The orange pericarp mutant of maize is a tryptophan auxotroph --Allen D. Wright, Karen C. Cone, Cynthia A. Moehlenkamp, and M. G. Neuffer Orange pericarp is a seedling lethal mutant caused by the duplicate unlinked recessive loci, orp1 and orp2. Several lines of evidence suggest that the mutant is a tryptophan auxotroph. Mutant plants benefit from tryptophan supplementation. Mutant seedlings accumulate indole and a glycoside of anthranilic acid. This phenotype would be expected if the mutant were deficient in tryptophan, which normally inhibits synthesis of anthranilate synthase by a feedback mechanism. Accumulation of indole implies that the last step in the tryptophan pathway (catalyzed by tryptophan synthase) is defective. In keeping with this interpretation, the mutant is more resistant to the indole analog 5-fluoroindole than normal plants, probably due to the inability of the mutant to convert 5-fluoroindole to the toxic product 5-fluorotryptophan (A. Wright, MNL, this issue). The tryptophan synthase complex is composed of two subunits A and B, both of which are required for the conversion of indole glycerol phosphate plus serine to tryptophan plus glyceraldehyde-3-phosphate. The function of the B subunit is to catalyze the condensation of serine and indole to form tryptophan. The accumulation of indole in the orp mutant suggests that the mutant is deficient in the B subunit of tryptophan synthase (trpB) and furthermore that maize has duplicate genes for trpB.

Recently, the presence of two trpB synthase genes in the Arabidopsis genome was reported (PNAS 86:4604-4608, 1989). Using a heterologous hybridization probe from the Arabidopsis gene (kindly provided by Robert Last, Cornell University), we isolated a maize cDNA clone for trpB. Preliminary sequence analysis indicates that the clone shows about 40% identity at the amino acid level to the Arabidopsis trpB sequence. The maize cDNA hybridizes to two bands in restriction digests of maize genomic DNA, consistent with the notion that maize has two trpB genes.

To determine if the maize trpB genes are indeed orp1 and orp2, we took two approaches. First, we used RFLP mapping to find out if the trpB genes map at the same locations as orp1 and orp2. Second, we used molecular segregation analyses to ask if the orange pericarp phenotype segregates with the trpB RFLP's present in the orp1 orp2 homozygote.

The map locations of the maize trpB genes were determined by RFLP mapping in the recombinant inbred family TxCM. DNAs were prepared from the members of the family, digested with a restriction enzyme that allowed us to distinguish all four parental alleles of trpB as discretely sized fragments. Hybridization patterns were scored and linkage was determined by comparison to the compiled data for the TxCM family (Ben Burr, Brookhaven National Laboratory). By RFLP analysis, orp1 maps equidistant between BNL markers 15.45 and 7.20L at position 82 on chromosome 4L. This is in fairly good agreement with genetic mapping that placed orp1 less than one map unit from su on 4S (J. Heredity 80:229-233, 1989). orp1 is uncovered by TB-4Sa, but not by TB-4Lf (MNL 61:44-45). The linkage of orp1 to molecular markers on 4L rather than on 4S seems to suggest that the placement of the centromere relative to these molecular markers may need to be re-evaluated. By RFLP analysis, orp2 maps on 10S near the centromere. orp2 maps at the same location as Pioneer marker 06003, i.e., at position 23. Genetic analysis of repulsion backcrosses had placed orp2 near position 45 on 10L between g1 and r1. This discrepancy may be due to suppression of recombination across the centromere between orp2 and the markers on 10L. In summary, the RFLP mapping data are in reasonably good agreement with the genetic data and place the maize trpB genes at the same locations as orp1 and orp2.

Molecular segregation analysis was performed on a population segregating for orange pericarp. The population was generated by backcrossing orp1 and orp2 into the inbred Mo17 background for two generations and then selfing to obtain an ear that segregated 15:1 normal: orange kernels. Plants were grown from 6 orange kernels and from 16 normal kernels. DNA was isolated from the individual plants, digested with a restriction enzyme that allowed all parental alleles to be distinguished, and hybridized with the maize trpB cDNA. If the orange pericarp phenotype is due to mutations in both trpB genes, then we should see a trpB hybridization pattern unique to the DNAs from plants grown from orange kernels. The results confirmed this expectation, i.e., a distinct RFLP pattern was present for all DNAs from orp plants and absent for DNAs from normal plants.

Taken together, the molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the B subunit of tryptophan synthase. Furthermore, these results confirm orp1 orp2 as a tryptophan auxotroph of maize.

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