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

Vascular plants have evolved with remarkable ways to form and protect the v asculature apparatus, ln certain woody shrubs. the secondary xylem can have within its center a hollowed pith surrounded by secondary xylem, whereas i n most trees there is a solid core of heartwood. Both types of woody system s have, however. the commonality of accumulating so-called 'secondary' meta bolites, albeit to different extents, whose roles are to protect and preser ve 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 e xpression of two lignan biosynthetic pathway enzymes. pinoresinol-laricires inol reductase (PLR) and phenylcoumaran benzylic ether reductase (PCBER), s oluble 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 accum ulated mainly in young stems, as well as in young roots and petioles. Furth ermore, PLR-specific DIG-labeled riboprobes established that in the stems i ts 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 s econdary xylem. In addition, immunocytochemical localization of PCBER in Pi nus taeda established that it was in the axial and radial parenchyma cells of secondary xylem of stems. That is, irrespective of whether the woody pla nts 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 entr y point to both the lignans and lignins), which appears to be more restrict ed to the vascular cambial regions. (C) 2001 Elsevier Science Ltd. All righ ts reserved.