A DELETION MUTATION AT THE EP LOCUS CAUSES LOW SEED COAT PEROXIDASE-ACTIVITY IN SOYBEAN

The Ep locus severely affects the amount of peroxidase enzyme in soybe an seed coats. Plants containing the dominant Ep allele accumulate lar ge amounts of peroxidase in the hourglass cells of the sub-epidermis. Homozygous recessive epep genotypes do not accumulate peroxidase in th e hourglass cells and are much reduced in total seed coat peroxidase a ctivity. To isolate the gene encoding the seed coat peroxidase and to determine whether it corresponds to the Ep locus, a cDNA library was c onstructed from developing seed coats and an abundant 1.3 kb peroxidas e transcript was cloned. The corresponding structural gene was also is olated from a genomic library. Sequence analysis shows that the seed c oat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa. Processing of a putative 26 amino acid signal sequence results in a mature protein of 326 residues with a calculated mass of 35 kDa and a pI of 4.4. Using probes derived from the cDNA, genomic DNA blot hybridization and polymerase chain reaction analysis detected polymorp hisms that distinguished EpEp and epep genotypes. Co-segregation of th e polymorphisms in an F-2 population from a cross of EpEp and epep pla nts shows that the Ep locus encodes the seed coat peroxidase protein. Comparison of Ep and ep alleles indicates that the recessive gene lack s 87 bp of sequence encompassing the translation start codon. Analysis by RNA blot hybridization shows that epep plants have drastically red uced amounts of peroxidase transcript compared with EpEp plants. The p eroxidase mRNA is abundant in seed coat tissues of EpEp plants during the late stages of seed maturation, and could also be detected in root tissues, but not in the flower, embryo, pod or leaf. The results indi cate that the lack of peroxidase accumulation in seed coats of homozyg ous recessive epep plants is due to a mutation of the structural gene that reduces transcript abundance.