Evolutionary History of Lignins

Dedication
The co-authors of this chapter would like to dedicate this work to the
memory of Professor Alfonso Ros Barceló, who died on 29 January 2012,
aged 51.
Alfonso was for us a true ‘MAESTRO’. He struggled with our blunders and
limitations in order to transform us into something approaching researchers.
But more important than his role as director was his unconditional friend-
ship. We now have to sail alone, without our captain. We will work hard to
follow the course he set out in the hope he would be proud of his crew.
Alfonso, te echamos de menos.
Evolutionary History of Lignins
ESTHER NOVO-UZAL,* FEDERICO POMAR,*,1

LAURA V. GÓMEZ ROS,{ JOSE M. ESPIÑEIRA* AND
ALFONSO ROS BARCELÓ{
*Department of Animal Biology, Plant Biology and Ecology, University

of A Coruña, A Coruña, Spain
{
Department of Plant Biology, University of Murcia, Murcia, Spain
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
A. Land Colonization and Appearance of a Lignified Vascular System .. 313
B. Composition and Cellular Distribution of Lignins........................ 316
II. Lignin Occurrence in the Different Phylogenetic Groups . . . . . . . . . . . . . . . . . . 318
A. Algae ............................................................................. 319
B. Embryophytes (Land Plants) ................................................. 321
III. Evolutionary View of S Lignin Biosynthetic Pathway and Transcriptional
Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
A. Enzymes.......................................................................... 336
B. Transcriptional Regulation ................................................... 338
IV. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
ABSTRACT
Lignin is a polymer resulting from the oxidative coupling of primarily three
p-hydroxycinnamyl alcohols (monolignols): p-coumaryl, coniferyl and sinapyl alcohols.
Lignin is deposited mainly in tracheids, vessels, fibres of xylem and phloem and
1
Corresponding author: E-mail: fpomar@udc.es
Advances in Botanical Research, Vol. 61 0065-2296/12 $35.00

Copyright 2012, Elsevier Ltd. All rights reserved. DOI: 10.1016/B978-0-12-416023-1.00009-4
312 ESTHER NOVO-UZAL ET AL.
sclerenchyma. However, lignin composition varies among species, phylogenetic groups,

cell types, developmental stages and even seasonal growth. In recent decades, the
number of species where lignins have been detected has notably increased, such as
the red alga Calliarthron, some Coleochaetale species and some bryophytes. Moreover,
the presence of syringyl lignins, which has been restricted solely to angiosperms, has been
confirmed also in most studied phylogenetic groups, from rhodophytes to gymnos-
perms, including non-vascular plants. The discovery in Selaginella of a novel enzyme
analogous to angiosperm ferulate-5-hydroxylase supports the existence of evolutionarily
convergent pathways that lead to syringyl lignin biosynthesis. In this review, the role of
lignins in the development of vascular system and the presence and composition of
lignins in different phylogenetic groups will be discussed, paying special attention to new
contributions and the evolutionary development of syringyl lignins.
I. INTRODUCTION
Lignins are abundant organic polymers, accounting for 25% of plant

biomass (Higuchi, 1990). They are important compounds of cell walls,
where they are imbibed in a polysaccharide matrix (Boudet et al., 1995).
These polymers impart water impermeability, including resistance against
tensile forces of the water columns, and confer structural support and flexural
stiffness to the aerial organs. Lignins can also provide protection against the
microbial degradation of cell walls (Ros Barceló, 1997).
Lignins are three-dimensional heteropolymers resulting from the oxidative
coupling of primarily the three p-hydroxycinnamyl alcohols (monolignols):
p-coumaryl, coniferyl and sinapyl alcohols. The cross-coupling reaction of
monolignol radicals produces a hydrophobic heteropolymer composed of
p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units (Vanholme et al.,
2010; Fig. 1).
Fig. 1. Structures of p-coumaryl (A), coniferyl (B), and sinapyl (C) alcohols,
precursors of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignins units,
respectively. O atoms are shaded in red, H atoms in pale grey and C atoms in dark
grey.
EVOLUTIONARY HISTORY OF LIGNINS 313
The lignin content and composition are closely related to plant cell wall
digestibility of (forage) plant cell walls. Unravelling the lignin biosynthetic
pathway will lead to a search for strategies to modify the lignin content and
composition to improve the digestibility of forage and for biofuel production.
A. LAND COLONIZATION AND APPEARANCE OF A LIGNIFIED

VASCULAR SYSTEM
One of the most important biological events in the history of life was the
colonization of land by plants and their rapid diversification during the early
Paleozoic period (Niklas, 1997; Raven and Edwards, 2001).
Land colonization meant plants had to cope with a new stress in addition
to adaptation to a gravitropic environment as, when taking CO2 from the
atmosphere, an inevitable loss of water occurred. To avoid such water loss,
plants developed different strategies to accumulate water in their tissues, to
supply it or to minimize its loss. In fact, cuticle and stomata were some of the
very first innovations in the first terrestrial plants (Fig. 2). When CO2 levels
were high (during the Silurian period), plants would lose less water molecules
than when CO2 levels decreased (late Carboniferous): water loss would have
been as much as 17 times higher for a Carboniferous plant (Sperry, 2003).
Thus, the adaptive radiation of plants by the early Carboniferous period was
probably due to very high atmospheric CO2 levels (Sperry, 2003). High CO2
concentrations encouraged plants with high capacitance and low hydraulic
conductance. In fact, the CO2 concentration seems to be an important factor
during plant evolution, not only in the diversification of a vascular system
but also in the origin of leaves. Until the CO2 levels decreased, plants were
not able to develop bladed photosynthetic structures because of lethal over-
heating (Beerling et al., 2001; Osborne et al., 2004). A second drop in CO2
took place during the Cretaceous period, and this was associated with
angiosperms diversification (McElwain et al., 2004).
However, a water supply system based on high capacitance prevents plants
from being bigger than a thin tallus. The first terrestrial plants had a cuticle
that avoided water loss and lacked a water-conducting system, and were
also small in size. These first land plants were poikilohydric, in which the
water potential was equilibrated with surrounding water sources (Raven,
1987). Those plants had high capacitance to accumulate water when the
environment was dry.
The first evolutionary radiation among land plants is related to the diver-
sification of tracheids (Kenrick and Crane, 1997; Knoll and Niklas, 1987).
This diversification originated three major clades, each with a specific type of
tracheid (Kenrick and Crane, 1997). True tracheids have been defined as
Fig. 2. Plant phylogenetic tree. Upper scale represents geological periods and time in millions of years ago (mya) according to the
International Commission of Stratigraphy. O, Ordovicean; S, Silurian; D, Devonian; Ca, Carboniferous; P, Permian; T, Triassic; J, Jurassic;
Cr, Cretacic. Vertical bars at right indicate different clades. A, angiosperms; G, gymnosperms. The presence and composition of lignins and
lignin-like compounds are denoted by circles at each branch—green circle: lignin-like compounds; red circle: H þ G þ S lignins; blue circle:
H þ G lignins. The question mark means lignin composition is unclear or unknown. Pink rectangles indicate the presence of vessels. {: Extinct
lineage. Based on Sperry (2003), Martone et al. (2009) and Weng and Chapple (2010).
single-celled conduits with lignified and ornamented walls, either banded or

pitted (Doyle, 1998; Kenrick and Crane, 1997).
S-type tracheids (named after the genus Sennicaulis) were present in the
members of Rhyniopsida, a group where initial diversification of vascular
plants is hypothesized to have occurred (Friedman and Cook, 2000). They
exhibited annular or helical thickenings made of spongy material. Adjacent
to this degradation-prone layer, S-type tracheids showed a thin degradation-
resistant layer next to the lumen (Friedman and Cook, 2000; Kenrick and
Crane, 1991, 1997).
G-type tracheids (after the genus Gosslingia) were found in the zostero-
phylls, an extinct group ancestral to extant lycophytes. These tracheids
showed annular or helical thickenings constituted by two different layers:
adjacent to the cell lumen there is a lignified zone with an unlignified hollow
core (Friedman and Cook, 2000).
P-type tracheids (after the genus Psilophyton) were typical of the earliest
members of euphyllophytes, which include modern ferns and spermatophytes.
These tracheids showed bordered pits with a degradation-prone layer
surrounded by a degradation-resistant layer (Friedman and Cook, 2000).
As regards these three types of tracheids, the degradation-resistant
layer seemed to be lignified, unlike the degradation-prone layer. However,
studies on tracheary elements have been done in extant spermatophytes, so
the available information is partial (Friedman and Cook, 2000). In extant
pteridophytes, an unlignified core at the base of secondary cell wall thicken-
ings has been described, which could be the missing degradation-prone layer
in early fossil tracheids (Bierhorst, 1960; Kenrick and Crane, 1991).
Developing a vascular system, together with the presence of stomata
(which are only absent in liverworts among land plants), allowed plants
to become homoiohydric rather than poikilohydric (Sperry, 2003). Plants
were able to regulate their water dependence, which involved a loss of
desiccation tolerance in vegetative cells (Oliver, 1996). Due to the change
into homoiohydric, plants were allowed to develop greater sizes, with
obvious advantages of escaping from shade and improving long-distance
aerial dispersal. This change also required a shift from hydrostatic support
to a lignified cell wall-based support (Niklas, 1994; Sperry, 2003), which
was accompanied by the evolution of sclerenchyma tissue surrounding the
vascular system (Raven, 1987). The diameter of tracheids increased gradually
until the Devonian period, reaching 80 mm (Niklas, 1985) but, due to physi-
cal restrictions, tracheids could not be wider (Zimmermann, 1983). This
problem was resolved by increasing the number of water-conducting cells,
the so-called vessels, allowing greater diameter and length than tracheids
(Zimmermann, 1983). Tracheids have a double function: they participate
both in water transport from roots to leaves and in mechanical support. In

the case of vessels, typical of angiosperms, there are two different types of
specialized cells: the vessel elements, which are responsible for water trans-
port, and the fibres, which provide mechanical support (Pittermann, 2010).
This division of functions provides vessels with higher conductivities and
makes them more efficient at maintaining such conductivities (Sperry, 2003).
In terms of development and physiology, the change from a tracheid-
producing cambium to one forming vessels may only have required a change
in auxin and gibberellin levels during differentiation (Aloni et al., 2000).
Vessels evolved independently in five different groups: selaginellales,
horsetails, ferns, gnetophytes and angiosperms (Baas and Wheeler, 1996;
Fig. 2), indicating the important advantages vessels represent in water
conduction. Despite the advantage represented by vessels, many widespread
plants lack vessels, including the gymnosperms. Some authors have sug-
gested that plants containing short tracheids may have greater cavitation
resistance because they can refill more easily (Ewers, 1985; Sperry, 2003),
which could explain the presence of gymnosperm woods in areas where
angiosperms have trouble surviving. Even so, the number of angiosperm
species far exceeds gymnosperm species (255,247 angiosperms vs. 831 gym-
nosperms). The number of gymnosperms species is even lower than that of
fern species (11,380) (Palmer et al., 2004). Given that gymnosperms and
angiosperms may exhibit similar hydraulic efficiencies (Pittermann et al.,
2005), it is surprising that the number of fern species exceeds that of
gymnosperms. All gymnosperms have tracheids as the water-conducting
system but, whereas most ferns have tracheids, some species such Pteridium
and the aquatic fern genus Ceratopteris are known to possess vessels
(Carlquist and Schneider, 2000, 2001). Tracheids in ferns only confer a
water transport system and are released from the need to provide mechani-
cal support, which is provided by a ring of sclerenchyma fibres peripheral to
the central axis of the frond (Rowe et al., 2004). This implies that tracheids
can be wider in ferns than in gymnosperms without the need for a rein-
forced cell wall. The wider and longer conduits of ferns allow a high degree
of hydraulic conductivity in the xylem, which can support the water loss in
the absence of an active hormone-controlled stomatal closure (Pittermann
et al., 2011).
B. COMPOSITION AND CELLULAR DISTRIBUTION OF LIGNINS
Lignin is deposited mainly in tracheids, vessels, fibres of the xylem and

phloem and sclerenchyma (Ros Barceló, 1997). However, lignin composition
varies among species, phylogenetic groups, cell types, development stage and
even seasonal growth. Different stresses provoke a change in lignin composi-

tion, and this shift depends on the stress which caused it (Moura et al., 2010).
The amount and composition of lignin also varies in the cell wall itself.
It has been reported that the middle lamella has a greater quantity of lignin
than the primary and secondary cell wall. In angiosperms, the middle lamella
contains 50–70% lignin, while the secondary cell wall contains 20% lignin.
In gymnosperms, lignin represents 50–70% of the middle lamella whereas the
secondary cell wall is composed of 16–19% lignin. The lignin percentage
differs between hardwoods and softwoods (Christiernin, 2006a). As regards
the overall content, lignin represents 20% of dry matter in angiosperms and
30% in gymnosperms. This difference translates into the higher efficiency of
angiosperms, since a lower lignin content and higher carbohydrate content
require less energy and carbon for growth (Amthor, 2003). As regards the
lignin composition of the different layers of the gymnosperm cell wall, the
middle lamella is supposed to be rich in p-hydroxyphenyl units, whereas
secondary cell wall layers contain mainly guaiacyl units (Terashima and
Fukushima, 1989). The main reported difference in angiosperm lignin com-
position among cell types is between vessels and fibres. Whereas vessels are
composed of G units, fibres are mainly composed of S units (Li et al., 2001).
Among the different taxa, the classical view is that angiosperms are com-
posed of a similar proportion of G and S units, whereas gymnosperm cell
walls mainly contain G units, with a lower percentage of H units (derived
from p-coumaryl alcohol monomer), except in compression woods where the
H unit level is significantly elevated (see below). The remaining groups of
vascular plants, Lycophyta and Pteridophyta are supposed to resemble
gymnosperm lignins, that is, composed of G units. However, recent and
more accurate data support the idea of the non-linear evolution of lignin
composition in plant history, as will be discussed later.
Differences between angiosperms and gymnosperms also occur in excep-
tional situations such as reaction wood synthesis. A clear difference between
angiosperms and gymnosperms exists in reaction wood. This type of wood is
produced in order to force leaning stems into the normal position. In angios-
perms, this situation is called tension wood and is characterized by having
fewer xylem vessels elements, a higher proportion of fibres, more cellulose
(including, in some species such as poplar, a filling of the lumena with
cellulose) and less lignin (Wilson and White, 1986). Tension wood is also
differentiated by a higher syringyl/guaiacyl ratio (Aoyama et al., 2001;
Joseleau et al., 2004; Yoshida et al., 2002). In contrast, compression wood
from gymnosperms is characterized by higher content of lignin and less
cellulose, small tracheids and the absence of S3 layer (Donaldson et al.,
2004; McDougall, 2000; Timell, 1986). In addition, compression wood has
a higher proportion of H units (Fukushima and Terashima, 1991) and

condensed linkages (Saito and Fukushima, 2005).
Besides the classical p-hydroxyphenyl, guaiacyl and syringyl monomers,
other molecules may participate in the formation of the lignin polymer, the so-
called non-canonical subunits. These include ferulates, hydroxycinnamalde-
hydes (coniferaldehyde and sinapaldehyde), dihydrocinnamyl alcohols, and
variously acylated monolignols (Ralph et al., 2004, 2008; Sederoff et al.,
1999), the presence and quantity of which is usually related to different taxa.
The complexity of lignin’s structure is not only determined by the mono-
lignol composition, but also by the bonds produced during lignification. The
main linkages are b-O-4, b-5, 5-5, 4-O-5, b-b and b-1 (Ralph et al., 2004). The
bond frequency varies among cell types, layers of the cell wall and plant
groups. Woody gymnosperms contain b-O-4 linkages as the major inter-unit
linkage, levels that are even higher in woody angiosperms. This implies that
lignin in gymnosperms contains more inter-unit C–C linkages than woody
angiosperm lignins. Sinapyl alcohol has two methoxy groups ortho to the
phenol; coupling therefore results in a more linear lignin polymer, with a
larger number of ether bonds (b-O-4). For the most part, the b-ether level
increases with increasing S/G.
II. LIGNIN OCCURRENCE IN THE DIFFERENT

PHYLOGENETIC GROUPS
This classical view of the distribution, composition and evolutionary history

of lignin is currently being reviewed, due to the new and more recent research
developed regarding diverse phylogenetic groups. These advances have been
enabled due to the development in the past decades of a number of analytical
techniques that are more accurate and sensitive, or more structurally reveal-
ing, than those available before:
. Anti-lignin labelling has not been widely used to study the presence of
lignin in many samples. The reason lies in the difficulty of obtaining lignin
free from other cell wall components to develop antibodies. However, this
problem has been solved by using antibodies against synthetic dehydro-
genative polymers (Joseleau et al., 2004; Müsel et al., 1997; Ruel et al.,
1994). This technique is usually accompanied by other lignin analyses
because it is not considered as determinant for lignin detection by itself.
. Nuclear magnetic resonance (NMR) provides useful information about
the structure of the lignin polymer, but it is not as sensitive as other
procedures. Recently, solution-state 2D NMR has emerged as a powerful
tool to study lignin structure and composition, even without requiring its
isolation from the cell wall (Kim et al., 2008). This technique produces
minimal structural modification in the cell wall (mainly due to the
requirement for fine milling), revealing structure and composition not
only of lignin, but also of polysaccharide components of the wall (Kim
and Ralph, 2010).
. Degradative methods: acidolysis, nitrobenzene oxidation and especially:
Thioacidolysis is an acid depolymerization procedure, which acts

through the cleavage of b-O-4 linkages. Only aryl-glycerol-b-aryl
ether linked units produce C6C3 phenylpropanoid monomers with
thioethylated chains. Thioacidolysis analysis allows the study of
both the composition and structure of the lignin polymer on whole
cell wall samples, that is, without requiring isolation of the lignin. The
most important limitation of this technique is the application to
samples with low contents of b-O-4 linkages. However, thioacidolysis
has been the most diagnostic method for lignin characterization
(Lapierre, 1993; Rolando et al., 1992).
Derivatization followed by reductive cleavage (DFRC) is a degradative
method that efficiently cleaves the a-ether and b-ether linkages in
lignins, releasing analysable monomers for quantification. The method
also provides some information on carbon–carbon-linked lignin (Lu
and Ralph, 1997). In the DFRC procedure, acetyl bromide produces
the derivatization and cell wall solubilization, in a similar way to lignin
determination based on acetyl bromide (Johnson et al., 1961). Reduc-
tive cleavage is performed in acidic medium and, following acetylation,
4-acetoxycinnamyl acetate monomers are quantified by GC. This tech-
nique provides data analogous to thioacidolysis, and offers an advan-
tage that it leaves native lignin esters intact (Lu and Ralph, 1997).
The application of these techniques to different plant groups has provided

important new findings and data, which make necessary an updated overview
of the occurrence and composition of lignins in the phylogenetic groups.
Here, we review the characteristics of lignins in the different plant groups,
with particular attention to the new contributions and the occurrence and
evolution of S lignins.
A. ALGAE
Green algae and bryophytes are paraphyletic groups, and some of the
characteristics that presented bryophytes with adaptations to life on land
have their precursors in Coloechaetales and Charales, which appear to be
preadapted to make the transition to land (Graham, 1993).
Plant cell walls arose as a barrier to cope with osmotic problems in the
aquatic environment (Gerhart and Kirschner, 1997). Usually, origins of cell
walls that will allow land colonization are attributed to the charophycean
green algae (Sørensen et al., 2010). These algae show some features regarding
cellulose synthase genes that are still retained in the embryophytes, although
cellulose biosynthesis has ancient cyanobacterial origins. Besides cellulose,
some components of cell wall from embryophytes, including homogalactur-
onan and xylans, are already present in charophycean algae (Carafa et al.,
2005; Popper and Tuohy, 2010; Sørensen et al., 2010). Other cell wall
components seem to be exclusive to land plants; xyloglucan is present in
the cell walls of all land plants but absent from charophytes (Popper and Fry,
2003; Sørensen et al., 2010).
Regarding the occurrence of lignins, historically algae were considered to
not have lignin polymers in their cell walls. However, recent papers question
the absence of lignins in this group.
Martone et al. (2009) described the detection of syringyl lignin in the red
alga Calliarthron cheilosporioides (Fig. 3). Some seaweed contain lignin-like
compounds (Delwiche et al., 1989; Gunnison and Alexander, 1975), but the
Fig. 3. Lignins in a red alga, Calliarthron. (A) Whole aspect: igc, intergenicular walls;
gc, genicular walls. Scale bars represent 1 mm. (B) Immunocytochemical localization of
S units in intergenicular walls. (C) Monomers released from Calliarthron intergenicular
walls and mass spectral fragmentation pattern of DFRC S-monomers released. Adapted
from Martone et al. (2009), reproduced with permission from Elsevier.
presence of lignin in algae has never been previously reported. Calliarthron

is a coralline alga exposed to strong waves and presents calcified fronds.
This alga has some joints called genicula where lignin has been discovered.
Lignin presence has been related to the need to prevent the breakage
caused by waves (although the tiny quantity makes this role questionable).
The presence of a secondary cell wall, with a pattern analogous to that of
secondary cell wall formation in angiosperms, has also been described.
Derivatization followed by reductive cleavage (DFRC) showed the pres-
ence of the three monolignols, in a proportion that varied depending on
the season, age or development stage, which has previously been observed
in some other species (Christiernin, 2006b). The ratio H:G:S varied from
1:1.5:trace to trace:1:1, but pointed to the occurrence of syringyl lignin in a
red algae that diverged from vascular plants more than one billion years
ago (Saunders and Hommersand, 2004). Although the presence of lignins
is validated in these samples, its origin remains unclear and finding the
genes that support monolignol synthesis are still under investigation.
Ligrone et al. (2008) also reported the occurrence of lignin (or lignin-like
compounds) in the charophycean alga Nitella flexilis, using immunocyto-
chemical analysis. However, the labelling intensity was very weak against
guaiacyl lignin-like polymers but surprisingly, though still weak, stronger
against guaiacyl/syringyl lignin. Moreover, the labelling was uniformly
distributed in the cell walls.
Sørensen et al. (2011) determined the composition of the cell walls in
charophycean green algae, ancestors of the first land plants (Graham,
1996), more specifically, Coleochaete nitellarum, Coleochaete orbicularis and
Coleochaete scutata cell walls released guaiacyl and syringyl monomers when
subjected to thioacidolysis (Fig. 4). Coleochaete represents a group of char-
ophycean green algae, with cell walls comparable to embryophyte primary
cell walls. The total amount of monomers was very low in the three species,
but the lignin or lignin-like nature of the released compounds was also
confirmed by the use of immunocytochemical analyses. Anti-lignin antibo-
dies labelled cell walls in all three Coleochaete species, suggesting that some
sort of phenylpropanoid metabolism emerged before land colonization.
Sørensen’s results differ from Ligrone’s, who reported no reaction with
antibodies against lignin in the alga C. scutata.
B. EMBRYOPHYTES (LAND PLANTS)
Land plants are also called embryophytes because they have a resting embryo
stage early in the life of the sporophyte. The embryophytes descended from a
charophyte-like ancestor and possess three important new features: alternation
Fig. 4. Lignin-like compounds in Coleochaetales. (A) Light microscopy image of

Coleochaete nitellarum. (B) A portion of the GC–MS total ion current profile of the
monomers released by thioacidolysis of C. scutata, C. orbicularis, and C. nitellarum.
(C) Thallus of C. scutata labelled with the anti-lignin antibody anti-S/C. (D) control
immunofluorescent labelling in which primary antibody was omitted. Anti-S/C is a
polyclonal antibody against a dehydrogenative polymer (DHP) made after polymeri-
zation of a mixture of guaiacyl/syringyl 1/5 monomers, and is directed against
non-condensed b-O-4 GS inter-unit linkages. Adapted from Sørensen et al. (2011),
reproduced with permission from Wiley-Blackwell.
between haploid and diploid generations; multicellular structures that produce

eggs and sperm and a protected embryo (Niklas and Kutschera, 2010).
The primary function of lignins in the first land plants is unclear and
probably was not related to structural support. Some authors point to the
fact that first functions of lignin was protection against pathogens and UV
radiation (Boyce et al., 2003; Ligrone et al., 2008; Raven, 1984; Speck and
Vogellehner, 1988). This hypothesis is supported by the deposition of lignin
in the cortex and not in water-conducting tissues in Early Devonian plants
like Aglaophyton, Rhynia and Asteroxylon (Boyce et al., 2003). Thus, cell wall
thickenings in tracheids of the first vascular plants were unlignified and only
later lignins were deposited in vascular tissues to form the modern tracheids
of extant vascular plants (Boyce et al., 2003). These results agree with the
anatomy of water-conducting cells of bryophytes, whose cell walls modify

their appearance according to adjacent living cells (Boyce et al., 2003;
Hébant, 1977). However, other reports suggest the primary function of lignin
was propping open tracheary elements (Raven, 1987). The first terrestrial
plants were able to stand only with hydrostatic support (Niklas, 1994;
Vincent and Jeronimidis, 1991), so the lignified central strand of water-
conducting wells would not be necessary to stand, nor to protect plants
from herbivores.
Consistent with the first hypothesis, polyphenols and lignin-like com-
pounds have been found in species without a true vascular system or even
with no water-conducting cells at all. The presence of lignin-like compounds
has been described in charophycean green algae, as we have described above
(Delwiche et al., 1989; Sørensen et al., 2011) and bryophytes (Kroken et al.,
1996; Ligrone et al., 2008). This implies that at least part of the phenylpro-
panoid pathway that eventually led to lignin biosynthesis was present in
algae, and the presence of lignin in tracheids may only have involved the
expression of those genes in a different type of cells (Boyce et al., 2003).
Traditionally, embryophytes are classified into bryophytes and vascular
plants, as will be reviewed in the following sections.
1. Bryophytes
The fossil record is poor regarding the first land plants because of the lack of
intact structures; mostly only the cuticle remains. However, some extant
plants, the bryophytes, are similar to those first poikilohydric plants.
There are three major lineages of bryophytes: liverworts, mosses and
hornworts. Bryophytes show some new features due to adaptations to land
colonization, such as cuticle and sporopollenin, pores in the epidermis or
stomata, and the accumulation of flavonoids.
The cell wall composition in bryophytes not only resembles that of vascular
plants in some characteristics but also presents important differences. For
example, mosses contain galactoglucomannans, also present in ferns, angios-
perms and gymnosperms, suggesting their presence prior to the divergence of
vascular plants; they may have provided tensile strength to cell walls
(Liepman et al., 2007). Rhamnogalacturonan II (RGII) exists in bryophytes
only at a level of 1% of the amount found in vascular plants (Matsunaga et al.,
2004). RGII is present in all tracheophytes in high concentrations, as it is
supposed to be related to the formation of lignified cell walls (Popper, 2008).
Xylan is also present in all vascular plants but is absent from some thickened
cell walls of bryophytes (Carafa et al., 2005). Bryophytes also lack cellulose
gene orthologues, so it appears that secondary cell wall is strictly restricted to
vascular plants (Popper, 2008). In contrast, 3-O-methylrhamnose is present in
bryophytes, as well as in charophytes, pteridophytes and gymnosperms, but

not in angiosperms (Matsunaga et al., 2004; Popper and Fry, 2004).
Some bryophytes show the presence of specialized water-conducting cells,
where the cytoplasmic content has been removed, and so water transport is
facilitated. It should be pointed out that water-conducting cells in bryophytes
may have evolved independently (Ligrone et al., 2000). There are not many
data to assure the presence of actual lignin in the cell walls of bryophytes.
However, the occurrence of lignin-like compounds in bryophytes has been
widely described (Delwiche et al., 1989).
Liverworts are usually regarded as the most basal group in bryophytes.
Among them, Marchantiales and Jungermanniales have no water-conduct-
ing cells (Hébant, 1977; Kobiyama and Crandall-Stotler, 1999; Ligrone et al.,
2000). However, an internal strand of water-conducting cells is present in
Calobryales and Metzgeriales (Burr et al., 1974; Hébant, 1977; Ligrone et al.,
2000). In this case, these specialized cells are dead and contain no cytoplas-
mic components, with cell walls containing pores derived from plasmodes-
mata (Ligrone et al., 2000). In Metzgeriales, the water-conducting cells show
thick cell walls with helicoidal pits, reminiscent of tracheids. However,
homology between the two types is highly unlikely because of their different
developmental programme (Ligrone et al., 2000). Liverworts are reported to
contain polyphenolic compounds as they show autofluorescence under UV
light (Graham et al., 2004). Ligrone et al. (2008) reported the presence of
guaiacyl–syringyl lignin in bryophytes, using immunocytochemical detec-
tion. Antibodies against G and GS synthetic lignin-like polymers strongly
labelled compounds not only from tracheophytes but also from bryophytes
such as liverworts and mosses. In the case of liverworts, a stronger signal for
GS lignin was labelled in Marchantia polymorpha and Conocephalum con-
icum. It has been established that syringyl lignin occurs in M. polymorpha, a
liverwort, belonging to terrestrial plants that lack a vascular system, as
discussed above. Specifically, Marchantiales are known to even lack the
central strand of water-conducting cells typical of other bryophytes. Never-
theless, by means of thioacidolysis and nitrobenzene oxidation, Espiñeira
et al. (2011) reported a H:G:S ratio of 14:80:6 in M. polymorpha (Fig. 5).
Mosses are known to contain specialized water-conducting cells, called
hydroids (Ligrone et al., 2000). These cells lack cytoplasmic content and are
highly elongated. The presence of lignin has not been detected, but hydroids
are assumed to contain polyphenols (Espiñeira et al., 2011; Miksche and
Yasuda, 1978; Scheirer, 1980).
Ligrone et al. (2008) used anti-lignin labelling to detect lignin in bryophytes.
The signal was stronger in the case of GS lignin antibodies than G lignin ones,
especially in the mosses Sphagnum cuspidatum and Dendroligotrichum
Fig. 5. Lignins in bryophytes. (A) Whole aspect of the thallus of Marchantia

polymorpha. (B) Mass spectrum and gas chromatography profiles (inner box) of the
thioethylated monomers (erythro and threo isomers) arising from aryl-glycerol-b-aryl
ether (b-O-4) structures derived from sinapyl alcohol from M. polymorpha lignins.
dendroides, whose signal given by the immunocytochemical detection was

similar to that in vascular plants. However, it was reported that phloroglucinol
(Wiesner reagent) staining, commonly used to detect lignin, was negative in
Sphagnum novozelandicum (Kremer et al., 2004). A similar outcome was
described by Ligrone et al. (2008), where the phloroglucinol test was negative
for several bryophytes, which, according to the authors, indicates the absence
of cinnamaldehydes, the main target of phloroglucinol (Pomar et al., 2002),
but a categorical absence of lignin cannot be ruled out (Ligrone et al., 2008).
It has been reported (Xu et al., 2009) that the first appearance of the entire
lignin biosynthetic pathway enzymes (excluding the pathway that leads to
syringyl lignin formation) from the catalysis of phenylalanine to coniferyl
alcohol formation, took place in mosses (Physcomitrella patens). However,
the presence of lignin in more basal plants cannot be ruled out, as the analysis
these authors performed was based on searching for sequences with Arabi-
dopsis gene homologs as templates for lignin biosynthetic gene identification
(Xu et al., 2009). Though this kind of study is highly useful, enzyme-coding
genes that could have arisen from convergent evolution are not considered.
Moreover, the number of fully sequenced species is very limited, most of
them are angiosperms, and only a few basal species have a fully sequenced
genome. As mentioned above, the presence of lignin has been reported in a
red alga and a marchantiopsid, neither of them fully sequenced species.
2. Lycophytes
Arising at least 400 mya, this lineage appeared in the fossil record very soon
after the first appearance of vascular plants. During the Carboniferous
period (345–290 mya), lycophytes were especially diverse and abundant,
dominating coastal swamps of tropical lowlands (Bateman et al., 1998).
The remains of these plants account for our major coal deposits.
Currently, there are some 1280 species of lycophytes belonging to three
major lines: Lycopodiaceae, which is homosporous, Selaginellaceae and
Isoetaceae, both of them heterosporous. Lycopodiaceae are terrestrial or
epiphytic, cosmopolitan and most diverse in tropical and alpine habitats.
Selaginella (Fig. 6) comprises 750 species, mostly terrestrial, herbaceous and
perennial, and mostly occurring in tropical forests, although many species
are able to survive long periods of drought. Isoetes is the only living remnant
of the clade that included the giant lycopods of the Carboniferous period.
Isoetes has retained the cambium and some secondary growth and it has
rootlets that resemble those of the extinct trees. This family has both terres-
trial and aquatic members, with a nearly cosmopolitan distribution (Judd
et al., 2008).
The distribution of syringyl lignins among extant lycophytes is not
uniform or entirely understood. Syringyl lignin derivatives cannot usually
be detected in extant Lycopodiaceae (i.e. Lycopodium) by chemical depoly-
merizing methods such as derivatization followed by reductive cleavage
(DFRC; Weng et al., 2008) or cupric oxide alkaline oxidation (Logan and
Thomas, 1987). It is perhaps evolutionarily significant that syringyl lignin
Fig. 6. Lignins in lycophytes. (A) Detail from Selaginella martensii frond.

(B) Maüle staining of transverse sections of Selaginella stems. Red and brown colours
indicate the presence of S and G lignins, respectively. Bars ¼ 300 mm. (C) Two-
dimensional HSQC NMR spectra of acetylated cellulolytic enzyme lignins from
Selaginella whole stems, revealing the presence of syringyl lignins. C box is taken
from Weng et al. (2011), reproduced with permission from ASPB.
derivatives have not been found in the extinct arborescent lycophyte Sigil-
laria ovata (Logan and Thomas, 1987). However, lignins, and more specifi-
cally, syringyl lignins, were reported by chemical degradative methods in the
herbaceous Isoetes and Huperzia (Towers and Gibbs, 1953; Towers and
Maass, 1965) and, more conclusively, by chemical depolymerizing methods
including thioacidolysis in Selaginella and Isoetes (Espiñeira et al., 2011).
The presence of S units in some lycopods is well known and has been
associated with the existence in the xylem of angiosperm-like vessels
(Schneider and Carlquist, 2000). Selaginella is a representative of the Lyco-
podiophyta and one of the earliest divergent clades of extant vascular plants
(Pryer et al., 2001). Using ozonation, acidolysis, infrared spectroscopy and

1
H NMR, Jin et al. (2005) reported the presence of S lignin in Selaginella
tamariscina, with an S/G ratio of 1.3. In Selaginella martensii, by means of
nitrobenzene oxidation, Gross (1980) detected 44% S units. Gómez Ros et al.
(2007a) reported 71% S units in S. martensii fronds, using thioacidolysis, with
an H:G:S ratio of 5:24:71. 2D NMR analysis revealed a 2:22:76 ratio, and a
remarkable distinction between the Selaginella cortex (with S/G of 3.53)
and the xylem (S/G 0.06) (Fig. 6).
Syringyl units in Isoetes histrix represented 27% of the total lignin building
blocks, a composition strongly recalling that of angiosperm lignins. The
presence of syringyl units in Isoetes lignin is, however, consistent with the
occurrence of angiosperm-like vessels (Schneider and Carlquist, 2000). How-
ever, although I. histrix showed syringyl lignins, this was not the case with
Isoetes fluitans, in which thioacidolysis revealed that lignins are exclusively
constituted by G units (Espiñeira et al., 2011). Clearly, the case of Isoetes
illustrates that the evolutionary gain of syringyl lignins is far from being
understood, at least according to the current models for lignin evolution.
3. Euphyllophytes
Living euphyllophytes belong to two major clades: the seed plants (sperma-
tophytes) and a clade that includes several ‘fern’ lineages, the horsetails and
the whisk ferns (monilophytes) (Pryer et al., 2004b; Rothwell and Nixon,
2006). The monilophytes include five major lineages: leptosporangiate ferns,
Marattiales, Ophioglossales, Psilotales and equisetophytes. The common
name ‘fern’ is applied to the members of Leptosporangiatae, Marattiales
and Ophioglossales (the last two constitute the so-called eusporangiate ferns;
Judd et al., 2008). Leptosporangiatae represent more than 12,000 living
species, whereas Marattiales represent 150 living species, mainly plants of
the wet tropics. The Ophioglossales contain 80 species, Psilotales include
about 15 species, as well as equisetophytes (horsetails; Judd et al., 2008).
Horsetails were present in the Devonian period but became much more
abundant and diverse in the Carboniferous period, when some of them
became impressive trees. The position of equisetophytes within the monilo-
phytes remains uncertain (Pryer et al., 2004a).
a. Ferns. Ferns are a monophyletic group, which include 300 genera and
9000 species ranging in size from a few centimetre to 20 m tall (Judd et al.,
2008). Ferns are widespread, especially in temperate regions and the tropics.
Monilophytes can be terrestrial, epiphytic or aquatic.
There are few data available concerning pteridophyte lignin, but the
presence of guaiacyl units has been confirmed in several ferns, such as
Pteridium and Phyllitis (Espiñeira et al., 2011). Interestingly, some fern

species do not follow this previously established pattern. For example, Azolla
caroliniana is an aquatic floating fern which has been described, by means of
pyrolysis and thermally assisted hydrolysis, as not containing lignin in leaves
or in roots (Nierop et al., 2011). However, some polyphenolic compounds
have been found, which show resemblance to tannins, and the authors
hypothesized these polyphenolic compounds might have a structural role
similar to lignin (Nierop et al., 2011).
Ceratopteris constitute a group of aquatic ferns that possess both trac-
heids and vessels in the conducting tissues (Carlquist and Schneider,
2000). The evolutionary and functional significance of the presence of
vessels in an aquatic fern with maximum availability of water still remains
unsolved. Espiñeira et al. (2011) studied the lignin composition, through
thioacidolysis and nitrobenzene oxidation analysis, of two species of the
genus Ceratopteris. Ceratopteris thalictroides contained only guaiacyl lig-
nin in their cell walls, whereas Ceratopteris cornuta also contained syr-
ingyl units (Fig. 7), reaching 48% of the total lignin building blocks.
However, C. cornuta is not the sole exception among ferns, as syringyl
lignins have been reported in Dennstaedtia bipinnata, a tree fern, by
means of the less diagnostic cupric oxide alkaline oxidation (Logan and
Thomas, 1985). Neither the habit nor the ecology of the species seems to
be, at first sight, connected with the distribution of this biochemical
characteristic.
Horsetails are a monophyletic group among monilophytes, with a vascular
system forming an eustela consisting of bundles surrounding a hypothetical
central axis. Equisetum telmateia, only showed lignins of the guaiacyl type
(Espiñeira et al., 2011), whereas other Equisetum species, such as Equisetum
fluviatile, have already been described as containing syringyl lignins (Logan
and Thomas, 1985).
The presence of non-expected compounds in Equisetum cell walls is also
reflected in polysaccharide composition. The mixed-linkage (1 ! 3),(1 ! 4)-
b-D-glucan (MLG) which was thought to be restricted to poales cell walls has
recently been found in the horsetail Equisetum arvense (Sørensen and Willats,
2008). MLG is thought to have emerged from convergent evolution (Popper
and Tuohy, 2010; Sørensen et al., 2010).
b. Spermatophytes (seed plants). The spermatophytes are by far the most

diverse lineage within vascular plants, with about 270,000 living species. All
the spermatophytes share the production of secondary xylem (at least ances-
trally) through the activity of the cambium (Judd et al., 2008). The first
spermatophytes appeared in the late Devonian period (more than 360 mya;
Fig. 7. Lignins in pteridophytes. (A) Detail of a Ceratopteris cornuta plant.

(B) Maüle staining of transverse sections of Ceratopteris stems. Red and brown
colours indicate the presence of S and G lignins, respectively. (C) Mass spectrum
and gas chromatography profiles (inner box) of the thioethylated monomers (erythro
and threo isomers) arising from aryl-glycerol-b-aryl ether (b-O-4) structures derived
from sinapyl alcohol from C. cornuta lignins.
Judd et al., 2008). There are two major lineages of spermatophytes: gymnos-
perms and angiosperms (flowering plants). Most of the spermatophyte diver-
sity is accounted for by just one subclade: the angiosperms.
Gymnosperms. Despite the large number of phylogenetic studies, the rela-

tionships between cycads, ginkgos, conifers and gnetophytes still remain
uncertain from morphological and molecular data (Doyle, 1998; Nixon
et al., 1994; Stefanovic et al., 1998). These lineages are encompassed under
the term ‘gymnosperms’ or ‘naked seed’. Some molecular data indicate that
these groups of naked seed plants form a paraphyletic clade, sister to angios-
perms. However, most molecular datasets support the monophyly of current
gymnosperms (Bowe et al., 2000; Chaw et al., 2000).
All gymnosperms except Gnetales have only tracheids in their xylem.

Angiosperms and Gnetales also have vessels. All gymnosperms are woody
and are the dominant vegetation in many colder and arctic regions.
Cycads are considered as the most basal gymnosperms, and currently they
are represented by 130 species, though they were more diverse during the
Mesozoic period (Rai et al., 2003).
Ginkgo biloba (Fig. 8) is the sole surviving species of Ginkgoales. During
the Mesozoic period it was most abundant, but today it is hardly known in
the wild. The most ancient fossils date from the late Permian period (260–
250 mya). G. biloba still maintains archaic reproductive characteristics, with
a type of fecundation that it shares with Cycadales.
Fig. 8. Lignins in an S-type gymnosperm, Ginkgo biloba. (A) Leaves from

G. biloba. (B) Cytochemical localization of lignins in suspension cell cultures of
G. biloba stained with acriflavine as seen by confocal laser scanning microscopy.
(C) Mass spectrum and gas chromatography profiles (inner box) of the thioethylated
monomers (erythro and threo isomers) arising from aryl-glycerol-b-aryl ether (b-O-4)
structures derived from sinapyl alcohol from G. biloba suspension cell culture lignins.
Conifers are the most diverse group among gymnosperms, with about 600
species.
Gnetales contain only 75 living species, divided into three lineages: Ephe-
dra (distributed in the deserts around the world), Gnetum (found in tropical
forests) and Welwitschia (restricted to south-western Africa). Gnetales share
some characteristics with angiosperms, such as opposite leaves, vessels, a
short life cycle and insect pollination. However, gnetophytes never became
a significant component of vegetation and underwent a dramatic decline
during the late Cretaceous period (Crane, 1996; Crane et al., 1995).
Until recent years, the general hypothesis was to consider that gymnosperms
contain mainly guaiacyl lignin, with a variable proportion of p-hydroxyphenyl
units. In gymnosperms, the presence of S lignin has been widely reported, but
in residual amounts, representing 1–2% of total lignin (Gross, 1980). However,
G. biloba suspension cell cultures contain S lignin in a proportion as high as
11%, as detected by thioacidolysis and nitrobenzene oxidation (Fig. 8).
However, the woody tissues of Ginkgo are not able to synthesize S lignin
(Novo-Uzal et al., 2009). A species belonging to Gnetopsida, Ephedra viridis,
has been reported to contain 60% S units and only traces of H units, a pattern
analogous to angiosperms (Gómez Ros et al., 2007a). This similarity has been
linked to the presence of angiosperm-like vessels in gnetales (Gross, 1980). The
xylem in gnetales emerged through convergent evolution (Chaw et al., 2000).
Angiosperms. Angiosperms (flowering plants) contain 257,000 species and

account for most of the green plants, land plants and seed plant diversity.
This monophyletic group underwent a major radiation starting in the early
Cretaceous period (Crane et al., 1995; Doyle and Donoghue, 1993). The
oldest angiosperm fossils are pollen grains from 135 mya. Angiosperms
developed some new and important features that probably had a deep impact
on flowering plants diversity: seeds covered with a carpel, reduced female
gametophyte, double fertilization and the presence of vessels as a water-
conducting system. However, the first angiosperms did not exhibit vessels,
as suggested by the presence of tracheids in the basal angiosperm Amborella
(Field et al., 2000). Angiosperms did not become widespread until the early
tertiary period, when the radiation of modern birds and mammals occurred;
the evolution of large colourful fruits and seeds are associated with the
evolution of these groups.
Angiosperm lignins are constituted mainly of guaiacyl and syringyl units,
with a minor proportion of p-hydroxyphenyl units. Thus, angiosperms,
whose radiation came after divergence between angiosperms and gymnos-
perms, have always been considered as the sole phylogenetic group containing
syringyl lignin. Nevertheless, in recent years many reports have seemed to
question this long held point of view. These data suggest syringyl lignins have
emerged at least five times during plant history by convergent evolution (in
algae, bryophytes, lycopods, gymnosperms and angiosperms) or that syringyl
lignins are an ancient feature that emerged in algae, even before land coloni-
zation and was lost a posteriori, except in some specific groups and angios-
perms. Recent data appear to point to convergent evolution, which suggests
that syringyl lignins play a key role in plant physiology, even though the
precise functions of S lignin in the plant have not been unravelled. In angios-
perms, most syringyl lignins are located in the vessel fibres, whereas xylem is
mainly composed of guaiacyl lignin, which suggests strong selective pressure
towards the presence of G lignin in conducting tissues (Peter and Neale, 2004).
In a similar fashion, the Selaginella xylem contains guaiacyl lignin, while
syringyl lignin is restricted to epidermal and subepidermial/cortical tissues
(Fig. 6). An analogous pattern has also been shown in some ferns, such as
Ceratopteris (Fig. 7). Some reports have associated the induction of S lignin
biosynthesis as a response against pathogens (Wuyts et al., 2006), whereas
other authors describe an increase in lignin amount after pathogen challeng-
ing, but due to an increase in H and G units, while S units remain unaltered
(Gayoso et al., 2010; Pomar et al., 2004). The structural differences between
G and S lignin are caused by the presence of the methoxyl group at the
5-position, which results in S lignin being more linear and less condensed.
Bonawitz and Chapple (2010) have suggested S lignin confers flexibility to
plants. The flexible polymer may be important for herbaceous plants, which
every year grow their aerial biomass and therefore must grow quickly, a
requirement woody angiosperms and gymnosperms do not have.
Some lignins are known to be acetylated at the g-carbon of the side chain.
Recently, it has been reported this acetylation occurred naturally in lignins,
to a greater extent than previously thought (del Rı́o et al., 2007; Ralph, 1996).
Acetylated lignin does not derive from acetylation of the growing polymer of
lignin, but from acetylated monolignols themselves, which are incorporated
into the lignin by the typical radical coupling polymerization process (Lu and
Ralph, 2002, 2008). del Rı́o et al. (2007) reported the occurrence of naturally
acetylated lignins in all studied angiosperms, both woody and herbaceous,
but did not detect it in either of two gymnosperms (pine and spruce). The
presence of acetylation was restricted to the g-carbon and occurred predom-
inantly on S units, whereas acetylated G units were barely detected, though
bamboo and eucalyptus were some exceptions (del Rı́o et al., 2007). Thus,
lignin acetylation was constrained to angiosperms and to species with a high
S/G ratio, as shown by the occurrence in abaca, kenaf and sisal (del Rı́o et al.,
2004). Lu and Ralph (2002, 2008) established g-acetylated monolignols alter
lignin structure because the g-OH group participates in some post-coupling
reactions, such as rearomatization of the quinone methide following b–b0

coupling and, if the g-OH group is acetylated, this reaction is no longer
possible resulting in the production of other novel b–b0 -coupled units in the
polymer. The physiological role of acetylated lignins remains unclear, al-
though their involvement in drought tolerance has been suggested, as the
acetylated lignin polymer is more hydrophobic than normal lignin, which
could make vascular tissues more hydrophobic reducing water loss. This
hypothesis is supported by the presence of acetylated lignin in some succulent
agaves (del Rı́o et al., 2007). However, the latter role is not widely accepted,
as there are plants with highly acetylated lignin that are not drought tolerant.
Monocotyledonous angiosperms. Usually, monocots show the most com-

plex lignin composition among the phylogenetic clades. Besides H, G and S
units, some monocotyledons contain important quantities of p-coumarate
esters (Ralph et al., 1994). For example, grasses have high concentrations of
ferulates and p-coumarates acylating cell wall polymers (Grabber and Lu,
2007), and in maize samples ferulate represents 4% of the lignin (even though
it solely acylates polysaccharides), whereas p-coumarate (which acylates both
polysaccharides and lignins) represents 3% (Hatfield et al., 1999; Saulnier
et al., 1999). Ferulate and diferulate cross-links contribute to cell wall stiff-
ening, the cessation of plant growth and reduced enzymatic hydrolysis
(Grabber et al., 1998; Schopfer, 1996) and may act as nucleation sites for
lignin formation (Grabber et al., 2002). Most p-coumarates acylate the side
chains of mainly S units in lignin and could reach up to 20% of lignin in some
C4 grasses (Ralph et al., 1994). p-Coumarate concentrations vary consider-
ably among tissues, with extremely low levels in pith parenchyma, scleren-
chyma and vascular tissues (Hatfield et al., 1999). Most p-coumarates remain
as terminal units with an unsaturated side chain and a free phenolic group
(Ralph et al., 1994), and are supposed to play a role in oxidative cross-linking
of cell walls in response to pathogen attack (Grabber and Lu, 2007).
The deposition of p-coumaroylated lignins is associated with growth ces-
sation in grasses (Müsel et al., 1997), but the role of p-coumarate in lignifica-
tion has not been fully ascertained (Grabber and Lu, 2007). Catalytic
amounts of p-hydroxycinnamic acids or their esters are reported to enhance
sinapyl alcohol oxidation by apoplastic peroxidases (Hatfield et al., 2008;
Takahama et al., 1996). In maize, an 8% increase in syringyl lignin has been
reported after adding catalytic amounts of methyl p-coumarate (Grabber
and Lu, 2007).
All the monocots studied have shown the presence of G and S units. In
some cases, the presence of lignin is of evolutionary importance. For exam-
ple, Posidonia oceanica is found in marine habitats, where it forms large
underwater meadows in the Mediterranean Sea. Posidonia belongs to a group

of land angiosperms that returned to the sea in the Cretacic period, 100 mya
(Klap et al., 2000; Larkum and Den Hartog, 1989). The P. oceanica lignin
composition mirrors the angiosperm pattern, with an H:G:S ratio of 8:30:62
(Espiñeira et al., 2011), despite its aquatic habitat (Fig. 9).
The higher complexity of monocot cell walls is not only restricted to lignin
composition but also polysaccharide compounds. In their primary cell wall,
poales possess more xylan and less polysaccharides derived from galactose and
fucose than other angiosperms. They also contain MLG, which was thought
to be a unique component of poales cell walls (Sørensen and Willats, 2008).
The concentration of expansin is much higher in cell walls from poales
than in dicots and is absent in bryophytes. This distribution links the pres-
ence of expansin to xylem development, and suggests a divergence of the
genes before the appearance of angiosperms (Carey and Cosgrove, 2007).
Fig. 9. Lignins in an aquatic monocotyledon, Posidonia oceanica. (A) View of

several Posidonia plants. (B) Mass spectrum and gas chromatography profiles (inner
box) of the thioethylated monomers (erythro and threo isomers) arising from aryl-
glycerol-b-aryl ether (b-O-4) structures derived from sinapyl alcohol from P. oceanica
lignins.
III. EVOLUTIONARY VIEW OF S LIGNIN

BIOSYNTHETIC PATHWAY AND TRANSCRIPTIONAL
REGULATION
A. ENZYMES
In recent years, several studies have deepened investigations into the presence
of S lignin biosynthetic pathway enzymes in different phylogenetic groups.
The obtained results may be crucial in assessing the evolutionary history of
syringyl lignins.
There are two enzymes that divert the flux of coniferyl alcohol to the
synthesis of S lignins: ferulate 5-hydroxylase (F5H) and caffeic acid/
5-hydroxyferulic acid O-methyltransferase (COMT). F5H catalyses the
5-hydroxylation of coniferaldehyde and coniferyl alcohol (Humphreys et al.,
1999; Meyer et al., 1998) and had only been identified in angiosperms. How
non-angiosperms were able to form syringyl lignin without the existence of a
F5H was a mystery until recently, when Weng et al. (2008) reported the
presence of a F5H-like enzyme in Selaginella moellendorffii. This enzyme
(SmF5H) is a cytochrome P450-dependent monooxygenase, like angiosperm
F5H, and it is able to complement Arabidopsis F5H-deficient mutants. Unlike
angiosperm F5H, SmF5H is able to also catalyse the 3-hydroxylation of
p-coumaraldehyde and p-coumaryl alcohol directly; complementation of the
Arabidopsis C3H mutant led to an intriguing lignin composed almost solely of S
and H units, practically devoid of G units. Independence from the need for the
enzymes that usually participate in the synthesis of coniferaldehyde and con-
iferyl alcohol (HCT and C30 H) points to a non-canonical lignin biosynthesis
pathway in Selaginella, where SmF5H has the capacity to form syringyl lignin
from p-coumaraldehyde and p-coumaryl alcohol (Weng et al., 2010). More-
over, SmF5H is not orthologous to angiosperm F5H, and has probably arisen
in a convergent evolution process (Weng et al., 2008, 2010). Hence, SmF5H is
functionally similar to angiosperm F5H but phylogenetically independent.
COMT catalyses the O-methylation of 5-hydroxyconiferaldehyde and
5-hydroxyconiferyl alcohol to form sinapaldehyde and sinapyl alcohol.
A novel COMT, distantly related to angiosperm COMTs, has been discov-
ered in S. moellendorffii. Interestingly, the SmCOMT encoding gene clusters
with SmF5H gene and shares an upstream regulatory region that may be
under the control of common cis-regulatory elements. This regulated coordi-
nation does not seem to exist in angiosperms (Weng et al., 2011).
The last step of lignin biosynthesis is the oxidation of monolignols, which
is driven by laccases (Berthet et al., 2011) and mainly peroxidases (Fagerstedt
et al., 2010). Both acidic and cationic peroxidases can oxidize p-coumaryl
and coniferyl alcohol (Ros Barceló et al., 2004). However, this situation is
not so clear in the case of sinapyl alcohol; in this case, typical acidic perox-
idases, with some exceptions (Christensen et al., 1998), are generally regarded
as poor catalysts.
Theoretical comparative models between Zinnia elegans S peroxidase and
Arabidopsis thaliana G peroxidase showed two important differences
(Fig. 10). Helix D0 in Arabidopsis fixes the motif which determines the
conformation and hydrophobicity of the substrate-binding site (Østergaard
et al., 2000) and it is absent in S peroxidases (Ros Barceló et al., 2007). On the
contrary, there is a novel b-strand in S peroxidases that influences the
catalytic centre of the enzymes. All of these factors are likely to condition
the substrate specificity of S peroxidases, determining the unique catalytic
properties (Gómez Ros et al., 2007a; Ros Barceló et al., 2007).
Gómez Ros et al. (2007a) determined the structural motifs of S peroxi-
dases, by alignment of the amino acid sequence of a set of peroxidases whose
capacity for oxidizing S moieties is well known (Gabaldón et al., 2005;
Quiroga et al., 2000; Sasaki et al., 2006; Takeda et al., 2003) with two typical
G peroxidases (Nielsen et al., 2001; Østergaard et al., 2000).
Searching through protein databases for other S peroxidases in angios-
perms showed that the determinants of S peroxidases have been conserved
Fig. 10. Predicted 3D structure of the Arabidopsis thaliana ATP A2 G peroxidase

(A) and the Zinnia elegans S peroxidase (B). Adapted from Ros Barceló et al. (2007),
reproduced with permission from Elsevier.
not only in angiosperms but also in Gnetales, which bear G,S lignins; in
Coniferales, such as Pinus sylvestris, a species that lacks S lignins; in basal
gymnosperms (Cycadales and Ginkgoales); in basal vascular plants, such as
the terrestrial lycopod S. moellendorffii and the aquatic fern Ceratopteris
richardii, both containing G,S lignins, in non-vascular plants such as
P. patens and M. polymorpha (Gómez Ros et al., 2007a; Logan and
Thomas, 1985; Ros Barceló et al., 2007). The presence of S peroxidases in
M. polymorpha is in concordance with the occurrence of S-type lignin and
lignin-like substances in the cell wall of liverworts (Espiñeira et al., 2011;
Logan and Thomas, 1985).
It has been suggested that S peroxidases are not as exceptional as previ-
ously assumed, and their presence predates not only the gymnosperm–
angiosperm divergence but also the radiation of tracheophytes itself. This
observation suggests that the genes coding these enzymes predate the appear-
ance of vascular plants (Duroux and Welinder, 2003; Gómez Ros et al.,
2007a), and might even be contemporaneous with the acquisition of the
most primitive short-distance water and nutrient transport systems that
coevolved with mosses and liverworts (Ligrone et al., 2000). Because the
main characteristic of the S peroxidases is the absence of steric restrictions
at the substrate-binding site, these observations support the existence of an
ancestral basic model of cell wall architecture and function, that would have
evolved before the evolutionary divergence of bryophytes, ferns and seed
plants (Gómez Ros et al., 2007a; Ligrone et al., 2002), and that have been
finely tuned in response to specific evolutionary pressures. In this model, G
peroxidases constitute the most evolved state, as would be expected from the
constraints that arise to increased enzyme specificity, during plant evolution.
B. TRANSCRIPTIONAL REGULATION
Lignified cells are an important carbon sink and unable to expand due to
lignin deposition, so lignification must occur after cell division. Given the
metabolic cost of building lignin polymer, along with the persistence and
properties of lignified cells, the timing and localization of lignification must
be strongly regulated (Rogers and Campbell, 2004). In recent years, many
advances have been made in the knowledge of the transcriptional regulation
of lignification. Some transcription factors that regulate the formation of
secondary cell wall formation have been identified, as well as regulators of
lignin biosynthesis. MYB46 regulates secondary cell wall formation in Ara-
bidopsis (Zhong et al., 2007) and upregulates two transcriptional factors,
MYB58 and MYB63, that are specifically involved in lignin biosynthetic
regulation in Arabidopsis (Zhou et al., 2009). Both MYB58 and MYB63
are able to regulate all the genes involved in the lignin biosynthetic pathway
except F5H, a key enzyme in S lignin biosynthesis (Zhou et al., 2009). All
these transcriptional factors bind to AC elements placed in the promoters of
the genes involved in lignin biosynthesis (Raes et al., 2003), except C4H,
COMT and F5H. However, C4H and COMT are regulated by a lignin-
specific MYB transcription factor, so their regulatory regions are supposed
to contain AC elements (Zhao and Dixon, 2011; Zhou et al., 2009). Never-
theless, the gene encoding F5H is not reported to contain AC elements.
Instead, F5H, and therefore syringyl lignin biosynthesis, is directly regulated
by NST1/SND1 (Zhao et al., 2010), which has previously been shown to
regulate MYB46, as well as other transcription factors involved in secondary
cell wall regulation (Zhong et al., 2006). It was suggested that MYB46 was
already present by the appearance of F5H, and NST1/SND1 evolved to
coordinately activate secondary cell wall machinery and F5H (Zhao et al.,
2010). There are no homologs of NST1/SND1 in gymnosperms, ferns,
mosses or algae, a fact that supports the evolution of F5H after
gymnosperm–angiosperm divergence (Zhao et al., 2010). However, the ge-
nome has been fully sequenced only in a few non-angiosperm species, so the
presence of both F5H and NST1/SND1 cannot be ruled out. On the other
hand, S. moellendorffii has been shown to possess an enzyme able to produce
syringyl lignin that is not homologous to angiosperm F5H (Weng et al.,
2008). Hence, this species (and perhaps other non-angiosperm species with
syringyl lignin in their cell walls) may have a different transcriptional net-
work regulating syringyl lignin biosynthesis.
IV. CONCLUSION
As discussed above, in the past decades the knowledge about lignins has
considerably increased. Lignin occurrence has been described in non-land
and non-vascular plants. Moreover, the presence of lignins in non-vascular
tissues of bryophytes and ferns, an observation with no counterpart in
gymnosperms or angiosperms (Gómez Ros et al., 2007a,b), supports the
view (Boyce et al., 2003; Friedman and Cook, 2000) that lignification might
have originated in the peripheral tissues of protracheophytes and was only
later co-opted to strengthen the tracheids in eutracheophytes. The presence
of lignins in peripheral cells of the red alga Calliarthron also seems to confirm
this hypothesis (Martone et al., 2009). As a whole, this scenario is reminiscent
of Gould’s exaptation concept (Gould, 2002), and probably reflects what
Donoghue (2005) termed ‘developmental enablers’, that is, early changes in
sequences that open up new design options. This working hypothesis is
supported by recent advances in knowledge of transcriptional regulation of

the biosynthesis of lignins. The AC elements are conserved cytosine- and
adenosine-rich motifs that are present in the promoters of genes encoding
enzymes of the phenylpropanoid pathway. AC elements contained in the
promoter region of lignin biosynthetic genes are thought to enhance their
expression in the xylem and, at the same time, to prevent their expression in
peripheral tissues (Peter and Neale, 2004). Since the deletion of the AC
element results in de-repression of these genes in tissues foreign to xylem
tissues (Raes et al., 2003), it has been suggested that a tissue-specific repressor
(transcription factor) is normally bound to the AC element, preventing
expression in cells foreign to the xylem tissues (Leyva et al., 1992).
The presence of such transcription factors in tissues other than the xylem
might reflect the evolution of AC-rich element-containing promoters from a
primitive non-specific to a specific vascular promoter through the introduc-
tion of a transcription factor that suppresses foreign tissue expression.
On the other hand, syringyl lignins are distributed with an unknown pattern
among plants, from algae to angiosperms. As far as is known, angiosperms and
Selaginella have evolved two enzymes (F5H and COMT) that catalyse the
production of syringyl lignins and, given the presence of syringyl lignins in
liverworts, lycopods, ferns and basal living gymnosperms, it is possible that the
pathway for sinapyl alcohol has evolved (independently or not) several times
during the evolution of land plants. Alternatively, sinapyl alcohol may have
been incorporated into the earliest land plants, and was subsequently lost or
repressed (Novo-Uzal et al., 2009) in some but not all of the extant diverging
basal liverworts, lycopods, equisetopsids, ferns and even algae. The recruitment
of sinapyl alcohol for lignin biosynthesis would thus be the ‘primitive state’.
All the technical research advances described in this review led to a better
understanding about why, when and how lignins appeared in plants. Hope-
fully in the following years, the analysis regarding the presence and compo-
sition of lignins in new species, the sequencing of genes encoding lignin
biosynthesis enzymes and their gene regulation in several new and distantly
related species will let us know and understand, step by step, the evolutionary
history of lignins.
ACKNOWLEDGEMENTS
The authors thank Dr. Ignacio Bárbara for providing Posidonia images and
Teresa Martı́nez for assistance in the histochemical analysis. This work was
supported in part by a grant from Xunta de Galicia (INCITE08P-
xIB103182PR) and Ministerio de Ciencia e Innovación (BFU2009-08151).
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Evolutionary History of Lignins

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Dedication

ESTHER NOVO-UZAL,* FEDERICO POMAR,*,1

*Department of Animal Biology, Plant Biology and Ecology, University

Advances in Botanical Research, Vol. 61 0065-2296/12 $35.00

sclerenchyma. However, lignin composition varies among species, phylogenetic groups,

Lignins are abundant organic polymers, accounting for 25% of plant

A. LAND COLONIZATION AND APPEARANCE OF A LIGNIFIED

single-celled conduits with lignified and ornamented walls, either banded or

both in water transport from roots to leaves and in mechanical support. In

B. COMPOSITION AND CELLULAR DISTRIBUTION OF LIGNINS

Lignin is deposited mainly in tracheids, vessels, fibres of the xylem and

even seasonal growth. Different stresses provoke a change in lignin composi-

a higher proportion of H units (Fukushima and Terashima, 1991) and

II. LIGNIN OCCURRENCE IN THE DIFFERENT

This classical view of the distribution, composition and evolutionary history

Thioacidolysis is an acid depolymerization procedure, which acts

The application of these techniques to different plant groups has provided

presence of lignin in algae has never been previously reported. Calliarthron

B. EMBRYOPHYTES (LAND PLANTS)

Fig. 4. Lignin-like compounds in Coleochaetales. (A) Light microscopy image of

between haploid and diploid generations; multicellular structures that produce

anatomy of water-conducting cells of bryophytes, whose cell walls modify

bryophytes, as well as in charophytes, pteridophytes and gymnosperms, but

Fig. 5. Lignins in bryophytes. (A) Whole aspect of the thallus of Marchantia

dendroides, whose signal given by the immunocytochemical detection was

Fig. 6. Lignins in lycophytes. (A) Detail from Selaginella martensii frond.

(Pryer et al., 2001). Using ozonation, acidolysis, infrared spectroscopy and

Pteridium and Phyllitis (Espiñeira et al., 2011). Interestingly, some fern

b. Spermatophytes (seed plants). The spermatophytes are by far the most

Fig. 7. Lignins in pteridophytes. (A) Detail of a Ceratopteris cornuta plant.

Gymnosperms. Despite the large number of phylogenetic studies, the rela-

All gymnosperms except Gnetales have only tracheids in their xylem.

Fig. 8. Lignins in an S-type gymnosperm, Ginkgo biloba. (A) Leaves from

Angiosperms. Angiosperms (flowering plants) contain 257,000 species and

reactions, such as rearomatization of the quinone methide following b–b0

Monocotyledonous angiosperms. Usually, monocots show the most com-

underwater meadows in the Mediterranean Sea. Posidonia belongs to a group

Fig. 9. Lignins in an aquatic monocotyledon, Posidonia oceanica. (A) View of

III. EVOLUTIONARY VIEW OF S LIGNIN

Fig. 10. Predicted 3D structure of the Arabidopsis thaliana ATP A2 G peroxidase

supported by recent advances in knowledge of transcriptional regulation of

Carlquist, S. and Schneider, E. L. (2000). SEM studies of vessels in ferns Ceratopteris.

Friedman, W. E. and Cook, M. E. (2000). The origin and evolution of tracheids in

Larkum, A. W. D. and Den Hartog, C. (1989). Evolution and biogeography of

Meyer, K., Shirley, A. M., Cusumano, J. C., Bell-Lelong, D. A. and Chapple, C.

Scheirer, D. C. (1980). Differentiation of bryophyte conducting tissues: Structure and

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