Characterisation of caffeic acid O -methyltransferase and cinnamyl alcohol dehydrogenase gene expression patterns by in situ hybridisation in Eucalyptus gunnii Hook. plantlets Simon Hawkins *, Alain Boudet, Jacqueline Grima-Pettenati Signaux et Messages Cellulaires Chez les Ve ´ge ´taux, UMR CNRS-UPS 5546, Po ˆle de Biotechnologie Ve ´ge ´tale, 24 Chemin de Borde Rouge, Auzeville BP17, 31326 Castanet Tolosan France Received 19 June 2002; received in revised form 19 August 2002; accepted 8 October 2002 Abstract The developmental tissue- and cell-specific expression pattern of two ‘lignification’ genes, caffeic acid O -methyltransferase (C- OMT) and cinnamyl alcohol dehydrogenase (CAD), was analysed by in situ hybridisation in leaf and stem samples of Eucalyptus plantlets. Both genes are expressed, in a coordinated, developmental fashion, in the same cell types */especially developing vessels */ of differentiating stem xylem tissue confirming their role in lignification and demonstrating that this process is under strict developmental control. C-OMT, but not CAD, transcripts were also localised to developing xylem vessels in the midribs of leaves. Histochemical analyses revealed that, in stem xylem tissues, C-OMT and CAD are expressed in cells poor in S-type lignin (primary xylem vessels and immature secondary xylem cells) and also in cells that later become rich in S-type lignin (mature secondary xylem cells). # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CAD; C-OMT; Eucalyptus; In situ hybridisation; Lignification; Lignin 1. Introduction Lignin is a complex phenolic polymer resulting from the oxidative polymerisation of hydroxycinnamyl alco- hols (p -coumaryl alcohol, coniferyl alcohol and sinapyl alcohol) [1] and which can constitute between 15 and 36% dry weight of wood [2]. Lignin reinforces the walls of certain cells in higher plants and is mainly found in the vascular tissues where its hydrophobicity water- proofs the conducting cells of the xylem and its rigidity strengthens the supporting fibre cells of both xylem and phloem. The chemical composition of lignin varies in function of a number of parameters including plant group, species, organ, tissue and cell-type. For example, gymnosperm lignin is usually composed of mainly guaiacyl units (G-units derived from coniferyl alcohol) whereas typical angiosperm lignin is composed of a mixed guaiacyl  /syringyl lignin (G- and S-units, derived from coniferyl alcohol and sinapyl alcohol, respectively). Similarly, in Arabidopsis thaliana , the lignin associated with vessel secondary cell walls in the xylem is composed mainly of guaiacyl units (G-units, derived from coniferyl alcohol) whereas the fibres are typically composed of a mixed guaiacyl  /syringyl lignin (G- and S-units, derived from coniferyl alcohol and sinapyl alcohol, respectively) [3]. As well as being of fundamental interest in a devel- opmental context, the composition of the lignin mole- Abbreviations: DTT, dithiothreitol; PBS, phosphate-buffered saline; SSC, sodium chloride/sodium citrate buffer; TBO, toludine blue-O. * Corresponding author. Present address: Biotechnologies de Mole ´cules Ve ´ge ´tales, Laboratoire de Biologie des Ligneux et des Grandes Cultures, UPRES EA 1207, Universite ´ d’Orle ´ans, Antenne Scientifique Universitaire de Chartres, 21 rue de Loigny la Bataille, 28000 Chartres, France. Tel.:  /33-237-309-944; fax:  /33-237-309-069 E-mail addresses: shawkins@mailhost.univ-orleans.fr, simon- hawkins@univ-orleans.fr (S. Hawkins). Plant Science 164 (2003) 165  /173 www.elsevier.com/locate/plantsci 0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(02)00361-8