Characterization of rice anthranilate synthase alpha-subunit genes OASA1 and OASA2. Tryptophan accumulation in transgenic rice expressing a feedback-insensitive mutant of OASA1

Abstract

Anthranilate synthase (AS) is a key enzyme in the synthesis of tryptophan (Trp), indole-3-acetic acid, and indole alkaloids. Two genes, OASA1 and OASA2, encoding AS alpha-subunits were isolated from a monocotyledonous plant, rice (Oryza sativa cv Nipponbare), and were characterized. A phylogenetic tree of AS alpha-subunits from various species revealed a close evolutionary relationship among OASA1 and Arabidopsis ASA2, Ruta graveolens AS alpha 2, and tobacco ASA2, whereas OASA2, Arabidopsis ASA1, and R. graveolens AS alpha 1 were more distantly related. OASA1 is expressed in all tissues tested, but the amount of its mRNA was greater in panicles than in leaves and roots. The abundance of OASA2 transcripts is similar among tissues and greater than that of OASA1 transcripts; furthermore, OASA2 expression was induced by a chitin heptamer, a potent elicitor, suggesting that OASA2 participates in secondary metabolism. Expression of wild-type OASA1 or OASA2 transgenes did not affect the Trp content of rice calli or plants. However, transformed calli and plants expressing a mutated OASA1 gene, OASA1(D323N), that encodes a protein in which aspartate-323 is replaced with asparagine manifested up to 180- and 35-fold increases, respectively, in Trp accumulation. These transgenic calli and plants were resistant to 300 microM 5-methyl-Trp, and AS activity of the calli showed a markedly reduced sensitivity to Trp. These results show that OASA1 is important in the regulation of free Trp concentration, and that mutation of OASA1 to render the encoded protein insensitive to feedback inhibition results in accumulation of Trp at high levels. The OASA1(D323N) transgene may prove useful for the generation of crops with an increased Trp content.

Figure 1

Alignment of the predicted amino acid sequences of rice OASA1 and OASA2 with those of other AS α-subunits. The sequences shown are encoded by the following genes: Os1 and Os2, rice OASA1 (GenBank accession no. AB022602), and OASA2 (AB022603), respectively; At1 and At2, Arabidopsis ASA1 (M92353) and ASA2 (M92354), respectively; Rg1 and Rg2, R. graveolens ASα1 (L34343) and ASα2 (L34344), respectively; Nt2, tobacco ASA2 (Song et al., 1998); Sc, S. cerevisiae TRP2 (X68327); and Ec, E. coli trpE (V00368). Hyphens indicate gaps introduced to optimize alignment. Residues shared by OASA1 and OASA2 are shaded, and residue numbers are shown on the right.

Figure 2

Phylogenetic relations among AS α-subunits. The amino acid sequences of OASA1 (residues 57–577), OASA2 (residues 84–606), Arabidopsis ASA1 (residues 73–595), Arabidopsis ASA2 (residues 88–621), ASα1 (residues 91–613), ASα2 (residues 88–609), tobacco (Nt) ASA2 (residues 60–618), TRP2 (residues 22–507), and TrpE (residues 1–520) were analyzed (Fig. 1). The phylogenetic tree was constructed from the evolutionary distance data derived by the neighbor-joining method (Saitou and Nei, 1987). The bootstrap procedure was sampled 1,000 times with replacement by CLUSTAL W (Thompson et al., 1994). The bar indicates the distance corresponding to 10 changes per 100 amino acid positions.

Figure 3

RNA gel-blot analysis of OASA1 and OASA2 expression. A, Tissue distribution of transcripts. Total RNA (10 μg) isolated from calli (lane 1), panicles (lane 2), roots (lane 3), and leaves (lane 4) of rice was subjected to RNA gel-blot analysis with digoxigenin-labeled riboprobes specific for OASA1 (left) or OASA2 (right) transcripts. The positions of the corresponding transcripts are indicated by arrows. B, Effects of an elicitor on transcript abundance. Suspension cultures of rice cells were incubated with chitin heptamer (1 μg mL⁻¹) for 0 min (lane 1), 30 min (lane 2), 120 min (lane 3), or 180 min (lane 4), after which total RNA was isolated and analyzed as in A.

Figure 4

Construction of binary vectors for rice transformation. A, Structure of the T region of each vector. pUASA1 was constructed for expression of wild-type OASA1 cDNA, pUASA1D for a mutated OASA1 cDNA [OASA1(D323N)] encoding a protein in which Asp-323 is replaced by Asn, pUASA1M for a mutated OASA1 cDNA [OASA1(M340T)] encoding a protein in which Met-340 is replaced by Thr, and pUASA2 for wild-type OASA2 cDNA. A 2-kb maize Ubi1 fragment including the promoter region (P_Ubil) and 1-kb intron of the 5′-untranslated region was ligated to each rice cDNA at a point immediately downstream of the 3′ side of the intron junction. Arrows indicate the direction of transcription. See “Materials and Methods” for further details. B, Alignment of the amino acid sequences surrounding the mutated residue (bold) of each transgene product. The mutated gene OASA1(D323N) and wild-type OASA2 each encode Asn at the corresponding positions 323 and 351, respectively.

Figure 5

Expression of OASA transgenes in transformants. A, Expression of wild-type or mutated OASA1 transgenes in transformed calli. Total RNA (10 μg) was subjected to RNA gel-blot analysis with a digoxigenin-labeled OASA1 riboprobe (top); the ethidium bromide-stained agarose gel is also shown (bottom). NB, Untransformed Nipponbare callus; lanes 1 through 7, Transformants expressing the wild-type OASA1 transgene (nos. W1, W2, W3, W4, W5, W9, and W10, respectively); lanes 8 through 17, Calli expressing the OASA1(D323N) transgene (nos. D5, D11, D13, D16, D17, D18, D19, D20, D25, and D26, respectively); lanes 18 through 24, Calli expressing the OASA1(M340T) transgene (nos. E1, E2, E3, E10, E12, E15, and E16, respectively). B, Expression of the OASA2 transgene in the leaves of pUASA2 transformants. Lanes 1 through 8, Regenerated plants from callus line numbers 25-2, 22 -1, 47-3, 38-1, 49-1a, 46-1, 28-2, and 49-1b, respectively. Arrows in A and B indicate the transgene-derived transcripts.

Figure 6

5MT resistance of transformed rice calli. Calli of NB (A), transformant number D10-5 expressing OASA1(D323N) (B), transformant number W9 expressing OASA1 (C), transformant number E1 expressing OASA1(M340T) (D), and transformants numbers 25-2 (sensitive) (E) and 49-1 (moderately resistant) (F) expressing OASA2 were photographed after cultivation for 3 weeks. The medium conditions are shown in G.

Figure 7

Segregation of 5MT resistance in progeny. Growth after 14 d of control cv Nipponbare seedlings (left two) and of segregants of transgenic line number D1-1-5 expressing OASA1(D323N) (right four) on medium containing 300 μm 5MT. The segregation ratio for resistance and sensitivity to 5MT in this line was 20:5. Bar = 2 cm.

Figure 8

Relative AS activities of transformants expressing OASA1, OASA1(D323N), or OASA2 transgenes. A, Untransformed calli (○) as well as calli of transformant number W2 expressing OASA1 (□), and transformant numbers D17 (●) and D13 (▪) expressing OASA1(D323N) were assayed for AS activity in the presence of various concentrations of Trp. B, Untransformed calli (○) and OASA2-expressing calli numbers 14-1 (▴), 25-2 (●), 46-2 (▪), and 49-1 (⋄) were assayed for AS activity. Data are expressed as picomoles of anthranilate produced per minute per milligram of protein, and are means of triplicates from a representative experiment.

Figure 9

HPLC Profiles of soluble amino acids in untransformed calli and calli expressing OASA1(D323N). Untransformed calli (A) and OASA1(D323N)-expressing calli (B) of transformant number D13 were analyzed for soluble amino acid content by HPLC. Retention times for each amino acid and anthranilate were determined with standard samples. Peak numbers: 1, Asp; 2, Glu; 3, Ser; 4, Asn; 5, Gly; 6, Gln; 7, His; 8, Thr; 9, Ala; 10, Arg; 11, Pro; 12, Tyr; 13, Val; 14, Met; 15, Cys; 16, anthranilate; 17, Ile; 18, Leu; 19, Phe; 20, Trp; and 21, Lys.