Tissue Culture-Specific Expression of a Naturally Occurring Tobacco Feedback-Insensitive Anthranilate Synthase1

Abstract

Abstract A cDNA and corresponding promoter region for a naturally occurring, feedback-insensitive anthranilate synthase (AS) α-subunit gene, ASA2, has been isolated from an unselected, but 5-methyl-tryptophan-resistant (5MTr), tobacco (Nicotiana tabacum) cell line (AB15–12-1). The ASA2 cDNA contains a putative transit peptide sequence, and Southern hybridization shows that more than one closely related sequence is present in the tobacco genome. TheASA2 cDNA complemented a trpE nonsense mutant Escherichia coli strain, allowing growth on 300 μm 5MT-containing minimal medium without tryptophan, and cell extracts contained feedback-insensitive AS activity. The 5MTr was lost when the E. coli strain was transformed with an ASA2 site-directed mutant (phenylalanine-107-arginine-108 → serine-107-glutamine-108). Identical nucleotide sequences encoding the phenylalanine-107-arginine-108 region have been found in polymerase chain reaction-amplified 326-bp ASA2 genomic fragments of wild-type (5-methyl-tryptophan-sensitive [5MTs]) tobacco and a progenitor species. High-level ASA2transcriptional expression was detected only in 5MTr-cultured cells, not in 5MTs cells or in plants. Promoter studies indicate that tissue specificity ofASA2 is controlled by the promoter region between −2252 and −607. Since the ASA2 promoter sequences are not substantially different in the 5MTr and 5MTslines, the increased levels of ASA2 mRNA in the 5MTr lines are most likely due to changes in a regulatory gene affecting ASA2 expression.