Anthranilate N-hydroxycinnamoyl/benzoyltransferase gene from carnation: rapid elicitation of transcription and promoter analysis.
Hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) catalyses the committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.). Three HCBT cDNAs were cloned previously from suspension-cultured carnation cells that had been induced with fungal elicitor. A rapid, transient induction of hcbt transcripts upon elicitation, reaching maximal abundances within about 0.5 h and returning to basal levels within 4 h, suggested the involvement of unusual cis elements. A DNA fragment of 3.8 kb, spanning the hcbt2 gene with the 5'-flanking region of roughly 1.8 kb, was cloned from carnation plants. The gene encodes one long open reading frame lacking introns. The DNA sequence revealed a conserved TATA box, three elicitor response elements (EREs) and a 9 bp direct repeat as well as an interrupted direct repeat of 11 bp in the TATA distal region. EMSA revealed the binding of elicitor-inducible nuclear factors to the promoter region from -377 to -326 spanning two of the EREs, and their functional relevance was confirmed by transient expression assays of hcbt2 promoter-GUS reporter gene constructs in parsley protoplasts. Furthermore, an oligo(A) segment was present immediately preceding the start of translation (+140 to +150). Transient expression analysis demonstrated that the sequence upstream to -1157 at least is required in context with the 5'-UTR, particularly including the poly(A) segment, for strong expression and full elicitor induction of the hcbt2 gene. The results suggested that several sequence motifs scattered over a wide range of the 5'-flanking region and into the exonic sequence are responsible for the full elicitor regulation of the hcbt2 gene.
Yang
Q
Q
Institut für Biologie II, Lehrstuhl für Biochemie der Pflanzen, Universität Freiburg, Germany.
Grimmig
B
B
Matern
U
U
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000096:biosynthesis; D018515:Plant Leaves; D010944:Plants / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D012717:Sesquiterpenes; D016385:TATA Box; D013729:Terpenes; D014158:Transcription, Genetic; D000094162:Phytoalexins
D018076:DNA, Complementary; D010936:Plant Extracts; D012717:Sesquiterpenes; D013729:Terpenes; D000217:Acyltransferases; C116404:anthranilate N-hydroxycinnamoyl benzoyltransferase; D000094162:Phytoalexins