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no impact on S-lignin biosynthesis (
Chen et al., 2006; Nakashima et al.,2008
),
suggesting that enzymes other than CCoAOMT are involved in this pathway,
at least in M. sativa.
ABSL assays with CCoAOMT-deficient pine TEs revealed, despite signifi-
cant suppression levels, only moderate reductions in lignin content of up to
20% (
Table I
;
Wagner et al., 2011
). This makes it likely that methyltrans-
ferases other than CCoAOMT exist in conifers that are capable of transfer-
ring methyl groups onto phenylpropanoid phenols. Conifers contain a
multifunctional methyltransferase called AEOMT that supports the methyl-
ation of caffeoyl-CoA in vitro (
Li et al., 1997
). However, the histological
expression pattern of a GUS reporter gene fused to the AEOMT promoter in
pine suggests that AEOMT is not involved in lignification (
Wagner and
Walter, 2004
). The expression profile of AEOMT during xylogenesis in
P. taeda also argues against an involvement of this enzyme in lignification
(
Anterola et al., 2002
). Methyltransferases encoded by COMT-like genes
with unknown function or CCoAOMT-like genes expressed in developing
xylem could potentially methylate caffeoyl-CoA in conifers, thus preventing
greater reductions in lignin content in CCoAOMT-RNAi experiments
(
Friedmann et al., 2007; Koutaniemi et al., 2007
).
Pyrolysis-GC/MS and 2D-NMR studies demonstrated that reductions in
lignin content in pine were due to a depletion of G-lignin. Correspondingly, the
proportion of H-lignin in CCoAOMT-deficient transgenic lines increased,
resulting in up to a 10-fold rise in the H:G-ratio relative to untransformed
controls (
Fig. 5
).
However, CCoAOMT suppression changed more than just the ratio of the
monolignols normally found in pine lignin. 2D-NMR experiments revealed
that CCR suppression also caused the incorporation of the caffeyl alcohol into
lignin, which resulted in a novel lignin polymer consisting of p-hydroxyphenyl
(H), catechyl (C) and guaiacyl (G) units (
Fig. 5
). The incorporation of caffeyl
alcohol into the lignin polymer provided experimental evidence for the pro-
posed enzymatic function of CCoAOMT in pine (
Fig. 2
) and the ability of
other lignin-related enzymes to utilise this novel metabolite. It is currently
unclear why suppression of CCoAOMT did not lead to the incorporation of
caffeyl alcohol into lignin in angiosperm species. A number of factors could
have interfered with the incorporation of caffeyl alcohol. For example, ortho-
diphenols such as caffeyl alcohol are highly reactive and likely to be subject
to oxidation by polyphenol oxidases and catechol dioxygenases (
Weng
and Chapple, 2010
), although this does not appear to be a problem with the
analogous 5-hydroxyconiferyl alcohol (
Morreel et al., 2004; Ralph et al.,
2001
). Also, caffeyl alcohol could form quinones, which interferes with
the radical coupling reactions required for the incorporation of monolignols
