RESUMEN
The switch from an out-crossing to a self-fertilizing mating system is one of the most prevalent evolutionary trends in plant reproduction and is thought to have occurred repeatedly in flowering plants. However, little is known about the evolution of self-fertility and the genetic architecture of selfing. Here, we establish Arabidopsis thaliana as a model for genetic analysis of the switch to self-fertility in the crucifer family, where the ancestral out-crossing mode of mating is determined by self-incompatibility (SI), a genetic system controlled by the S locus. We show that A. thaliana ecotypes exhibit S-locus polymorphisms and differ in their ability to express the SI trait upon transformation with S-locus genes derived from the obligate out-crosser Arabidopsis lyrata. Remarkably, at least one ecotype was reverted to a stable, self-incompatible phenotype identical to that of naturally self-incompatible species. These ecotype differences are heritable and reflect the fixation in different A. thaliana populations of independent mutations that caused or enforced the switch to self-fertility. Their continued analysis promises to identify the loci that were the targets of natural selection for selfing and to contribute to a mechanistic understanding of the SI response.
Asunto(s)
Arabidopsis/genética , Arabidopsis/fisiología , Evolución Biológica , Arabidopsis/clasificación , Arabidopsis/crecimiento & desarrollo , Secuencia de Bases , Cruzamientos Genéticos , ADN de Plantas/genética , Genes de Plantas , Variación Genética , Plantas Modificadas Genéticamente , Polimorfismo Genético , Reproducción/genética , Especificidad de la Especie , Transformación GenéticaRESUMEN
Genetic self-incompatibility in Brassica is determined by alleles of the transmembrane serine-threonine kinase SRK, which functions in the stigma epidermis, and of the cysteine-rich peptide SCR, which functions in pollen. Using tagged versions of SRK and SCR as well as endogenous stigma and pollen proteins, we show that SCR binds the SRK ectodomain and that this binding is allele specific. Thus, SRK and SCR function as a receptor-ligand pair in the recognition of self pollen. Specificity in the self-incompatibility response derives from allele-specific formation of SRK-SCR complexes at the pollen-stigma interface.
Asunto(s)
Alelos , Brassica/genética , Brassica/metabolismo , Proteínas de Plantas/metabolismo , Estructuras de las Plantas/metabolismo , Polen/metabolismo , Proteínas Quinasas/metabolismo , Sitios de Unión , Fertilización/fisiología , Ligandos , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Plantas Tóxicas , Unión Proteica , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Especificidad por Sustrato , NicotianaRESUMEN
As a starting point for a phylogenetic study of self-incompatibility (SI) in crucifers and to elucidate the genetic basis of transitions between outcrossing and self-fertilizing mating systems in this family, we investigated the SI system of Arabidopsis lyrata. A. lyrata is an outcrossing close relative of the self-fertile A. thaliana and is thought to have diverged from A. thaliana approximately 5 million years ago and from Brassica spp 15 to 20 million years ago. Analysis of two S (sterility) locus haplotypes demonstrates that the A. lyrata S locus contains tightly linked orthologs of the S locus receptor kinase (SRK) gene and the S locus cysteine-rich protein (SCR) gene, which are the determinants of SI specificity in stigma and pollen, respectively, but lacks an S locus glycoprotein gene. As described previously in Brassica, the S haplotypes of A. lyrata differ by the rearranged order of their genes and by their variable physical sizes. Comparative mapping of the A. lyrata and Brassica S loci indicates that the S locus of crucifers is a dynamic locus that has undergone several duplication events since the Arabidopsis--Brassica split and was translocated as a unit between two distant chromosomal locations during diversification of the two taxa. Furthermore, comparative analysis of the S locus region of A. lyrata and its homeolog in self-fertile A. thaliana identified orthologs of the SRK and SCR genes and demonstrated that self-compatibility in this species is associated with inactivation of SI specificity genes.
Asunto(s)
Arabidopsis/genética , Brassica/genética , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Brassicaceae/genética , Mapeo Cromosómico , Clonación Molecular , Intercambio Genético , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Genes de Plantas , Glicoproteínas , Haplotipos , Región de Control de Posición , Datos de Secuencia Molecular , Polen/fisiología , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Proteínas Quinasas , Proteínas Recombinantes , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
It is estimated that 5 million years of evolution separate Arabidopsis thaliana from its close relative Arabidopsis lyrata. The two taxa differ by many characteristics, and together they exemplify the differentiation of angiosperms into self-fertilizing and cross-fertilizing species as well as annual and perennial species. Despite their disparate life histories, the two species can be crossed to produce viable and vigorous hybrids exhibiting heterotic effects. Although pollen sterile, the hybrids produce viable ovules and were used as female parent in backcrosses to both parental species. The resulting backcross plants exhibited transgressive variation for a number of interesting developmental and growth traits as well as negative nuclear/cytoplasmic interactions. Moreover, the genesis of a fertile amphidiploid neospecies, apparently by spontaneous somatic doubling in an interspecific hybrid, was observed in the laboratory. The mechanisms responsible for the generation of amphiploids and the subsequent evolution of amphiploid genomes can now be studied through direct observation using the large arsenal of molecular tools available for Arabidopsis.
Asunto(s)
Arabidopsis/genética , Evolución Molecular , Hibridación Genética/genética , Ploidias , Arabidopsis/crecimiento & desarrollo , Segregación Cromosómica , Cruzamientos Genéticos , Variación Genética , Vigor Híbrido/genética , Especificidad de la EspecieAsunto(s)
Brassicaceae/citología , Proteínas de Plantas/genética , Polen/citología , Transducción de Señal , Secuencia de Aminoácidos , Brassicaceae/genética , Brassicaceae/fisiología , Haplotipos , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Polen/fisiología , Polen/ultraestructura , Polimorfismo Genético , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Significant progress towards understanding the molecular basis of self recognition in the self-incompatibility response of Brassica has been made during the past two years. The highly polymorphic molecules that determine the specificity of this interaction in the pollen and stigma have been identified. The structural features of these molecules suggest that a ligand-receptor-kinase interaction triggers the initiation of a signaling cascade within the stigma epidermis and the subsequent arrest of self-pollination.
Asunto(s)
Brassica/fisiología , Glicoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Comunicación Celular , Haplotipos , Datos de Secuencia Molecular , Polen , ReproducciónRESUMEN
The S-locus-encoded S receptor kinase (SRK) is an intrinsic plasma membrane protein that is viewed as the primary stigma determinant of specificity in the self-incompatibility response of Brassica spp. We analyzed two self-compatible mutant strains that express low levels of the S-locus glycoprotein (SLG), a cell wall-localized protein also encoded at the S locus that is coordinately expressed with SRK. We found that mutant stigmas synthesized wild-type levels of SRK transcripts but failed to produce SRK protein at any of the developmental stages analyzed. Furthermore, SRK was shown to form aberrant high-molecular mass aggregates when expressed alone in transgenic tobacco (Nicotiana tabacum) plants. This aggregation was prevented in tobacco plants that co-expressed SRK and SLG, but not in tobacco plants that co-expressed SRK and SLR1, an SLG-related secreted protein not encoded at the S locus. In analyses of protein extracts under reducing and non-reducing conditions, evidence of intermolecular association was obtained only for SLG, a fraction of which formed disulfide-linked oligomers and was membrane associated. The data indicate that, at least in plants carrying the S haplotypes we analyzed, SRK is an inherently unstable protein and that SLG facilitates its accumulation to physiologically relevant levels in Brassica stigmas.
Asunto(s)
Brassica/genética , Glicoproteínas/genética , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Tóxicas , Proteínas Quinasas/genética , ARN Mensajero/metabolismo , Brassica/metabolismo , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica de las Plantas , Glicoproteínas/metabolismo , Immunoblotting , Proteínas de Plantas/metabolismo , Estructuras de las Plantas/genética , Estructuras de las Plantas/metabolismo , Plantas Modificadas Genéticamente , Isoformas de Proteínas , Proteínas Quinasas/metabolismo , Nicotiana/metabolismo , Transcripción GenéticaRESUMEN
A genetic analysis was performed to study the frequency of recombination for intervals across the Brassica S locus region. No recombination was observed between the S locus glycoprotein gene and the S receptor kinase gene in the segregating populations that we analyzed. However, a number of recombination breakpoints in regions flanking these genes were identified, allowing the construction of an integrated genetic and physical map of the genomic region encompassing one S haplotype. We identified, based on the pollination phenotype of plants homozygous for recombinant S haplotypes, a 50-kb region that encompasses all specificity functions in the S haplotype that we analyzed. Mechanisms that might operate to preserve the tight linkage of self-incompatibility specificity genes within the S locus complex are discussed in light of the relatively uniform recombination frequencies that we observed across the S locus region and of the structural heteromorphisms that characterize different S haplotypes.
Asunto(s)
Brassica/genética , Mapeo Cromosómico , Genes de Plantas , Haplotipos , Familia de Multigenes , Fenotipo , Polen/genética , Recombinación GenéticaRESUMEN
In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor.
Asunto(s)
Brassica/fisiología , Genes de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Polen/fisiología , Alelos , Secuencia de Aminoácidos , Brassica/genética , Cisteína/química , Germinación , Glicoproteínas/genética , Glicoproteínas/metabolismo , Haplotipos , Ligandos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estructuras de las Plantas/genética , Estructuras de las Plantas/fisiología , Polen/genética , Polimorfismo Genético , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Alineación de Secuencia , Transformación GenéticaRESUMEN
The crucifer family includes self-incompatible genera, such as Brassica, and self-fertile genera, such as Arabidopsis. To gain insight into mechanisms underlying the evolution of mating systems in this family, we used a selective comparative mapping approach between Brassica campestris plants homozygous for the S8 haplotype and Arabidopsis. Starting with markers flanking the self-incompatibility genes in Brassica, we identified the homeologous region in Arabidopsis as a previously uncharacterized segment of chromosome 1 in the immediate vicinity of the ethylene response gene ETR1. A total of 26 genomic and 21 cDNA markers derived from Arabidopsis yeast artificial and bacterial artificial chromosome clones were used to analyze this region in the two genomes. Approximately half of the cDNAs isolated from the region represent novel expressed sequence tags that do not match entries in the DNA and protein databases. The physical maps that we derived by using these markers as well as markers isolated from bacteriophage clones spanning the S8 haplotype revealed a high degree of synteny at the submegabase scale between the two homeologous regions. However, no sequences similar to the Brassica S locus genes that are known to be required for the self-incompatibility response were detected within this interval or other regions of the Arabidopsis genome. This observation is consistent with deletion of self-recognition genes as a mechanism for the evolution of autogamy in the Arabidopsis lineage.
Asunto(s)
Arabidopsis/genética , Evolución Biológica , Brassica/genética , Brassicaceae/genética , Mapeo Cromosómico , Arabidopsis/fisiología , Brassica/fisiología , Brassicaceae/fisiología , Cromosomas Artificiales de Levadura , Ligamiento Genético , Marcadores Genéticos , Haplotipos , Homocigoto , Reproducción , Mapeo RestrictivoRESUMEN
Differential display has proven to be a very successful technique for isolating differentially expressed transcripts. We sought to expand the capabilities of the technique by attempting to isolate cDNAs from specific genomic loci. Two loci of interest to us are the S locus and the MOD locus, both involved in the self-incompatibility phenomenon of Brassica. The S locus is a complex locus for which molecular markers have previously been isolated, and the MOD locus is a single-gene locus for which no markers are available. We used plant material from F2 populations that segregate for two allelic variants of each locus to create two bulks or pools of plants for each differential display screen. Pooling F2 individuals effectively homogenizes background polymorphisms found in the parent plants. RNA was prepared from each bulk and differential display was performed using a kit from GenHunter Corporation (Nashville, TN). One cDNA that segregated completely with the target locus was isolated from each screen. Multiple cDNAs that were linked to each locus were also identified. We have demonstrated that differential display, when used in conjunction with bulked segregants, is a very powerful technique for isolating cDNAs from genomic loci of interest.
Asunto(s)
Brassica/genética , Clonación Molecular/métodos , Genes de Plantas/genética , Cruzamientos Genéticos , ADN Complementario/genética , Marcadores Genéticos/genética , Germinación/genética , Glicoproteínas/genética , Haplotipos/genética , Canales Iónicos/genética , Proteínas de Plantas/genética , Proteínas Quinasas/genéticaRESUMEN
Self-incompatibility in Brassica refers to the rejection of self-related pollen and is mediated by a receptor protein kinase localized to the plasma membrane of the stigma epidermis in the flower. The recessive mutation mod eliminates self-incompatibility in the stigma. In mod mutants, self-compatibility was shown to be associated with the absence of transcripts encoded by an aquaporin-related gene. This observation suggests that a water channel is required for the self-incompatibility response of Brassica, which is consistent with the concept that regulation of water transfer from the stigma to pollen is a checkpoint in the early events of pollination in the crucifer family.
Asunto(s)
Brassica/fisiología , Genes de Plantas , Canales Iónicos/fisiología , Proteínas de Plantas/fisiología , Secuencia de Aminoácidos , Brassica/genética , Canales Iónicos/genética , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Polen , Reproducción , Agua/fisiologíaRESUMEN
In Brassica, the recognition of self-related pollen by the stigma is controlled by the highly polymorphic S locus that encodes several linked and coadapted genes and can span several hundred kilobases. We used pulsed-field gel electrophoresis to analyze the structure of different S haplotypes. We show that the S2 and S13 haplotypes of Brassica oleracea contain extensive sequence divergence and rearrangement relative to each other. In contrast, haplotypic configuration is more conserved between B. oleracea S13 and B. campestris S8, two haplotypes that have been proposed to be derived from a common ancestral haplotype based on sequence comparisons. These results support the view that extensive restructuring of the S locus preceded speciation in Brassica. This structural heteromorphism, together with haplotype-specific sequences, may suppress recombination within the S locus complex, potentially providing a mechanism for maintaining the linkage of coadapted allelic combinations of genes over time.
Asunto(s)
Brassica/genética , Glicoproteínas/genética , Haploidia , Proteínas de Plantas/genética , Electroforesis en Gel de Campo Pulsado , Mapeo Restrictivo , Fase SAsunto(s)
Brassica/fisiología , Glicoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Polen , Proteínas Quinasas/metabolismo , Transducción de Señal , Brassica/genética , Comunicación Celular , Evolución Molecular , Glicoproteínas/genética , Haplotipos , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Proteínas Quinasas/genéticaRESUMEN
ARK1 is a vegetatively expressed receptor protein kinase gene isolated from Arabidopsis thaliana based on its sequence similarity to Brassica genes involved in pollen-stigma signaling and the self-incompatibility response. This paper shows that the kinase domain of ARK1 autophosphorylates on serine and threonine residues when expressed as a recombinant fusion protein. ARK1 produces a 2.9 kb transcript encoding a transmembrane receptor protein kinase and a 1.4 kb transcript encoding the receptor domain alone. Constitutive high-level expression of ARK1 transcripts in transgenic Arabidopsis resulted in severe stunting and also disrupted normal cellular expansion and differentiation.
Asunto(s)
Arabidopsis/genética , Genes de Plantas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Mensajero/genética , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Fenotipo , Fosforilación , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Transcripción GenéticaRESUMEN
The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.
Asunto(s)
Brassica/enzimología , Regiones Promotoras Genéticas , Proteínas Quinasas/biosíntesis , Secuencia de Bases , Brassica/genética , Membrana Celular/enzimología , Clonación Molecular , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Ligamiento Genético , Vectores Genéticos , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas de Plantas/biosíntesis , Plantas Modificadas Genéticamente , Plantas Tóxicas , Proteínas Quinasas/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Homología de Secuencia de Ácido Nucleico , Nicotiana/enzimologíaRESUMEN
The self-incompatibility locus of Brassica consists of a coadapted gene complex that contains at least two genes required for the recognition and inhibition of pollen by the stigma when self-pollinated. Here, we report the identification of a third S locus-linked gene from the S2 haplotype of Brassica oleracea. This gene, which we designated SLA (for S Locus Anther), is a novel gene with an unusual structure. SLA is transcribed from two promoters to produce two complementary anther-specific transcripts, one spliced and the other unspliced, that accumulate in an antiparallel manner in developing microspores and anthers. The sequence of the spliced transcript showed the presence of two open reading frames that predict proteins of 10 and 7.5 kD. Neither transcript was produced in a self-compatible B. napus strain carrying an S2-like haplotype, indicating that the SLA gene in this strain is nonfunctional. Interestingly, sequences related to SLA were not detected in DNA or RNA from plants carrying S haplotypes other than S2. The haplotype specificity of SLA, its anther-specific expression, and its physical linkage to the S locus are properties expected for a gene that encodes a determinant of S2 specificity in pollen.