Recombinant Pinoresinol-Lariciresinol Reductases from Western Red Cedar (Thuja plicata) Catalyze Opposite Enantiospecific Conversions

Abstract

Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (؉)-Pinoresinol-(؉)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with ␤-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (؎)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (؉)-pinoresinol into (-)-secoisolariciresinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (؉)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development.

Enhancing lignan biosynthesis by over‐expressing pinoresinol lariciresinol reductase in transgenic wheat

“…They occur naturally in a number of plant families, including the gramineae and oleaceae which contain the monocots and eudicots, respectively [3,4]. In monocots such as wheat, lignans are mostly located in the aleurone layer of seeds [5], and in eudicots such as forsthysia, lignans occur in the fruits and stems [6].…”

“…The biosynthetic pathways to SDG occur via coupling of two coniferyl alcohol molecules to afford pinoresinol ( Figure 1). Then pinoresinol undergoes sequential reduction by pinoresinol-lariciresinol reductase (PLR) to generate lariciresinol and secoisolariciresinol [3,22]. Since PLR catalyzes the last steps of the lignan biosynthesis, it is postulated that over-expression of PLR gene by genetic engineering may enhance lignan contents.…”

“…Since PLR catalyzes the last steps of the lignan biosynthesis, it is postulated that over-expression of PLR gene by genetic engineering may enhance lignan contents. Although PLR gene had already been isolated from various woody plants such as Forsthysia intermedia [3,[23][24], the only known isolated PLR enzyme in monocots has been found from flaxseeds [25]. In wheat, however, the PLR gene and corresponding protein(s) have not been reported yet.…”

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism

“…This compound is formed from pinoresinol via the action of pinoresinol reductase (Fujita et al, 1999). Two genes encoding putative pinoresinol reductase were induced in lignin-forming conditions, and one gene had a constant expression in both conditions (Fig.…”

“…8B). This enzyme catalyzes the reduction of pinoresinol to lariciresinol and, in some species, also the further reduction of lariciresinol to secoisolariciresinol (Fujita et al, 1999). Native Norway spruce lignin contains pinoresinol and secoisolariciresinol structures .…”