The myb -homologous p1 gene regulates the synthesis of flavonoid pigments in maize kernel pericarp and cob; these floral organs are greatly modified in size and shape compared with their counterparts in teosinte, the progenitor of maize. To elucidate the molecular evolution of the p1 gene in relation to its expression and possible functions in maize and teosinte, we have isolated a second maize gene ( p2 ) that is highly homologous with the p1 gene, and a related gene ( p2-t ) from Zea mays subsp parviglumis . We present evidence that the maize p1 and p2 genes were generated by duplication of an ancestral p gene ( p pre ) and its downstream sequences; the duplicated 3 Ј flanking sequences were inserted upstream of the p pre gene, thereby changing its transcription pattern. This model accounts for the structural organization and the observed differential expression of the p1 and p2 genes: p1 transcripts accumulate in kernel pericarp, cob, tassel glumes, and silk, whereas p2 transcripts are found in developing anther and silk. The duplication is estimated to have occurred 2.75 million years ago; subsequently, multiple retroelements have been inserted between the p1 and p2 genes. Our results demonstrate the evolution of a single gene into a compound locus containing two component genes with different tissue specificities. Expression of the p1 gene in the kernel pericarp may have provided a selective advantage during the evolution of maize kernel morphology. INTRODUCTIONEukaryotic genomes have been shaped to a large extent by gene duplications. Changes in ploidy are common in plants and give rise to immediate whole-genome duplications. Local gene duplications are commonly observed in genomic sequence analysis; for example, a 1.9-Mb stretch of Arabidopsis genomic sequence contains eight pairs of related genes, located adjacent to each other and in the same orientation (Bevan et al., 1998). These local sequence repeats apparently are produced by segmental duplications of discreet chromosome intervals. By whatever mechanism they occur, duplications can have a fundamentally important role in evolution. A complete gene duplication produces two identical copies, which then may undergo one of several alternative fates. Both gene copies may retain their original function, enabling the organism to produce a greater quantity of RNA or proteins. Or one of the copies may retain the original function, whereas the other copy may mutate to a functionless state (pseudogene) or acquire mutations that generate a new function (neomorph) (Li and Graur, 1991). Finally, one copy may retain the same coding sequence function but acquire new regulatory elements that specify a different pattern of expression. The latter process is best exemplified in the evolution of genes that regulate biosynthesis of flavonoid pigments in plants. In maize, anthocyanin biosynthesis is controlled by the combined function of two groups of regulatory genes: the c1/pl genes, which encode Myb-homologous transcriptional activators, and the r/b gene family, which ...

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In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3′H encoding gene (Zmf3′h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3′H1 promoter–gene construct established that the encoded protein product was sufficient to perform a 3′-hydroxylation reaction. The Zmf3′h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5′-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize.

In Sorghum bicolor, a group of phytoalexins are induced at the site of infection by Colletotrichum sublineolum, the anthracnose fungus. These compounds, classified as 3-deoxyanthocyanidins, have structural similarities to the precursors of phlobaphenes. Sorghum yellow seed1 (y1) encodes a MYB transcription factor that regulates phlobaphene biosynthesis. Using the candystripe1 transposon mutagenesis system in sorghum, we have isolated functional revertants as well as loss-of-function alleles of y1. These near-isogenic lines of sorghum show that, compared to functionally revertant alleles, loss of y1 lines do not accumulate phlobaphenes. Molecular characterization of two null y1 alleles shows a partial internal deletion in the y1 sequence. These null alleles, designated as y1-ww1 and y1-ww4, do not accumulate 3-deoxyanthocyanidins when challenged with the nonpathogenic fungus Cochliobolus heterostrophus. Further, as compared to the wild-type allele, both y1-ww1 and y1-ww4 show greater susceptibility to the pathogenic fungus C. sublineolum. In fungal-inoculated wild-type seedlings, y1 and its target flavonoid structural genes are coordinately expressed. However, in y1-ww1 and y1-ww4 seedlings where y1 is not expressed, steady-state transcripts of its target genes could not be detected. Cosegregation analysis showed that the functional y1 gene is genetically linked with resistance to C. sublineolum. Overall results demonstrate that the accumulation of sorghum 3-deoxyanthocyanidin phytoalexins and resistance to C. sublineolum in sorghum require a functional y1 gene.

SummaryThe maize P gene encodes a Myb-like transcription factor that regulates synthesis of red¯avonoid pigments in¯oral organs. To study the transcriptional regulation of the P gene, candidate regulatory sequences of the P1-rr gene promoter were identi®ed by Ac insertional mutagenesis and subjected to functional testing in transgenic maize plants. The results indicate that a 561 bp fragment (Pb) encompassing the transcription start site (±235 to +326) supports weak expression of a GUS reporter gene in¯oral organs, including husk, silk, kernel pericarp, cob and male in¯orescence. Two other fragments, located approximately 1 and 5 kb 5¢ of the transcription start site, increased the levels of GUS activity in¯oral tissues and thus appear to contain enhancer elements. All of the tested constructs gave similar patterns of GUS expression, suggesting that the 561 bp Pb fragment that is common among the transgene constructs contains regulatory elements that promote activation in¯oral organs. The basal promoter and proximal enhancer fragments contain putative binding sites for bZip regulatory factors, and a complex arrangement of palindromes including a large inverted repeat of two tRNA-like genes. Possibly, interconversions between linear and cruciform conformations of the palindromes may affect protein/DNA interactions and thereby modulate P1-rr expression.