cDNA Cloning and Molecular Characterization of 4-Coumarate: Coenzyme A Ligase in Eucalyptus camaldulensi

The 4-coumarate: coenzyme A ligases (4CLs) are a group of enzymes very important to the biosynthesis of plant phenylpropanoids including lignin, which is 1 of 3 major wood components. To gradually approach the genetic manipulation of the lignin content in Eucalyptus trees, we report on the cloning of two 4CL cDNAs designated Euc4CLE3 and Euc4CL1 (GenBank accession no., DQ147001) from secondary developing xylem tissues of E. camaldulensis Blakely. The Euc4CLE3 fragment containing 823-bp cDNA was a partial sequence of the open reading frame (ORF) encoding 274 amino acid residues. The other 1953-bp clone from nucleic acid sequencing revealed a full length of 4CL cDNA containing an entire ORF of 1632-bp cDNA, which encoded a polypeptide of 544 amino acid residues with a calculated molecular weight of 59,470. In comparisons of amino acid sequences, the Euc4CLE3 and Euc4CL1 clones were classified into distinct classes of 4CLs. Alignment of the deduced Euc4CL1 protein with other plant 4CLs in class I showed that Euc4CL1 was a member of the 4CL family. Recombinant protein analyses indicated that the enzymatic activity and substrate preference of Euc4CL1 differed from those of lignin precursors, implying its involvement in lignification. Southern blot analysis confirmed the existence of native Euc4CL1 in the chromosomal genome of E. camaldulensis. Northern and western hybridizations verified that the Euc4CL1 gene was clearly expressed at the mRNA and protein levels in secondary developing xylem tissues, further verifying that Euc4CL1 is tightly associated with lignin biosynthesis and xylem development.