4-Coumarate:coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes

摘要

Developing xylem, young leaves and cell suspensions of hybrid poplar (Populus deltoides X P. trichocarpa [P. interamericana]) H11 were examined for the presence of 4-coumarate:coenzyme A ligase (4CL) isoforms by fast-protein liquid chromatography, and a minimum of three 4CL isoforms were detected. These isoforms shared similar hydroxycinnamic acid substrate utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CLcDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity towards cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.