RESUMO
The maize (Zea mays) b1 gene encodes a transcription factor that regulates the anthocyanin pigment pathway. Of the b1 alleles with distinct tissue-specific expression, B-Peru and B-Bolivia are the only alleles that confer seed pigmentation. B-Bolivia produces variable and weaker seed expression but darker, more regular plant expression relative to B-Peru. Our experiments demonstrated that B-Bolivia is not expressed in the seed when transmitted through the male. When transmitted through the female the proportion of kernels pigmented and the intensity of pigment varied. Molecular characterization of B-Bolivia demonstrated that it shares the first 530 bp of the upstream region with B-Peru, a region sufficient for seed expression. Immediately upstream of 530 bp, B-Bolivia is completely divergent from B-Peru. These sequences share sequence similarity to retrotransposons. Transient expression assays of various promoter constructs identified a 33-bp region in B-Bolivia that can account for the reduced aleurone pigment amounts (40%) observed with B-Bolivia relative to B-Peru. Transgenic plants carrying the B-Bolivia promoter proximal region produced pigmented seeds. Similar to native B-Bolivia, some transgene loci are variably expressed in seeds. In contrast to native B-Bolivia, the transgene loci are expressed in seeds when transmitted through both the male and female. Some transgenic lines produced pigment in vegetative tissues, but the tissue-specificity was different from B-Bolivia, suggesting the introduced sequences do not contain the B-Bolivia plant-specific regulatory sequences. We hypothesize that the chromatin context of the B-Bolivia allele controls its epigenetic seed expression properties, which could be influenced by the adjacent highly repeated retrotransposon sequence.
Assuntos
Alelos , Genes de Plantas , Retroelementos , Zea mays/genética , Dados de Sequência Molecular , Filogenia , Pigmentação/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , SementesRESUMO
The B-Peru allele of the maize b regulatory gene is unusual relative to most b alleles in that it is expressed in the aleurone layer of the seed. It is also expressed in a subset of plant vegetative tissues. Transgenic maize plants containing the B-Peru gene with the first 710 bases of upstream sequence conferred the same levels of aleurone expression as nontransgenic B-Peru plants, but no pigment was made in vegetative tissues. Transient transformation assays in aleurone tissue localized the aleurone-specific promoter to the first 176 bases of the B-Peru upstream region and identified two critically important regions within this fragment. Mutation of either region alone reduced expression greater than fivefold. Surprisingly, the double mutation actually increased expression to twice the native promoter level. Our results suggest that these two critical sequences, which lie close together in the promoter, may form a negative regulatory element. Several lines of evidence suggest that the B-Peru promoter arose through the translocation of an existing aleurone-specific promoter to the b locus. Immediately upstream of the aleurone-specific promoter elements and in the opposite orientation to the b coding sequence is a pseudogene sequence with strong similarity to a known class of proteins. Our findings that novel aleurone-specific promoter sequences of the B-Peru transcription factor are found adjacent to part of another gene in a small insertion are quite unexpected and have interesting evolutionary implications.
Assuntos
Genes de Plantas , Genes Reguladores/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Translocação Genética , Zea mays/genética , Alelos , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Éxons , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Mutagênese Insercional , Especificidade de Órgãos/genética , Proteínas de Plantas/biossíntese , Plantas Geneticamente Modificadas , Pseudogenes , Sementes/genética , Fatores de Transcrição/biossíntese , Zea mays/embriologiaRESUMO
The b locus of Zea mays encodes a transcriptional activator of the anthocyanin biosynthetic pathway. The B-Peru allele is expressed in the aleurone layer of the seed, which results in dark purple pigmentation of this tissue. An unstable Mutator-induced B-Peru mutant allele, b-Perum220, displays weak, variable pigment and a high germinal reversion rate not characteristic of other Mutator insertions. Characterization of relevant regions of b-Perum220 revealed a Mu2 element insertion in one copy of a 534 bp sequence. This 534 bp sequence is tandemly triplicated in the progenitor B-Peru allele, upstream of the B-Peru transcription start site. In addition to the Mu2 insertion, the b-Perum220 allele contains a newly formed large tandem duplication of 4.0 kb, which includes the promoter region and the first three exons of the B-Peru gene. The Mu2 element does not reside at any of the duplication breakpoints. The molecular study of eleven independent germinal revertants revealed five structural classes including structures in which the 4.0 kb tandem duplication is partially or completely deleted, the Mu2 element is partially or completely deleted, or a combination of these events has occurred. We hypothesize that most of the revertants arose by unequal recombination between the duplicated regions. Based on these structural analyses, models are discussed to explain the reduced b gene expression in b-Perum220.
Assuntos
Elementos de DNA Transponíveis/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Fatores de Transcrição , Zea mays/genética , Alelos , Antocianinas/biossíntese , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Clonagem Molecular , Éxons/genética , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Mapeamento por Restrição , Sementes/genética , Análise de Sequência de DNA , Transcrição GênicaAssuntos
Antocianinas/genética , Genes Reguladores , Proteínas de Plantas/genética , Fatores de Transcrição , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Clonagem Molecular , Dados de Sequência Molecular , RNA Mensageiro/genética , Alinhamento de SequênciaRESUMO
The B locus in maize is required for the accumulation of anthocyanin pigments. Numerous B alleles have been described: each determines a particular pattern of pigment synthesis with respect to the tissues that are pigmented and the time during development that pigment synthesis begins. We report here a genetic analysis of one B allele, B-Peru, which regulates synthesis of pigments in both kernel and plant tissues. We used stocks with active Mutator transposable elements to produce eight mutations in B-Peru. All eight alter pigment synthesis in all the kernel and plant tissues pigmented by B-Peru, suggesting that each mutation has disrupted a region of the gene required for expression in all tissues. Six of the mutations cause a colorless phenotype, while two cause a reduction in pigment in both kernel and plant tissues. Four of the mutations are unstable, and four are stable upon self-pollination. Multiple independent revertants were isolated from each unstable allele. DNA blot analysis demonstrated that all eight mutants are the result of insertions within an approximately 5-kb region that encodes the B-Peru transcript. One of the four unstable alleles contains a Mu element, Mu1.7. Two others contain insertions related to the Spm family of transposable elements. Thus, our Mutator stocks also contained active transposable elements from the unrelated Spm family. Our experiments suggest that the B-Peru allele is not complex, but contains a single coding region that regulates pigment synthesis in multiple tissues.