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.