4-coumarate:CoA ligase gene family in Rubus idaeus: cDNA structures, evolution, and expression

The enzyme 4-coumarate:CoA ligase (4CL) activates cinnamic acid and its hydroxylated derivatives by forming the corresponding CoA thioesters. These serve as substrates for biosynthesis of phenylpropanoid-derived end-products that are important determinants of fruit quality in raspberry (Rubus idaeus L.). In higher plants, 4CL is typically encoded by a gene family. To investigate the participation of distinct 4CL genes in the process of fruit ripening, we have characterized this gene family in raspberry. By complementing a PCR-based homology search with low-stringency cDNA library screening, we have isolated three classes of raspberry 4CL cDNAs (Ri4CL1, Ri4CL2, and Ri4CL3). Phylogenetic analysis places the three raspberry 4CL gene family members into two distinct groups, a pattern consistent with an ancient divergence from an ancestral progenitor. Quantitative RT-PCR assay reveals a differential pattern of transcription of each of the three genes in various organs, as well as distinct temporal patterns of expression during flower and fruit development. The regulatory elements thus appear to have evolved independently of the genes themselves. Based on phylogenetic classification, expression patterns and recombinant protein activities the different Ri4CL genes are likely to participate in different biosynthetic pathways leading to the various phenylpropanoid-derived metabolites that help create flavor and color in raspberry fruit.