Pinoresinol-lariciresinol Reductase Research Articles

In situ hybridization and immunolocalization of lignan reductases in woody tissues: implications for heartwood formation and other forms of vascular tissue preservation

Vascular plants have evolved with remarkable ways to form and protect the vasculature apparatus. In certain woody shrubs, the secondary xylem can have within its center a hollowed pith surrounded by secondary xylem, whereas in most trees there is a solid core of heartwood. Both types of woody systems have, however, the commonality of accumulating so-called ‘secondary’ metabolites, albeit to different extents, whose roles are to protect and preserve the vascular (lignified) tissues. This investigation had as its purpose establishing the nature of the cells involved in the biosynthesis of these specialized ‘secondary’ metabolites in plants forming heartwood and hollow piths, respectively. This was achieved by determining the tissue-specific expression of two lignan biosynthetic pathway enzymes: pinoresinol-lariciresinol reductase (PLR) and phenylcoumaran benzylic ether reductase (PCBER), soluble enzymes which catalyze analogous benzylic ether reductions of 8–8′ and 8–5′ linked lignans, respectively. Using Forsythia intermedia, reverse transcription-PCR (RT–PCR) and northern blots revealed that PLR mRNA accumulated mainly in young stems, as well as in young roots and petioles. Furthermore, PLR-specific DIG-labeled riboprobes established that in the stems its mRNA accumulated in the radial parenchyma cells [and to a lesser extent in the developing vessels], as well as in the cambial cells of developing secondary xylem. In addition, immunocytochemical localization of PCBER in Pinus taeda established that it was in the axial and radial parenchyma cells of secondary xylem of stems. That is, irrespective of whether the woody plants formed hollowed piths or heartwood, the ‘secondary’ metabolite pathways leading to the protective lignans predominantly involved axial and radial parenchyma cells. This is in contrast to monolignol coupling (i.e. the entry point to both the lignans and lignins), which appears to be more restricted to the vascular cambial regions.

Evolution of Plant Defense Mechanisms: RELATIONSHIPS OF PHENYLCOUMARAN BENZYLIC ETHER REDUCTASES TO PINORESINOL-LARICIRESINOL AND ISOFLAVONE REDUCTASES

Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

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 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 beta-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.