Characterization of cis-elements required for vascular expression of the Cinnamoyl CoA Reductase gene and for protein–DNA complex formation

Authors

  • Eric Lacombe,

    1. Signaux et Messages Cellulaires chez les Végétaux, UMR CNRS-UPS 5546, Pôle de Biotechnologie Végétale, BP 17, Auzeville, 31 320 Castanet Tolosan, France, and
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      The first two authors contributed equally to this work.
  • Jan Van Doorsselaere,

    1. Signaux et Messages Cellulaires chez les Végétaux, UMR CNRS-UPS 5546, Pôle de Biotechnologie Végétale, BP 17, Auzeville, 31 320 Castanet Tolosan, France, and
    2. Laboratorium voor Genetica, Department of Plant Genetics, Flanders Interuniversity Institute for Biotechnology, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000, Belgium
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      The first two authors contributed equally to this work.
  • Wout Boerjan,

    1. Laboratorium voor Genetica, Department of Plant Genetics, Flanders Interuniversity Institute for Biotechnology, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000, Belgium
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  • Alain M. Boudet,

    1. Signaux et Messages Cellulaires chez les Végétaux, UMR CNRS-UPS 5546, Pôle de Biotechnologie Végétale, BP 17, Auzeville, 31 320 Castanet Tolosan, France, and
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  • Jacqueline Grima-Pettenati

    Corresponding author
    1. Signaux et Messages Cellulaires chez les Végétaux, UMR CNRS-UPS 5546, Pôle de Biotechnologie Végétale, BP 17, Auzeville, 31 320 Castanet Tolosan, France, and
      For correspondence (fax +33 5 62 19 35 02; e-mail
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  • The first two authors contributed equally to this work.

    The EMBL accession number for the sequence of the EgCCR promoter is AJ132750.

For correspondence (fax +33 5 62 19 35 02; e-mail grima@smcv.ups-tlse.fr).

Summary

Cinnamoyl-CoA reductase (CCR) catalyses the first specific step in the biosynthesis of monolignols, the monomeric units of lignins. We examined the developmental regulation of the Eucalyptus gunnii EgCCR promoter by analysing the expression of EgCCRGUS fusions in tobacco. EgCCR promoter activity was strongest in lignified organs (stems and roots) consistent with the EgCCR mRNA level in these organs. Histochemical analysis showed expression in vascular tissues (cambium, young differentiating xylem, ray cells, internal and external phloem) of stems and roots in agreement with in situ hybridization data. Promoter deletion analysis and gain-of-function experiments identified the sequences between positions −119 and −77 as necessary and sufficient for expression in vascular tissues of stems. Electrophoretic mobility-shift assays showed that this region is specifically recognized by nuclear proteins present in tobacco stems, giving rise to two retarded complexes, LMC1 and LMC2. Using overlapping EgCCR fragments and mutated oligonucleotides as competitors in gel-shift assays, two DNA–protein interaction sites were mapped. Finally, the role of protein–protein interactions in the formation of the LMC1 and LMC2 complexes was investigated using the detergent sodium deoxycholate, and protein fractionation onto a heparin Sepharose column.

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