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bm6-3 is mutated in the highly conserved 188GXGGV(L)G193 motif essen-
tial for NADPH binding. The genotyping analysis of bm6ref mutant revealed
a premature STOP codon resulting in impaired nucleotide binding sites in
addition to many of the residues that form the substrate binding sites. It was
shown that bm6-27 has nucleotide insertions that cause a frameshift resulting
in the formation of a protein with non-conservative substitutions in the last
31C-terminal residues. Bm6 mutants show up to 22% less KL ( Pillonel et al.,
1991 ). Enzymatic saccharification of bm6 stover results in a 7% increase in
glucose yield. This example exemplifies how genome sequencing may help to
speed up the cloning of bm mutant.
CAD mutants have also been characterized in C3 grasses. The rice gold hull
and internode (gh2, first observed in 1917) mutant was identified recently as a
lignin-deficient mutant by a map-based cloning approach ( Zhang et al., 2006 ).
GH2 locus encodes a CAD gene that differs by one amino acid (554G/554A)
substitution near the NADPH binding site. Although the mutant shows
dramatic differences in residual CAD activity, the S/G ratio is not substantially
changed between gh2 and WT. KL analysis showed that the total lignin
content of the gh2 plants was reduced by 5-6% compared with the WT plants.
Interestingly, Li et al.(2009) reported a new mutant named T-DNA-tagged
ricemutant flexible culm1 (fc1) whose phenotype displays a dramatic reduction
in mechanical strength. fc1 carries a T-DNA in OsCAD7 (Loc_Os04g52280), a
member of a clade not described previously (and different from the GH2 clade).
This mutant shows a slight decrease in height and fertility. Lignin is strongly
reduced to about 82% of WT levels as well as a 14% reduction of cellulose
content. As observed in gh2 mutants, the S/G ratio was not substantially
reduced however, H units were greatly reduced by 36.4%. Noteworthy, despite
a significant reduction of CAD activity possibly linked to its delay in its
development, fc1 does not show any reddish-brown color in stem as observed
in other CAD mutants suggesting an absence of accumulation of cinnamalde-
hydes. Therefore, it is plausible that this gene is not completely redundant with
GH2 and may be involved in an unknown function in lignin biosynthesis.
More recently, RNA-mediated silencing of CAD was induced in switch-
grass. In the transgenic plants, CAD activity in stems of silenced lines was
significantly reduced ( Saathoff et al., 2011 ). Although lignin was not studied
in detail, quantity of overall lignin in addition to cutin was found significant-
ly below WT levels. Recent unpublished data suggest that Brachypodium
mutants obtained after chemical mutagenesis and modified in the genomic
sequence of a putative CAD gene (Bradi3g1653) show similar lignin char-
acteristics as bm1 or/and bm6 lines ( Bouvier d'Yvoire, 2011 ). The two CAD
Brachypodium mutants first identified for their brown-stem phenotypes and
high saccharification yield show a strong decrease in the S/G ratio, drastic
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