ORIGINAL ARTICLE Cloning and expression analysis of candidate genes involved in wax deposition along the growing barley (Hordeum vulgare) leaf Andrew Richardson Æ Alexandre Boscari Æ Lukas Schreiber Æ Gerhard Kerstiens Æ Mike Jarvis Æ Pawel Herzyk Æ Wieland Fricke Received: 15 June 2007 / Accepted: 3 July 2007 / Published online: 28 July 2007 Ó Springer-Verlag 2007 Abstract The aim of the present study was to isolate clones of genes which are likely to be involved in wax deposition on barley leaves. Of particular interest were those genes which encode proteins that take part in the synthesis and further modification of very long chain fatty acids (VLCFAs), the precursors of waxes. Previously, it had been shown that wax deposition commences within a spatially well-defined developmental zone along the growing barley leaf (Richardson et al. in Planta 222:472– 483, 2005). In the present study, a barley microarray ap- proach was used to screen for candidate contig-sequences (www.barleybase.org) that are expressed particularly in those leaf zones where wax deposition occurs and which are expressed specifically within the epidermis, the site of wax synthesis. Candidate contigs were used to screen an established in-house cDNA library of barley. Six full- length coding sequences clones were isolated. Based on sequence homologies, three clones were related to Ara- bidopsis CER6/CUT1, and these clones were termed HvCUT1;1, HvCUT1;2 and HvCUT1;3. A fourth clone, which was related to Arabidopsis Fiddlehead (FDH), was termed HvFDH1;1. These clones are likely to be involved in synthesis of VLCFAs. A fifth and sixth clone were related to Arabidopsis CER1, and were termed HvCER1;1 and HvCER1;2. These clones are likely to be involved in the decarbonylation pathway of VLCFAs. Semi-quantita- tive RT-PCR confirmed microarray expression data. In addition, expression analyses at 10-mm resolution along the blade suggest that HvCUT1;1 (and possibly HvCUT1;2) and HvCER1;1 are involved in commencement of wax deposition during barley leaf epidermal cell development. W. Fricke (&) UCD School of Biology and Environmental Science, UCD Science Centre West, University College Dublin, Belfield, Dublin 4, Ireland e-mail: wieland02fricke@yahoo.co.uk Present Address: A. Richardson Millipore, Gemini Crescent, Dundee Technology Park, Dundee DD2 1SW, UK Present Address: A. Boscari Interactions Plantes Microorganismes et Sante ´ Ve ´ge ´tale, UMR INRA 1064/Universite ´ de Nice-Sophia Antipolis/CNRS 6192, 400 Route des Chappes, B. P. 167, 06903 Sophia Antipolis Cedex, France Electronic supplementary material The online version of this article (doi:10.1007/s00425-007-0585-0) contains supplementary material, which is available to authorized users. A. Richardson Á A. Boscari Á W. Fricke Division of Biological Sciences, University of Paisley, Paisley PA1 2BE, Scotland, UK L. Schreiber Department of Ecophysiology, IZMB, University of Bonn, 53115 Bonn, Germany G. Kerstiens Department of Biological Sciences, Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK M. Jarvis Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, UK P. Herzyk Sir Henry Wellcome Functional Genomics Facility, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK 123 Planta (2007) 226:1459–1473 DOI 10.1007/s00425-007-0585-0