153
A 20th century roller coaster ride: a short account of lignification
Norman G Lewis
Addresses
Institute of Biological Chemistry, Washington State University, Pullman,
WA 99164-6340, USA; e-mail: [email protected]
Figure 1
R1
8
http://biomednet.com/elecref/1369526600200153
6
© Elsevier Science Ltd ISSN 1369-5266
Abbreviation
PRP
proline-rich protein
R1
R1
7
R1
1
R1
2
R1
3
R1
R
4
4
R1
OR 2
R1
9
Current Opinion in Plant Biology 1999, 2:153–162
R3
5
=
=
=
=
=
=
=
=
CH
R = H,
p-Coumaryl alcoho
2OH, R2 = 3R= 4
CH
R= H, 4R = OMe, Coniferyl a
2OH, R2 = 3
CH
R = OMe, Sinapyl alc
2OH, R2 = H,3 R= 4
CO
R = 4R = H,
p-Coumaric acid
2H, R
2 = 3
CO
R = H,4 R= OMe, Ferulic acid
2H, R
2 = 3
CO
R = OMe, Sinapic acid
2H, R
2 = H,3 R= 4
Me,2 R= 3R = H,4 R= OMe, Isoeugenol
CH
R H,4 R= OMe, Coniferi
2OH, R2 = Glc,
3 =
Current Opinion in Plant Biology
Introduction
There can be very few natural products that have evoked
as much scientific controversy as that associated with the
constitution and macromolecular assembly of the lignin
biopolymers, which are second in abundance only to cellulose. That such a situation could have arisen stems from a
number of confounding factors, misconceptions, and missteps that have plagued the lignin field for nearly half a
century. Today there are two quite disparate schools of
thought as to how macromolecular lignin configurations are
created in vivo. The one favored by the author is that of
full biochemical control over the outcome of phenoxy radical coupling in vivo, in harmony with that of other
biological systems. Another view contends that lignins are
not only randomly assembled but can freely exchange their
monomeric precursor units. How indeed could two such
diametrically opposite views co-exist at the present time?
This synopsis briefly reviews and explains the historical
development leading to both views, together with how the
discovery of dirigent proteins has shed light on the hitherto unknown biochemical control mechanisms involved in
radical-radical coupling reactions. As the next millennium
beckons, it seems that a beginning finally has been made
in identifying the fundamental mechanism through which
macromolecular lignin configurations are created in vivo.
Occurrence of lignins
Lignins are absent from algae, fungi, and mosses
(bryophytes) [1]. They apparently first emerged with the
appearance of ferns (pteridophytes), clubmosses, and
horsetails, but it was the evolution of the woody gymnosperms and angiosperms where they became most
abundant constituting from 20–30% of the entire plant
mass. Indeed, the term lignin was coined from the Latin
lignum (wood) and was used to describe the non-cellulosic
encrusting substances present in wood [2].
The lignins from woody gymnosperms are mainly coniferyl
alcohol derived together with small amounts of p-coumaryl
alcohol, whereas those from woody angiosperms mostly contain coniferyl and sinapyl alcohols as well as lower levels of
p-coumaryl alcohol [3] (Figure 1). Being ostensibly threedimensional structural biopolymers, they cannot readily be
Monolignols and related phenylpropanoids.
liberated from woody plant cell walls without substantial
bond-cleavage reactions first occurring. Interestingly, lignins
are nearly colorless. For example, sound spruce wood, which
contains ~28% lignin, is of an off-white color, and typically
lacks any significant amount of the colored non-lignin, nonstructural, heartwood infusions that are characteristic of
many other woody species. Grasses and herbaceous plants
also contain lignins, these being complicated by the presence
of hydroxycinnamic acids, such as p-coumaric and ferulic
acids, as well as other non-lignin phenolic substances [3].
The constitution of lignin
The first clue to lignin constitution was obtained in 1874
by Tiemann and Haarmann [4], who correctly determined
the chemical structures of both coniferyl alcohol and its
β-glucoside, coniferin. In 1933, Erdtman [5] made a farreaching conclusion, that was to be of central importance in
lignin biochemistry — namely that lignin resulted from the
dehydrogenation of its monomeric precursors. This
hypothesis was made following a re-investigation of an earlier study by Cousin and Hérissey in 1908 [6] on the nature
of the products obtained through the one-electron oxidation of isoeugenol. From this study, he correctly surmised
that lignins were formed by the dehydrogenation of phenolic precursors like coniferyl alcohol via a free-radical
coupling process. Interestingly, the existence of free-radicals had been determined in an unrelated study by M
Gomberg some thirty three years earlier [7], who immortalized his contribution with the statement “This work will
be continued as I wish to reserve the field for myself’’.
Freudenberg subsequently showed that the one-electron
oxidation of coniferyl alcohol in vitro gave a resonancestabilized intermediate which could undergo radical–radical
coupling to give, at least initially, the three racemic dilignol
products, dehydrodiconiferyl alcohol (8–5′ linked),
pinoresinol (8–8′ linked), and guaiacylglycerol 8–O–4′
coniferyl alcohol ether (8–O–4′ linked), with the latter being
formed in small amount (∼9%) [8] (Figure 2). Sinapyl alcohol, on the other hand, afforded essentially only the 8–8′
154
Discussion point
Figure 2
5
4
MeO
O
4′
O
7′′
8′′
OH
8
OMe
7
Dibenzodioxocin interunit linkage
5–5′ and 8–O–4′ coupling and
intramolecular cyclization (18-20%)
1
2
6
3
(R)
O
OH
9
5′
5
4
OMe
O
OMe
5 radical
8 radical
4–O radical
OH
MeO
OMe
HO
OH
O
OH
OH
OH
R
OMe
O
OMe
OMe
8–5′ coupling
O
R
OMe
O
8–O–4′ coupling
Intramolecular
cyclization
H2O
8–8′ coupling
O
Intramolecular
cyclization
MeO
OH
OH
HO
OH
HO
5′
OH
O
8
O
4′
OMe
RO
R = H, (±)-Dehydrodiconiferyl alcohols
R=
, 8–5′ interunit linkage (9-12%)
R
8′
8
OMe
OMe
OMe
O
8
R
O
HO
OR
OMe
R = H, (±)-erythro/threo Guaiacylglycerol
8–O–4′-coniferyl alcohol ethers
R=
, 8–O–4′ interunit linkage (50-70%)
R = H, (±)-Pinoresinols
R = OMe, (±)-Syringaresinols
Current Opinion in Plant Biology
Depiction of the main free-radical coupling reactions in vitro to give the corresponding dilignol dimers; estimated frequencies of linkages in lignins
are shown in parentheses.
linked syringaresinol [9]. At around the same time, Adler
and Miksche established the predominance of the 8–O–4′
interunit linkage in both gymnosperm and angiosperm
lignins, these approximating 50 and 70% respectively [10].
Subsequent attempts to satisfactorily duplicate lignin structure in vitro using monolignols and oxidative enzymes
(laccase and peroxidases) failed, with the ‘synthetic lignins’
having, for example, very low frequencies of 8–O–4′ interunit linkages. Indeed, this deficiency may have prompted
Kyosti Sarkanen in 1971 to propose that lignin biosynthesis
in vivo must occur through an end-wise polymerization
process, that is, whereby the 8–O–4′ linkage was preferentially formed by addition of coniferyl alcohol radicals to the
growing lignin chain [11]. Even today, the primary structure(s) of the lignins has not yet been fully established.
Brunow, however, recently discovered that the dibenzodioxocin subunit is a major component of plant lignins [12]
Indeed, it is now thought that, next to the 8–O–4′ inter-unit
linkage, both dibenzodioxocin and dehydrodiconiferyl
A short account of lignification Lewis
alcohol sub-units are the most frequent in natural lignins.
The question that thus arises is — how do plants control the
outcome of radical–radical coupling in such a way as to attain
the primary structure of natural lignins?
Cell wall initiation sites implicated in
lignin biosynthesis
In specific cells targeted for lignification (for example,
those ultimately leading to tracheids, vessels, phloem
fibers), both structural biopolymeric carbohydrates (cellulose, hemicelluloses, and pectins) and structural proteins
are laid down prior to lignin deposition. This occurs in such
a way as to establish the overall architecture of the secondary cell wall. Lignin deposition subsequently begins at
sites far removed from the plasma membrane, namely in
the cell corners and primary wall/S1 outer layer regions,
and then extends into the middle lamella and secondary
cell wall regions until completed.
Patterns of lignification are determined by a well-defined
distribution of lignin initiation sites within the cell wall
regions (see Donaldson [13]). Significantly, the lignin
domains ‘growing’ at each initiation site expand uniformly,
but at apparent constant density, until the neighboring
domains coalesce. This observation is contrary to an earlier view by Freudenberg that lignin formation occurs via
random, diffusion-driven, collisions of monomeric, dimeric, and higher oligomeric forms. Instead, domain growth is
indicative of a self-replicating mechanism which continues
once the primary structure has been established.
Lignin monomers are also differentially laid down in discrete regions of various lignifying cell wall types, which
suggests that some mechanism is in effect permitting discrimination between the incoming monolignols. This
differential deposition was discovered by Goring and coworkers using UV microscopy, where it was observed that
the lignin in birch wood vessel cell walls was mainly
derived from coniferyl alcohol, whereas in the fiber wall,
both sinapyl and coniferyl alcohols were incorporated [14].
Moreover, in a subsequent study of spruce wood, it was
concluded that middle lamella lignin embodies more
p-coumaryl alcohol units, in comparison to the secondary
wall lignin which was mainly coniferyl alcohol derived
[15]. These findings were independently confirmed and
extended through the deployment of radiolabeled monolignol precursors into developing xylem, with subsequent
microautoradiography of the resulting lignified sections
[16]. This again indicated that p-coumaryl alcohol was
preferentially laid down in the middle lamella/cell corners,
whereas coniferyl alcohol was mainly located in the secondary wall. Additionally, deposition of monolignols in the
vessels and fibers of angiosperms followed a similar trend
to that previously noted by Goring and co-workers.
Other lines of evidence support the concept that patterns
of lignin deposition, in terms of both monomeric constituents and presumed defined sequence(s) of inter-unit
155
linkages (primary structure), are fully pre-determined for
particular cell wall regions and cell wall types. For example, various lignin antibodies were raised against synthetic
lignin preparations, which differed in terms of the overall
frequencies of the 8–O–4′ inter-unit linkages relative to
that of other bond types; these were then employed to
ascertain if there were any notable differences in the antibody recognition of the lignins in discrete cell wall layers.
Although the precise structural ramifications were not
determined, two of the antibodies differentially crossreacted with the lignins present in the various cell wall
layers of a wheat metaxylem vessel [17]. This observation
can tentatively be considered to be indicative of different
primary lignin structures within discrete regions of the cell
wall. Moreover, in various grasses and herbaceous plant
species, the sinapyl alcohol content of the lignin
increases during maturation of the lignifying tissues and
p-coumarate residues also appear to be linked exclusively to
(the 9-hydroxyl group of) sinapyl alcohol moieties [18]. All
of these observations seem to be in keeping with a rather
precise mechanism of macromolecular assembly leading to
well-defined lignin configurations in vivo.
Lignin primary structures and dirigent protein
(arrays)
The observed patterning of lignins, in terms of both
monomeric constituents and presumed distinct primary
structures in discrete cell wall layers, raises a number of
important questions that need to be explicitly resolved:
first, what is the biochemical basis for the initiation sites?
Second, how are they able to discriminate between the
various monolignols? Third, how do they stipulate the
sequence of interunit linkages along the chain of the growing biopolymer. Fourth, how do the chains replicate?
Initially, it was considered that definition of the catalytic
properties of the (per)oxidase involved in monolignol oneelectron oxidation would resolve these matters. Because of
the very facile ability of these enzymes to catalyze the oneelectron oxidation of monolignols, however, at least five
different oxidative enzymes (peroxidases, laccases,
polyphenol oxidases, cytochrome oxidases and coniferyl
alcohol oxidases) became implicated as all being involved
in lignification (reviewed in [19]). None, however, faithfully reproduced native lignin structure(s) through in vitro
coupling/polymerization. Indeed, to our knowledge, there
is no other biological system that is claimed to involve at
least five different enzymes for the same catalytic step.
Dirigent proteins
Attention has more recently focused upon cell-wall glycoproteins, and whether they have any possible role in
stipulating the outcome of radical–radical coupling
processes to give lignins and related substances. This
interest stemmed from two perspectives: first, certain glycoproteins were noted to be translocated into cell walls at
points which temporally and spatially preceded the onset
of lignification [20,21]. Second, a glycoprotein from
156
Discussion point
Figure 3
OH
OH
OH
HO
OH
H
OH
H
H
OMe
H
O
coupling
oxidase
OH
O
O
OMe
OMe
OMe
O
Coniferyl alcoholproposed binding and orientation
of radicals to dirigent protein
OMe MeO
O
HO
O
OMe
(+)-Pinoresinol
Current Opinion in Plant Biology
Oxidase catalyzed generation of free-radicals and dirigent protein stipulation of outcome of radical–radical coupling.
Forsythia species was discovered which was able to, provided that external oxidative capacity was supplied (e.g. by
laccase), control both the regio- and stereochemical outcome of coniferyl alcohol derived phenoxy radical coupling
reactions [22]. The term, dirigent protein (from the Latin
dirigere, to guide and align) was introduced to describe this
phenomenon. That is, if a one-electron oxidase (such as
laccase) was used, only the corresponding racemic dilignols
would be formed. When the dirigent protein was also present, however, only stereoselective coupling at the 8 and 8′
positions was observed to give (+)-pinoresinol (Figure 3).
This finding was the first demonstration of a proteinaceous
system stipulating precisely the outcome of bimolecular
phenoxy radical coupling in vitro, and which also appeared
able to productively exploit the non-specific oxidative
nature of one-electron oxidants, such as laccase. The
mechanism presumed operative is unique, involving capture by the dirigent protein of free-radical intermediates,
which are bound and oriented in such a manner as to stipulate the outcome of radical–radical coupling [22].
The gene encoding the 18 kDa dirigent protein subunit
was obtained, and found to have no homology to any other
protein of known function; a finding in harmony with its
unique biochemical mode of action [23]. Moreover,
expression of the recombinant dirigent protein provided a
fully functional glycoprotein capable of engendering (+)pinoresinol formation when an oxidase (laccase) was
provided. The pinoresinol dirigent protein has a
Mr∼78 kDa, with a SDS PAGE subunit of ∼27 kDa rather
than 18 kDa; this difference is due to glycosylation.
Significantly, however, the regio- and stereochemical control of radical–radical coupling only occurred using
coniferyl alcohol, and not with either p-coumaryl or sinapyl
alcohols, indicating that the monomer binding site was
able to discriminate between the different monolignol
(radicals) [22]. It will be of much interest to establish
indeed whether there is one or two monomer binding sites
per the ~18 kDa non-glycosylated subunit. Interestingly,
dirigent protein genes have since been obtained from a
number of plant species (including loblolly pine [Pinus
taeda]) and are being examined for their biological roles.
([23]; and NG Lewis, unpublished data). There is every
indication that a class of these proteins exists stipulating
the outcome of various phenolic coupling reactions in vivo.
Origin of lignin primary structure
Consideration of how primary structures of lignins might
be propagated, must take into account the considerable
lignin heterogeneity within plant cell walls, and thus how
the variations in sequences of inter-unit linkages of lignins
are attained [23,24]. It is quite unlikely that the same dirigent proteins, governing regio- and stereo-selective
coupling, leading to (+)-pinoresinol, can directly participate in monolignol dehydrogenation to give the polymeric
lignins. This is because, in order to achieve what is
presently known about lignin structure, monomer dehydrogenative coupling must be able to occur with the
growing macromolecular chain.
Protein(s) determining macromolecular lignin configuration would be expected to contain arrays of adjacent lignin
binding sites which could stipulate both the linkages to be
engendered and the monolignol radical to be bound. The
primary structure of lignin, when defined in this manner,
could then undergo self-replication through a template
mechanism; indeed, preliminary evidence for lignin template polymerization has been obtained in vitro [25]. The
question is, therefore, whether there is any evidence for
proteinaceous dirigent arrays in lignifying cell walls. It is
known, for example, that a 33 kDa molecular weight
proline-rich protein (PRP) is both temporally and spatially
coincident with the sites of lignin deposition in developing
cell walls of Zea mays coleoptiles, as revealed using antibodies raised against both PRP epitopes and lignins [20]. A
A short account of lignification Lewis
comparable situation also holds for the differentiating protoxylem elements in Glycine max hypocotyls [21].
Accordingly, it has been considered that PRP’s might act as
‘scaffolds’ for lignification. Whether these encode arrays of
monomeric binding motifs stipulating lignin configurations, however, has not been investigated.
Other indirect evidence that structural glycoproteins might
possibly dictate how macromolecular lignin assembly occurs
has more recently been obtained using polyclonal antibodies raised against the Forsythia dirigent protein ([23];
V Burlat, M Kwon, LB Davin, NG Lewis, unpublished
data). It was anticipated that the antibodies might embody
sufficient flexibility to recognize both individual dirigent
proteins affording the lignans, as well as that which would
be part of a dirigent protein array involved in lignification.
Indeed, it was found that the dirigent protein polyclonal
antibodies recognized vascular tissues associated with lignification. These same tissues were also immunolocalized
using lignin antibodies. Most importantly, immunolabeling
of dirigent protein epitopes resulted in the identification of
two distinct sites of antibody recognition at the subcellular
level; the dirigent protein epitopes were mainly evident in
the S1 outer part [S1 layer] of F. intermedia xylem cell walls,
this being considered to be an initiation site for lignin
biosynthesis. Dirigent protein epitopes were also observed
in ray (living) cells, which more likely are mainly involved in
lignan formation as discussed later.
Taken together, these data indicate that dirigent protein
epitopes are laid down in specific subcellular locations
which are implicated in initiation of lignin (biopolymer)
and lignan (dimer) biosynthesis, respectively. Moreover,
the presence of the proposed dirigent protein arrays would
afford a novel mechanism for how macromolecular lignin
chains are initially created in vivo. Indeed, even apparent
lack of optical activity in lignins could also be explained in
this manner, if, for example, complementary chains were
produced via template polymerization. Accordingly, these
data suggest that a beginning has been made in identifying
how lignin primary structure is achieved, although much
remains to be done regarding fully delineating the biochemical mechanisms and processes involved.
Nevertheless, it would appear that lignin biosynthesis
proper can no longer be denied the involvement of proteins in determining the outcome of macromolecular
assembly from its monomeric precursors.
The random coupling concept: a
fundamental misconception
The random coupling concept is not viable on the basis of
various lines of reasoning [19] including those summarized
above. Yet the complexity of the lignin problem had
allowed such a view to prevail virtually unchallenged until
quite recently. As in all scientific endeavors, however, it
must be recognized that just because a biochemical solution to the question of free-radical coupling could not be
imagined some fifty years ago, it did not, of course, mean
157
that one did not exist. Indeed, it is worth noting that
Erdtman, who proposed in 1933 that lignins resulted from
monolignol dehydrogenation [5], had warned researchers
some 24 years later to exercise judgment in their studies of
lignin formation [26]. He clearly sensed that the scope of
lignin investigations (biosynthesis and structure) were so
restricted as to be incapable of distinguishing between
alternative working hypotheses.
Erdtman’s remarks turned out to be prophetic for a number
of reasons. First, in the early 1950s, Freudenberg had incorrectly asserted that synthetic ‘lignin’ preparations, obtained
by random coupling of monolignols in vitro were identical
to natural lignins. Moreover, this claim was repeatedly
made by Freudenberg over a span of nearly two decades, in
articles spanning numerous journals and languages [27–29]
and this view pervaded the field until quite recently.
The first clue that something was amiss with the random
coupling concept came with the series of unexplained revisions, in various reviews, of the actual amounts of the
8–O–4′ dilignol formed from coniferyl alcohol in vitro, relative to that of the 8–5′ linked dilignols. Over the space of
nearly two decades, an upward revision of its amount
increased from circa 10 to 60% without any identifiable,
experimental corroboration [30,31]: Yet, all previous [8]
and subsequent [32] studies showed that the 8–5′ linkage
actually prevailed in dilignol formation. These revisions
did, however, occur at the same time period as when
Miksche and Adler found that the frequency of 8–O–4′
linkages in naturally occurring lignins ranged from 50 to
70% (see Adler [33] for a review).
Freudenberg had also portrayed that monolignol precursors
and dimeric lignans accumulated in the cambial regions of
various tree species, prior to diffusing into adjacent lignifying
cells, although full experimental details were not forthcoming. Goldschmidt and Hergert [34], however, were unable to
confirm these findings, in spite of identifying numerous
other phenolic substances. The results of the Freudenberg
study are now even more puzzling, given Donaldson’s observations of growth of lignin domains in discrete regions of the
cell walls, which argues strongly against such a diffusiondriven process for macromolecular assembly.
Lignins were also claimed to be present in mosses [35], in
algae, and in fungi, with the former purportedly having a
lignin derived from p-coumaryl alcohol (as reviewed in
[1,3]). This was, however, not the case, and the phenolic
constituents of mosses [36] must be formed through quite
different biochemical pathways. Additionally, not only do
algae not contain lignins [37], there was no evidence for
monolignol forming pathway! Nor did fungal fruiting bodies
contain lignins; their metabolites were instead styrylpyrone-derived substances [38]. Thus, while recognizing that
there is no necessary connection between monomer identity and randomness of coupling of monomeric units, any
determination of the outcome of monomer coupling would
158
Discussion point
be difficult to bring to a convincing conclusion if the
monomers themselves could not even be identified.
Perhaps the most telling account of the Freudenberg random coupling concept is recorded in the putative monomer
flexibility of the hemiparasitic mistletoe plant to synthesize
its lignin [31,35]. It was claimed, again with no tangible
experimental corroboration, that mistletoe growing on gymnosperms formed a coniferyl alcohol derived lignin, whereas
when sustained on angiosperms, its lignin was composed of
both coniferyl and sinapyl alcohol units. Indeed,
Freudenberg’s acceptance of this remarkable account, given
to him by a member of his own research group, resulted in
him concluding that ‘these are examples of roles that lignins
play in taxonomy’. That is, he believed that the hemiparasite was able to suck up, from the host plant’s cambial sap,
the corresponding monolignol precursors. Mistletoe has
since, however, been demonstrated to biosynthesize a rather
unexceptional angiosperm lignin through its own biosynthetic processes [39]. Thus, the assertions originally made
for mistletoe lignin biosynthesis were untenable.
The Freudenberg group had, however, made the important
structural determinations of the various possible dilignol
structures, as well as subsequently showing that addition of
a monolignol radical to a dilignol radical in vitro generally
introduced a new 8–O–4′ linkage during trimer formation
[31]. However, their studies had not made even a beginning
in determining: whether a primary structure for lignin could
be established; what the molecular weight ranges of the natural lignins really were; and how the configurations of the
lignins were established in vivo. Instead, their in vitro experiments incorrectly led them to the belief that the properties
of lignin could be represented through a random coupling
regimen; today, this could be likened to the polymerization
of a peptide molecule in vitro and claiming the outcome to
be that of a naturally occurring protein or enzyme.
The misidentification of other metabolic
products as lignins
It was quite unexpected to recently note that the random
coupling concept for lignin macromolecular assembly
had been extended beyond the original definition of the
scope of randomness [40–42]. It was claimed that macromolecular lignin assembly could utilize other precursors
if normal monolignol biosynthesis was somehow
blocked, and the term ‘abnormal lignin’ was introduced
to describe this supposed phenomenon.
This claim resulted from a very preliminary and incomplete analysis of a loblolly pine (Pinus taeda) plant, which
was said to have mutated in such a way that it harbored an
‘abnormal lignin’ incorporating 2-methoxybenzaldehyde
and dihydroconiferyl alcohol units (Figure 4a). Although
the authors had not definitively determined what the
mutations actually were, it was perceived that it had resulted in the repression of cinnamyl alcohol dehydrogenase
which catalyzes the final step of monolignol (coniferyl
alcohol) biosynthesis [43]. The notion was next entertained [40–42] that the ‘plant simply needs a polymer with
required properties and that lignin’s composition is not
particularly significant’ [42]. Curiously, this perspective
was extended to the biosynthesis of hemicelluloses, which
were also claimed to be randomly assembled on the basis
‘that there may never be two (hemicellulose) molecules
that are identical’. Prior to that, it had been proposed there
could be as many as 1066 isomers in a lignin of Mr ~21,500,
this particular assertion being estimated as approximating
the number of atoms in the galaxy [44].
Such hypotheses, if ever widely adopted, would drastically
change current perceptions of how macromolecular cell
wall assembly might be attained, and accordingly also the
strategies to employ for achieving numerous biotechnological goals. There is, however, no known precedent for the
free interchange of monomeric units in any biopolymer
assembly, then or now, and no biochemical evidence for
any of these assertions was presented to document this
contention. As indicated at the beginning of the article,
this concept of the free-interchange of lignin monomeric
units is diametrically opposite to the working hypotheses
that guide our own research undertakings. Indeed, it is the
opinion of this writer that defining the actual mechanisms
associated with cell wall assembly, including the initiation,
polymerization, and termination steps in cellulose, hemicellulose and lignin biosynthesis, represents some of the
most important challenges facing plant biology as we
approach and enter the next millennium.
How, therefore, can such divergent viewpoints be reconciled, and is there any other explanation to account for the
claims of ‘abnormal lignins’? Actually, there are several,
none of which involve anything other than that already
known previously from the scientific literature (reviewed
in Gang et al. [45]).
As indicated earlier, the ‘novel lignin’ that was claimed to
be formed in the putative loblolly pine mutant purportedly had incorporated 2-methoxybenzaldehyde and ~30% of
dihydroconiferyl alcohol units. Further, it was proposed
that dihydroconiferyl alcohol formation occurred through a
speculative 1,4- and 1,2-reduction of coniferyl aldehyde
(Figure 4a), for which no experimental support was
offered. It has also been repeatedly stated that cinnamyl
alcohol dehydrogenase in P. taeda is encoded by a single
gene, although there is no convincing proof for this.
Indeed, a similar assertion had previously been made by
some of these researchers for phenylalanine ammonialyase in P. taeda [46]; this seems highly unlikely given that
the closely related jack pine (P. banksiana) contains at least
five classes of PAL genes [47].
Actually, the purported presence of 2-methoxybenzaldehyde
in the P. taeda ‘lignin’ resulted from the misassignment of
NMR spectral signals, and this particular claim has since
been fully retracted [48]. The dihydroconiferyl alcohol
A short account of lignification Lewis
159
Figure 4
(a)
(b)
H
OH
OH
O
OH
OH
"1,4-reduction"
followed by
"1,2-reduction"
free-radical
coupling
?
OMe
OMe
OH
Coniferyl
aldehyde
OH
Dihydroconiferyl
alcohol
OMe
OMe
Dihydrodehydrodiconiferyl
alcohol
OH
OH
OH
OH
OH
free-radical
coupling
reduction
O
x
OMe
OH
Coniferyl
alcohol
OMe
HO
OH
Coniferyl Dihydroconiferyl
alcohol
alcohol
(c)
2
O
+
OMe
OH
OH
O
OMe
HO
OMe
HO
OMe
Dehydrodiconiferyl
alcohol
OMe
Dihydrodehydrodiconiferyl
alcohol
Current Opinion in Plant Biology
(a) A recent postulate attempting to account for dihydroconiferyl
alcohol formation [40]. (b,c) More plausible biochemical
explanations (this paper) affording dihydrodehydrodiconiferyl
alcohol, through either (b) heterologous coupling of coniferyl alcohol
and dihydroconiferyl alcohols or (c) reduction of preformed
dehydrodiconiferyl alcohol.
component, on the other hand, was not detected as such, but
instead as part of a dihydrodehydrodiconiferyl alcohol substructure, a substance that we had already previously
described in P. taeda some years earlier [49]. Indeed, this and
related dihydrodilignols (e.g., from reduction of guaiacylglycerol 8–O–4′ coniferyl alcohol ether) and other dihydro
derivatives are well-known constituents of the Pinaceae [50].
Non-lignin, non-structural phenolic infusions
in woody plant tissues
Furthermore, the biosynthesis of coniferyl alcohol itself
could hardly have been blocked (Figures 4b,4c) and hence
there was no need to invoke a new pathway for lignin
biopolymer formation on this basis. The researchers had
failed to recognize that formation of dihydrodehydrodiconiferyl alcohol results from dehydrogenative dimerization
of a least one, and more probably two, coniferyl alcohol
molecules, depending on when reduction takes place.
Indeed, an ∼40 kDa enzyme, capable of catalyzing the
allylic bond reduction of dehydrodiconiferyl alcohol, to
afford dihydrodehydrodiconiferyl alcohol, has been purified to apparent homogeneity by my research group and its
properties will be described elsewhere. Moreover, lignans
modified as such will not be able to participate in the polymerization process leading to lignins. This is because of
differences in, for example, redox potential, as well as the
loss of reactive centers and the presumed inability to bind
dirigent protein (like) binding sites.
What, therefore, is the most plausible explanation for the
presence of the so-called ‘abnormal lignins’? To answer
this question, the reader must first recognize that wood is
heterogenous. It can contain sapwood, reaction wood,
heartwood, diseased wood, and discontinuities such as
those engendered by knots and branches, as well as having
imperfections and damage caused by herbivores,
pathogens, and other stresses. More importantly, however,
the non-structural phenolic constituents present in those
different tissues can vary substantially, in both type and
amount, varying not only within individual members of a
particular species, but also between plant species as well.
Unfortunately, there is a tendency to disregard such differences, as in the case of the ‘abnormal lignins’ [40–43]. In
that case, the entire wood sample, which was clearly heterogenous, was ball-milled into a fine powder, and then
treated as if it had been homogenous to begin with. More
then twenty years earlier, Hergert had cautioned against
such practices [51]. The reason for discussing this heterogeneity lies in the fact that, in addition to the structural
lignin biopolymers in secondary cell walls, woody plants also
evolved the means to form other specialized tissues and
metabolic products essential for prolonged survival. These
160
Discussion point
Figure 5
Cross section of an ebony wood stem, showing light colored sapwood
and black heartwood. (Photograph provided by L Shain and WE Hillis.)
include, for example, bark tissue as well as highly variable,
distinctive, heartwoods. A striking example is ebony
(Diospyrus species) which contains black-colored heartwood
and yellowish sapwood (Figure 5). It is the deposition of the
non-lignin phenolic constituents, which fulfill important
protective functions, that help enable woody plants to attain
lifespans ranging from decades to thousands of years.
Non-lignin, non-structural phenolic infusions (e.g. in heartwood) are sometimes erroneously described as ‘extractives’,
due to the fact that a portion can be removed through solvent extraction with the remainder being solubilized under
conditions normally used for lignin dissolution. Additionally,
it is often overlooked that heartwood constituents, which
make up the bulk of such non-lignin phenolics, are biosynthesized as a post-lignification, non-structural infusion
process. For example, the jet black phenolic constituents in
ebony heartwood are formed at some undetermined time
during growth and development, when lignification has
been completed. These substances first appear in the pith
region, but are successively deposited until circa 95% of the
sapwood is encompassed. Non-structural infusions can also
in some cases [e.g. Western red cedar (Thuja plicata)] constitute up to 20% of the dry weight of woody plants, and can
over a period of time become difficult to solubilize, and be
misidentified as lignins. In that case, even though fulfilling
no structural function, they can have Mrs >1000–9000 [52].
In the Pinaceae, such non-structural heartwood phenolic
polymeric/oligomeric substances have been known for
some time (reviewed in [50,53]). These encompass many,
if not all, of the characteristics of the claimed ‘abnormal
lignins’. Accordingly, the Ralph and Sederoff groups [48]
have now substantially modified their original claims of
having ‘abnormal and novel lignins’ by indicating that
“Whether this altered lignin is a true structural component
of the cell wall remains to be determined. Indeed, it has
been suggested (Gang et al. [45]) that the isolated lignins
[in particular for the mutant pine previously studied] may
represent partially polymerized phenolic extractives, similar to those that occur in heartwood.” The latter, however,
are not lignins, from either a biochemical, chemical or
physical (functional) point of view. They are distinct natural products from other biochemical pathways, and it
serves no use to describe them as lignins.
In this context, in 1949, Chattaway reported that heartwood
formation occurred through extrusion of substances from living (ray) parenchyma cells into the already pre-lignified
(dead) tracheary elements (vessels, fibers, etc.) [53]. This
deposition is generally initiated in the pith, but then over
time centripetally expands across the diameter of the woody
tissue, as those substances are biosynthesized at (or near to)
the expanding heartwood-sapwood transition zone interface.
The composition of the heartwood constituents, however, is
highly variable between species, but can contain (oligomeric) lignans, flavonoids, isoflavonoids, terpenoids and
alkaloids in various proportions and complexities of mixtures.
Indeed, their differential deposition helps to substantially
define the overall quality, color, odor, durability, and texture
of particular heartwoods of woody plant species. Indeed,
depending upon the heartwood, it could be anticipated that
typical lignin isolation protocols used would give rise to different ‘abnormal lignins’ for each species examined!
That such metabolites are definitively not lignins awaited
the onset of our own biochemical studies (reviewed in
[45,49,54,55]). In this regard, various members of the
Pinaceae (e.g., loblolly pine [55], Cryptomeria japonica, and
the Cupressaceae [e.g., Western red cedar] [54] utilize
coniferyl alcohol in metabolic pathways other than those
only leading to the lignin structural biopolymers. Instead,
they are also directed to pathways resulting in the formation of (oligomeric) lignans. These, in turn, can be utilized
to afford the corresponding heartwood constituents characteristic of their species.
Non-lignin phenolic substances, however, are not restricted to heartwood. Comparable, but more localized,
depositions can also occur in sapwood, when woody plants
are stressed or challenged by, for example, encroaching
pathogens and herbivores.
Lignins in transgenic plants
As with the need for circumspection in the interpretation
of the analyses of woody tissues, just as much care must
also be given to the study of transgenic plants that have
supposedly been lignin modified. The reasons for this are
as follows: firstly, it is often assumed by various researchers that down-regulation of a presumed lignin-specific
A short account of lignification Lewis
enzymatic step will only impact lignin composition and
content. This may be an incorrect assumption, however,
given that we have identified more than ten distinct
enzymes (and their corresponding genes) that metabolize
both monolignols and dilignols in processes other than
those leading to the lignins [23,50,54,55]. Indeed, most if
not all, plants contain (oligomeric) lignans in their flowers,
seed, stems and other plant parts, and thus any claim of a
lignin-specific enzyme requires particular scrutiny.
Secondly, given the wide range of phenylpropanoid
(acetate) pathway products formed in the plant kingdom,
care must also be exercised that it is the monolignol
biosynthetic pathway that is even being modulated.
Thirdly, perhaps the greatest need for caution resides in
the general application of existing protocols, such as those
originally designed for the partial dissolution of lignins
from true woody plant species, to herbaceous transgenic
plants, such as those obtained from tobacco. Such lignin
dissolution procedures can involve extensive ball-milling
of plant material over several days (e.g. in the presence of
dioxane-H20), and may also involve prolonged enzymatic
(e.g. cellulase) digestion treatment prior to or following
ball-milling [48]. Clearly, such treatments could lead to
artifacts as discussed elsewhere [45].
In the recent analysis [48] of presumed lignin-modified
tobacco transgenic plants, the isolation procedure gave a
preparation containing lignins, as well as small amounts of
feruloyltyramine-derived constituents and ∼15% hemicelluloses. However, only about 8.5% of the original lignin
was accounted for, and much would have to be done in
order to distinguish whether presence of feruloyltyramine
was an artifact. Feruloyltyramine is a well known metabolite in (stressed) tomato plants, as well as being a presumed
constituent of (suberized) cell walls in Solanaceous species
[50]. Accordingly, its presence in the partially purified
lignin preparations could result from its dissolution from a
non-lignified portion of the plant, with subsequent artifact
formation occurring through covalent linkages to the presumed lignin. It may even be present as an impurity, given
the ~15% hemicellulose content of the lignin sample.
Moreover, since its biosynthesis is quite distinct from that
leading to the monolignols, in much the same way as hemicelluloses and celluloses differ, it is not useful to suggest
that it could be construed as forming lignins.
Conclusions
The preceding discussions hopefully illustrate just some of
the obstacles encountered in lignin research over the past
fifty years. As the twentieth century comes to a close, it is
evident that this field was once quite unique in terms of
the perceived mode of a random macromolecular assembly
of the lignins. With the advances now being made in clearly delineating between distinct monolignol metabolic
pathways, leading to lignins and oligomeric lignans, and
the roles of cell-wall glycoproteins, it appears that much
progress can now be made in accurately delineating the
161
biochemical mechanisms involved. Put more succinctly,
the field is at a turning point.
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