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Nevertheless, it is crucial to rigorously identify any new classes of compounds, such as the
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tricin-oligolignols and their variously acylated counterparts implicated here, by absolute
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authentication via their independent chemical syntheses. We have accomplished that via the
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syntheses of eight authentic compounds (Figure 1), including seven 4'–O–β cross-coupled dimers
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3H, 3G, 3S, 3G', 3S', 3G'', and 3S'', and a trimer 4SG. The fragmentation patterns of these
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compounds then provided sufficient support to confidently characterize analogous tricin-
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containing metabolites extracted from maize. In addition, the synthetic compounds helped in
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verifying whether any compounds extracted from the maize internodes had escaped the CSPP
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network algorithm.
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The syntheses of 3H, 3G, 3S, and 4SG were described in detail in a previous study (Lan et al.,
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2015). The products 3G'/S' and 3G"/S" (Figure 2) of tricin’s coupling with acetylated and p-
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coumaroylated monolignols were prepared by modifications to the syntheses of their parent
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compounds. The preparation begins with the coupling of bromo-ketones 7 to a suitably protected
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tricin derivative 6, followed by retro-aldol addition of formaldehyde to create the lignin unit’s 3-
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carbon sidechain, the synthesis of which has been described (Lan et al., 2015). At this point, the
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product is suitably protected but bears the free γ-OH to allow acylation with p-coumarate or
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acetate to provide the acylated precursors 11 and 12. Deprotection of phenolic acetyl and
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methoxymethyl groups and reduction of the benzylic ketone then affords the required conjugates
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3G'/S' and 3G"/S". Full synthetic details, along with the NMR characterization are provided in
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the Experimental Section and Supporting Information (Data S1).
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Metabolite authentication and isomers analysis
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The eight synthetic compounds were used to authenticate the maize metabolites, and six of them
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could be identified in the methanol extracts from maize. Interestingly, whereas the trimer 4SG
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could be authenticated, its parent compound 3S, along with the analog 3S' from sinapyl acetate,
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were below the detection limit in the maize extract (Figure 3 and Figure 4). In addition, tricin
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coupled with the sinapyl p-coumarate conjugate, dimer 3S", could be authenticated. These
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observations presumably reflect the relative coupling propensities of the various dimers,
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suggesting that coniferyl alcohol, for example, may couple faster with dimer 3S than with the p-
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coumaroylated dimer 3S".
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We examined the distribution of diastereomers of the tricin-containing dimers and their
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acetate and p-coumarate derivatives (Figure 3), for both the synthetic products and their
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counterparts in maize metabolites. Such dimeric compounds were each separable as two peaks in
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an HPLC chromatogram, which respectively represent syn and anti isomers, indicated by
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comparing the retention times of authentic compounds and the MS2 spectra from the two peaks
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(Figure 3). In addition, as can be seen from Figure 4, most of the trimeric compounds revealed
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more than four (usually more than 6) peaks, and it is reasonable to conclude that the larger peaks
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are from two overlapping isomers. It is therefore evident that such spectra from compounds 4
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result from the eight possible isomers in each case; there are four optical centers affording 24
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optical isomers and half that number (23 = 8) of physically distinct isomers. The β–5-trimer
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labeled 5GS" in Figure 4 should have only 4 isomers, so the observation of at least 6 distinct
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peaks, two of which are again larger and are likely from two overlapping isomers, suggests that
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both regioisomers (5GS" and 5G"S) appear in the same area of the chromatogram here (see Table
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1). We further examined the enantiomers of the tricin-oligolignol dimers from maize, using 3H
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and 3G as examples, using an LC-MS equipped with a chiral column and the multiple reaction
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monitoring (MRM) MS technique; the MRM mode has the advantage of selectivity and
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improved quantitation for trace amounts of compounds. The chiral chromatogram (Figure 5)
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shows three peaks, one of which corresponds to the syn isomers (with 2 enantiomers
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overlapping), and the other two with similar peak areas originating from the anti isomers (two
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separated enantiomers), indicating that both the syn and anti isomers are racemic, just like their
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synthesized analogs.
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DISCUSSION
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Tricin couples with monolignol acetate and p-coumarate conjugates
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One of the most interesting findings from this maize metabolite profiling is not just the presence
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of tricin-oligolignols but also of their acetate and p-coumarate analogs (Figure 1, Figure 3, and
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Figure 4). p-Coumarate has long been a known feature of monocot lignins where it is found
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acylating the γ-OH of lignin sidechains (Ralph et al., 1994; Grabber et al., 1996; Ralph, 2010).
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Such acylation has now been compellingly demonstrated to arise via lignification with
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biosynthesized monolignol conjugates 2" (Lu and Ralph, 2008; Ralph, 2010; Lan et al., 2015; Lu
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et al., 2015). The monolignol: p-coumaroyl-CoA transferase (PMT) enzyme and the PMT gene
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involved have been identified and the function proven via knock-out, down-regulation, and over-
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expression (Withers et al., 2012; Marita et al., 2014; Petrik et al., 2014). Acetates are also well-
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known to acylate monolignols in various plant lines (Ralph, 1996; del Río et al., 2007; del Río et
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al., 2008; Lu and Ralph, 2008; Martinez et al., 2008; del Río et al., 2012a; del Río et al., 2012b;
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Rencoret et al., 2013), although the responsible transferase protein and the corresponding gene
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have not been unambiguously identified to date. Monocots have more extensive lignin
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acetylation than previously realized (del Río et al., 2007; del Río et al., 2008; Martinez et al.,
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2008; del Río et al., 2012a; del Río et al., 2012b; Rencoret et al., 2013; del Río et al., 2015). The
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products 3G' from tricin’s cross-coupling with acetylated monolignols 2' here (Figure 1) further
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confirm the involvement of monolignol acetate conjugates in maize (and other monocot)
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lignification.
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Structural analyses of tricin-oligolignols support the combinatorial radical coupling theory
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The above finding that monolignols 2 as well as their acetate and p-coumarate conjugates 2' and
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2" are all coupling with tricin (Figure 3 and Figure 4) has deeper consequences regarding
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lignification. As in the established theory (Harkin, 1967; Freudenberg and Neish, 1968), and as
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increasingly evidenced (Ralph et al., 2004b; Ralph et al., 2008; Vanholme et al., 2010), lignins
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are the products of simple, but combinatorial, radical coupling chemistry. They are consequently
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racemic polymers, are characterized by being products with a huge number of possible isomers,
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and have no defined sequence or (repeating) structure, a position that was heatedly debated after
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notions of absolute proteinaceous control over lignin structure were championed for a period
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(Davin and Lewis, 2005). Such impressions continue to be eroded as evidence accumulates from
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various structural studies of natural plants along with the rich variety of monolignol biosynthetic
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pathway mutants and transgenics, reestablishing the validity of the original theory (Ralph et al.,
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2008). The results here further add to the evidence.
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Another key argument supporting the combinatorial coupling theory follows from the
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separation and identification of the enantiomers of 3G and 3H. Compound 3G was previously
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isolated from Avena sativa by capillary electrophoresis; as both of the diastereomers were
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enantiomerically pure, they were termed flavonolignans (Wenzig et al., 2005). In our study,
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however, the two enantiomers of anti 3G and of anti 3H isolated from maize were successfully
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separated using chiral-column HPLC, as confirmed by multiple reaction monitoring (MRM) on
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MS that is able to accurately track compounds at low levels; the similar peak areas (Figure 5),
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indicate the racemic nature of both, just as has been reported for various lignin units (Ralph et
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al., 1999; Akiyama et al., 2015). Importantly, therefore, these dimeric compounds 3, resulting
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from the coupling of tricin and a monolignol, can not be termed flavonolignans (that, like their
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component lignan moieties (Umezawa, 2004), would logically be optically active); they should
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therefore be considered to be oligomers that are destined for the fully racemic lignins, and are
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therefore suggested to be generally termed flavonolignin oligomers or, specifically, tricin-
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oligolignols. Optical activity determinations are not always carried out, so it is not always
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possible to determine whether extracted components are (optically active) lignans or (racemic)
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di- and oligolignols, as discussed briefly previously (Dima et al., 2015); the same is true here for
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these flavonolignans vs flavonolignols.
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The variety of tricin-oligolignols and their variously acylated counterparts, and the
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identification of their diastereomers and enantiomers, provide compelling new evidence for the
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combinatorial nature of lignification – available monomer radicals, including those from tricin
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and the monolignol conjugates, will couple and cross-couple subject only to their chemical
311
propensities for doing so. We therefore observe certain combinatorial possibilities for the cross-
312
coupling of both coniferyl and sinapyl acetates and p-coumarates, along with the parent
313
monolignols, with tricin, as shown schematically in Figure 1. The trimers and tetramers then
314
attest to the chain extension via further coupling from among the available monolignols and their
315
conjugates. Importantly, the products observed here also provide evidence for the growth of the
316
polymer in the ‘endwise coupling’ sense (Freudenberg, 1956; Ralph et al., 2004b), in which
317
chain extension is via monomer addition to the phenolic end of the growing oligomer, and are
318
not consistent with the notion of the tricin-containing polymeric units being derived from
319
preformed flavonolignans. All that perhaps remains surprising is that all of these monomeric
320
entities must be present at the same time and space, an observation that might not have been
321
expected; in dicots, for example, sinapyl alcohol enters lignification later in cell wall
322
development, preceded by p-coumaryl alcohol and then coniferyl alcohol, although there is
323
overlap (Terashima et al., 1993). Essentially nothing is known about the temporal (or spatial)
324
nature of monolignol conjugate incorporation into monocot lignins.
325
Finally, again given that tricin can only start a chain, and given that it is present at significant
326
levels (currently estimated to be 1.5% of the lignin in the wild-type maize internode samples
327
analyzed here, according to the thioacidolysis method (Unpublished data)), it must be nucleating
328
a fraction of the lignin chains. The identification of compound 3H, the coupling product of tricin
329
with p-coumaryl alcohol, suggests that tricin is present early in lignification. Tricin is noted to be
330
higher in concentration in younger and less lignified tissues (del Río et al., 2015) but it is not yet
331
clear if it is biosynthesized throughout wall development. These revelations regarding tricin in
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332
lignins contributed to the resolution of a monocot-lignin structural dilemma that has existed for
333
decades – that monocot lignins, unlike other syringyl-guaiacyl lignins in dicots/hardwoods, have
334
essentially no, or very low levels of, resinols, syringaresinol and pinoresinol (Marita et al., 2003;
335
Lan et al., 2015). Such β–β-linked units are produced only as the result of monolignol (sinapyl
336
alcohol) dimerization and are the obvious mechanism for starting a lignin chain. Maize whole-
337
cell-wall or lignin NMR spectra had little evidence until recently of anything but β-ether units,
338
with only a paucity of the other units [resinol (β–β), phenylcoumaran (β–5), dibenzodioxocin (5–
339
5/4–O–β), and spirodienone (β–1)] seen in dicot lignins with a comparable S:G distribution (Lan
340
et al., 2015). However, we recently disclosed the preponderance of sinapyl p-coumarate
341
homodimerization units in maize lignins (Lan et al., 2015). Although this product is from β–β-
342
coupling, the γ-acylation does not allow resinol formation (Ralph, 2010), so its presence had
343
been missed. Also, when the lignin chain is nucleated by another unit, such as tricin here (and as
344
assumed for ferulate previously (Ralph et al., 1995), and in addition to it), lignification does not
345
need to start with a dimerization reaction. The near absence of such resinol units in maize and
346
some other monocots is now recognized as being a consequence of the nucleation of lignin
347
chains by tricin and ferulate, as well as from the surprising prevalence of acylated monolignol
348
dimerization events in such lignins. Such features of the previously puzzling spectra of monocot
349
lignins are now therefore consistent with the evolving paradigms for the unique nature of
350
lignification in monocots.
351
352
CONCLUSIONS
353
Following analysis of maize metabolites by UHPLC-MS, we have identified and characterized
354
12 tricin-oligolignols (Figure 1), in some 42 resolved peaks (Table 1) that include various
355
diastereomers, the structures of which were further supported by comparison with independently
356
synthesized authentic compounds. The maize metabolites include the 4'–O–β-cross-coupling
357
products between tricin and monolignols as well as their acetate and p-coumarate conjugates.
358
Chiral chromatography of tricin-(4'–O–β)-p-coumaryl alcohol and tricin-(4'–O–β)-coniferyl
359
alcohol coupling products from maize showed that the flavonolignols are fully racemic. The
360
above findings provide compelling new evidence: 1) for the natural cross-coupling of tricin with
361
monolignols and monolignol conjugates into flavonolignol dimers in planta; 2) that such dimers
362
undergo further endwise coupling with additional monomers to form oligomers that are destined
363
for lignin polymers in which chains are started by tricin; and 3) for the combinatorial nature of
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Combinatorial Tricin-Oligolignols in Maize
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364
lignification, i.e., supporting the theory that lignin polymers are formed by combinatorial radical
365
coupling chemistry independent of proteinaceous control.
366
367
EXPERIMENTAL
368
General
369
All chemicals and solvents used in this study were from commercial sources and used without
370
further purification. Preparative thin layer chromatography plates (1 or 2 mm thickness, 20 cm ×
371
20 cm, normal-phase) were purchased from Analtech (Newark, DE). Flash chromatography was
372
conducted on an Isolera One instrument (Biotage, Charlottesville, VA) with Biotage snap silica
373
cartridges. The eluent for chromatography was hexane/ethyl acetate or methanol/dichloro-
374
methane as described. NMR spectra were recorded at 25 °C on a Bruker Biospin (Billerica, MA)
375
AVANCE 500 MHz or 700 MHz spectrometer fitted with a cryogenically cooled 5 mm TCI (500
376
MHz) or TXI (700 MHz) gradient probe with inverse geometry (proton coil closest to the
377
sample). Bruker’s Topspin 3.1 (Mac) software was used to process spectra. The central solvent
378
peak was used as internal reference (δC/δH: acetone-d6, 29.84/2.04). The standard Bruker
379
implementations of 1D and 2D [gradient-selected correlation spectroscopy (COSY),
380
heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation
381
(HMBC)] NMR experiments were used for routine structural assignments of newly synthesized
382
compounds.
383
384
Syntheses of arylglycerol-β–O–4'-tricin ethers 3 and 4
385
Compounds 3H, 3G, 3S, and 4SG were synthesized as recently described (Lan et al., 2015).
386
387
Syntheses of γ-acylated arylglycerol-β–O–4'-tricin ethers 3', 3"
388
Figure 2 outlines the synthetic procedure for both the normal and γ-acylated arylglycerol-β-O-4'-
389
tricin ethers 3' and 3"; details are provided in the following.
390
391
Compounds 6, 7G/S, 8G/S, 9G/S, and 10G/S were synthesized according to the methods
previously described (Lan et al., 2015).
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Combinatorial Tricin-Oligolignols in Maize
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392
Compound 11G/S. Compound 10G (50 mg, 95.3 μmol) was first acetylated in pyridine/acetic
393
anhydride (v/v, 2:1, 5 mL) at room temperature for 2 h. The solution was extracted with ethyl
394
acetate (EtOAc, 25 mL) and acidic water (pH = 2.0, 25 mL). EtOAc layers were combined and
395
washed with saturated ammonium chloride (NH4Cl) solution (50 mL) and dried over anhydrous
396
magnesium sulfate (MgSO4). After condensation, the acetylated product and ammonium acetate
397
(146.9 mg, 1.90 mmol) were added to methanol (MeOH, 20 mL) and heated at 50 °C to remove
398
the phenolic acetate group. When the starting material had totally disappeared (~8 h, monitored
399
by TLC), the solvent was evaporated under reduced pressure and the resulting material was
400
subjected to TLC purification to give 11G (82% yield). 11S was synthesized analogously in 80%
401
yield.
402
Compound 3G'/S'. Borane-tert-butylamine complex (19.2 mg, 220.6 μmol) and 11G (25.0 mg,
403
44.1 μmol) were dissolved in CH2Cl2 (5 mL) at room temperature. The solvent was removed
404
under reduced pressure after the reaction was completed (~12 h, monitored by TLC). The
405
product was dissolved in EtOAc/water (25 mL, v/v, 10:1) with 1 mL of 6 M HCl solution, and
406
stirred for 1 h to break down the borate intermediates. The mixture solution was washed with
407
water and saturated NH4Cl solution (50 mL). The EtOAc layer was separated, dried over
408
anhydrous MgSO4, filtered, and the solvent removed under reduced pressure to produce 3G'
409
(92%). 3S' was prepared analogously in 90% yield.
410
Compound 12G/S. Acylation of 9G/S was catalyzed by 4-dimethylaminopyridine (DMAP) in
411
CH2Cl2. A detailed procedure is given using 12G as example. 9G (250 mg, 409.4 μmol) was
412
dissolved in CH2Cl2 (10 mL), to which fresh-made acetylated p-coumaroyl chloride 16 (110 mg,
413
490.5 μmol) and DMAP (50 mg, 409.3 μmol) was added. After 1 h, the solution was washed
414
with 0.5 M HCl solvent (3×50 mL) to remove DMAP. The CH2Cl2 solution was dried over
415
anhydrous MgSO4, filtered, and the solvent was removed under reduced pressure. TLC
416
purification using CH2Cl2 and MeOH (v/v, 40:1) as eluent was conducted, giving 67%/84% yield
417
of 12G/S.
418
Compound 13G/S. Selective deprotection of the methoxylmethyl group was achieved in
419
ethylene glycol as previously described (Miyake et al., 2004). 12G/S (100 mg, 125.2 μmol for G,
420
120.7 μmol for S) was mixed with ethylene glycol (25 mL) and heated at 120 °C for 3 h. Then
421
water (25 mL) was added to quench the reaction. EtOAc (3×25 mL) was used to extract the
422
products. The combined EtOAc fraction was dried over anhydrous MgSO4, filtered, and the
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Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
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423
solvent was evaporated under reduced pressure. TLC purification using CH2Cl2 and MeOH (v/v,
424
40:1) as eluent yielded 13G/S (43%/56%).
425
426
Compound 14G/S. The phenolic acetate group of 13G/S was eliminated using ammonium
acetate in MeOH as described in the synthesis of 11G/S. The yield of 14G/S was 64%/71%.
427
Compound 3G"/S". Borane-tert-butylamine complex was used to reduce the α-ketone in
428
13G/S to its alcohol, as above for the synthesis of 3G'/S'. The yield of 3G"/S" was 80%/75%.
429
Compound 16. p-Coumaric acid 15 (1 g, 4.8 mmol) was first acetylated in pyridine/acetic
430
anhydride (v/v, 2:1, 15 mL). The acetylated product and thionyl chloride (1 mL, 13.8 mmol)
431
were added to toluene (10 mL) and heated, with stirring, to 100 °C until the material was
432
completely dissolved (~1 h). Then the solvent was evaporated under reduced pressure. The
433
product was dissolved in toluene (50 mL) and evaporated again. This procedure was repeated
434
several times to eliminate residual thionyl chloride. The final acyl chloride product 16 was
435
obtained as a white powder in 96% yield.
436
437
Growth conditions and extraction
438
Maize plants (inbred line B104) were grown in a greenhouse (16 h light; minimum temperature
439
of 25 °C and 23 °C during day and night, respectively). Supplementary light was added using
440
high-pressure sodium vapor lamps when natural light intensity dropped below 200 W m-2.
441
Fertilizer was added with the water supply (Ec = 1 mS cm-1, NPK = 20:5:20, MgO = 3). The 9th
442
internode (internode just below the cob) was dissected from 22 plants harvested 7 days after
443
silking. At this time, the plants had reached a height of 2 m. Internode samples were ground
444
using liquid-nitrogen-cooled Retch Grinding Jars MM 400 Stainless Steel 50 ml. A volume of
445
500 μL of powder was extracted with 1 mL of methanol at 70 °C for 15 min. Following
446
evaporation, the pellet was dissolved in 300 μL MilliQ water/cyclohexane (2:1, v/v). 10 μL of
447
the aqueous phase was used for metabolite profiling.
448
449
Metabolite profiling
450
Reversed-phase ultrahigh-performance liquid chromatography (UHPLC) coupled to Fourier
451
Transform-Ion Cyclotron Resonance-Mass Spectrometry (FT-ICR-MS) was performed on a
452
previously described platform (Accela coupled to a LTQ FT Ultra, Thermo Electron
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Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
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453
Corporation, Bremen, Germany) using the conditions previously described (Morreel et al., 2014)
454
with some modifications. The applied gradient was: 0 min 95% A, 30 min 55% A, 35 min 0% A
455
(solvents A and B were aqueous 0.1% acetic acid and acetonitrile/water, 99/1, v/v, respectively).
456
Electrospray Ionization (ESI) source conditions were: spray voltage 3.5 kV, sheath gas 10 (arb),
457
aux gas 15 (arb). Full FT-MS spectra were recorded between 120 and 1200 m/z. Data-dependent
458
MS2 spectra of the four most abundant ions in the previous full MS spectra were recorded using
459
the ion-trap analyzer. Integration, alignment, grouping of m/z features derived from the same
460
compound, and generation of the Candidate Substrate Product Pair (CSPP) network were
461
performed as previously described (Morreel et al., 2014). Structural characterization of the tricin-
462
oligolignols was further aided via MSn-based oligolignol sequencing (Morreel et al., 2010a;
463
Morreel et al., 2010b). The latter method unveiled some typical characteristics in the gas-phase
464
fragmentation of tricin-oligolignols. A full analysis of the CSPP network characteristics of these
465
and other monocot-specific compounds will be published elsewhere.
466
467
MS2 Analysis of tricin-oligolignols 3 and 4
468
Upon collision-induced dissociation (CID), (4'–O–β)-type oligolignols undergo characteristic gas
469
phase fragmentation channels involving the 7-OH function (Morreel et al., 2010a; Morreel et al.,
470
2010b). One series of fragmentations (called type I fragmentations) yield small neutral losses of
471
which the 48 Da loss often leads to the base peak in the CID spectrum, especially in the case of a
472
threo configuration of the 4'–O–β-linkage (Morreel et al., 2004a). The 48 Da loss results from
473
expelling the 7- and 9-OH groups as water and formaldehyde, respectively (Morreel et al.,
474
2010a). Type II fragmentations lead to the cleavage of the 4'–O–β-linkage and allow
475
characterization of the units connected by it (Morreel et al., 2010a). The precursor ions of all 4'–
476
O–β-coupled tricin-type oligolignols are subjected to similar type I and II gas phase
477
fragmentations as described for traditional oligolignols. Nevertheless, their MS2 spectra were
478
quite often dominated by tricin product ion (m/z 329) resulting from a type II fragmentation.
479
Clearly, this fragmentation channel is favored as the charge of the ion can be readily delocalized
480
across the extensive conjugated π-system.
481
482
Chiral chromatography of synthetic dimers and their maize-derived counterparts
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Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
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483
Separation of the enantiomers of 3H and 3G (for both synthetic compounds and methanol
484
extracts from maize) was accomplished on an LC-MS system (Shimadzu, USA), equipped with 2
485
LC-20AD pumps, a SIL-20AC HT autosampler, a CTO-20A column oven, a CBM-20A
486
controller, and LCMS-8040 triple-quadrupole mass spectrometer, using a Lux Cellulose-1
487
(150×4.60 mm, 5 μm, Phenomenex, USA) column at 40 °C. A Dual Ion Source (DUIS) method
488
was applied for ionization. The mobile phase is water (solvent A) and methanol (solvent B) with
489
0.1% (v/v) formic acid in each solution and 60% of solvent B was used as eluant method. The
490
injection volume was 1 μL and the flow rate was 0.7 mL min-1. Detection was achieved using
491
Multiple Reaction Monitoring (MRM) mode, in which the first quadrupole was conducted a
492
single ion monitoring (SIM) mode for a set m/z value, and the target ion (precursor) becomes
493
broken down into fragments (products) using an optimal collision energy before entering the
494
second quadrupole region, followed by a SIM in the third quadrupole to track the fragments. The
495
chiral chromatogram was analyzed in Origin 9.1 for multiple peak fitting using Gaussian
496
function.
497
498
ACKNOWLEDGEMENTS
499
The authors thank the China Scholarship Council, State Education Department, for supporting
500
living expenses for Wu Lan’s PhD Program in the Department of Biological System
501
Engineering, University of Wisconsin, Madison. WL, FL and JR were funded by the DOE Great
502
Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494), and JR,
503
KM, and WB were funded by Stanford’s Global Climate and Energy Program (GCEP). WB and
504
KM additionally acknowledge the Institute for Promotion of Innovation through Science and
505
Technology in Flanders (IWT) for funding the SBO project ‘Bioleum’.
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Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
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506
507
SUPPORTING INFORMATION
508
Additional Supporting Information may be found in the online version of this article.
509
510
Figure S1. LC-MS trace and the MS2 spectra of each peak (isomer or isomer mixture) for the
511
tricin-(4'–O–β)-coniferyl alcohol-(4–O–β)-coniferyl alcohol coupling product, trimer 4GG and
512
tricin-(4'–O–β)-sinapyl alcohol-(4–O–β)-coniferyl alcohol coupling product, trimer 4SG, run
513
under slightly different (and slightly better resolving) conditions than those in Figure 4. Besides
514
the 6 resolved 4GG isomers that were verified by MS2, two other isomers represented by the
515
peaks at 21.6 and 21.7 shown in dark gray do not appear in the chromatogram run for Figure 4
516
and are therefore unlikely to be the same tricin-containing compounds; the other two peaks at
517
18.3 and 19.2 (light gray) also have the same nominal mass but not the same exact mass as the
518
other peaks and are therefore likely not tricin-containing compounds. In the case of 4SG, only 5
519
of the isomers could be clearly resolved; again, the peak at 23.4 min (dark gray) is not likely to
520
be a tricin-containing isomer of compound 4SG.
521
522
Data S1. 1H and 13C NMR data for synthetic compounds.
523
524
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Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
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525
Table 1. Tricin-oligolignols detected in maize.
#
Shorthand Name
Elucidation levela
-1.5
T-(4–O–βt)-H
Identified
-1.2
T-(4–O–βe)-H
Identified
C27H25O11
-2.2
T-(4–O–βt)-G
Identified
tR
m/z
Formula
∆ppm
3H
19.9
495.12891
C26H23O10
3H
20.6
495.12907
C26H23O10
3G
20.4
525.13906
Dimers
3G
21.2
525.13889
C27H25O11
-2.6
T-(4–O–βe)-G
Identified
3G'
24.7
567.14943
C29H27O12
-2.4
T-(4–O–βt)-G'
Identified
3G'
25.0
567.15031
C29H27O12
-0.8
T-(4–O–βe)-G'
Identified
3G"
25.7
671.17480
C36H31O13
-3.3
T-(4–O–βt)-G''
Identified
3G"
25.9
671.17447
C36H31O13
-3.8
T-(4–O–βe)-G''
Identified
3S"
25.5
701.18628
C37H33O14
-1.9
T-(4–O–βt)-S''
Identified
3S"
25.7
701.18532
C37H33O14
-3.2
T-(4–O–βe)-S''
Identified
3GαOMe
24.7
539.15522
C28H27O11
-1.2
T-(4–O–β)-GαOMe
Annotated
4GG
18.9
721.21084
C37H37O15
-4.1
T-(4–O–β)-G-(4–O–β)-G
Annotated
4GG
19.2
721.21151
C37H37O15
-3.2
T-(4–O–β)-G-(4–O–β)-G
Annotated
4GG
19.4
721.21334
C37H37O15
-0.6
T-(4–O–β)-G-(4–O–β)-G
Annotated
4GG
19.6
721.21185
C37H37O15
-2.7
T-(4–O–β)-G-(4–O–β)-G
Characterized
4GG
19.8
721.21251
C37H37O15
-1.8
T-(4–O–β)-G-(4–O–β)-G
Annotated
4GG
20.0
721.21093
C37H37O15
-4
T-(4–O–β)-G-(4–O–β)-G
Annotated
4SG
19.8
751.22302
C38H39O16
-1.8
T-(4–O–βt)-S-(4–O–βt)-G
Identified
4SG
20.3
751.22338
C38H39O16
-1.3
T-(4–O–βe)-S-(4–O–βt)-G
Identified
4SG
20.6
751.22316
C38H39O16
-1.6
T-(4–O–β)-S-(4–O–β)-G
Characterized
4SG
21.0
751.22285
C38H39O16
-2
T-(4–O–β)-S-(4–O–β)-G
Characterized
4GG'
22.7
763.22264
C39H39O16
-2.3
T-(4–O–β)-G-(4–O–β)-G'
Annotated
4G'G
23.2
763.22198
C39H39O16
-3.1
T-(4–O–β)-G'-(4–O–β)-G
Annotated
4G'G
23.5
763.22264
C39H39O16
-2.2
T-(4–O–β)-G'-(4–O–β)-G
Annotated
4G'G
24.0
763.22147
C39H39O16
-3.8
T-(4–O–β)-G'-(4–O–β)-G
Annotated
5GS"
24.3
879.24808
C47H43O17
-2.8
T-(4–O–β)-G-(5–β)-S''
Characterized/Annotated
Characterized
Trimers
5GS"
[5G"S]
25.2
879.24942
C47H43O17
-1.3
T-(4–O–β)-G-(5–β)-S''
[T-(4–O–β)-G''-(5–β)-S]
5GS"
25.7
879.24975
C47H43O17
-0.9
T-(4–O–β)-G-(5–β)-S''
Characterized/Annotated
Characterized
5GS"
[5G"S]
26.3
879.24928
C47H43O17
-1.5
T-(4–O–β)-G-(5–β)-S''
[T-(4–O–β)-G''-(5–β)-S]
5GS"
[5G"S]
26.9
879.24915
C47H43O17
-1.6
T-(4–O–β)-G-(5–β)-S''
[T-(4–O–β)-G''-(5–β)-S]
Characterized
5GS"
[5G"S]
27.4
879.24823
C47H43O17
-2.7
T-(4–O–β)-G-(5–β)-S''
[T-(4–O–β)-G''-(5–β)-S]
Characterized
4G"G
[4GG"]
24.8
867.24907
C46H43O17
-1.7
T-(4–O–β)-G''-(4–O–β)-G
[T-(4–O–β)-G-(4–O–β)-G'']
Characterized
4G"G
[4GG"]
25.0
867.24993
C46H43O17
-0.7
T-(4–O–β)-G''-(4–O–β)-G
[T-(4–O–β)-G-(4–O–β)-G'']
Characterized
4G"G
[4GG"]
25.7
867.24982
C46H43O17
-0.9
T-(4–O–β)-G''-(4–O–β)-G
[T-(4–O–β)-G-(4–O–β)-G'']
Annotated
4GG"
26.0
867.24835
C46H43O17
-2.6
T-(4–O–β)-G-(4–O–β)-G''
Annotated
-1.4
T-(4–O–β)-G''-(4–O–β)-G /
T-(4–O–β)-G-(4–O–β)-G''
Characterized
4G"G
[4GG"]
26.3
867.24938
C46H43O17
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8
Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
Revision following review, highlighting removed, March 31, 2016
526
527
528
529
530
531
532
533
a
4G"G
[4GG"]
26.5
867.24895
C46H43O17
-1.9
T-(4–O–β)-G''-(4–O–β)-G /
T-(4–O–β)-G-(4–O–β)-G''
Characterized
4GS"
26.2
897.25787
C47H45O18
-3.6
T-(4–O–β)-G-(4–O–β)-S''
Annotated
4GS"
26.4
897.25903
C47H45O18
-2.3
T-(4–O–β)-G-(4–O–β)-S''
Annotated
4GS"
26.5
897.25803
C47H45O18
-3.5
T-(4–O–β)-G-(4–O–β)-S''
Annotated
4GS"
26.7
897.25813
C47H45O18
-3.4
T-(4–O–β)-G-(4–O–β)-S''
Annotated
4GS"
26.9
897.25816
C47H45O18
-3.3
T-(4–O–β)-G-(4–O–β)-S''
Annotated
‘Identified’ indicates a structure confirmed by comparison with the synthesized authentic
compound; ‘Annotated’ indicates a rather firm structural elucidation based on comparison of
the MSn spectrum with those of identified structural analogs and on the accurate m/z value;
‘Characterized’ indicates a fairly high degree of certainty in the structural elucidation that is
based on the MSn spectral interpretation, and accurate m/z value, and data available from
literature and public databases.
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9
Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
Revision following review, highlighting removed, March 31, 2016
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535
FIGURE CAPTIONS
536
Figure 1. Tricin 1 and its oxidative coupling with monolignols 2 and monolignol conjugates 2'
537
and 2" to produce tricin-oligolignols 3 (dimers), 4 (trimers), and 5 (trimers). Primes are used to
538
indicate the acylation of monolignols and derived units in the polymer, and small upper-case
539
letters H, G, and S, are used to designate the p-hydroxyphenyl, guaiacyl, or syringyl nature of the
540
aromatic rings (and therefore the moiety’s derivation from its monolignol, p-coumaryl, coniferyl,
541
or sinapyl alcohol); we also refer to the A and B ring, as shown, in trimers 4. For example, the
542
hypothetical compound formed by coupling of coniferyl acetate 2G' with tricin, followed by
543
further chain extension by coupling the product dimer with sinapyl p-coumarate 2S" would be
544
designated as 4G'S"; in various tables, we also designate this with the more descriptive shorthand
545
– T-(4–O–β)-G'-(4–O–β)-S" – that indicates the coupling modes from the starting tricin to the
546
final sinapyl p-coumarate, in this case. The two asterisked structures were synthesized authentic
547
compounds that were not found among the maize metabolites.
548
549
Figure 2. Synthesis of tricin-monolignol, tricin-monolignol acetate and tricin-monolignol p-
550
coumarate dimeric products 3G'/S' and 3G"/S". (i) K2CO3, DMF; (ii) Formaldehyde, K2CO3,
551
dioxane; (iii) MeOH/CHCl3, HCl; (iv) Pyridine/acetic anhydride; (v) Ammonium acetate,
552
MeOH; (vi) Borane-tert-butylamine complex, CH2Cl2; (vii) Acetylated p-coumaroyl chloride,
553
DMAP, CH2Cl2; (viii) Ethylene glycol, 120 °C; (ix) Toluene, thionyl chloride.
554
555
Figure 3. LC-MS of monolignol and acylated monolignol coupling products with tricin (dimers
556
3).
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Figure 4. LC-MS of trimeric compounds 4 and 5 formed between tricin and various monolignols
559
and acylated monolignols.
560
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Figure 5. Chiral chromatography of 3H and 3G in maize extracts, by LC-MS using multiple
562
reaction monitoring mode (MRM) detection, showing their racemic nature.
563
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Combinatorial Tricin-Oligolignols in Maize
Plant Physiology #PP2015-02012
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564
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1
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