Download Understanding Lignification: Challenges Beyond

Future Perspectives in Plant Biology
Understanding Lignification: Challenges Beyond
Monolignol Biosynthesis1
Xu Li and Clint Chapple*
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
Lignin, a major component of vascular plant cell
wall, provides mechanical support for plants to stand
upright and enables xylems to withstand the negative
pressure generated during water transport. Although
important for plant growth, the presence of lignin
limits access to cell wall polysaccharides and thereby
negatively affects human utilization of biomass such
as its use as livestock feed, in paper manufacturing,
and in lignocellulosic biofuel production. Because of
its significant economic impact, lignin has been one of
the most intensively studied subjects in plant biochemistry.
Lignin is a heterogeneous phenolic polymer largely
composed of three major types of monomers (monolignols), p-coumaryl, coniferyl, and sinapyl alcohols.
During lignin deposition, monolignols are synthesized
in the cytoplasm, translocated to the apoplast, and
polymerized into lignin. Over the last two decades, the
biosynthesis of monolignols has been a major focus of
research on lignification. We now believe that the
monolignol biosynthetic pathway has been relatively
well elucidated, at least in angiosperms. Based upon
this knowledge, genetic engineering approaches have
been used to successfully modify lignin content and/
or composition in a variety of plant species (for review,
see Boerjan et al., 2003; Li et al., 2008).
Despite these advances, there remain significant
gaps in our knowledge of the lignification process.
Little is known about how monolignols are transported through the cell membrane to their polymerization sites. Neither are the catalysts involved in the
polymerization process itself well understood. It is
also unknown how lignification is restricted to certain
regions within the cell wall of a single cell. In addition,
it is still unclear how and why perturbation of lignification affects plant growth, an issue that must be
addressed if lignin modification is to be successfully
applied to the improvement of biofuel crops. Recent
reports of the presence of lignin in nonvascular plants
and red algae, as well as the identification of alternative routes for lignin monomer biosynthesis in nonangiosperms raise important questions about lignin
evolution (Martone et al., 2009; Espiñeira et al., 2010;
1
This work was supported by the Office of Science (Biological
and Environmental Research), U.S. Department of Energy (grant no.
DE–FG02–06ER6430).
* Corresponding author; e-mail [email protected].
www.plantphysiol.org/cgi/doi/10.1104/pp.110.162842
Weng et al., 2010). In summary, there are still great
challenges in lignin research. In this article, we will
focus our discussion on important topics that are
poised to become the new frontiers in lignin research.
MONOLIGNOL TRANSPORT
AND POLYMERIZATION
After their biosynthesis, monolignols must be transported to the cell wall where they undergo oxidation
and polymerization to form lignin. In contrast to our
detailed knowledge of monolignol biosynthesis, we
know little about how these compounds are moved
from the cytoplasm to the cell wall. The translocation
of small molecules across the cell membrane may
occur by at least three different mechanisms: exocytosis, transporter-mediated export, and diffusion. Golgiderived vesicles are known to be involved in exporting
some other cell wall components such as hemicelluloses to cell wall (Cosgrove, 2005). Whereas observations from some early feeding experiments have
suggested the involvement of Golgi and associated
vesicles in monolignol transport (Pickett-Heaps, 1968),
results from a recent autoradiography study aiming to
trace the spatial distribution of monolignols during
secondary cell wall development in lodgepole pine
(Pinus contorta) do not support the hypothesis that
monolignols are exported by this mechanism (Kaneda
et al., 2008). Considering that ATP-binding cassette
transporters are responsible for the translocation of
various secondary metabolites in plant cells (Yazaki,
2006; Rea, 2007), current thinking in the field favors the
idea that monolignols are exported to the cell wall by
membrane-bound transporters (Kaneda et al., 2008),
although in vitro partition experiments suggest that
monolignols may also be able to pass the cell membrane by diffusion (Boija and Johansson, 2006).
Once transported to the cell wall, monolignols are
oxidized to phenolic radicals that undergo polymerization by chemical coupling. Laccases and peroxidases
are thought to be the catalysts responsible for the
oxidation of monolignols (Boerjan et al., 2003). Indeed,
global transcript profiling and coexpression analysis
revealed several ATP-binding cassette transporter
genes and a set of laccase and peroxidase genes that
are coordinately expressed with monolignol biosynthetic genes in Arabidopsis (Arabidopsis thaliana) developing inflorescence stems (Ehlting et al., 2005). The
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Li and Chapple
recent identification of MYB58 and MYB63, two Arabidopsis transcription activators specific for lignin
biosynthesis (Zhou et al., 2009), highlights an alternative approach to discover candidate genes involved
in lignifications such as monolignol transporter and
oxidase genes. Overexpression of MYB58 or MYB63
under the control of the 35S promoter in Arabidopsis
leads to ectopic lignification of epidermal and mesophyll cells that are normally nonlignified, suggesting
that in addition to monolignol biosynthetic genes,
monolignol transporter and oxidase genes may also
be directly activated by these transcription factors.
Indeed, the expression of a laccase gene (LAC4), which
was among the 22 candidate oxidase genes identified
from the aforementioned gene coexpression study
(Ehlting et al., 2005), was found to be induced by
these two MYB transcription factors. It would be
interesting to test if disruption of LAC4 abolishes the
ectopic lignification exhibited in the above MYB58 or
MYB63 overexpression lines. In the future, similar
analysis may also facilitate the identification of monolignol transporter genes. The candidate genes identified from the above approaches hold great promise to
reveal the identity of the transporters and the oxidases
involved in monolignol transport and polymerization,
although genetic redundancy may hinder the reverse
genetic analysis of their functions.
It would also be interesting to determine by what
mechanisms lignin is directed to specific sites within
the cell wall including cell corners and regions that
undergo secondary thickening. Indeed, the regulation
of this deposition is so specific that the wall-thickening
pattern of tracheary elements can be used to help
identify in paleontological and forensic studies the
plant species from which tissue remnants are derived
(Lane et al., 1990). During secondary wall thickening,
microtubule bundles guide the positioning and movement of cellulose synthase on the plasma membrane to
deposit cellulose microfibrils at specific sites within
the wall where subsequent lignification occurs (Gardiner
et al., 2003; Wightman and Turner, 2008; Gutierrez et al.,
2009; Pesquet et al., 2010). It is unknown how lignin
deposition is directed to these sites. Future identification of monolignol transporters and oxidases may allow
us to monitor their localization in live cells using the
Arabidopsis in vitro tracheary element differentiation
system (Oda et al., 2005).
LIGNIN DEFICIENCY AND PLANT GROWTH
Lignin-deficient mutants or transgenic plants often
show reduced growth and, in severe cases, dwarfing
(Jones et al., 2001; Franke et al., 2002; Hoffmann et al.,
2004; Chen and Dixon, 2007). This growth defect has
been assumed to be a direct consequence of the lignindeficient xylem failing to support water transport and
deemed as an inherent limitation of the lignin reduction approach for biomass improvement. A recent
physiological study on the transgenic poplar (Populus
spp.) trees in which the p-coumaroyl shikimate 3#hydroxylase gene was silenced showed that their
xylem indeed has reduced hydraulic conductivity
and is more prone to collapse and cavitation; however,
instead of having increased water-use efficiency,
which is expected for drought-stressed plants, these
low-lignin plants have reduced water-use efficiency,
suggesting some other mechanisms may be involved
in plant response to lignin perturbation (Coleman
et al., 2008).
Recently, it was reported that blocking flavonoid
biosynthesis by RNAi silencing of the chalcone synthase gene (CHS) alleviates the dwarf phenotype that
results from RNAi silencing of a lignin biosynthetic
gene encoding hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase in Arabidopsis, suggesting that flavonoid-mediated inhibition of auxin
transport may be responsible for the growth defects
exhibited by lignin-deficient plants (Besseau et al.,
2007). In contrast, a recent study using the flavonoiddeficient Arabidopsis CHS null mutant instead of
CHS-RNAi demonstrated that the growth inhibition of
hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl
transferase-deficient plants as well as plants having
low levels of p-coumaroyl shikimate 3#-hydroxylase
activity is independent of flavonoid accumulation (Li
et al., 2010). As a result, the mechanism that connects
defects in monolignol biosynthesis and plant growth
inhibition remains an open question.
One possible explanation for the dwarf phenotypes
seen in lignin down-regulated plants may relate to the
fact that in addition to being polymerized to form
lignin, coniferyl alcohol is also a precursor of dehydrodiconiferyl alcohol glucosides, soluble compounds
shown cell-division promoting activity (Lynn et al.,
1987; Tamagnone et al., 1998). It is possible that suppression of monolignol biosynthesis results in deficiency of dehydrodiconiferyl alcohol glucosides that in
turn affect plant growth. Another interesting hypothesis is that the perturbation of the cell wall in lignindeficient plants may trigger a cell wall surveillance
system and elicit a general growth response. It is well
known that in yeast a cell wall integrity signaling
pathway orchestrates responses to cell wall changes
during growth and development (Levin, 2005). Like
lignin biosynthetic mutants, several Arabidopsis irregular xylem mutants that are defective in cellulose or
hemicellulose biosynthesis also have collapsed xylem
and abnormal growth (Brown et al., 2005). The recent
discovery that the growth inhibition exhibited by some
Arabidopsis cellulose-deficient mutants can be alleviated by knocking out a receptor-like kinase gene
THESEUS1 provides evidence for the involvement of
a cell wall integrity sensing and signaling system in
mediating growth response to cell wall defects in
plants (Hematy et al., 2007; Seifert and Blaukopf,
2010). Perhaps a similar mechanism is responsible
for the growth defects of lignin-deficient plants. Elucidating the details of the processes that result in
lignin-related dwarfing may open the door to much
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Future Challenges for Lignin Research
more extensive modification of lignification in biomass
crops than is currently possible and may significantly
improve the processing of plants grown for biofuel.
EVOLUTION OF LIGNIFICATION
The emergence of lignified water-conducting cells
has been considered as an important adaptation for
vascular plants to be able to thrive in terrestrial environments. The recent discovery of lignin in a bryophyte, the liverwort Marchantia polymorpha, expands
the distribution of lignification to nonvascular plants
(Espiñeira et al., 2010). Even more strikingly, lignin
was recently found in the cell wall of the red alga
Calliathron cheilosporioides, which shares a common
ancestor with vascular plants over 1 billion years ago
(Martone et al., 2009). These observations raise new
questions about the evolutionary origin and history of
lignification.
Similarly, recent research on the distribution of
lignin monomers in the plant kingdom challenges
our perception about the evolution of syringyl (S)
lignin monomer biosynthesis (Fig. 1). The incorporation of p-coumaryl, coniferyl, and sinapyl alcohols
gives rise to the three major types of lignin units found
in nature, p-hydroxyphenyl, guaiacyl, and S lignin,
respectively. Despite reports in older literature
(Towers and Gibbs, 1953; Erickson and Miksche,
1974; Logan and Thomas, 1985), S lignin has been
generally regarded to be characteristic of the angiosperms; however, recent studies using modern diagnostic methods confirmed that S lignin is also present
in gymnosperms and some basal vascular plants such
as lycophytes and ferns (Weng et al., 2008a, 2008b;
Espiñeira et al., 2010). Moreover, S lignin is also
detected in liverworts and red algae, which are more
distantly related to angiosperms (Martone et al., 2009;
Espiñeira et al., 2010).
The occurrence of lignification in liverworts and red
algae suggests that the genes required for lignin deposition evolved in their common ancestor before the
divergence of specific lineages (e.g. red algae or liverworts) and were subsequently lost in certain lineages
(such as green algae and mosses). Alternatively, red
algae and liverworts lineages could have independently evolved the biochemical pathways for lignification. Similar evolutionary scenarios can also be
envisaged to explain the phylogenetic distribution
pattern of S lignin. Convergent evolution of S lignin
biosynthesis between the lycophyte Selaginella and
angiosperms has been demonstrated by characterization of an S lignin biosynthetic enzyme, ferulic acid
5-hydroxylase (F5H) from the lycophyte Selaginella moellendorffii. The Selaginella F5H belongs to a phylogenetic
clad distinct from the angiosperm F5Hs, indicating
independent evolution of these enzymes (Weng et al.,
2008b, 2010). Obviously, additional research on the
genes, enzymes, and the pathways of monolignol synthesis in other plant lineages is needed to shed more
light on the evolution of this important process. The
ever-decreasing cost of genome sequencing will soon
make possible deep and broad comparisons of the
lignification toolkit at nodes of the plant family tree.
These studies will provide significant insight into how
and when phenylpropanoid metabolism in general
and lignification in particular arose and evolved.
CONCLUSION
The potential to reduce lignin’s negative impacts on
human uses of biomass has been and will continue to
be a major force that propels lignin research. Our
current understanding of lignification is mainly limited to the biosynthesis of the building blocks for lignin
in angiosperms. Future research on lignin biosynthesis
needs to be focused on the identification of the genes
Figure 1. Phylogenetic tree showing the distribution of lignification and lignin monomer composition among major plant lineages. It is evident
that S lignin is not restricted to angiosperms. The
distribution of S lignin within lycophytes, fern,
and gymnosperms is not uniform. Black squares
indicate no lignification. G, Guaiacyl lignin; H,
p-hydroxyphenyl lignin.
Plant Physiol. Vol. 154, 2010
451
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Li and Chapple
involved in monolignol transport and polymerization,
the mechanisms that connect lignin biochemistry and
plant growth and development, and a broadening of
our understanding of evolutionary aspects of lignification. This knowledge will further improve our ability to manipulate lignification in biomass feedstocks
for human uses and give us a better understanding of
how this important polymer contributed to the dominance of vascular plants in terrestrial environments.
Received July 15, 2010; accepted July 20, 2010; published October 6, 2010.
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