Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships

Abstract

SummaryCinnamoyl CoA:NADP oxidoreductase (CCR, EC 1.2.1.44) catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, i.e. the first specific step in the synthesis of the lignin monomers. The cloning of a cDNA encoding CCR in Eucalyptus gunnii (EUCCR) is reported here. The identity of the EUCCR eDNA was demonstrated by comparison with peptide sequence data from purified CCR and functional expression of the recombinant enzyme in Escherichia coil Sequence analysis revealed remarkable homologies with dihydroflavonol-4-reductase (DFR), the first enzyme of the anthocyanin biosynthetic pathway. Moreover, significant similarities were found with mammalian 3~-hydroxysteroid dehydrogenase and bacterial UDP-galactose-4-epimerase, suggesting that CCR shared a common ancestor with these enzymes and can therefore be considered as a new member of the mammalian 3p-hydroxysteroid dehydrogenase/ plant dihydroflavonol reductase superfamily. In Eucalyptus gunnii, CCR is encoded by one gene containing four introns whose positions are similar to those of introns I, II, III and V in DFR genes from dicots. In agreement with the involvement of CCR in lignification, the CGR transcript was shown to be expressed in lignified organs, i.e. root and stem tissues, and was localized mainly in young differentiating xylem. On the other hand, its abundance in Eucalyptus leaves suggests that monolignols may be precursors of end products other than lignins. This first characterization of a gene corresponding to CCR opens new possibilities to genetically engineer plants with lower lignin content. This is particularly important for woody plants such as Eucalyptus which are used for pulp making.

Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily

“…Neither did we find any reactivity towards cinnamaldehydes (p-coumaraldehyde, coniferaldehyde, and sinamaldehyde), the natural substrates for CADs. Interestingly, the sequences around the proposed substrate-binding site in CCR [3] are divergent from the CRLs, and these sequences can be separated into the same four groups as is shown in the tree (compare Fig. 2B,C).…”

“…A cDNA encoding CCR in Eucalyptus gunnii has been isolated [3] and shown to be expressed in lignified tissue, consistent with a role in lignification. Recently, two CCR-encoding cDNAs were cloned from Zea mays and found to be differentially regulated [4].…”

“…2B,C). [3]), AtDFR (A. thaliana dfr [12]), InDFR (Ipomoea nil dfr [13]), ZmDFR (Zea mays dfr [14]), HvDFR (Hordeum 6ulgare dfr [15]), and AtRD14.1 (A. thaliana rd14 similar to EUCAD1 and CPRD14 [8,9]). Length of exons (grey boxes) are denoted in base pairs, and intron Roman numbering is above (A) and (B).…”

“…The CCRs identified to date exhibit similarity to plant dihydroflavonol-4-reductase (DFR) (EC 1.1.1.219), the enzyme mediating the first step of the anthocyanin biosynthetic pathway [3,5]. DFRs are encoded by gene families, and their expression is most pronounced in spatial and temporal patterns during corolla and inflorescence development [6,7].…”

“…The patterns of regulation and developmental expression of genes in the plant DFR superfamily are beginning to emerge [3,4,7,8], and expression of a EUCAD1 homologue from cowpea (CPRD14) was found to be responsive to drought and high salinity [9]. The exon -intron pattern of the genomic members of the superfamily, including DFRs, CCRs, and the EUCAD1-like proteins, is highly conserved [3,8].…”