Tissue- and cell-specific expression of a cinnamyl alcohol dehydrogenase promoter in transgenic poplar plants

Cinnamyl alcohol dehydrogenase (CAD) which catalyses the synthesis of the cinnamyl alcohols, the immediate precursors of lignins, from the corresponding cinnamaldehydes is considered to be a highly specific marker for lignification. We have isolated and characterized a CAD genomic clone from eucalyptus, a woody species of economic importance. The full-length promoter (EuCAD, 2.5 kb) and a series of 5' deletions were fused to the beta-glucuronidase (GUS) reporter gene. These constructs were tested in a homologous transient expression system of eucalyptus protoplasts which enabled the identification of several regions involved in transcriptional control. In order to study the spatial and developmental regulation of the CAD gene, the chimeric gene fusion (EuCAD-GUS) was then transferred via Agrobacterium tumefaciens-mediated transformation into poplar, an easily transformable woody angiosperm. Quantitative fluorometric assays conducted on eight independent in vitro transformants showed that GUS activity was highest in roots followed thereafter by stems and leaves. Histochemical staining for GUS activity on both in vitro primary transformants and more mature greenhouse-grown plants indicated a specific expression in the vascular tissues of stems, roots, petioles and leaves. At the onset of xylem differentiation, GUS activity was detected in parenchyma cells differentiating between the xylem-conducting elements. After secondary growth has occurred, GUS activity was localized in xylem ray cells and parenchyma cells surrounding the lignified phloem and sclerenchyma fibers. This first characterization of a woody angiosperm CAD promoter provides functional evidence for the role of CAD in lignification and suggests that parenchyma cells expressing CAD may provide lignin precursors to the adjacent lignified elements (vessels and fibres).