#6

Cinnamoyl CoA reductase (CCR)

Originally, the cinnamoyl CoA reductases (CCRs) in various plant species have been considered to catalyze the NADPH-dependent conversions of p-coumaroyl CoA, feruloyl CoA and sinapoyl CoA into the corresponding aldehydes. Analysis of the Arabidopsis database, however, indicates that there are actually 11 (and possibly 12) putative CCR homologues annotated with greatly differing similarities and identities from 85.8 – 40.9% and 82.8 – 32.8%, respectively, relative to CCR1 (1). All of these are presumed to be cytosolic, since they have no signal peptide targeting sequences. Of these, only CCR1 and CCR2 have been partially characterized in Arabidopsis (24), with the former being implicated with a role in lignin biosynthesis. This is based on the following: a) its homology (75% identity) to a lignin-specific Eucalyptus gunnii CCR (24); b) its high affinity for feruloyl over sinapoyl CoA, as compared to CCR2; c) Northern blot analyses of CCR1 transcripts, which revealed its presence in stem and floral tissues, and to a much lesser extent, in leaf tissue; and d) a mutant defective in the CCR1 gene, which gave phenotypes with severely (~50%) reduced lignin contents (25). In contrast, CCR2 was strongly (albeit transiently) expressed in leaf tissue upon infection with Xanthomonas campestris (24) suggesting a role in plant defense.

The EST database entries indicated that only eight of the 12 putative CCR homologues could be detected in various tissues and organs, but not CCR3, 4 5 and 5a. Of these, CCR1 was apparently expressed at very similar levels in all tissues examined (including above-ground organs), this perhaps being consistent with it proposed role in lignification. On the other hand, the CCR2 homologue was present in root tissue, in contrast to its induction in leaf tissue upon infection as noted above. Additionally, CCR6 was only found in developing green siliques, and to a much lesser extent in mixed tissues, whereas CCR7 was mainly expressed in green siliques and developing seeds, with lower transcript levels in the roots, above-ground organs and mixed plant tissue. CCR8 was also present in most organs (except root tissue), whereas CCR9/10 and 12 were found in developing seeds and CCR11 was in the above-ground organs and mixed tissues.

Thus, as before, in spite of their annotations as cinnamoyl CoA reductases, it should be evident that much remains to be done to a) define the patterns of gene expression for each of these homologues and b) their precise metabolic roles in vivo. To this point, however, the vast majority of these genes have no known physiological functions or one(s) that can readily be deduced based on the information currently available.

For these reasons, the same strategy was adopted as before: (i) comprehensive characterization of the 11-12 member putative CCR multigene family, and (ii) comprehensive definition of expression patterns of the individual putative CCR promoters (GUS and/or GFP fusion) constructs and analysis of CCR gene “knockouts”. Progress is shown in Tables 11 – 13.