UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells

The first enzyme of the shikimate pathway catalyzes the condensation of phosphoenolpyruvate and erythrose-4-phosphate to yield DAHP. DAHP synthase is reportedly induced by abiotic stresses such as mechanical wounding [1]. The first evidence of metabolic regulation of a plant DAHP synthase came from experiments with suspension cultured potato cells, exposed to glyphosate [2]. Metabolic regulation of DAHP synthase in plants appears to occur preferentially at the transcriptional level. DAHP synthase transcript was found to accumulate in response to several environmental stimuli that also induced phenylalanine ammonia lyase (PAL) mRNA [1,3]. This suggests that the synthesis of aromatic amino acids might be regulated in concert at the transcriptional level. Several isozymes specific to cytosolic and plastid have been reported for plant DAHP synthase [4]. The elicitor treatment of parsley cell suspensions or wounding of potato tubers induced DAHP synthase isoenzyme specific to plastid but not the putative cytosolic form [5,6]. Isolation and characterization of cDNA that encodes DAHP synthase from Catharanthus roseus and accumlation of its transcript and the signaling components involved have been described in this study.A homology-based PCR cloning strategy was used to clone DAHP synthase by amplifying a partial DAHP cDNA sequence using the UV-B induced C. roseus suspension cell λZAP cDNA library as template and two DAHP gene-specific oligonucleotide primers. The PCR fragment of the expected size was cloned and sequenced. The deduced amino acid sequence of the PCR fragment showed strong homology to known DAHP synthase primary structure. The PCR fragment was used as a probe to screen the C. roseus cDNA library (2 × 105 plaque forming units). This led to the isolation of a full-length cDNA clone.Its insert DNA was completely sequenced. The cDNA contains an open reading frame of 1361 nucleotides and encodes a deduced protein of 446 amino acid residues with a calculated molec