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Lignin structure and its engineering
John Ralph1,2, Catherine Lapierre3 and Wout Boerjan4,5
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 toward 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.
Addresses
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, Universite’
Paris-Saclay, 78000, Versailles, France
4
Ghent University, Department of Plant Biotechnology and
Bioinformatics, Technologiepark 71, B-9052, Gent, Belgium
5
VIB Center for Plant Systems Biology, Technologiepark 927, B-9052,
Gent, Belgium
Corresponding author: Ralph, John (jralph@wisc.edu)
Current Opinion in Biotechnology 2019, 56:240–249
This review comes from a themed issue on Plant biotechnology
Edited by Wout Boerjan and John Ralph
For a complete overview see the Issue and the Editorial
Available online 25th March 2019
https://doi.org/10.1016/j.copbio.2019.02.019
0958-1669/ã 2018?Elsevier Inc. All rights reserved.
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 dis-
covered structural features, and instrumental methods, par-
ticularly 2D NMR [1], allowed structural details to be teased
from the complex polymer. Exciting ‘new’ structures in
Current Opinion in Biotechnology 2019, 56:240–249
lignins were revealed, including the dibenzodioxocins D
(DBDOX, Figure 1), in which most 5–5-linked units find
themselves [1,2], and the b–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
,5,6
,7–9,10
]. 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 [9,10
,11], the per-
turbation 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 toward its simpler removal from polysaccharides
or for enhanced value. This short review is challenged with
doing justice to the extraordinary range and depth of 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 Figure 1). Lignification, primarily via the ‘endwise’
coupling of a monomer (radical) with the growing poly-
mer (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, coni-
feryl, and sinapyl alcohols), couple at their b-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. Figure 1 shows our somewhat rep-
resentative 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
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 Lignin structure and its engineering Ralph, Lapierre and Boerjan 241

Figure 1

OH
(a) OMe
(b) A β-ether (β–O–4) (c) HO
OH OH
G B phenylcoumaran (β–5) H OH
OH O
C resinol (β–β) MeO 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 HO
OH
HO MeO
OH E biphenyl ether (5–O–4)
O
OMe
HO
O
F spirodienone (β–1) O OMe
T OMe

G X cinnamyl alcohol endgroup

OMe Gymnosperm/ MeO
S
OMe
pCA p-coumarate
MeO S
OH
MeO O

O
OH Softwood OH
pBA p-hydroxybenzoate MeO O
O
HO OH

HO
OMe HO OMe FA ferulate Me G
G H p-Hydroxyphenyl unit O S OMe O HO
Ac acetate O OMe
O S
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
OMe
G S
HO
O Angiosperm/ OH
MeO
OH

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

HO
O

OH
O S
OMe
Monocot S
O
O
HO
O
OMe

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

Current Opinion in Biotechnology

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 b–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 b-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 (b–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 (b–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 Figure 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
Figure 2d) because it remains, as highlighted, free-phenolic; 1 Dibenzodioxocin D (5–5/4–O–b, 5%, estimated at some 9–12% in G-lignins, so perhaps
low); 12 b-Ether units A (b–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 (b–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 (b–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 Figure 2d); 0 Dibenzodioxocins D (5–5/4–O–b, 0%, even though the G-units in lignin allow a modest level of
such structures, perhaps 3%); 16 b-Ether units A (b–O–4, 80%, depending on how they are counted, about the right level for a 65%-S lignin). Note

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242 Plant biotechnology

resonance forms drawn to rationalize the radical cou-
pling 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 Figure 1 caption,
underappreciated stereochemical complexity [5,6
].

p-Hydroxyphenyl (H-unit, Figure 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 pheno-
lics 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 abbre-
viations 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 deg-
radative analytical methods (thioacidolysis, DFRC), in
which the cleavage of b-ether units is required to
produce the diagnostic products [25,26].

Non-cyclic benzyl ethers J (a-aryl ethers, Figure 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, Figure 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 b–1 unit F does not represent a branchpoint, either,
because one of its (latent) phenolic moieties always
remains non-etherified, Figure 2b. Currently there is
no structural evidence for branching.

Upregulation and downregulation of ferulate 5-hydrox-
ylase (F5H) provide the most straightforward way to alter
the S:G ratio in angiosperms (dicots and grasses). Knock-
ing 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

,33,34]. Such
high-S lignins are linear with essentially only two types of
units, the b–b-dimeric unit that starts the polymeriza-
tion, and b–O–4-ethers emanating from both phenolic
ends of that dimeric unit, Figure 3h. The b-ether content
is consequently high allowing for depolymerization reac-
tions to produce high levels of monomers: 69%, 93%, and
78% from DFRC, nitrobenzene oxidation, and hydro-
genolysis [33,34].

H-enriched lignins can be produced when genes
encoding the enzymes associated with the 3-hydroxyl-
ation steps are targeted, including C3H, HCT [35–
37,38

,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 attri-
bute [38

]. H-lignins are surprisingly b-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].

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.

Hydroxycinnamaldehydes (HCAs), the immediate pre-
cursors of monolignols, are exported to the wall and
used for lignification in CAD-downregulated plants (but
see a limitation for softwoods, noted in Figure 2e).
Viable arabidopsis plants could be formed using

(Figure 1 Legend Continued) 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 (b–5, 10%, a bit high for a lignin with this S-level); 1 Tetrahydrofuran C0 (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 g-OH is acylated; 0 Spirodienones F (b–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 Figure 2d);
0 Dibenzodioxocins D (5–5/4–O–b, 0%, even though the G-units in lignin allow a modest level of such structures, perhaps 3%); 15 b-Ether units A
(b–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 g-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.

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 Lignin structure and its engineering Ralph, Lapierre and Boerjan 243

Figure 2

(a)

J A B D
(α-aryl ether, α–O–4) (β–O–4, β-ether) (β–5, phenylcoumaran) (5–5/4–O–β, DBDOX)

(b)

F′e F F′f F′′

(β–1, open) (β–1, spirodienone) (β–1, open) (glyceraldehyde-
(etherified) (free-phenolic) 2-aryl ether)

(c)

De Df
(5–5/4–O–β, DBDOX) (5–5/4–O–β, DBDOX)
(etherified) (free-phenolic)

(f)
(d)

N1 N2
Ee Ef (β–6) (β–O–γ)
(4–O–5, biphenyl ether) (4–O–5, biphenyl ether)
(etherified) (free-phenolic) N4
(γ–O–γ)

(e)
N3
(β–α)

KGʹ-G KGʹ-S KSʹ-G/S

(8–O–4, β-ether) (8–O–4, β-ether) (8–O–4, β-ether) N5 N6
(Gʹ-G) (Gʹ-S) (Sʹ-G/S) (5–5) (4–O–5)

Current Opinion in Biotechnology

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 a-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’ b–1 units in lignin are often shown in the open form F0 , and often with etherification of the B-ring F0 e;
they have now been shown to exist in lignins as spirodienones F from intramolecular trapping of the quinone methide intermediate, explaining why

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244 Plant biotechnology

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
,54–
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 engineer-
ing targets as they are completely linear and essentially
100% mono-structural, Figure 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 essen-
tially a linear homogeneous b–O–4-polymer of benzo-
dioxane units, Figure 3i, in vitro as well as in vivo. As an
all-ether-linked polymer, it can be quantitatively con-
verted 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 conju-
gates 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], Figure 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 b–b-coupled components such as C0
(Figure 1c) in grass lignins; these mono-tetrahydro-
furans were produced because protection of the
g-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-couma-
rate has been discovered [62,63].

p-Hydroxybenzoylated lignins (Figure 1b) and acety-
lated lignins (Figure 1c) similarly derive from mono-
lignol p-hydroxybenzoate and acetate conjugates
[1,17,18,26,64]. Other monolignol conjugates have
been recently discovered, including benzoates, vanil-
lates, and ferulates, even in the same plant line [65].
Not all of the genes/enzymes have yet been unambig-
uously identified.

Monolignol ferulates were discovered to be naturally
incorporated into lignins in a range of plants [66]
subsequent to their (assumed) de novo strategic engi-
neering into transgenics [67

]. This special case is
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 pretreat-
ment and saccharification [17,68]. A particularly promis-
ing 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 mono-
lignol p-coumarate and p-hydroxybenzoate conjugates
were becoming established as lignification ‘monomers’
and; 3) It was obvious that monolignols could be (par-
tially) replaced by various other compatible phenolics to
produce polymers that were not fully derived from the
canonical monolignols [6
]. A required feruloyl transfer-
ase gene was transformed into arabidopsis and poplar
[67

], which did indeed result in the introduction of
ester linkages (‘zips’) into the lignin backbone (Figure 4).
Encouraged by the improved performance after a variety
of pretreatments [69–71], current research is aimed at

(Figure 2 Legend Continued) B-ring etherification is impossible [3]; treatment in acid does release the open forms F0 f, with the free-phenolic B-
ring only, and also explains the origin of the glyceraldehyde-2-aryl ether structures F00 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 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 KG0 -G cannot be found [4
,30,31]; coniferaldehyde can so-couple with syringyl units,
though, forming KG0 -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 does not 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].

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 Lignin structure and its engineering Ralph, Lapierre and Boerjan 245

Figure 3

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
MeO
(h) OH OH (i)
O O
MeO OH OH
radical coupling n
O HO
cross-coupling OMe
O radical coupling
OMe
MeO 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
Current Opinion in Biotechnology

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 (Figure 4a). (b) Feruloyl amides such as

www.sciencedirect.com Current Opinion in Biotechnology 2019, 56:240–249

246 Plant biotechnology

Figure 4

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 OH
OMe G from CA-FA 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 OH
HO MeO OMe S′ OMe OH
OMe G OH MeO O
OH
O
OH
S MeO O OMe OH
OMe
HO OH G OMe OH
S′ OMe

G OMe G HO
O HO G O

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

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

MeO G
HO

Current Opinion in Biotechnology

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-cross-coupling, 5–
b-cross-coupling, 4–O–b-cross-coupling, 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 Figure 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–b) as well as in 8–8-homocoupled units. Each sinapaldehyde-
derived S0 unit is colored red, and the bonds formed by radical coupling are colored as in Figure 1.

elevating the Zip-lignin levels and developing superior perhaps several times independently [66]. Grasses utilize
lines in commercially relevant tree species. mainly sinapyl ferulate, and many hardwoods (but no
softwoods) utilize mainly coniferyl ferulate in their ligni-
Since engineering plants to utilize monolignol ferulates fication. The advantage or pressure that gave rise to the
for lignification to produce Zip-lignins, it was discovered evolution of the pathway remains unclear, but many
that such capabilities had already evolved naturally, related genes and enzymes are now in contention for

(Figure 3 Legend Continued) 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 b-ethers. I) 5-Hydroxyconiferyl alcohol and, surprisingly, caffeyl alcohol, couple
essentially only by b–O–4-coupling to produce linear homopolymers of benzodioxane units [47,48,49
] 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 Figure 1).

Current Opinion in Biotechnology 2019, 56:240–249 www.sciencedirect.com

 Lignin structure and its engineering Ralph, Lapierre and Boerjan 247
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 com-
promised (see Figure 4).
Monomers from beyond the monolignol biosynthetic
pathway
Even more striking was the recent discovery that plants
could conscript phenolics from other biochemical path-
ways 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 (Figure 3g), a valuable flavonoid, derived from a
combination of the shikimate-derived and acetate/mal-
onate-derived polyketide pathways, was the first phe-
nolic 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
b-position.

Hydroxystilbenes, including piceatannol (Figure 3f),
isorhapontigenin, and resveratrol (Figure 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,
Figure 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 b–O–4-cou-
pling, 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-based 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 b–O–4-coupling requirement: monolignols, mono-
lignol conjugates, incompletely methylated monolignols
(caffeyl, 5-hydroxyconiferyl, and, in principle, gallyl alco-
hols), and hydroxycinnamaldehydes and hydroxycinna-
mate esters. A weird and wonderful assortment of possi-
bilities for monomers was described in a review [9], and
some of these are being more systematically tested [73].
Additionally, some monomers have significant value.
Monolignol p-coumarate conjugates increase the
www.sciencedirect.com
frequency of free phenolic groups in lignins, which makes
them more susceptible to alkaline or oxidative treat-
ments. 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 incorpo-
ration 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-neu-
tral biomass. Marveled for their biochemical flexibility
that can be capitalized upon by biotechnological meth-
ods, lignins are transitioning from being regarded as
problematic recalcitrant polymers to resources with
boundless potential.
Conflict of interest statement
Nothing declared.
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’.
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:

of special interest


of outstanding interest
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 Lett 1995, 36:169-170.
3. Zhang L, Gellerstedt G, Ralph J, Lu F: NMR studies on the
occurrence of spirodienone structures in lignins. J Wood Chem
Technol 2006, 26:65-79.
4. Sederoff RR, MacKay JJ, Ralph J, Hatfield RD: Unexpected

variation in lignin. Curr Opin Plant Biol 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. Phytochem Rev 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, , vol 1. Edited by Daayf F, El Hadrami A, Adam L,
Ballance GM. Wiley-Blackwell Publishing; 2008:36-66.
Current Opinion in Biotechnology 2019, 56:240–249
248 Plant biotechnology
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.
Curr Opin Plant Biol 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 Biol 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 Phytol 2012, 196:978-1000.
10. Mottiar Y, Vanholme R, Boerjan W, Ralph J, Mansfield SD:

Designer lignins: harnessing the plasticity of lignification. Curr
Opin Biotechnol 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. Angew Chem Int Ed 2016, 55:8164-8215.
12. Boerjan W, Ralph J, Baucher M: Lignin biosynthesis. Ann Rev
Plant Biol 2003, 54:519-546.
13. Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W: Lignin
biosynthesis and structure. Plant Physiol 2010, 153:895-905.
14. Ralph J, Peng J, Lu F, Hatfield RD, Helm RF: Are lignins optically
active? J Agric Food Chem 1999, 47:2991-2996.
15. Akiyama T, Magara K, Meshitsuka G, Lundquist K, Matsumoto Y:
Absolute configuration of b- and a-asymmetric carbons
within b-O-4-structures in hardwood lignin. J Wood Chem
Technol 2015, 35:8-16.
16. Ramos Barbosa IC, Rojas-Murcia N, Geldner N: The Casparian
strip – one ring to bring cell biology to lignification? Curr Opin
Biotechnol 2019, 56:121-129.
17. Ralph J: Hydroxycinnamates in lignification. Phytochem Rev
2010, 9:65-83.
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 Res 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.
Phytochem Rev 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. J Agric Food Chem 1995, 43:2746-2751.
21. del Rı́o JC, Rencoret J, Prinsen P, Martı́nez ÁT, Ralph J,
Gutiérrez A: Structural characterization of wheat straw lignin
as revealed by analytical pyrolysis, 2D-NMR, and reductive
cleavage methods. J Agric Food Chem 2012, 60:5922-5935.
22. Ralph J, Schatz PF, Lu F, Kim H, Akiyama T, Nelsen SF: Quinone
methides in lignification. In Quinone Methides, , vol 1. Edited by
Rokita S. Wiley-Blackwell; 2009:385-420 Reactive Intermediates
in Chemistry and Biology.
23. Vanholme R, De Meester B, Ralph J, Boerjan W: Lignin
biosynthesis and its integration into metabolism. Curr Opin
Biotechnol 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.
Current Opinion in Biotechnology 2019, 56:240–249
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. J Agric Food Chem 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 (4O5 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. Org Biomol Chem 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 Physiol 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. Proc Nat Acad Sci U S A 1998, 95:6619-6623.
One of the first papers to show that massive changes in the composition
of lignins’ normalH, G, and S units could be elicited in viable plants.
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 Physiol 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 J
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. Proc Nat Acad
Sci U S A 2007, 104:11856-11861.
37. Coleman HD, Park J-Y, Nair R, Chapple C, Mansfield SD: RNAi-
mediated suppression of p-coumaroyl-CoA 30 -hydroxylase in
hybrid poplar impacts lignin deposition and soluble secondary
metabolism. Proc Nat Acad Sci U S A 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 down-
regulation 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 Res 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 sig-
nificant 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
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 Lignin structure and its engineering Ralph, Lapierre and Boerjan 249
microbial biofuel production in drought-stressed switchgrass
hydrolysate. Biotechnol Biofuels 2016, 9.
42. Ralph J, Grabber JH, Hatfield RD: Lignin-ferulate crosslinks in
grasses: active incorporation of ferulate polysaccharide
esters into ryegrass lignins. Carbohydr Res 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. J Chem Soc Perkin Trans 1994, 1:3485-3498.
44. del Rı́o 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. J Agric Food Chem 2018, 66:4402-4413.
45. del Rı́o JC, Rencoret J, Gutiérrez A, Kim H, Ralph J:
Hydroxystilbenes are monomers in palm fruit endocarp
lignins. Plant Physiol 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. Plant J 2016, 88:1046-1057.
After the initial discovery of the first lignin monomer, tricin (a flavone), from
outside the lignin biosynthetic pathway (Ref. [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. Plant Cell 2013, 25:2587-2600.
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. Plant J 2013, 73:201-211.
49. Chen F, Tobimatsu Y, Havkin-Frenkel D, Dixon RA, Ralph J: A

polymer of caffeyl alcohol in plant seeds. Proc Nat Acad Sci U S
A 2012, 109:1772-1777.
The discovery ofC-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. Sci Adv 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. Proc Nat Acad Sci U S A 2013,
110:13660-13665.
53. Lapierre C, Tollier MT, Monties B: A new type of constitutive unit

in lignins from the corn bm3 mutant. C R Acad Sci. 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.
J Agric Food Chem 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. J Chem
Soc Perkin Trans 2001, 1:2939-2945.
57. Vanholme R, Ralph J, Akiyama T, Lu F, Rencoret Pazo J,
Christensen J, Rohde A, Morreel K, DeRycke R, Kim H et al.:
www.sciencedirect.com
Engineering traditional monolignols out of lignins by
concomitant up-regulation F5H1 and down-regulation of
COMT in Arabidopsis. Plant J 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. Plant J 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. Plant J 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 Physiol 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 Physiol 2016, 170:1358-1366.
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.
Plant J 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 J 2014, 78:850-864.
64. Lu F, Ralph J: Novel tetrahydrofuran structures derived from
b–b-coupling reactions involving sinapyl acetate in Kenaf
lignins. Org Biomol Chem 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 Physiol 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. Sci Adv 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
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