Seed coat color in soybean is determined by four alleles of the classically defined I (inhibitor) locus that controls the presence or absence as well as the spatial distribution of anthocyanin pigments in the seed coat. By analyzing spontaneous mutations of the I locus, we demonstrated that the I locus is a region of chalcone synthase (CHS) gene dupllcations. Paradoxically, deletions of CHS gene sequences allow higher levels of CHS mRNAs and restore pigmentation to the seed coat. The unusual nature of the I locus suggests that its dominant alleles may represent naturally occurring examples of homology-dependent gene silenclng and that the spontaneous deletions erase the gene-silencing phenomena. Specifically, mutations from the dominant i' allele (yellow seed coats wlth pigmented hila) to the recessive i allele (fully pigmented) can be associated wlth the absence of a 2.3-W Hindlll fragment that carries CHS4, a member of the multigene CHS family. Seven independent mutatlons exhibit deletions in the CHS4 promoter mgion. The dominant I allele (yellow seed coats) exhibits an extra 12.1-kb Hindlll fragment that hybridlzes with both the CHS coding region and CHSí promoter-specific probes. Mutatlons of the dominant I allele to the mcessive i allele (pigmented seed coats) give rise to 10.4-or 9.6-kb Hindlll CHS fragments that have lost the duplicated CHSí promoter. Finally, gene expression analysis demonstrated that heterozygous plants (//i) wlth yellow seed coats have reduced mRNA levels, indlcatlng that the 12.1-kb Hindlll CHS fragment associated with the dominant I allele inhibits pigmentation in a trans-dominant manner. Moreover, CHS gene-specific expression in seed coats shows that multiple CHS genes are expressed ln seed coats.

The dominant I gene inhibits accumulation of anthocyanin pigments in the epidermal layer of soybean (Crycine max) seed coats.Seed-coat color i s also influenced by the R locus and by the pubescence color alleles (T, tawny; f, gray). Protein and RNA from cultivars with black (i,R,T) and brown (i,r,T) seed coats are difficult to extract. To determine the nature of the interfering plant products, we examined seed-coat extracts from Clark isogenic lines for flavonoids, anthocyanins, and possible proanthocyanidins by thinlayer chromatography. We show that yellow seed-coat varieties ( I ) do not accumulate anthocyanins (anthocyanidin glycosides) or proanthocyanidins (polymeric anthocyanidins). Mature, black (i,R,T) and imperfect-black (i,R,f) seed coats contained anthocyanins, whereas mature, brown (i,r,r) and buff (i,r,f) seed coats did not contain anthocyanins. In contrast, all colored ( i ) genotypes tested positive for the presence of proanthocyanidins by butanol/ HCI and 0.5% vanillin assays. Immature, black (/R,T) and brown (&r,T) seed coats contained significant amounts of procyanidin, a 3',4'-hydroxylated proanthocyanidin. Immature, black (i,R,T) or brown (i,r,r) seed-coat extracts also tested positive for the ability to precipitate proteins in a radial diffusion assay and to bind RNA in vitro. Imperfect-black (i,R,f) or buff (i,r,f) seed coats contained lesser amounts of propelargonidin, a 4'-hydroxylated proanthocyanidin. Seed-coat extracts from these genotypes did not have the ability to precipitate protein or bind to RNA. In summary, the dominant I gene controls inhibition of not only anthocyanins but also proanthocyanidins in soybean seed coats. In homozygous recessive i genotypes, the T-f gene pair determines the types of proanthocyanidins present, which is consistent with the hypothesis that the T locus encodes a microsomal 3'-flavonoid hydroxylase.Commercially grown soybean (Glycine max) varieties have yellow seed. However, some soybean varieties are selfcolored and accumulate anthocyanins within the epidermal layer of the seed coat. The I locus and the R locus along with the pleiotropic T locus have major effects on seed-coat color (reviewed by Bemard and Weiss, 1973;Palmer and Kilen, 1987). The 1 locus (inhibitor) has four alleles that determine the absence or presence of pigment along with the spatial distribution of the pigments. Unlike other species in which the presence of pigment is usually the dominant phenotype, the absence of anthocyanin pigments in soybean seed coats is dominant because of the 1 allele, which inhibits pigment The anthocyanins (anthocyanidin glycosides) of the mature, black seed coat (i,R,T) have been identified as cyanidin-

Seed coat color in soybean is determined by four alleles of the classically defined / (inhibitor) locus that controls the presence or absence as well as the spatial distribution of anthocyanin pigments in the seed coat. By analyzing spontaneous mutations of the / locus, we demonstrated that the / locus is a region of chalcone synthase (CHS) gene duplications. Paradoxically, deletions of CHS gene sequences allow higher levels of CHS mRNAs and restore pigmentation to the seed coat. The unusual nature of the / locus suggests that its dominant alleles may represent naturally occurring examples of homology-dependent gene silencing and that the spontaneous deletions erase the gene-silencing phenomena. Specifically, mutations from the dominant ii allele (yellow seed coats with pigmented hila) to the recessive i allele (fully pigmented) can be associated with the absence of a 2.3-kb Hindlll fragment that carries CHS4, a member of the multigene CHS family. Seven independent mutations exhibit deletions in the CHS4 promoter region. The dominant / allele (yellow seed coats) exhibits an extra 12.1-kb Hindlll fragment that hybridizes with both the CHS coding region and CHS1 promoter-specific probes. Mutations of the dominant / allele to the recessive i allele (pigmented seed coats) give rise to 10.4- or 9.6-kb Hindlll CHS fragments that have lost the duplicated CHS1 promoter. Finally, gene expression analysis demonstrated that heterozygous plants (I/i) with yellow seed coats have reduced mRNA levels, indicating that the 12.1-kb Hindlll CHS fragment associated with the dominant / allele inhibits pigmentation in a trans-dominant manner. Moreover, CHS gene-specific expression in seed coats shows that multiple CHS genes are expressed in seed coats.

The seed of all wild Clycine accessions have black or brown pigments because of the homozygous recessive i allele in combination with alleles at the R and T loci. In contrast, nearly all commercial soybean (GIycine max) varieties are yellow due to the presence of a dominant allele of the I locus (either I or rl) that inhibits pigmentation in the seed coats. Spontaneous mutations to the recessive i allele occur in these varieties and result in pigmented seed coats. We have isolated a clone for a soybean dihydroflavonol reductase (DFR) gene using polymerase chain reaction. We examined expression of DFR and two other genes of the flavonoid pathway during soybean seed coat development in a series of nearisogenic isolines that vary in pigmentation as specified by combinations of alleles of the I , R, and T loci. The expression of phenylalanine ammonia-lyase and DFR mRNAs was similar in all of the gene combinations at each stage of seed coat development. In contrast, chalcone synthase (CHS) mRNA was barely detectable at all stages of development in seed coats that carry the dominant I allele that results in yellow seed coats. CHS activity in yellow seed coats (I) was also 7-to 10-fold less than in the pigmented seed coats that have the homozygous recessive i allele. It appears that the dominant I allele results in reduction of CHS mRNA, leading to reduction of CHS activity as the basis for inhibition of anthocyanin and proanthocyanin synthesis in soybean seed coats. A further connection between CHS and the I locus is indicated by the occurrence of multiple restriction site polymorphisms in genomic DNA blots of the CHS gene family in near-isogenic lines containing alleles of the I locus.Pigmentation of soybean (Glycine max) seed coats is influenced by at least three genes, I, R, and T. The I gene (for inhibitor) controls synthesis and spatial occurrence of pigments in the epidermal layer of the seed coat, whereas R and T are responsible for the specific color (reviewed in Bemard and Weiss, 1973;Palmer and Kilen, 1987). The dominant I allele inhibits pigment accumulation, resulting in a yellow seed coat color at maturity, whereas the homozygous, recessive i allele specifies full pigmentation across the entire seedWe are grateful for a graduate fellowship award from the. Na- Recently, we showed that polymeric proanthocyanidins are also synthesized in pigmented seed coats with the homozygous recessive i genotype (Todd and Vodkin, 1993). Proanthocyanidins are not precursors of anthocyanins but are synthesized as polymeric condensation products of leucoanthocyanins, the precursors of anthocyanins. Brown (irT) and buff (irt) seed coats do not contain anthocyanins but they do synthesize proanthocyanidins. The mechanism of inhibition of anthocyanin and proanthocyanidin synthesis by the I allele is unknown.Spontaneous mutations of the dominant I alleles ( I or 2%) to the recessive i allele have occurred in many soybean varieties, resulting in isogenic lines with pigmented seed coats. A Prorich cell wall protein, PRP1, and its ...