ABSTRACT
The enzyme encoded by the strictosidine synthase (Str) gene catalyses a key step in the biosynthesis of therapeutically valuable terpenoid indole alkaloids. In Catharanthus roseus the Str gene was shown to be regulated by a wide variety of signals including auxin, methyl jasmonate and fungal elicitors in cell suspension cultures and by tissue-specific control in plant organs. The Str promoter contains a functional G-box (CACGTG) cis-regulatory sequence. In order to understand better the mechanisms involved in the regulation of Str gene expression, we isolated the C. roseus cDNAs encoding G-box binding factors Crgbf1 and Crgbf2. The binding specificity of their protein products CrGBF1 and CrGBF2 was analysed by competitive electrophoresis mobility and saturation binding assays. CrGBF1 had a high binding specificity for class I G-boxes including the Str G-box. CrGBF1 showed a lower affinity for class II G-boxes and for the G-box-like element (AACGTG) found in the tryptophan decarboxylase (Tdc) gene which encodes another enzyme involved in TIA biosynthesis. CrGBF2 showed a high affinity for all types of G-boxes tested and to a lesser extent for the Tdc G-box-like element. Transient bombardment experiments demonstrated that both CrGBF1 and CrGBF2 can act in vivo as transcriptional repressors of the Str promoter via direct interaction with the G-box. These data indicate that GBFs may play functional role in the regulation of expression of the terpenoid indole alkaloid biosynthetic gene Str.
Subject(s)
Carbon-Nitrogen Lyases/genetics , DNA-Binding Proteins/genetics , Plants/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Binding Sites/genetics , Binding, Competitive , Blotting, Northern , Blotting, Southern , DNA/genetics , DNA/metabolism , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA, Plant/genetics , DNA-Binding Proteins/metabolism , G-Box Binding Factors , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Molecular Sequence Data , Plant Cells , Promoter Regions, Genetic , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Tissue Distribution , Transcription Factors/metabolismABSTRACT
Omission of 2,4-D from culture medium during one subculture of Catharanthus roseus cells, strain C20, resulted in an increased alkaloid accumulation, without effect on growth. Alkaloid accumulation, rather than growth, seemed to be more sensitive to 2,4-D. 2,4-D inhibited alkaloid accumulation essentially during growth phase, but its inhibitory effect during this period was partially reversible. As this reversibility was underlined only during the stationary phase, this suggested that this action could be situated upstream in a terpenoid non-specific pathway. 2,4-D feeding showed that inhibition is weaker and weaker as the alkaloid accumulation period proceeds. Auxin action during this period could take place downstream in specific alkaloid pathways. The lower alkaloid accumulation obtained after loganic acid feeding compared to that obtained with secologanin and loganin could indicate that loganic acid methylation should be one of the 2,4-D target(s).