The Zea mays P gene has been postulated to regulate the biosynthetic pathway of a flavonoid-derived pigment in certain floral tissues [Styles, E. D. & Ceska, 0. (1977) Can. J. Genet. Cytol. 19,[289][290][291][292][293][294][295][296][297][298][299][300][301][302]. We have characterized two P transcripts that are alternatively spliced at their 3' ends. One message of 1802 nucleotides encodes a 43.7-kDa protein with an N-terminal region showing -40% homology to the DNAbinding domain of several members of the myb family of protooncogene proteins. A second message of 945 nucleotides encodes a 17.3-kDa protein that contains most of the mybhomologous domain but differs from the first protein at the C terminus. The deduced P-encoded proteins show an even higher homology (70%) in the myb-homologous domain to the maize regulatory gene C1. Additionally, the P and Cl genes are structurally similar in the sizes and positions of the first and second exons and first intron. We show that P is required for accumulation in the pericarp of transcripts of two genes (Al and C2) encoding enzymes for flavonoid biosynthesis genes also regulated by Cl in the aleurone.Coordinated development of multicellular organisms requires precisely regulated differential gene expression; yet the molecular mechanisms involved in organ-and cellspecific regulation remain largely unknown. Analysis of regulatory genes and their targets is difficult if the genes' products are essential for viability. Flavonoid pigment biosynthesis in plants is an ideal system for studying gene regulation in higher eukaryotes, because the presence or absence of pigment has no deleterious effects, pigmentation is a convenient visual indicator of gene expression, and the biochemical steps to pigment synthesis are well defined (1).The flavonoid pigments commonly found in maize are either anthocyanins or phlobaphenes; although these two pigments have their initial synthetic steps in common, there are significant differences between them. Anthocyanins are derived from flavan-3,4-diol and can be produced in most tissues of the maize plant. Phlobaphenes are formed by the nonenzymatic polymerization of flavan-4-ol and are found only in certain floral tissues, including the pericarp and the glumes of the cob (2). [The pericarp is the outer covering of the maize kernel, derived from the ovary wall; the cob glumes are floral bracts that subtend the kernel (3)]. Four genes (R, B, Cl, and Po) are known to regulate anthocyanin biosynthesis in specific parts of the plant (recently reviewed in ref.4). The R gene family is involved in anthocyanin pigmentation in the aleurone, scutellum, coleoptile, roots, and anthers (1); R-like proteins have homology to the helix-loop-helix domain of the myc oncogene products (4). The B gene family, which is required for anthocyanin pigmentation in several other plant parts, has homology with R (5). The Cl gene is required for anthocyanin pigmentation of the kernel aleurone and embryo, while the Pi gene, which is homologous to Cl (6), is requi...

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The maize P gene is a transcriptional regulator of genes encoding enzymes for flavonoid biosynthesis in the pathway leading to the production of a red phlobaphene pigment. Multiple alleles of the P gene confer distinct patterns of pigmentation to specific floral organs, such as the kernel pericarp and cob tissues. To determine the basis of allele-specific pigmentation, we have characterized the gene products and transcript accumulation patterns of the P-wr allele, which specifies colorless pericarps and red cob tissues. RNA transcripts of P-wr are present in colorless pericarps as well as in the colored cob tissues; however, the expression of P-wr in pericarp does not induce the accumulation of transcripts from the C2 and A1 genes, which encode enzymes for flavonoid pigment biosynthesis. The coding sequences of P-wr were compared with the P-rr allele, which specifies red pericarp and red cob. The P-wr and P-rr cDNA sequences are very similar in their 5' regions. There are only two nucleotide changes that result in amino acid differences; both are outside of the Myb-homologous DNA binding domain. In contrast, the 3' coding region of P-rr is replaced by a unique 210-bp sequence in P-wr. The predicted P-wr protein has a C-terminal sequence resembling a cysteine-containing metal binding domain that is not present in the P-rr protein. These results indicate that the differential pericarp pigmentation specified by the P-rr and P-wr alleles does not result from an absence of P-wr transcripts in pericarps. Rather, the allele-specific patterns of P-rr and P-wr pigmentation may be associated with structural differences in the proteins encoded by each allele.

The maize P gene encodes a Myb-homologous transcriptional regulator of flavonoid pigmentation in floral organs, and different P gene alleles condition precise tissue- and organ-specific pigmentation patterns. To determine the molecular basis for allele-specific expression patterns, we have isolated and compared two natural alleles of the P gene which differ in expression, structure and copy number. The P-rr allele is associated with pigmentation of most floral tissues and contains a single copy of the P gene. In contrast, the P-wr allele restricts pigmentation to a subset of floral tissues, and is composed of six gene copies arranged in a tandem head-to-tail array. Each of the six repeats contains a single P gene, including regulatory and coding sequences. Despite the six-fold tandem repetition of P-wr gene copies, P-wr mRNA levels in kernel pericarp are much reduced compared to mRNA levels from the single-copy P-rr gene. Moreover, the P-wr multicopy complex is hypermethylated relative to P-rr. Thus, maize P gene alleles may represent a natural system for studying the effects of methylation and gene copy number on tissue-specific gene expression. We discuss the possibility that somatic pairing of repeated gene copies may be involved in regulating gene expression.

The maize P gene is a transcriptional regulator of genes encoding enzymes for flavonoid biosynthesis in the pathway leading to the production of a red phlobaphene pigment. Multiple alleles of the P gene confer distinct patterns of pigmentation to specific floral organs, such as the kernel pericarp and cob tissues. To determine the basis of allele-specific pigmentation, we have characterized the gene products and transcript accumulation patterns of the P-wr allele, which specifies colorless pericarps and red cob tissues. RNA transcripts of P-wr are present in colorless pericarps as well as in the colored cob tissues; however, the expression of P-wr in pericarp does not induce the accumulation of transcripts from the C2 and A1 genes, which encode enzymes for flavonoid pigment biosynthesis. The coding sequences of P-wr were compared with the P-rr allele, which specifies red pericarp and red cob. The P-wr and P-rr cDNA sequences are very similar in their 5' regions. There are only two nucleotide changes that result in amino acid differences; both are outside of the Myb-homologous DNA binding domain. In contrast, the 3' coding region of P-rr is replaced by a unique 210-bp sequence in P-wr. The predicted P-wr protein has a C-terminal sequence resembling a cysteine-containing metal binding domain that is not present in the P-rr protein. These results indicate that the differential pericarp pigmentation specified by the P-rr and P-wr alleles does not result from an absence of P-wr transcripts in pericarps. Rather, the allele-specific patterns of P-rr and P-wr pigmentation may be associated with structural differences in the proteins encoded by each allele.