Tissue culture-specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase

Abstract

A cDNA and corresponding promoter region for a naturally occurring, feedback-insensitive anthranilate synthase (AS) alpha-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. The ASA2 cDNA complemented a trpE nonsense mutant Escherichia coli strain, allowing growth on 300 microm 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 ASA2 transcriptional expression was detected only in 5MTr-cultured cells, not in 5MTs cells or in plants. Promoter studies indicate that tissue specificity of ASA2 is controlled by the promoter region between -2252 and -607. Since the ASA2 promoter sequences are not substantially different in the 5MTr and 5MTs lines, the increased levels of ASA2 mRNA in the 5MTr lines are most likely due to changes in a regulatory gene affecting ASA2 expression.

Figure 1

Nucleotide and the predicted amino acid sequence of the ASA2 cDNA. The coding region corresponds to amino acid sequence 1 (ATG) to 616 (TAG). The putative translation start and stop codons are indicated in boldface. The 89 and 205 bp of the upstream and downstream coding region represent 5′ and 3′ UTRs, respectively.

Figure 2

Genomic DNA-blot analysis. A, Twenty micrograms of AB15–12-1 genomic DNA was digested with nine restriction enzymes. Lanes from left to right correspond to BamHI, EcoRI, EcoRV, HincII, HindIII, KpnI, PstI, ScaI, and XbaI. B, Ten micrograms of each genomic DNA obtained from leaves of N. sylvestris, N. tomentosiformis, N. tabacum, and suspension-cultured cells of the 5MT^s and 5MT^r tobacco cell line (AB15–12-1) was digested with KpnI. The full-length ASA2 cDNA clone was used as a probe in both A and B. Fragment sizes were determined by a 1-kb ladder (GIBCO-BRL).

Figure 3

Amino acid sequence alignment of AS genes from plants and a prokaryote. TASA2, RASA1, RASA2, AASA1, AASA2, and CTRPE correspond to N. tabacum ASA2, R. graveolens ASα1 and ASα2, A. thaliana ASA1 and ASA2, and C. thermocellum trpE cDNA clones, respectively. Dashes within sequences indicate gaps. Asterisks under the sequence represent identical amino acids among these six different AS sequences. Dots under the sequence indicate similar amino acids. Two consensus motifs, Leu-Leu-Glu-Phe-X₁₀-Ser and Asn-Pro-Ser-Pro-Tyr-Met, affecting feedback inhibition based on microorganisms and yeast, are indicated in boldface. The amino acid(s) substitutions Phe-Arg and Arg, which caused feedback insensitivity in N. tabacum and R. graveolens, are indicated in boldface with a diamond mark at positions 107 and 108, respectively. A single amino acid change in an Arabidopsis mutant (Asp to Asn) is indicated in boldface with a dagger at position 326. A consensus sequence motif for plant chloroplast transit peptides in the tobacco ASA2 is underlined at positions 58 to 61. The amino acid numbers indicated are based on the tobacco ASA2 amino acid sequence.

Figure 4

Northern analysis. Twenty micrograms of total RNA per lane was used for northern hybridization. A full-length ASA2 cDNA clone (ASA2) and rRNA (rRNA) were used as probes for hybridization. A, Total RNAs were prepared from 1-week-old N. tabacum suspension-cultured cells (SC) of four 5MT^s (s) and four 5MT^r (r) cell lines, 5MT^s N. tabacum leaves (L) harvested from 3-week-old AB15–12-1 shoot cultures, and 1-week-old 5MT^r N. sylvestris suspension-cultured cells. B, Different plant organs were used to determine tissue specificity. AB15–12-1 leaves, roots, and stems harvested from 3-week-old shoot cultures and dried mature seeds and suspension-cultured 5MT^s cells (TXD) and 5MT^r cells (AB15–12-1) were used to prepare total RNA.

Figure 5

Complementation of E. coli trpE5972 by the tobacco ASA2 and a site-directed mutant. The pQE30/ASA2 and pQE30/ASA2 mutants were transformed into the trpE nonsense mutant E. coli strain (trpE5972) and plated on M9 minimal medium containing 100 μg/mL ampicillin (A) and 0.1 mm IPTG without and with 300 μm 5MT (B). The picture was taken 2 d after streaking.

Figure 6

Kinetics of AS from wild-type (□) and 5MT-selected (▪) tobacco cells and from E. coli transformed with the Arabidopsis ASA1 gene (⋄) or the tobacco ASA2 gene (♦). A, Relative AS activity in the presence of Trp was measured as described in Methods (with 100 μm chorismate) and is expressed as a percentage of the activity with no Trp added. The specific activity with no Trp for AS from each tobacco line was 73 and 169 nmol anthranilate min⁻¹ mg⁻¹ protein, respectively. B. Lineweaver-Burk plot of AS activity. Velocity is expressed as nanomoles per minute per milligram of protein.

Figure 7

Nucleotide sequence alignment of ASA2 genomic DNA fragments. The 326-bp ASA2 genomic DNA fragments, corresponding to the ASA2 cDNA sequence from +181 to +392, were amplified from the genomic DNAs of tobacco leaves (ASA2) harvested from AB15–12-1 shoot cultures and three wild-type (5MT^s) Nicotiana sp. cultured cell lines, N. tabacum (TXD), N. tomentosiformis (Nto), and N. sylvestris (Ns) using primers 4 (sense) and 10 (antisense), which are shown in bold. These genomic DNA fragments contain a 115-bp intron, which is underlined. Eight nucleotides, indicated by (+) above the sequence, are different in the N. sylvestris genomic fragment compared with those in N. tabacum and N. tomentosiformis. The nucleotide sequences corresponding to Phe-107-Arg-108 are indicated in boldface and underlined with the one-letter amino acid codon abbreviations F and R above the sequence.

Figure 8

The nucleotide sequence of the ASA2 promoter region. The first possible TATA box (TATAAA), translation start codon (ATG), is indicated in boldface. The 98 bp at the 3′ end of the ASA2 promoter region, which is identical to the nucleotide sequence at 5′ end of the ASA2 cDNA, is underlined.

Figure 9

Chimeric ASA2 promoter-GUS constructs and transient GUS assays with tobacco leaves and suspension-cultured cells. Four deleted promoter fragments were fused to the GUS reporter gene with NOS terminator in pUC19 and designated 2252, 1356, 606, and 370. These chimeric constructs and the CaMV 35S-GUS construct as a control were bombarded into 2-d-old AB15–12-1 cultured cells (black bars) and leaves (white bars) harvested from 3-week-old shoot cultures. GUS transient expression was measured using the MUG assay 3 d after bombardment. The average values after subtracting the background from four separate experiments with two replicates are presented. GUS-specific activity was determined as picomoles of 4-methylumbelliferone per hour per milligram of protein.