From Plant Cell Walls to Biofuels—Arabidopsis thaliana Model - Bioenergy Crop Plants - page 81

be efficiently activating sinapate in addition to the regular 4CL substrates such as 4-coumarate,
caffeate, and ferulate. Studies are underway to fully elucidate the functions of all four At4CL gene
family and other genes with homologous sequences in Arabidopsis . A notable study in this regard
is the expression and functional characterization of 11 genes (including the four At4CL members)
that were identified to be a putative multigene 4-coumarate:CoA ligase network that could be tak-
ing part in the formation of syringyl lignin and a sinapate/sinapyl alcohol derivative (Costa et al.
2005). Another enzyme functional characterization study by Ehlting et al. (2001) investigated the
domain structures in At4CL1 and two isoforms and the functions of these domains. These studies
led to the identification of two adjoining putative functional domains, substrate-binding domains
I (sbd I) and II (sbd II), in these isoforms and to the understanding of their roles in substrate rec-
ognition and binding. Few studies have been done toward elucidating the expression patterns of
the A. thaliana 4CL gene family members except for the report by Soltani et al. (2006), which
describes the regulatory roles played by multiple cis-regulatory elements in complex patterns of
4CL expressions developmentally or up on induction by wounds.
The first step in lignin monomer synthesis in the lignin branch biosynthetic pathway is catalyzed
by cinnamoyl CoA reductase (CCR). Isolation of CCR cDNA was first carried out from Eucalyptus
gunnii (Boudet and Grima-Pettenati 1996). The same group later carried out its cloning, expression,
and in-depth phylogenetic relationship analysis (Lacombe et al. 1997). In A. thaliana , two different
cDNA clones, namely AtCCR1 and AtCCR2 , were reported later (Lauvergeat et al. 2001). AtCCR1
and AtCCR2 proteins shared approximately more than 80% sequence identity. The A. thaliana
CCR isoform, AtCCR1 , is engaged in the constitutive lignification process and AtCCR2 is impor-
tant for disease resistance in plants by its possible roles in the biosynthesis of phenolics (Lauvergeat
et al. 2001). The AtCCR1 Arabidopsis mutants [i.e., irregular xylem4 ( irx4 ) mutants] are severely
lignin-deficient, with a dramatic wall phenotype that affects the mechanical properties of the stem
(Jones et al. 2001). This Arabidopsis irx4 mutant has also been reported to have a much delayed but
normal lignification process (Laskar et al. 2006). More recent studies using Arabidopsis transfer
DNA (T-DNA) knockout mutants for CCR1 emphasize phenotype differences in these mutants (Mir
Derikvand et al. 2007). Interestingly, the phenylpropanoid pathway in these mutants is redirected
to feruloyl malate, resulting in the formation of lignin with altered structure. These results, from
a bioenergy research point of view, are significant because they show that the phenylpropanoid
metabolism in Arabidopsis is flexible, thereby encouraging strategies for developing plants with
modified and less recalcitrant lignin structures.
The last step in the biosynthesis of the lignin precursors is the conversion of cinnamaldehydes to
the corresponding alcohols. This step is catalyzed by the enzyme cinnamyl alcohol dehydrogenase
(CAD). Isolation of the full-length cDNA of CAD was first reported by Walter et al. (1988) from
elicitor-treated cell cultures of bean ( Phaseolus vulgaris L.). Later, using poplar cDNA sequences,
Baucher et al. (1995) screened the Arabidopsis genome to report the genomic nucleotide sequence of
an AtCAD gene encoding a CAD. The most popular Arabidopsis genome database, The Arabidopsis
Information Resource (TAIR), annotates approximately 17 genes with putative CAD functions that
belong to a CAD multigene family. However, subsequent studies by Kim et al. (2004) functionally
reclassified this putative Arabidopsis gene family and demonstrated that only 9 of these 17 mem-
bers could actually be considered as members of the AtCAD family. These studies further explored
the functions of all nine AtCADs and found that the two Arabidopsis CAD genes, AtCAD5 and
AtCAD4, had the peak activity and shared approximately 83% homology with previously charac-
terized CADs from various plant species. Additionally, functional redundancy of various AtCADs
in Arabidopsis lignin biosynthetic pathways was confirmed using studies with AtCAD5, AtCAD6,
and AtCAD9 knockout mutants for various growth and developmental stages (Kim et al. 2004). The
reports on such functional redundancy in a set of CAD genes thus directs the attempts of making
lignin modified plants (that may turn out as less recalcitrant crops suitable to the biofuel industry) by
focusing on targeting multiple CAD knockouts to attain a significant change in the lignin composi-
tion. More functional studies on AtCAD4 and AtCAD5 genes hint as to their roles as main genes