Biology Reference
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case of Arabidopsis, where point mutations in the coding region should affect
C4H activity equally in all cell types. Other possible explanations beyond
experimental differences include differential feedforward effects on later down-
stream enzymes, metabolic channelling of precursors into distinct pathways
mediated by specific isoforms of the general phenylpropanoid pathway, or
sensing of precursor pool sizes and adjustment of branch-pathway activities on
the transcriptional or posttranscriptional level. However, none of these spec-
ulations has been convincingly addressed experimentally to date.
B. AN APPARENT DETOUR TO S- AND G-LIGNIN: THE
3-HYDROXYLASE OF THE AROMATIC RING
meta- or 3-hydroxylation of phenylpropanoid precursors is necessary not
only for the biosynthesis of G- and S-units of lignin but also for the genera-
tion of UV-absorbing compounds such as sinapoyl malate and for the
formation of many other bioactive compounds, for example, chlorogenic
acid, rosmarinic acid, or coumarins (
Vogt, 2010
).
1. A surprising twist in the lignin pathway
Originally, it was believed that the 3-hydroxylation occurs on free 4-coumaric
acid yielding caffeic acid, or on the level of the corresponding CoA-thioesters
(
Fig. 1
). Multiple classes of enzymes were proposed to catalyse the reaction,
but none had been characterized down to a purified enzyme (for review, see
Ehlting et al., 2006
). Among them, P450 enzymes have been suggested to
catalyse the 3-hydroxylation of quinate and shikimate esters of 4-coumarate
yielding chlorogenic acid and caffeoyl-shikimate, respectively (
Heller and
K¨hnl, 1985; K¨hnl et al., 1987
). But only in the early 2000s, these enzymes
were characterized at the molecular level and an involvement in lignin
monomer biosynthesis was elucidated: the CYP98A3 gene form A. thaliana
was identified independently by functional genomics and classical genetic
approaches to encode the 3-hydroxylase of the phenylpropanoid pathway.
Schoch et al. (2001)
and
Nair et al. (2002)
employed a candidate gene
approach based on sequence and expression similarity to C4H, while
Franke et al. (2002a)
identified CYP98A3 via map-based cloning of the
reduced epidermal fluorescence 8 (ref8) mutant, which had been identified
based on the lack of fluorescence caused by sinapate ester in wild-type Arabi-
dopsis leaves. This forward genetic screen proved to be very efficient in
identifying mutants in several genes of the phenylpropanoid pathway. The
CYP98A3 gene was shown to be expressed predominantly in lignifying tissues,
similar to other phenylpropanoid genes and recombinant protein expressed
in yeast showed that the shikimate and quinate esters of 4-coumaric acid are
