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Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

Lignin Structure and its Engineering

John Ralph,1,2 Catherine Lapierre,3 and Wout Boerjan4,5
1
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA
2
Department of Energy Great Lakes Bioenergy Research Center, the Wisconsin Energy Institute,
University of Wisconsin, Madison, WI 53726, USA
3
Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, 78000 Versailles,
France
4
Ghent University, Department of Plant Biotechnology and Bioinformatics, Technologiepark 927, B-
9052 Gent, Belgium
5
VIB Center for Plant Systems Biology, Technologiepark 927, B-9052 Gent, Belgium

Correspondence
e-mail: jralph@wisc.edu

Abstract
Studies on lignin structure and its engineering are inextricably and bidirectionally linked. Perturbations of
genes on the lignin biosynthetic pathway may result in striking compositional and structural changes that
in turn suggest novel approaches for altering lignin and even ‘designing’ the polymer to enhance its value
or with a view towards its simpler removal from the cell wall polysaccharides. Basic structural studies on
various native lignins increasingly refine our knowledge of lignin structure, and examining lignins in
different species reveals the extent to which evolution and natural variation have resulted in the
incorporation of “non-traditional” phenolic monomers, including phenolics from beyond the monolignol
biosynthetic pathway. As a result, the very definition of lignin continues to be expanded and refined.

Highlights
• New monomers have been discovered, and structural aspects of the lignin polymer have been refined.
• The definition of a lignin monomer has been broadened.
• Lignification is strikingly metabolically plastic.
• Actual lignin ‘design’ can be contemplated.
• Findings portend improved utilization potential and enhanced value for lignin.

Introduction
Lignin structural studies historically focused on how the polymerization occurred, on the structural
ramifications of that process, and on how structural alterations affected lignin processing. The
combinatorial nature of the radical coupling reactions fascinated chemists. New analytical methods
improved the characterization of traditional and newly discovered structural features, and instrumental
methods, particularly 2D NMR [1], allowed structural details to be teased from the complex polymer.
Exciting ‘new’ structures in lignins were revealed, including the dibenzodioxocins D (DBDOX, Fig. 1) in
which most 5–5-linked units find themselves [1,2], and the β–1-linked structures in the form of
spirodienones F [1,3]. Structural analysis became even more captivating after the biogenetic age
introduced the possibility of perturbing lignification in more exquisitely targeted ways. Transgenic plants
with, initially, single-gene manipulations revealed the incredible metabolic flexibility of lignification [4-
10]. We also came to realize that evolution had produced many such pathway manipulations. As studies
on lignin structure and on its engineering are inextricably and bidirectionally linked [9-11], the
perturbation of genes on the pathway became vigorously explored as a way to engender striking
compositional and structural changes, and led to the realization that it was possible to actually
contemplate ‘designing’ the polymer with a view towards its simpler removal from polysaccharides or for
enhanced value. This short review is challenged with doing justice to the extraordinary range and depth of

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Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

new insights into lignification and lignin structure.

Advances in understanding ‘conventional lignin’ structure (aided by transgenics)

Lignin is often touted as being structurally complex. Indeed, it is. Many phenolics are now recognized as
‘lignin monomers’ [4,5,7,9,12], and they radically couple and cross-couple combinatorially, resulting in a
racemic polymer with bewildering complexity [5,13] (see caption to Fig. 1). Lignification, primarily via
the ‘endwise’ coupling of a monomer (radical) with the growing polymer (radical), is a simple process.
Once the radicals are generated, polymerization is purely chemical, not controlled by enzymes or proteins
[6]. New chiral centers are created each time monolignols, the primary lignin 4-hydroxycinnamyl alcohol
monomers (p-coumaryl, coniferyl, and sinapyl alcohols), couple at their β-positions, but the products have
no discernable optical activity [5,6,14,15]; see in this issue a possible exception in the Casparian strip
[16]. Nor is chirality apparently induced by the optically active polysaccharides in the cell wall.
Models for various types of lignin can be constructed, but the process needs to be carried out
carefully following rigorous mechanistic principles. Fig. 1 shows our somewhat representative lignin
structures for three major plant classes.
Several other misconceptions exist regarding the lignin structure and the process of lignification.
• There is only a single phenolic radical formed from a monolignol or the growing polymer [22]. The
resonance forms drawn to rationalize the radical coupling regiochemistry are often mistaken for
chemically different structures – they are not.
• Radical coupling is not enzymatically controlled in cell wall lignification producing, as noted in the Fig.
1 caption, underappreciated stereochemical complexity [5,6].
• p-Hydroxyphenyl (H-unit, Fig. 1) levels have often been wildly exaggerated due to misinterpretation of
data obtained from analytical methods that produce H-unit signature compounds from other cell-wall
phenolics such as p-coumarate and p-hydroxybenzoate. Reported values as high as 30% are simply
incorrect (except in certain so-targeted transgenics deficient in C3H, HCT or CSE – enzyme names for
these abbreviations can be found elsewhere in this issue [23]). H-units are strictly defined as deriving
solely from p-coumaryl alcohol; the level is rarely above 5% as can be determined by NMR [24], or
more diagnostic degradative analytical methods (thioacidolysis, DFRC) in which the cleavage of β-
ether units is required to produce the diagnostic products [25,26].
• Non-cyclic benzyl ethers J (α-aryl ethers, Fig. 2A) remain prominent in literature lignin models and in
low-molecular-mass model synthetic lignins, but there is little evidence for them in plants [27].
• The widely stated concept of a “highly condensed 3D structure” is now questioned. “Y-type” branches
were always assumed when two lignin chains connect via 5–5 or 4–O–5 radical coupling, Fig. 2C,D.
However, such structures appear to be mostly free-phenolic, in which case lignins may be less branched
and more linear than commonly thought [28,29]. It was long ago realized that a β–1 unit F does not
represent a branchpoint, either, because one of its (latent) phenolic moieties always remains non-
etherified, Fig. 2B. Currently there is no structural evidence for branching.

Structural insights from engineering of conventional monolignol production

Misregulation of several genes throughout the pathway gives entirely predictable results and can be
exploited to markedly alter the H:G:S distribution.
• Up- and downregulation of ferulate 5-hydroxylase (F5H) provides the most straightforward way to alter
the S:G ratio in angiosperms (dicots and grasses). Knocking out F5H produces plants with solely G-
lignins [32]. More strikingly, upregulation of F5H, when driven by a strong promoter, produces lignins
with extraordinarily high syringyl content (up to ~98% S) [32-34]. Such high-S lignins are linear with
essentially only two types of units, the β–β-dimeric unit that starts the polymerization, and β–O–4-
ethers emanating from both phenolic ends of that dimeric unit, Fig. 3H. The β-ether content is
consequently high allowing for depolymerization reactions to produce high levels of monomers: 69%,
93%, and 78% from DFRC, nitrobenzene oxidation, and hydrogenolysis [33,34].
• H-enriched lignins can be produced when genes encoding the enzymes associated with the 3-
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

hydroxylation steps are targeted, including C3H, HCT [35-39], and the more recently discovered CSE
[40,41]. Arabidopsis c3h (or ref8) mutants are stunted, tempting an inference that H-lignins are unable
to satisfy the lignin requirements of the plant cell wall [38]. However, when certain mediator complex
genes were also downregulated, the plants recovered to near-WT stature while retaining their 100% H-
lignin attribute [38]. H-lignins are surprisingly β-ether-rich, but may have higher dibenzodioxocin D
and biaryl ether E contents.

Engineering monolignol pathway intermediates into lignin polymers

One of the earliest revelations from studying monolignol pathway mutants and transgenics was that
products of incomplete monolignol biosynthesis could be utilized by plants to produce more or less
functional polymers that allowed their survival [4,12].
• Hydroxycinnamaldehydes (HCAs), the immediate precursors of monolignols, are exported to the wall
and used for lignification in CAD-downregulated plants (but see a limitation for softwoods, noted in
Fig. 2E). Viable arabidopsis plants could be formed using ~100% HCA monomers (and therefore
essentially no monolignols) [51], and a Medicago truncatula mutant had a lignin that was derived from
some 95% HCAs [52]. Plants can therefore survive reasonably well when their lignins are highly or
completely derived from HCAs; such lignins are fascinating because of the rich chemistry available to
aldehyde groups.
• 5-Hydroxyconiferyl alcohol is incorporated integrally into COMT-deficient mutants and transgenics
[53-56]. A 70% level, with some 90% benzodioxane units, was attained in Arabidopsis [57,58], and a
seedcoat entirely from it was discovered in three Escobaria species [48]. The latter lignins are attractive
engineering targets as they are completely linear and essentially 100% mono-structural, Fig. 3I.
• Caffeyl alcohol has only been marginally successfully engineered to any significant level in
gymnosperms [59], but comprises 100% of the ‘C-lignin’ in various seedcoats [48,49]. Despite the
expectation that caffeyl alcohol, analogously to coniferyl alcohol, could couple with various
regiochemistries, it also produced essentially a linear homogeneous β–O–4-polymer of benzodioxane
units, Fig. 3I, in vitro as well as in vivo. As an all-ether-linked polymer, it can be quantitatively
converted to monomers hydrogenolytically [50], and in high proportion to a single monomer, a feat that
is all but inconceivable for conventional lignins. Renewed effort is therefore directed at attempting to
engineer large-scale biomass crops with C-lignins.

Esters and monolignol conjugates

Various esters and, importantly, monolignol ester conjugates are now well established as being involved
in lignification.
• Ferulates merit being regarded as lignin (monomeric) precursors because they couple and cross-couple
by the same radical coupling mechanisms in the same time and space, and become thereby inextricably
integrated into the lignin polymer [17], Fig. 3A.
• Monolignol p-coumarate conjugates were implicated mainly in monocot lignification. When substantial
enough, their participation makes lignins richer in free phenolic groups and more alkali-soluble [60,61].
In addition, model studies led to the elucidation of novel β–β-coupled components such as C´ (Fig. 1C)
in grass lignins; these mono-tetrahydrofurans were produced because protection of the γ-OH precluded
the possibility of rearomatizing by intramolecular cyclization that otherwise produced characteristic
resinols C [17]. The gene/enzyme responsible for acylating monolignols with p-coumarate has been
discovered [62,63].
• p-Hydroxybenzoylated lignins (Fig. 1B) and acetylated lignins (Fig. 1C) similarly derive from
monolignol p-hydroxybenzoate and acetate conjugates [1,17,18,26,64]. Other monolignol conjugates
have been recently discovered, including benzoates, vanillates, and ferulates, even in the same plant line
[65]. Not all of the genes/enzymes have yet been unambiguously identified.
• Monolignol ferulates were discovered to be naturally incorporated into lignins in a range of plants [66]
subsequent to their (assumed) de novo strategic engineering into transgenics [67]. This special case is
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

covered below.

Monolignol ferulates in lignification – ‘Zip-lignins’

Developing a way to engineer readily cleavable bonds into the very backbone of the lignin polymer would
enable its more ready depolymerization and enhance the ease of processing, from pulping to biomass
pretreatment and saccharification [17,68]. A particularly promising idea was to attempt to utilize
monolignol ferulates as monomer-augmenting precursors for lignification. The rationale was three-tiered:
1) Ferulates were known to participate integrally in lignification; 2) Analogous monolignol p-coumarate
and p-hydroxybenzoate conjugates were becoming established as lignification ‘monomers’ and; 3) It was
obvious that monolignols could be (partially) replaced by various other compatible phenolics to produce
polymers that were not fully derived from the canonical monolignols [6]. A required feruloyl transferase
gene was transformed into arabidopsis and poplar [67], which did indeed result in the introduction of ester
linkages (‘zips’) into the lignin backbone (Fig. 4). Encouraged by the improved performance after a
variety of pretreatments [69-71], current research is aimed at elevating the Zip-lignin levels and
developing superior lines in commercially relevant tree species.
Since engineering plants to utilize monolignol ferulates for lignification to produce Zip-lignins, it was
discovered that such capabilities had already evolved naturally, perhaps several times independently [66].
Grasses utilize mainly sinapyl ferulate, and many hardwoods (but no softwoods) utilize mainly coniferyl
ferulate in their lignification. The advantage or pressure that gave rise to the evolution of the pathway
remains unclear, but many related genes and enzymes are now in contention for use in Zip-lignin plants.
It seems evident that we have not yet hit the limiting level to which plants can tolerate various conjugates
without becoming agronomically compromised (see Fig. 4).

Monomers from beyond the monolignol biosynthetic pathway

Even more striking was the recent discovery that plants could conscript phenolics from other biochemical
pathways to utilize for lignification. Examples afford not only fresh insight into additional modifications
that may be contemplated for lignin, but also portend significantly enhanced value to the polymer.
• Tricin (Fig. 3G), a valuable flavonoid, derived from a combination of the shikimate- and
acetate/malonate-derived polyketide pathways, was the first phenolic from distinctly outside the
canonical pathway. Found in ‘all’ grasses [21,46], it can function only as a polymerization starting
point; it is therefore located only at the beginning end of the polymer, resulting from its initial 4–O-
coupling with a monolignol at its β-position.
• Hydroxystilbenes, including piceatannol (Fig. 3F), isorhapontigenin, and resveratrol (Fig. 3E), the
assumed health-active component in red wines, were next discovered in the lignins of various palm fruit
endocarps [45].
• Diferuloylputrescine has recently been implicated as a lignin precursor in corn pericarp tissues [44],
joining tyramine ferulate [12] as a nitrogenous monomer, Fig. 3B.

Conclusions. What constitutes an ideal lignin monomer, and what are the prospects for exploiting
lignin structure?
A 2006 book chapter pondered the question: “What makes a good monolignol substitute?” [72]. The most
important requirement appears to be a feature of the evolved monolignols, the ability to undergo β–O–4-
coupling, as it allows the monomer to couple into the growing polymer; monomers without the
conjugated sidechain position are relegated to solely initiating chains or to 5–5- or 5–O–4-based
branching but, as noted above, these may not produce real branching and therefore also represent end-
units. The following monomers all satisfy the β–O–4-coupling requirement: monolignols, monolignol
conjugates, incompletely methylated monolignols (caffeyl, 5-hydroxyconiferyl, and, in principle, gallyl
alcohols), and hydroxycinnamaldehydes and hydroxycinnamate esters. A weird and wonderful assortment
of possibilities for monomers was described in a review [9], and some of these are being more
systematically tested.
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

Additionally, some monomers have significant value. Monolignol p-coumarate conjugates increase
the frequency of free phenolic groups in lignins, which makes them more susceptible to alkaline or
oxidative treatments. Others such as in the Zip-lignin strategy allow the polymer to be more readily
cleaved (depolymerized), an obvious benefit to processes designed to remove the lignin from the
polysaccharide components. We are also now able to contemplate the biosynthesis and incorporation of
components with substantially higher value to extract or derive from the polymer. In principle, such
components might be available in enormous quantities, making them valuable as commodity chemical
feedstocks for an industry that will someday have no choice but to source itself from sustainable and
renewable carbon-neutral biomass. Marveled for their biochemical flexibility that can be capitalized upon
by biotechnological methods, lignins are transforming from being regarded as problematic recalcitrant
polymers to resources with boundless potential.

Acknowledgements
A great many other researchers have cooperated on the topics covered in this review; some have their
own articles in this special issue. Current funding to JR was largely from the DOE Great Lakes Bioenergy
Research Center (DOE Office of Science BER DE-FC02-07ER64494 and DE-SC0018409); Funding to
CL was largely from INRA support and also from LabEx Saclay Plant Sciences-SPS (grant no. ANR–
10–LABX–0040–SPS to the Institut Jean-Pierre Bourgin); WB was funded by the IWT-SBO project
BIOLEUM (grant no 130039) and the IWT-FISH-SBO project ARBOREF (grant no. 140894), and by
FWO projects GOC1914N and G020618N; WB and JR were also funded by Stanford University’s Global
Climate and Energy Program (GCEP), project ‘Engineering novel lignin types.’

FIGURE CAPTIONS

Fig. 1. Lignin model structures. Model lignin 20-mers are shown for: A) A gymnosperm/softwood, B)
An angiosperm/dicot/hardwood, and C) A (commelinid) monocot. Note that, unlike many models in the
literature, structural details have been drawn based on the best current information and with rigorously
followed concepts. That said, although some attempt is made to represent the various units at roughly the
levels that they are found analytically, there are limitations in trying to fit such data into a 20-mer; for
example, the β–1-unit level is only 1-2%, but one such spirodienone unit F is drawn in (only) the
softwood structure in A). The convention used here, although impossible to make totally rigorous, is that
a structure is colored by the type of interunit linkage in which it is involved; where one unit is involved in
two types of units, these are shown as split structures; complete unambiguity remains difficult as, for
example, a spirodienone F (as in A) derives from the coupling of a monolignol to an existing β-ether
phenolic end-unit A – we nevertheless colored it differently to reflect its presence in the spirodienone F.
Bonds formed in the important radical coupling steps are shown in red; oxygen or hydroxy groups that
derive from water during quinone methide rearomatization, and the bonds to them, are in gray. Note also
that, due to the under-appreciated racemic nature of lignins, each of these structures represents many
billions (we calculate 1.07B, 17.2B, and 8.6B for the three structures drawn) of physically distinct
possible isomers [5,6]. Attributes of each of the models include the following…
A) Gymnosperm/Softwood lignin: 20 Monomeric units, all G-units derived from coniferyl alcohol; 2
cinnamyl alcohol endgroup units X from the chain-starting dimerization reactions, both phenylcoumarans
B (β–5, 10%, approximately the level in G-lignins); 1 Resinol C (5%, about the right level) that is also a
monolignol dimerization chain starting point – the 20-mer lignin chain has three starting-points because
of the oligomer-oligomer coupling in the D and E structures; 1 Spirodienone F (β–1, 5%, too high for the
1-2% in lignin, but we wanted to show this unit in one structure) highlighting why one (latent) phenolic
group is never etherified in lignin – it is not in fact a phenol but a conjugated ketone (see Fig. 2B); 1
Biphenyl ether E (4–O–5, 5%, a bit high for the ~1% in the lignins but, again, we wanted to represent this
cross-coupling and illustrate that it may not form a Y-type branchpoint (see Fig. 2D) because it remains,
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

as highlighted, free-phenolic; 1 Dibenzodioxocin D (5–5/4–O–β, 5%, estimated at some 9-12% in G-

lignins, so perhaps low); 12 β-Ether units A (β–O–4, ~60%, depending on how they are counted (when
there is also one in the D-unit), about the right level).
B) Angiosperm/Dicot/Hardwood lignin: 20 Monomeric units, 13 S and 7 G (S:G 65:35); 1 Cinnamyl
alcohol endgroup unit X from a chain-starting dimerization reaction; 1 Phenylcoumaran B (β–5, 5%,
approximately the level in S-G lignins); 1 Resinol C (5%, about the right level) that is also a monolignol
dimerization chain starting point – the 20-mer lignin chain has two starting-points because of the
oligomer-oligomer coupling in the E structure; 0 Spirodienones F (β–1, 0%, even though the levels are
slightly higher in S-G lignins, 1-2%); 1 Biphenyl ether E (4–O–5, 5%, a bit high for the ~1-2% in the
lignins but, again, we wanted to represent this cross-coupling and illustrate that it may not form a Y-type
branchpoint because it remains, as highlighted, free-phenolic, see Fig. 2D); 0 Dibenzodioxocins D (5–
5/4–O–β, 0%, even though the G-units in lignin allow a modest level of such structures, perhaps ~3%); 16
β-Ether units A (β–O–4, ~80%, depending on how they are counted, about the right level for a 65%-S
lignin). Note that 2 p-hydroxybenzoates pBA (10% on an S+G basis) are also shown; these derive from
monolignol (mainly, as both are here, sinapyl) p-hydroxybenzoate conjugates, but only on
willow/aspen/poplar/palms and not on other dicots [17,18]; their phenolic groups remain largely free-
phenolic (un-etherified) because their radicals preferentially undergo radical transfer reactions rather than
radical coupling [19].
C) (Commelinid) Monocot lignin: 20 Monomeric units, 13 S and 6 G (S:G 68:32) with one ferulate FA
or tricin T unit; 0 Cinnamyl alcohol endgroup units X from a chain-starting dimerization reaction, even
though there likely should be one – we chose to illustrate the chain initiation via either ferulate FA or
tricin T, features specific to commelinid monocots, in either case first coupled with coniferyl alcohol to
give a G unit (which is usually the case [17,20,21]); 2 Phenylcoumarans B (β–5, 10%, a bit high for a
lignin with this S-level); 1 Tetrahydrofuran C´ (5%, about the right level) that is also a monolignol
dimerization chain starting point but, unlike the resinol C in the dicot, this derives (as it often does) from
dimerization of two sinapyl p-coumarate conjugates – the rearomatization pathway differs when the γ-OH
is acylated; 0 Spirodienones F (β–1, 0%, even though the levels are slightly higher in S-G lignins, 1-2%);
1 Biphenyl ether E (4–O–5, 5%, a bit high for the ~1-2% in the lignins but, again, we wanted to represent
this cross-coupling and illustrate that it may not form a Y-type branchpoint because it remains, as
highlighted, free-phenolic, see Fig. 2D); 0 Dibenzodioxocins D (5–5/4–O–β, 0%, even though the G-units
in lignin allow a modest level of such structures, perhaps ~3%); 15 β-Ether units A (β–O–4, ~75%,
depending on how they are counted, about the right level for a 68%-S lignin). Note that 4 p-coumarates
pCA (20% of an S+G unit basis) are also shown; these derive from monolignol (mainly, as all are here,
sinapyl) p-coumarate conjugates; their phenolic groups remain largely free-phenolic (un-etherified)
because their radicals preferentially undergo radical transfer reactions rather than radical coupling [19].
Two γ-acetates are shown; acetates, mainly on S-units, are quite common on monocot as well as some
dicot lignins (not shown in B) and likely not on gymnosperm lignin.

Fig. 2. Structural elements mistakenly thought to be in lignins, and their corrections. Many
structures are often shown in the literature as being in lignins, but are either simply not present or are
incorrect. A) Non-cyclic α-aryl ethers J are prominent in some synthetic lignins but are simply
undetectable in most plant lignins [27]; their implied existence often results from misrepresentation of
cyclic ethers B and D, especially before the latter were discovered. B) The ‘traditional’ β–1 units in lignin
are often shown in the open form F', and often with etherification of the B-ring F'e; they have now been
shown to exist in lignins as spirodienones F from intramolecular trapping of the quinone methide
intermediate, explaining why B-ring etherification is impossible [3]; treatment in acid does release the
open forms F'f, with the free-phenolic B-ring only, and also explains the origin of the glyceraldehyde-2-
aryl ether structures F'' noted in acid-treated lignins. C) Dibenzodioxocins D are often drawn as effecting
‘Y-type’ branching (highlighted insets) in lignins but they appear to not etherify; to date, evidence for
only the free phenolic versions Df can be found, not the etherified versions De, which therefore represent
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

little more than ‘U-type’ connections, and not really branchpoints. Note that DBDOX structures D are
shown in two representations, neither particularly pleasing as the cyclooctane ring and the 90° disposition
of the two 5–5-connected aromatic rings makes it difficult to represent the structure in two dimensions
while keeping bond angles and bond lengths normal. D) Similarly, etherified 4–O–5 structures Ee cannot
be validated; only free-phenolic versions Ef have been evidenced [28,29]. E) There are some structures
that, despite being reasonable on paper, simply do not form. For example, coniferaldehyde will not 8–O–
4-cross-couple with G units, in vivo or in vitro, so such structures KG΄-G cannot be found [4,30,31];
coniferaldehyde can so-couple with syringyl units, though, forming KG΄-S, and sinapaldehyde can cross-
couple with G or S units, so hydroxycinnamaldehydes are well-incorporated into S-G lignins; there is
some evidence for coniferaldehyde’s cross-coupling with more S-like G structures, such as those with 5-
substitution. F) The literature also abounds with structures such as N1-N4 that are mechanistically
impossible in native lignin because such coupling cannot occur; structures N5 and N6 also do not occur in
lignins because coniferyl alcohol simply doesn’t couple this way; 5–5 D and 4–O–5 E structures derive
only from the coupling of oligomer chains, not from monomers [5,6,12].

Fig. 3. Coupling and cross-coupling of novel monomers into lignins. Various other phenolics act as
monomers during lignification. A) Ferulates, acylating arabinoxylans in monocots or acylating
monolignols at low levels, couple and cross-couple integrally into lignins [17,42]. Ferulate dimerization is
the basis of polysaccharide cross-linking in monocots [17]. Ferulate-monolignol coupling products have
been discovered [17,20], and their radical cross-coupling into lignins results in lignin-polysaccharide
cross-linking [19,43]. Diferulates (and higher oligomers) can also enter lignification (not shown).
Monolignol ferulates, in which the ferulate moieties can couple as shown, produce Zip-lignins (Fig. 4A).
B) Feruloyl amides such as tyramine ferulate in Solanaceae [12], and the recently discovered
diferuloylputrescine in maize kernels [44], are nitrogenous phenolics that also act as lignin monomers. C)
Coniferaldehyde and D) Sinapaldehyde cross-couple into lignins in ‘normal’ WT plants and, more
prevalently, in CAD-deficient mutants and transgenics. E) Resveratrol and F) Piceatannol are
hydroxystilbenes recently discovered as phenolics from outside the monolignol biosynthetic pathway that
can couple integrally into the polymer [45]. G) Tricin, a flavone, was the first non-pathway phenolic to be
discovered in lignins [21,46]. H) Lignins derived essentially entirely from the monolignol sinapyl alcohol
produce a linear polymer containing basically only two types of units, resinols and β-ethers. I) 5-
Hydroxyconiferyl alcohol and, surprisingly, caffeyl alcohol, couple essentially only by β–O–4-coupling
to produce linear homopolymers of benzodioxane units [47-49] that are more resistant to pulping, for
example, but can be hydrogenolytically degraded to monomers in high yield [50]. Note: in all structures,
the bonds potentially formed by radical (cross-)coupling are shown dotted, in black; those that can only
arise from post-coupling reactions are in gray (as in Fig. 1).

Fig. 4. Models of novel lignin from mutants/transgenics. A) A hypothetical Zip-lignin derived from
lignification of a gymnosperm (monomer: coniferyl alcohol) with the coniferyl ferulate conjugate. The
ferulate moiety couples integrally into the polymer, shown here via 8–O–4-, 5–β-, 4–O–β-, and 5–5-cross-
coupling, along with 8–8-homocoupling. In general, coupling of n monolignol ferulates into a polymer
can result in up to n+1 fragments after cleavage of the ester in mild base [66]; in this case, even with the
extensive coupling shown for the two ferulates near the center, with n=4 here, n+1=5 fragments are still
released. The coniferyl alcohol and the ferulate moieties of the units derived from the coniferyl ferulate
conjugates are each colored separately, but not the other units; however, the bonds formed by radical
coupling are colored as for the respective units in Fig. 1. CA, coniferyl alcohol; FA, ferulate. B) A model
of a CAD-deficient plant’s lignin in which most (all) of the incorporated hydroxycinnamaldehyde
monomers are sinapaldehyde, as in a recently analyzed poplar transgenic [31]. The sinapaldehyde units
are shown as being involved in various (cross-)couplings (8–5, 8–O–4, 4–O–β) as well as in 8–8-
homocoupled units. Each sinapaldehyde-derived S´ unit is colored red, and the bonds formed by radical
coupling are colored as in Fig. 1.
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

References
1. Ralph J, Landucci LL: NMR of lignins. In Lignin and Lignans; Advances in Chemistry. Edited by
Heitner C, Dimmel DR, Schmidt JA: CRC Press (Taylor & Francis Group); 2010:137-234.
2. Karhunen P, Rummakko P, Sipilä J, Brunow G, Kilpeläinen I: Dibenzodioxocins; a novel type of
linkage in softwood lignins. Tetrahedron Letters 1995, 36:169-170.
3. Zhang L, Gellerstedt G, Ralph J, Lu F: NMR studies on the occurrence of spirodienone
structures in lignins. Journal of Wood Chemistry and Technology 2006, 26:65-79.
4.• Sederoff RR, MacKay JJ, Ralph J, Hatfield RD: Unexpected variation in lignin. Current Opinion
in Plant Biology 1999, 2:145-152.
The first paper recognizing the flexibility of lignification, a revelation arising from early studies on lignin-
biosynthetic-pathway mutants and transgenics.
5. Ralph J, Lundquist K, Brunow G, Lu F, Kim H, Schatz PF, Marita JM, Hatfield RD, Ralph SA,
Christensen JH, et al.: Lignins: natural polymers from oxidative coupling of 4-
hydroxyphenylpropanoids. Phytochemistry Reviews 2004, 3:29-60.
6.• Ralph J, Brunow G, Harris PJ, Dixon RA, Schatz PF, Boerjan W: Lignification: Are lignins
biosynthesized via simple combinatorial chemistry or via proteinaceous control and template
replication? In Recent Advances in Polyphenol Research. Edited by Daayf F, El Hadrami A, Adam
L, Ballance GM: Wiley-Blackwell Publishing; 2008:36-66. vol 1.]
A rationale for developmental lignin’s being biosynthesized via simple radical coupling rather than
from under proteinaceous control.
7. Vanholme R, Morreel K, Ralph J, Boerjan W: Lignin engineering. Current Opinion in Plant
Biology 2008, 11:278-285.
8. Grabber JH, Schatz PF, Kim H, Lu F, Ralph J: Identifying new lignin bioengineering targets: 1.
Monolignol substitute impacts on lignin formation and cell wall fermentability. BMC Plant
Biology 2010, 10:1-13.
9. Vanholme R, Morreel K, Darrah C, Oyarce P, Grabber JH, Ralph J, Boerjan W: Metabolic
engineering of novel lignin in biomass crops. New Phytologist 2012, 196:978-1000.
10.• Mottiar Y, Vanholme R, Boerjan W, Ralph J, Mansfield SD: Designer lignins: Harnessing the
plasticity of lignification. Current Opinion in Biotechnology 2016, 37:190-200.
Advancing the idea of actually rationally designing lignins for improved processing or value
11. Rinaldi R, Jastrzebshi R, Clough MT, Ralph J, Kennema M, Bruijnincx PCA, Weckhuysen BM:
Paving the way for lignin valorisation: Recent advances in bioengineering, biorefining and
catalysis. Angewandte Chemie (International Edition) 2016, 55:8164-8215.
12. Boerjan W, Ralph J, Baucher M: Lignin biosynthesis. Annual Reviews in Plant Biology 2003,
54:519-546.
13. Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W: Lignin biosynthesis and structure.
Plant Physiology 2010, 153:895-905.
14. Ralph J, Peng J, Lu F, Hatfield RD, Helm RF: Are lignins optically active? Journal of
Agricultural and Food Chemistry 1999, 47:2991-2996.
15. Akiyama T, Magara K, Meshitsuka G, Lundquist K, Matsumoto Y: Absolute configuration of β-
and α-asymmetric carbons within β-O-4-structures in hardwood lignin. Journal of Wood
Chemistry and Technology 2015, 35:8-16.
16. Ramos Barbosa IC, Rojas-Murcia N, Geldner N: The Casparian strip – One ring to bring cell
biology to lignification? Current Opinion in Biotechnology, Special issue on Lignin Engineering
2019: in press (COBIOT_2018_2043).
17. Ralph J: Hydroxycinnamates in lignification. Phytochemistry Reviews 2010, 9:65-83.
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

18. Lu F, Karlen SD, Regner M, Kim H, Ralph SA, Sun R-c, Kuroda K-i, Augustin MA, Mawson R,
Sabarez H, et al.: Naturally p-hydroxybenzoylated lignins in palms. BioEnergy Research 2015,
8:934-952.
19. Ralph J, Bunzel M, Marita JM, Hatfield RD, Lu F, Kim H, Schatz PF, Grabber JH, Steinhart H:
Peroxidase-dependent cross-linking reactions of p-hydroxycinnamates in plant cell walls.
Phytochemistry Reviews 2004, 3:79-96.
20. Jacquet G, Pollet B, Lapierre C, Mhamdi F, Rolando C: New ether-linked ferulic acid-coniferyl
alcohol dimers identified in grass straws. Journal of Agricultural and Food Chemistry 1995,
43:2746-2751.
21. del Río JC, Rencoret J, Prinsen P, Martínez ÁT, Ralph J, Gutiérrez A: Structural characterization
of wheat straw lignin as revealed by analytical pyrolysis, 2D-NMR, and reductive cleavage
methods. Journal of Agricultural and Food Chemistry 2012, 60:5922-5935.
22. Ralph J, Schatz PF, Lu F, Kim H, Akiyama T, Nelsen SF: Quinone methides in lignification. In
Quinone Methides. Edited by Rokita S: Wiley-Blackwell; 2009:385-420. Reactive Intermediates in
Chemistry and Biology, vol 1.]
23. Vanholme R, De Meester B, Ralph J, Boerjan W: Lignin biosynthesis and its integration into
metabolism. Current Opinion in Biotechnology, Special issue on Lignin Engineering 2019: in
press.
24. Kim H, Padmakshan D, Li Y, Rencoret J, Hatfield RD, Ralph J: Characterization and elimination
of undesirable protein residues in plant cell wall materials for enhancing lignin analysis by
solution-state NMR. Biomacromolecules 2017, 18:4184–4195.
25. Lapierre C: Application of new methods for the investigation of lignin structure. In Forage Cell
Wall Structure and Digestibility. Edited by Jung HG, Buxton DR, Hatfield RD, Ralph J: American
Society of Agronomy, Crop Science Society of America, Soil Science Society of America;
1993:133-166.
26. Lu F, Ralph J: The DFRC (Derivatization Followed by Reductive Cleavage) method and its
applications for lignin characterization. In Lignin: Structural Analysis, Applications in
Biomaterials, and Ecological Significance. Edited by Lu F: Nova Science Publishers, Inc; 2014:27-
65.
27. Kilpeläinen I, Sipilä J, Brunow G, Lundquist K, Ede RM: Application of two-dimensional NMR
spectroscopy to wood lignin determination; Identification of some minor structural units of
hard- and softwood lignins. Journal of Agricultural and Food Chemistry 1994, 42:2790-2794.
28. Yue F, Lu F, Ralph S, Ralph J: Identification of 4–O–5-units in softwood lignins via definitive
lignin models and NMR. Biomacromolecules 2016, 17:1909-1920.
29. Li Y, Akiyama T, Yokoyama T, Matsumoto Y: NMR assignment for diaryl ether structures
(4−O−5 structures) in pine wood lignin. Biomacromolecules 2016, 17:1921-1929.
30. Kim H, Ralph J, Lu F, Ralph SA, Boudet A-M, MacKay JJ, Sederoff RR, Ito T, Kawai S, Ohashi
H, et al.: NMR Analysis of Lignins in CAD-deficient Plants. Part 1. Incorporation of
hydroxycinnamaldehydes and hydroxybenzaldehydes into lignins. Organic and Biomolecular
Chemistry 2003, 1:268-281.
31. Van Acker R, Déjardin A, Desmet S, Hoengenaert L, Vanholme R, Morreel K, Laurans F, Kim H,
Santoro N, Foster C, et al.: Different routes for conifer- and sinapaldehyde and higher
saccharification upon deficiency in the DEHYDROGENASE CAD1. Plant Physiology 2017,
175:1018-1039.
32.•• Meyer K, Shirley AM, Cusumano JC, Bell-Lelong DA, Chapple C: Lignin monomer composition
is determined by the expression of a cytochrome P450-dependent monooxygenase in
Arabidopsis. Proceedings of the National Academy of Sciences, USA 1998, 95:6619-6623.
One of the first papers to show that massive changes in the composition of lignins’ normal H, G,
and S units could be elicited in viable plants.
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

33. Stewart JJ, Akiyama T, Chapple CC, Ralph J, Mansfield SD: The effects on lignin structure of
overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Physiology 2009, 150:621-635.
34. Shuai L, Amiri MT, Questell-Santiago YM, Héroguel F, Li Y, Kim H, Meilan R, Chapple C, Ralph
J, Luterbacher JS: Formaldehyde stabilization facilitates lignin monomer production during
biomass depolymerization. Science 2016, 354:329-333.
35. Franke R, Hemm MR, Denault JW, Ruegger MO, Humphreys JM, Chapple C: Changes in
secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis.
Plant Journal 2002, 30:47-59.
36. Wagner A, Ralph J, Akiyama T, Flint H, Phillips L, Torr KM, Nanayakkara B, Te Kiri L:
Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate
hydroxycinnamoyltransferase in Pinus radiata. Proceedings of the National Academy of
Sciences, USA 2007, 104:11856-11861.
37. Coleman HD, Park J-Y, Nair R, Chapple C, Mansfield SD: RNAi-mediated suppression of p-
coumaroyl-CoA 3´-hydroxylase in hybrid poplar impacts lignin deposition and soluble
secondary metabolism. Proceedings of the National Academy of Sciences 2008, 105:4501-4506.
38.•• Bonawitz ND, Kim JI, Tobimatsu Y, Ciesielski PN, Anderson NA, Ximenes E, Maeda J, Ralph J,
Donohoe BS, Ladisch M, et al.: Disruption of Mediator rescues the stunted growth of a lignin-
deficient Arabidopsis mutant. Nature 2014, 509:376-380.
Describes the striking finding that the biomass yield penalty upon downregulation of C3H in
Arabidopsis, and originally assumed to be because of the inadequacy of the H-only lignin structure,
could be largely mitigated by downregulating two other genes.
39. Ralph J, Akiyama T, Coleman HD, Mansfield SD: Effects on lignin structure of coumarate 3-
hydroxylase downregulation in Poplar. BioEnergy Research 2012, 5:1009-1019.
40.• Vanholme R, Cesarino I, Rataj K, Xiao Y, Sundin L, Goeminne G, Kim H, Cross J, Morreel K,
Araujo P, et al.: Caffeoyl shikimate esterase (CSE), a newly discovered gene in the lignin
biosynthetic pathway. Science 2013, 341:1103-1106.
A new gene discovered in the lignin biosynthetic pathway, having significant implications for plant
engineering.
41. Ong RG, Higbee A, Bottoms S, Dickinson Q, Xie D, Smith SA, Serate J, Pohlmann E, Jones AD,
Coon JJ, et al.: Inhibition of microbial biofuel production in drought-stressed switchgrass
hydrolysate. Biotechnology for Biofuels 2016, 9.
42. Ralph J, Grabber JH, Hatfield RD: Lignin-ferulate crosslinks in grasses: active incorporation of
ferulate polysaccharide esters into ryegrass lignins. Carbohydrate Research 1995, 275:167-178.
43. Ralph J, Quideau S, Grabber JH, Hatfield RD: Identification and synthesis of new ferulic acid
dehydrodimers present in grass cell walls. Journal of the Chemical Society, Perkin Transactions
1 1994:3485-3498.
44. del Río JC, Rencoret J, Gutierrez A, Kim H, Ralph J: Structural characterization of lignin from
maize (Zea mays L.) fibers: Evidence for diferuloylputrescine incorporated into the lignin
polymer in maize kernels. Journal of Agricultural & Food Chemistry 2018, 66:4402-4413.
45. del Río JC, Rencoret J, Gutiérrez A, Kim H, Ralph J: Hydroxystilbenes are monomers in palm
fruit endocarp lignins. Plant Physiology 2017, 174:2072-2082.
46.• Lan W, Rencoret J, Lu F, Karlen SD, Smith BG, Harris PJ, del Rio JC, Ralph J: Tricin-lignins:
Occurrence and quantitation of tricin in relation to phylogeny. The Plant Journal 2016,
88:1046-1057.
After the initial discovery of the first lignin monomer, tricin (a flavone), from outside the lignin
biosynthetic pathway (Reference 21), the paper provided a method for its quantification and
examined phylogenic aspects.
47. Tobimatsu Y, Chen F, Nakashima J, Jackson L, Escamilla-Treviño LL, Dixon RA, Ralph J:
Coexistence but independent biosynthesis of catechyl and guaiacyl/syringyl lignins in plant
seeds. The Plant Cell 2013, 25:2587-2600.
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

48. Chen F, Tobimatsu Y, Jackson L, Nakashima J, Ralph J, Dixon RA: Novel seed coat lignins in the
Cactaceae: structure, distribution and implications for the evolution of lignin diversity. The
Plant Journal 2013, 73:201-211.
49.• Chen F, Tobimatsu Y, Havkin-Frenkel D, Dixon RA, Ralph J: A polymer of caffeyl alcohol in
plant seeds. Proceedings of the National Academy of Sciences of the United States of America
2012, 109:1772-1777.
The discovery of C-lignin, a new class of homogeneous linear lignin polymers that can be useful in
its own right and converted to phenolic monomers in high yield (see ref. 50).
50. Li Y, Shuai L, Kim H, Motagamwala AH, Mobley JK, Yue F, Tobimatsu Y, Havkin-Frenkel D,
Chen F, Dixon RA, et al.: An “ideal lignin” facilitates full biomass utilization. Science Advances
2018, 4:eaau2968, 2961-2910.
51. Anderson NA, Tobimatsu Y, Ciesielski PN, Ximenes E, Ralph J, Donohoe BS, Ladisch M, Chapple
C: Manipulation of guaiacyl and syringyl monomer synthesis in an Arabidopsis Cinnamyl
Alcohol Dehydrogenase mutant results in atypical lignin biosynthesis and modified cell wall
structure. Plant Cell 2015, 27:2195-2209.
52. Zhao Q, Tobimatsu Y, Zhou R, Pattathil S, Gallego-Giraldo L, Fu C, Jackson LA, Hahn MG, Kim
H, Chen F, et al.: Loss of function of Cinnamyl Alcohol Dehydrogenase 1 causes accumulation
of an unconventional lignin and a temperature-sensitive growth defect in Medicago
truncatula. Proceedings of the National Academy of Sciences of the United States of America 2013,
110:13660-13665.
53.• Lapierre C, Tollier MT, Monties B: A new type of constitutive unit in lignins from the corn bm3
mutant. Comptes rendus de l’Académie des sciences. Série III 1988, 307:723-728.
The first discovery of a product of incomplete monolignol biosynthesis, 5-hydroxyconiferyl
alcohol, incorporated into lignin.
54. Ralph J, Lapierre C, Marita J, Kim H, Lu F, Hatfield RD, Ralph SA, Chapple C, Franke R, Hemm
MR, et al.: Elucidation of new structures in lignins of CAD- and COMT-deficient plants by
NMR. Phytochemistry 2001, 57:993-1003.
55. Ralph J, Lapierre C, Lu F, Marita JM, Pilate G, Van Doorsselaere J, Boerjan W, Jouanin L: NMR
evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl
alcohol into lignins of O-methyl-transferase-deficient poplars. Journal of Agricultural and Food
Chemistry 2001, 49:86-91.
56. Marita JM, Ralph J, Lapierre C, Jouanin L, Boerjan W: NMR characterization of lignins from
transgenic poplars with suppressed caffeic acid O-methyltransferase activity. Journal of the
Chemical Society, Perkin Transactions 1 2001:2939-2945.
57. Vanholme R, Ralph J, Akiyama T, Lu F, Rencoret Pazo J, Christensen J, Rohde A, Morreel K,
DeRycke R, Kim H, et al.: Engineering traditional monolignols out of lignins by concomitant
up-regulation F5H1 and down-regulation of COMT in Arabidopsis. The Plant Journal 2010,
64:885-897.
58. Weng J-K, Mo H, Chapple C: Over-expression of F5H in COMT-deficient Arabidopsis leads to
enrichment of an unusual lignin and disruption of pollen wall formation. The Plant Journal
2010, 64:898-911.
59. Wagner A, Tobimatsu Y, Phillips L, Flint H, Torr K, Donaldson L, Piers L, Ralph J: CCoAOMT
suppression modifies lignin composition in Pinus radiata. The Plant Journal 2011, 67:119-129.
60. Smith RA, Gonzales-Vigil E, Karlen SD, Park J-Y, Lu F, Wilkerson CG, Samuels L, Mansfield SD,
Ralph J: Engineering monolignol p-coumarate conjugates into Poplar and Arabidopsis lignins.
Plant Physiology 2015, 169:2992-3001.
61. Sibout R, Le Bris P, Legee F, Cezard L, Renault H, Lapierre C: Structural redesigning
Arabidopsis lignins into alkali-soluble lignins through the expression of p-coumaroyl-
CoA:monolignol transferase PMT. Plant Physiology 2016, 170:1358-1366.
Lignin Engineering – Special issue for Current Opinion in Biotechnology vs 2018-11-29

62. Petrik DL, Karlen SD, Cass CL, Padmakshan D, Lu F, Liu S, Le Bris P, Antelme S, Santoro N,
Wilkerson CG, et al.: p-Coumaroyl-CoA:Monolignol Transferase (PMT) acts specifically in the
lignin biosynthetic pathway in Brachypodium distachyon. The Plant Journal 2014, 77:713-726.
63. Marita JM, Hatfield RD, Rancour DM, Frost KE: Identification and suppression of the p-
coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L. Plant Journal 2014,
78:850-864.
64. Lu F, Ralph J: Novel tetrahydrofuran structures derived from β–β-coupling reactions
involving sinapyl acetate in Kenaf lignins. Organic & Biomolecular Chemistry 2008, 6:3681-
3694.
65. Karlen SD, Smith RA, Kim H, Padmakshan D, Bartuce A, Mobley JK, Free HCA, Smith BG,
Harris PJ, Ralph J: Highly decorated lignins occur in leaf base cell walls of the Canary Island
date palm Phoenix canariensis. Plant Physiology 2017, 175:1058-1067.
66. Karlen SD, Zhang C, Peck ML, Smith RA, Padmakshan D, Helmich KE, Free HCA, Lee S, Smith
BG, Lu F, et al.: Monolignol ferulate conjugates are naturally incorporated into plant lignins.
Science Advances 2016, 2:e1600393: 1600391-1600399.
67.•• Wilkerson CG, Mansfield SD, Lu F, Withers S, Park J-Y, Karlen SD, Gonzales-Vigil E,
Padmakshan D, Unda F, Rencoret J, et al.: Monolignol ferulate transferase introduces
chemically labile linkages into the lignin backbone. Science 2014, 344:90-93.
Perhaps the first paper attempting to introduce a new gene activity to modify lignins; the so-called
Zip-genes produced monolignol ferulate conjugates that, when incorporated into lignin, resulted in
the introduction of readily chemically cleavable ester bonds into the backbone of the polymer. It
was later discovered that Nature is already making such lignins at low levels – see reference 66.
68. Grabber JH, Hatfield RD, Lu F, Ralph J: Coniferyl ferulate incorporation into lignin enhances
the alkaline delignification and enzymatic degradation of maize cell walls. Biomacromolecules
2008, 9:2510-2516.
69. Bhalla A, Bansal N, Pattathil S, Li M, Shen W, Particka CA, Semaan R, Gonzales-Vigil E, Karlen
SD, Ralph J, et al.: Engineered lignin in poplar biomass facilitates Cu-AHP pretreatment. ACS
Sustainable Chemistry & Engineering 2018, 6:2932-2941.
70. Zhou S, Runge T, Karlen SD, Ralph J, Gonzales-Vigil E, Mansfield SD: Chemical pulping
advantages of Zip-lignin hybrid poplar. ChemSusChem 2017, 10:3565-3573.
71. Kim KH, Dutta T, Ralph J, Mansfield SD, Simmons BA, Singh S: Impact of lignin polymer
backbone esters on ionic liquid pretreatment of poplar. Biotechnology for Biofuels 2017,
10:101: 101-110.
72. Ralph J: What makes a good monolignol substitute? In The Science and Lore of the Plant Cell
Wall Biosynthesis, Structure and Function. Edited by Hayashi T: Universal Publishers
(BrownWalker Press); 2006:285-293.
A) B) C)
OH
OH OMe A β-ether (β–O–4) HO OH
G B phenylcoumaran (β–5) H OH
OH
C resinol (β–β) MeO O
OH
HO
HO
G HO OMe C´ tetrahydrofuran (β–β) S OMe
O
HO O OMe D dibenzodioxocin (5–5/4–O–β) O O

G S OH
HO MeO
OH E biphenyl ether (5–O–4) HO
O
OMe
HO F spirodienone (β–1) O OMe
T OMe

Gymnosperm/
O
G MeO OMe X cinnamyl alcohol endgroup MeO S MeO O
OMe S pCA p-coumarate
Softwood
OH HO OH
O OH
OH pBA p-hydroxybenzoate MeO O
O
HO
OMe HO OMe FA ferulate Me G
G H p-Hydroxyphenyl unit O S OMe HO
O S Ac acetate O
O OMe
OH G Guaiacyl unit MeO
OH
T tricin HO
OH

OH S Syringyl unit HO
O OH O OMe Ara-Xyl
G OMe O O

Angiosperm/
OMe
G MeO S
O OH OH

Dicot/
HO
OMe MeO OH G OMe O
HO MeO OH FA
Hardwood
G
Monocot
OH O
HO O OMe
O
O S S O
HO
O
OMe

HO OH OMe O
HO G O MeO S HO O
G
O OMe
G G OMe
MeO
O
HO MeO HO

OH S HO
O OMe
S pCA HO
O OMe
MeO O O O
G OMe O
HO OMe
O
HO OMe OH
O
OH S OMe pBA OH O S OMe
HO OH O O pCA OH
G OH OH pCA
MeO O OH MeO O
O OMe OH OMe S OH
S OH
MeO OMe MeO OMe
O
G O
O
OH MeO S O
OH MeO S
O G O OMe OMe
HO O OMe HO O OMe
MeO G G Me
O HO OH O HO O
O OH G OMe
HO HO O

G OMe HO O S OMe S OMe

OH S OMe OH S OMe OH
OH MeO O OH MeO O
O OH
MeO O OH MeO O OH
OH
OH OH
MeO G MeO S MeO S
MeO G MeO G MeO G
O OH
MeO O OMe MeO O OMe
O OH
O O OH O O OH
HO
HO
HO pBA HO O G OH HO O G OH
O OH HO pCA O OH
G OMe
S OMe OH S OMe OH
G HO
G O OH
MeO O G MeO O G
MeO O
OH OMe
HO OMe HO OMe
 X
A) HO MeO HO MeO
OMe
OMe
O HO O MeO OMe
O O O O

, OH
,
MeO OMe
OMe
⇒ MeO OMe
OMe

MeO OMe
MeO HO

O O O OH OMe
J A B D

X
(α-aryl ether, α–O–4) (β–O–4, β-ether) (β–5, phenylcoumaran) (5–5/4–O–β, DBDOX)

B) OMe
O OH
MeO
MeO OMe MeO O O MeO OMe

HO OMe
HO

OH
⇒ O

OH
H+ HO

OH
+
MeO O
OMe

MeO OMe MeO OMe MeO OMe HO H

O O O O
F'e F F'f F''
(β–1, open) (β–1, spirodienone) (β–1, open) (glyceraldehyde-

X X
(etherified) (free-phenolic) 2-aryl ether)

C) OH
‘~U’
OH
‘Y’
O OMe O OMe

MeO OMe
O O

=
MeO O
⇒ MeO
O O
OMe

=
MeO O

MeO HO
MeO HO
MeO OMe MeO OMe
O OMe
O HO OMe OH

X
De Df
(5–5/4–O–β, DBDOX) (5–5/4–O–β, DBDOX)

X X
(etherified) (free-phenolic)

D) F) HO MeO
O
OMe

OMe OMe HO HO
‘Y’ ‘U’
HO

OMe
O
O
OMe
⇒ OMe
O
OH
OMe
MeO
O
O
OMe
OMe O

N1 MeO OMe N2
Ee Ef

X X
(β–6) O (β–O–γ)
(4–O–5, biphenyl ether) (4–O–5, biphenyl ether) OMe
(etherified) (free-phenolic)

X
O N4
OMe (γ–O–γ)
HO O MeO
O MeO

E)
HO
OMe
HO
OH O
MeO MeO OMe

X X
H O H O H O
S G/S MeO OMe N3
G O (β–α) MeO OMe
O O O OH
O
OMe OMe
,
OH

G
OMe
OMe
⇒ G
OMe MeO
S
OMe O
OH

OMe
OMe
O O O MeO
O O OMe
KG´-G KG´-S KS´-G/S OMe O

(8–O–4, β-ether) (8–O–4, β-ether) (8–O–4, β-ether) N5 N6

(G΄-G) (G΄-S) (S΄-G/S) HO (5–5) (4–O–5)
 H H
A) O O O O B) N O N O

radical coupling radical coupling

cross-coupling cross-coupling

OMe OMe OMe OMe

OH O OH O
Ferulate ester Feruloyl amide

C) H O H O D) H O H O

radical coupling radical coupling

cross-coupling cross-coupling
OMe OMe MeO OMe MeO OMe
OH O OH O
Coniferaldehyde Sinapaldehyde

HO OH HO OH HO OH HO OH

E) F)
radical coupling radical coupling
cross-coupling cross-coupling

OH O
OH O OH O
Resveratrol Piceatannol
OMe OMe OH
G) OH O OMe

HO O HO O
OMe radical coupling OMe OR O
cross-coupling OMe

OH O OH O
Tricin OH
O
MeO OMe

H) I)
MeO
OH OH
O O
MeO OH OH
radical coupling n
O HO
cross-coupling OMe
O radical coupling
MeO OMe OMe
cross-coupling O O
OH n
MeO MeO OH
Sinapyl O OH
alcohol OH MeO
O Caffeyl alcohol
HO OMe 5-Hydroxyconiferyl alcohol HO OH

OH

MeO OMe
O
n
 OH
OH

A) G OMe B) MeO
HO
G OH
MeO
O
O
O G
OH O OMe
MeO
A β–O–4/8–O–4 HO
G
MeO
OH S′ MeO S′ H
MeO
G B β–5/8–5 O O
H
O
O
O G OH
C β–β/8–8 HO OMe
HO O MeO O HO OMe
OMe
HO
G OMe D 5–5 OH
HO
G
HO
OCH3 E 5–O–4 S S OMe G
MeO OMe
O
O
OH
FA from CA-FA O HO OH OH
HO
OMe G from CA-FA OH
OMe
O
O O FA S′ sinapaldehyde HO
O OMe OH

OMe G S G OMe

HO MeO OH OMe
O MeO S′
OMe OH O HO
G OH O H O OMe
O
FA O O G G HO G HO
OMe
O
G
OMe O OH
HO
H MeO S
O OMe O O
O
HO
OH
MeO OMe S′ OMe OH
OMe G OH MeO O
OH
O
OH
S MeO O OMe OH
HO OH G OMe OH OMe
S′ OMe

G OMe G HO
O HO G O

MeO O OH O OMe S′ HO
O
HO MeO
OH OMe O H
G MeO
O
OMe
G O
H

OMe MeO OH
MeO O
G FA O OMe
O
G
HO
G
O
OH
OH
S OMe
O OH O
O
OMe OH
MeO
O
OH
O S′
FA OMe OH
O H
OH

MeO G
HO
 OH OH OH OH OH
(Hydroxycinnamyl Alcohols) OMe O Ara-Xyl
HO HO O
MeO O
OH
Monolignols

OH
MeO O
H G S C 5HG OMe
OMe MeO OMe OH MeO OH OH O
O O
O OMe
OH OH OH OH OH
OMe
p-Coumaryl Coniferyl Sinapyl Caffeyl 5-Hydroxy- OMe
O O
Alcohol Alcohol Alcohol Alcohol coniferyl Alcohol O
O O O
OH
O
OH OH O
OH HO
pCA FA HO
O O
OH O
OMe HO

O Me O
pBA O O
OH O OMe
OMe MeO HO
Monolignol
Conjugates

l l l O
no tes no
lig es no OH O
O l O
lig oa O
no arat
O lig
no no enz no tes HO O OMe Me
lig Mo oum Mo erula
OMe
no tes Moroxyb OH O O
Mo ceta F O
p-C
d HO O
ML A ML p- Hy ML ML HO O OMe MeO O
MeO HN OH
MeO OMe MeO OMe MeO OMe MeO OMe
O HO O
OH OH OH OH O O
O MeO
HO OH OMe H OMe HO
O O
H O OMe
O O H O N O
Xyl-Ara OMe O O
Other Monomers

OH MeO OH
R = H: Resveratrol O O
R = OH: Piceatannol MeO
HO O
T R = OMe: Isorhapontigenin OMe
OMe
FA HS MeO OH
OMe MeO OMe OMe R = H, OH, OMe Hypothetical Lignin Model
OH OH OH OH O OH HO OMe (Showing example structures incorporating the various monomers)
Ferulate Hydroxy- Feruloyl Tricin Hydroxystilbenes
on Arabinoxylan cinnamaldehydes Amides (a flavone)