Environmental Engineering Reference
In-Depth Information
The pathways, models, and mechanisms in lignin biosynthesis were delineated by earlier studies
on phenylpropanoid metabolism by dynamic groups such as Hahlbrock's laboratory in Germany
and others as reviewed earlier (Hahlbrock and Scheel 1989). Recent studies with mutants in the
A.
thaliana
lignin biosynthesis pathway have revised and refined these models.
Cinnamate-4-hydroxylase (C4H) and ferulate-5-hydroxylase (F5H) are the two cytochrome
P450 (CytP450)-dependent monooxygenases that catalyze hydroxylation reactions in the plant
phenylpropanoid pathway (Meyer et al. 1996). The first CytP450-dependent monooxygenase of the
phenylpropanoid pathway in
Arabidopsis
is C4H. The expression patterns of
C4H
had been thor-
oughly studied earlier in
A. thaliana
(Bell-Lelong et al. 1997). These studies reported that
C4H
is extensively expressed in different tissues of
Arabidopsis
.
C4H
expression is specifically high in
roots and cells undergoing lignification (Bell-Lelong et al. 1997). Furthermore, accumulation of
C4H message (mRNA level), according to these studies, was observed to be dependent on light.
Another interesting observation made by Bell-Lelong et al. (1997) is the sequence similarity of
several putative regulatory motifs in the
C4H
promoter with motifs in promoters of other phenyl-
propanoid pathway genes. More recently, Chen et al. (2007) successfully cloned two genes encod-
ing C4H from oilseed rape (
Brassica napus
) using sequence identity analysis of the corresponding
Arabidopsis
C4H
gene.
Studies show that the expression of the other CytP450-dependent monooxygenase, F5H (that
catalyzes an irreversible step of the hydroxylation reaction in the lignin biosynthesis pathway redi-
recting ferulic acid away from G lignin biosynthesis to sinapic acid and S lignin), does have an
influence on the monomeric composition of lignin. Evidence of this was provided by ectopic F5H
overexpression studies in
A. thaliana
(Meyer et al. 1998). It was found that tissue specificity of
lignin monomer accumulation was negatively influenced by ectopic F5H overexpression (Meyer
et al. 1998). Again, when F5H was overexpressed under the control of a lignification-linked pro-
moter (C4H promoter) instead of the more routinely used 35S constitutive promoter, a lignin with
almost entirely syringylpropane units was made (Meyer et al. 1998). Structural studies using nuclear
magnetic resonance (NMR) imaging on isolated lignins from
Arabidopsis
mutants deficient in F5H
(
f5h
) and
f5h
mutants overexpressing the
F5H
gene were done earlier (Marita et al. 1999) and con-
firmed the compositional and structural differences between these isolated lignins. These studies
indicate that altering F5H expression in biofuel crops could result in plants containing lignin with
an altered composition that might possess less recalcitrant traits.
Another phenylpropanoid pathway enzyme that has not been fully functionally characterized
in plants yet is
p
-coumarate-3-hydroxylase (C3H) or CYP98A3. Franke et al. (2002) reported that
lack of C3H function resulted in the reduced epidermal fluorescence phenotype (“ref” phenotype).
These mutants were called
ref8
mutants. The
ref8
plants contained lignin with significantly altered
composition causing developmental defects in plants and susceptibility to fungal pathogen attack
(Franke et al. 2002). These results therefore suggest that the phenylpropanoid pathway products
downstream of C3H do hold significance in normal plant development and disease resistance.
4-Coumarate:coenzymeA ligase (4CL) is a critical enzyme in the lignin biosynthesis pathway
that is involved in the synthesis of precursor molecules such as CoA thiol esters of 4-coumarate
and additional hydroxycinnamates. A
4CL
cDNA was first cloned from Parsley using enzyme
purification and a cDNA library screening approach (Ragg et al. 1981). Studies on stress and
developmentally regulated expression of the
A. thaliana
4-coumarate:CoA ligase (
4CL
) gene and
the identification of its cDNA sequence opened the way to the discovery of other family members
of this gene family (Lee et al. 1995). Subsequently, Ehlting et al. (1999) reported the cloning
and characterization of three members of the
4CL
gene family (namely
At4CL1
,
At4CL2
, and
At4CL3
) from
A. thaliana
. Their studies further established that these three members belonged
to two divergent evolutionary classes and encode isozymes with different substrate preferences
and specificities. More recently, Hamberger and Hahlbrock (2004) identified the fourth and final
member of the
At4CL
gene family that encodes the enzyme At4CL4. According to these stud-
ies, At4CL4 might be carrying out a distinct metabolic function because this enzyme seems to
