Recombinant pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) catalyze opposite enantiospecific conversions

Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biologi cal processes leading to its formation are initiated and regulated. Accordi ngly, a reverse transcription-polymerase chain reaction-guided cloning stra tegy was employed to obtain genes encoding pinoresinol-lariciresinol reduct ases from western red cedar (Thuja plicata) as a means to initiate the stud y of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase f rom Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, whe n followed by homologous hybridization cloning, resulted in the isolation o f two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expresse d as a fusion protein with P-galactosidase and assayed for enzymatic activi ty. Using both deuterated and radiolabeled (+/-)-pinoresinols as substrates , it was established that each class of cDNA encoded a pinoresinol-laricire sinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (-)-secoisolarici resinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises imp ortant questions about the role of each of these individual reductases in h eartwood formation, such as whether they are expressed in different cells/t issues or at different stages during heartwood development.