Identification of UV-B light-responsive regions in the promoter of the tryptophan decarboxylase gene from Catharanthus roseus.
The tryptophan decarboxylase (Tdc) gene encodes a key enzyme in the biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus. TIAs absorb ultraviolet light (UV) and putative functions in plants include a role as UV protectants. In support of this possible function we demonstrate here that UV light induces accumulation of several TIAs as well as expression of the Tdc gene in C. roseus. In addition, in tobacco a Tdc-gusA construct was found to be specifically induced by UV-B light. Lack of induction by UV-A or other wavelengths of light indicate that Tdc expression is regulated by a specific UV-B receptor and corresponding signal transduction pathway. To identify UV-responsive Tdc promoter elements, a loss-of-function analysis was performed, in which deletion derivatives were fused to the gusA reporter gene and analysed in transgenic tobacco plants. Truncation of the Tdc promoter from -1818 (relative to the start of transcription) to -160 reduced expression levels two-fold without affecting the qualitative UV response. Deletion to -37 further reduced expression levels five-fold, but the delta37 promoter also remained UV-responsive. Subsequently, the -160 to -37 region was further studied by gain-of-function experiments, in which the transcriptional activities of tetramerized subfragments fused to truncated promoters were analysed. Combination of the data identified several functional regions in the -160 to +198 promoter. The - 160 to -99 region acts as the main transcriptional enhancer. UV-responsive elements appeared to be redundant in the -160 Tdc promoter and to reside between -99 and -37 and between -37 and + 198.
Ouwerkerk
P B
PB
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, The Netherlands.
Hallard
D
D
Verpoorte
R
R
Memelink
J
J
D000470:Alkaloids / Q000378:metabolism; D000595:Amino Acid Sequence; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics*; D001483:Base Sequence; D019759:Carbon-Nitrogen Lyases / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000528:radiation effects; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics / Q000528:radiation effects*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000528:radiation effects; D014026:Nicotiana / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays
D000470:Alkaloids; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase; D001142:Aromatic-L-Amino-Acid Decarboxylases; D019759:Carbon-Nitrogen Lyases; C022684:strictosidine synthetase