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the primary substrates for 3-hydroxylation of the phenolic moiety. In contrast,
4-coumarate, 4-coumaroyl-CoA, or the corresponding aldehyde and alcohol
were poorly or not metabolized (
Franke et al.,2002a;Nairet al.,2002;Schoch
et al.,2001
). The Arabidopsis CYP98A3 converts the shikimate ester most
efficiently, but the quinate ester of 4-coumarate is also converted with appre-
ciable activity. This defined CYP98A3 as 4-coumaroyl-shikimate/quinate-
3
0
-hydroxylase (C3
0
H, because quinate and shikimate are cyclic as well, the
phenolic moiety becomes annotated as the prime [
0
]ring).Thus,C3
0
Halso
catalyses the final step of the biosynthesis of chlorogenic acid (caffeoyl-
quinate), an abundant antioxidant that accumulates in many plants
(
Petersen et al.,2009
). However, functional proof that C3
0
H is also the central
3-hydroxylase of the phenylpropanoid pathway came from a phenotypic
analyses of the ref8 mutant: Soluble sinapoyl malate and sinapoyl choline
levels are drastically reduced in the mutant leaves and seeds, respectively. In
the ref8 mutant, total lignin content was reduced to 20-40% of wild-type levels
and both G- and S-units were found only in trace amounts (
Franke et al.,
2002b
). Instead, the mutant accumulates almost exclusively 4-coumarate-
derived H-units, which are found only in minute amounts in wild-type lignin.
Instead of incorporating H-lignin into cell walls that normally produce G/S
lignin, it now appears that regular H-lignin biosynthesis is taking place early in
inflorescence stem development of the ref8 mutant, while only small amounts
of H monolignols are incorporated into walls that normally would produce S-
or G-lignins (
Patten et al.,2010
). The inability of the ref8 mutant to produce
G- and S-lignins clearly established that the 3-hydroxylation of the monolignol
pathway occurs at the level of the shikimate ester of 4-coumarate in Arabi-
dopsis rather than on the level of the free acid or CoA-ester. In parallel, a
transferase belonging to the BAHD superfamily had been identified in tobacco
that was shown to catalyse the synthesis of 4-coumaroyl-shikimate (and
-quinate) from 4-coumaroyl-CoA (
Hoffmann et al.,2003
). The same enzyme
also efficiently catalyses the interconversion between caffeoyl-shikimate and
caffeoyl-CoA and was thus named hydroxycinnamoyl-CoA: shikimate/qui-
nate hydroxycinnamoyltransferase (HCT). HCT downregulation also causes
reduction of G- and S-lignins in several plants and leads to a lignin mainly
composed of H-units (
Besseau et al.,2007;Puet al.,2009;Shadleet al.,2007
).
Together, the discovery of HCT and C3
0
H immediately suggested that
caffeoyl-CoA is synthesized from 4-coumaroyl-CoA via coumaroyl-shikimate
and caffeoyl-shikimate instead of from free caffeic acid (
Fig. 1
). An involve-
ment of C3
0
H in both G- and S-lignin units has been confirmed in other species
both on the enzyme activity level and by reverse genetic approaches. Down-
regulation of C3
0
H in alfalfa (M. sativa) and hybrid poplar (Populus grand-
identata
alba) resulted in strong reduction in total
lignin and a drastic