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Characterization of cis ‐elements required for vascular expression of the Cinnamoyl CoA Reductase gene and for protein–DNA complex formation
Author(s) -
Lacombe Eric,
Van Doorsselaere Jan,
Boerjan Wout,
Boudet Alain M.,
GrimaPettenati Jacqueline
Publication year - 2000
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2000.00838.x
Subject(s) - gene , dna , gene expression , reductase , chemistry , biology , genetics , biochemistry , enzyme
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 EgCCR – GUS 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.