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NMR and pyrolysis-GC/MS analyses revealed that reductions in lignin
content in 4CL-RNAi plants and TEs were mainly due to depletion of
G-lignin, which resulted in increased H:G-ratios in transgenic material
(
Table I
;
Wagner et al., 2009
). This result should however not lead to the
conclusion that conifers contain a 4CL unigene solely responsible for the
biosynthesis of H-lignin. The 4CL gene targeted for suppression in P. radiata
is the homologue of the P. taeda 4CL gene that was up-regulated during
compression wood formation, a wood type extremely rich in H-lignin
(
Nanayakkara et al., 2009
). 2D-NMR experiments revealed that changes in
monolignol composition in transgenic pine plants also caused modifications
in the lignin interunit linkage distribution. This included elevated levels of
b
-aryl ether (
b
-O-4) and spirodienone (
b
-1) units, which were accompanied
by lower levels of phenylcoumaran (
b
-5), resinol (
b
-
b
), and dibenzodioxocin
b
(5-5/4-O-
) units. A sharp depletion in the level of saturated (dihydroconi-
feryl alcohol) end groups was also observed.
Severe suppression of 4CL in pine plants also affected carbohydrate me-
tabolism. Monosaccharide analysis revealed an increase in galactose content
in affected plants (
Wagner et al., 2009
). However, similar increases in galac-
tose content could, despite substantial reductions in lignin content, not be
observed in 4CL-RNAi TEs. This phenotype was therefore identified as a
pleiotropic effect associated with reduced wood stiffness in plant stems,
which triggered compression wood and therefore galactan formation in
affected transgenic plants (
Wagner et al., 2009
).
Most surprisingly, transgenic plants with severe 4CL suppression displayed a
much (almost fivefold) higher proportion of bark relative to wood. In addition,
severely affected 4CL-RNAi plants contained, within the woody part of the
stem bands, flavonoid-rich axial parenchyma. LC-MS/MS experiments identi-
fied elevated levels of quercetin, quercetin methyl ether and kaempferol in
extracts of 4CL-RNAi plants (
Wagner et al.,2009
), and HPLC-based experi-
ments revealed elevated levels of quercetin glycoside and cyanidin glycoside
in developing TEs with suppressed 4CL levels (Wagner et al., unpublished
results). From a biosynthetic perspective it seems confusing that suppression
of 4CL in pine resulted in the stimulation of flavonoid biosynthesis (
Fig. 3
), as
the biosynthesis of flavonoids and proanthocyanidins requires 4CL activity
(
Boudet, 2007; Dixon et al., 2005; Ku and Mun, 2007
). However, as conifers
contain a small 4CL gene family (
Friedmann et al.,2007;Koutaniemiet al.,
2007
), it is quite possible that different 4CL unigenes are responsible for
monolignol and flavonoid biosynthesis in conifers. This would also explain
why suppression of the lignin-related 4CL gene did not interfere with flavonoid
biosynthesis in pine plants (
Wagner et al.,2009
). However, it is still surprising
that the redirection of the metabolic flux triggered in 4CL-RNAi plants caused
