Cloning and characterization of a ryegrass ( Lolium perenne) gene encoding cinnamoyl-CoA reductase (CCR)

Abstract

Cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) catalyzes the reduction of cinnamoyl acid-CoA esters into their corresponding aldehydes, the first step in the biosynthesis of lignin. The synthesis of lignin monomers is strictly associated with the activity of this enzyme which occurs in significant concentrations in lignin producing tissues. A 1207 bp cDNA ( LpCCR) encoding a polypeptide of 344 amino acids with a predicted molecular mass of 37.4 kDa was isolated from a ryegrass ( Lolium perenne) stem cDNA library. The identity of the LpCCR was established by comparison of the deduced polypeptide sequence with other isolated plant CCR enzymes. A motif, NWYCY, representing a putative CCR active site is conserved in the encoded LpCCR amino acid sequence. The encoded polypeptide exhibits sequence similarity to CCRs from different plants, the highest identities (87 and 86%, respectively) being to CCRs from Festuca arundicinea and Hordeum vulgare. Phylogenetic analysis shows that LpCCR and CCR from other monocotyledons form a group distinct from dicot CCRs. Genomic Southern blot hybridization demonstrated that LpCCR probably is represented as a single-copy gene in the ryegrass genome. The isolation of a single genomic CCR clone ( gLpCCR) from a ryegrass leaf library further supports this observation. The gLpCCR contains four introns, the same number found in the Eucalyptus gunnii gCCR. Computer analysis of a 1333 bp 5′-flanking region of gLpCCR suggests the presence of binding sites for a MYB transcription factor and a binding site for a P-box factor of the Dof class transcription factors. In agreement with the involvement of CCR in lignification, the ryegrass CCR mRNA was detected in stem tissue only. The isolation and characterization of gLpCCR provides tools for genetically modifying plants with reduced lignin content thereby increasing forage digestibility.