RESUMEN
Cereal grain develops from fertilised florets. Alterations in floret and grain development greatly influence grain yield and quality. Despite this, little is known about the underlying genetic control of these processes, especially in key temperate cereals such as barley and wheat. Using a combination of near-isogenic mutant comparisons, gene editing and genetic analyses, we reveal that HvAPETALA2 (HvAP2) controls floret organ identity, floret boundaries, and maternal tissue differentiation and elimination during grain development. These new roles of HvAP2 correlate with changes in grain size and HvAP2-dependent expression of specific HvMADS-box genes, including the B-sister gene, HvMADS29 Consistent with this, gene editing demonstrates that HvMADS29 shares roles with HvAP2 in maternal tissue differentiation. We also discovered that a gain-of-function HvAP2 allele masks changes in floret organ identity and grain size due to loss of barley LAXATUM.A/BLADE-ON-PETIOLE2 (HvBOP2) gene function. Taken together, we reveal novel pleiotropic roles and regulatory interactions for an AP2-like gene controlling floret and grain development in a temperate cereal.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Hordeum/metabolismo , Proteínas de Dominio MADS/metabolismo , Proteínas de Plantas/metabolismo , Alelos , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Grano Comestible/anatomía & histología , Grano Comestible/metabolismo , Flores/crecimiento & desarrollo , Flores/metabolismo , Edición Génica , Regulación de la Expresión Génica de las Plantas , Genotipo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Hordeum/crecimiento & desarrollo , Proteínas de Dominio MADS/genética , Mutagénesis , Fenotipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The normal developmental sequence in a grass grain entails the death of several maternal and filial tissues in a genetically regulated process termed programmed cell death (PCD). The progression and molecular aspects of PCD in developing grains have been reported for domesticated species such as barley, rice, maize and wheat. Here, we report a detailed investigation of PCD in the developing grain of the wild model species Brachypodium distachyon. We detected PCD in developing Brachypodium grains using molecular and histological approaches. We also identified in Brachypodium the orthologs of protease genes known to contribute to grain PCD and surveyed their expression. We found that, similar to cereals, PCD in the Brachypodium nucellus occurs in a centrifugal pattern following anthesis. However, compared to cereals, the rate of post-mortem clearance in the Brachypodium nucellus is slower. However, compared to wheat and barley, mesocarp PCD in Brachypodium proceeds more rapidly in lateral cells. Remarkably, Brachypodium mesocarp PCD is not coordinated with endosperm development. Phylogenetic analysis suggests that barley and wheat possess more vacuolar processing enzymes that drive nucellar PCD compared to Brachypodium and rice. Our expression analysis highlighted putative grain-specific PCD proteases in Brachypodium. Combined with existing knowledge on grain PCD, our study suggests that the rate of nucellar PCD moderates grain size and that the pattern of mesocarp PCD influences grain shape.
Asunto(s)
Apoptosis/genética , Brachypodium/genética , Grano Comestible/genética , Cisteína Endopeptidasas/genética , Endospermo/genética , Hordeum/genética , Oryza/genética , Filogenia , Proteínas de Plantas/genética , Semillas/genética , Triticum/genéticaRESUMEN
Fruits come in an impressive array of shapes, sizes, and consistencies, and also display a huge diversity in biochemical/metabolite profiles, wherein lies their value as rich sources of food, nutrition, and pharmaceuticals. This is in addition to their fundamental function in supporting and dispersing the developing and mature seeds for the next generation. Understanding developmental processes such as fruit development and ripening, particularly at the genetic level, was once largely restricted to model and crop systems for practical and commercial reasons, but with the expansion of developmental genetic and evo-devo tools/analyses we can now investigate and compare aspects of fruit development in species spanning the angiosperms. We can superimpose recent genetic discoveries onto the detailed characterization of fruit development and ripening conducted with primary considerations such as yield and harvesting efficiency in mind, as well as on the detailed description of taxonomically relevant characters. Based on our own experience we focus on two very morphologically distinct and evolutionary distant fruits: the capsule of opium poppy, and the grain or caryopsis of cereals. Both are of massive economic value, but because of very different constituents; alkaloids of varied pharmaceutical value derived from secondary metabolism in opium poppy capsules, and calorific energy fuel derived from primary metabolism in cereal grains. Through comparative analyses in these and other fruit types, interesting patterns of regulatory gene function diversification and conservation are beginning to emerge.
Asunto(s)
Frutas/crecimiento & desarrollo , Frutas/genética , Grano Comestible/genética , Grano Comestible/crecimiento & desarrollo , Genes de Plantas , Papaver/genética , Papaver/crecimiento & desarrollo , Filogenia , Proteínas de Plantas/metabolismoRESUMEN
The plant-specific YABBY genes were initially defined by their roles in determining abaxial/adaxial cell fate in lateral organs of eudicots, and repressing meristematic genes in differentiating tissues such as leaves. In Arabidopsis thaliana FILAMENTOUS FLOWER (FIL) is also required for inflorescence and floral meristem establishment and flower development in a pathway involving the floral transition and identity genes. Here we describe the characterization of a FIL orthologue from the basal eudicot, Papaver somniferum (the opium poppy), and demonstrate a role for the gene in patterning the highly lobed leaf of the poppy. Silencing of PapsFIL using viral-induced gene silencing resulted in leaves of reduced laminar area, more pronounced margin serration and, in some cases, leaf bifurcation. In contrast, the gene does not appear to affect the development of the flower, and these variations in function are discussed in relation to its taxonomic position as a basal eudicot and its determinate growth habit.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Papaver/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Agrobacterium/genética , Agrobacterium/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Clonación Molecular , Flores/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Silenciador del Gen , Genes de Plantas , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Datos de Secuencia Molecular , Papaver/crecimiento & desarrollo , Papaver/metabolismo , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Transformación GenéticaRESUMEN
BACKGROUND: Bidirectional promoters are common in genomes but under-studied experimentally, particularly in plants. We describe a targeted identification and selection of a subset of putative bidirectional promoters to identify genes involved in seed development and to investigate possible coordinated responses of gene pairs to conditions important in seed maturation such as desiccation and ABA-regulation. RESULTS: We combined a search for 100-600 bp intergenic regions in the Arabidopsis genome with a cis-element based selection for those containing multiple copies of the G-box motif, CACGTG. One of the putative bidirectional promoters identified also contained a CE3 coupling element 5 bp downstream of one G-box and is identical to that characterized previously in the HVA1 promoter of barley. CE3 elements are significantly under-represented and under-studied in Arabidopsis. We further characterized the pair of genes associated with this promoter and uncovered roles for two small, previously uncharacterized, plant-specific proteins in Arabidopsis seed development and stress responses. CONCLUSIONS: Using bioinformatics we identified putative bidirectional promoters involved in seed development and analysed expression patterns for a pair of plant-specific genes in various tissues and in response to hormones/stress. We also present preliminary functional analysis of these genes that is suggestive of roles in seed development.
Asunto(s)
Arabidopsis/genética , Arabidopsis/fisiología , Regiones Promotoras Genéticas , Semillas/crecimiento & desarrollo , Semillas/genética , Ácido Abscísico/farmacología , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Emparejamiento Base/genética , Secuencia de Bases , ADN Bacteriano/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Heterocigoto , Homocigoto , Datos de Secuencia Molecular , Reguladores del Crecimiento de las Plantas/farmacología , Semillas/efectos de los fármacos , Especificidad de la Especie , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Members of the core pooids represent the most important crops in temperate zones including wheat, barley, and oats. Their importance as crops is largely due to the grain, particularly the storage capabilities of the endosperm. In this study, a comprehensive survey of grain morphology and endosperm organization in representatives of wild and cultivated species throughout the core pooids was performed. As sister to the core pooid tribes Poeae, Aveneae, Triticeae, and Bromeae within the Pooideae subfamily, Brachypodium provides a taxonomically relevant reference point. Using macroscopic, histological, and molecular analyses distinct patterns of grain tissue organization in these species, focusing on the peripheral and modified aleurone, are described. The results indicate that aleurone organization is correlated with conventional grain quality characters such as grain shape and starch content. In addition to morphological and organizational variation, expression patterns of candidate gene markers underpinning this variation were examined. Features commonly associated with grains are largely defined by analyses on lineages within the Triticeae and knowledge of grain structure may be skewed as a result of the focus on wheat and barley. Specifically, the data suggest that the modified aleurone is largely restricted to species in the Triticeae tribe.
Asunto(s)
Grano Comestible/anatomía & histología , Endospermo/anatomía & histología , Poaceae/anatomía & histología , Brachypodium/anatomía & histología , Brachypodium/genética , Brachypodium/metabolismo , Tamaño de la Célula , Pared Celular , Grano Comestible/genética , Grano Comestible/metabolismo , Endospermo/genética , Endospermo/metabolismo , Hordeum/anatomía & histología , Hordeum/genética , Hordeum/metabolismo , Indicadores y Reactivos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poaceae/genética , Poaceae/metabolismo , ARN Mensajero/genética , ARN de Planta/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Almidón/metabolismo , Sales de Tetrazolio , Cloruro de Tolonio , Triticum/anatomía & histología , Triticum/genética , Triticum/metabolismoRESUMEN
Grain development and its evolution in grasses remains poorly understood, despite cereals being our most important source of food. The grain, for which many grass species have been domesticated, is a single-seeded fruit with prominent and persistent endosperm. Brachypodium distachyon, a small wild grass, is being posited as a new model system for the temperate small grain cereals, but little is known about its endosperm development and how this compares with that of the domesticated cereals. A cellular and molecular map of domains within the developing Brachypodium endosperm is constructed. This provides the first detailed description of grain development in Brachypodium for the reference strain, Bd21, that will be useful for future genetic and comparative studies. Development of Brachypodium grains is compared with that of wheat. Notably, the aleurone is not regionally differentiated as in wheat, suggesting that the modified aleurone region may be a feature of only a subset of cereals. Also, the central endosperm and the nucellar epidermis contain unusually prominent cell walls that may act as a storage material. The composition of these cell walls is more closely related to those of barley and oats than to those of wheat. Therefore, although endosperm development is broadly similar to that of temperate small grain cereals, there are significant differences that may reflect its phylogenetic position between the Triticeae and rice.
Asunto(s)
Brachypodium/embriología , Endospermo/embriología , Brachypodium/anatomía & histología , Brachypodium/genética , Grano Comestible/anatomía & histología , Grano Comestible/embriología , Grano Comestible/genética , Endospermo/anatomía & histología , Triticum/anatomía & histología , Triticum/embriología , Triticum/genéticaRESUMEN
BACKGROUND AND AIMS: The MADS-box transcription factor AGAMOUS (AG) is an important regulator of stamen and fruit identity as well as floral meristem determinacy in a number of core eudicots and monocots. However, its role outside of these groups has not been assessed explicitly. Examining its role in opium poppy, a basal eudicot, could uncover much about the evolution and development of flower and fruit development in the angiosperms. METHODS: AG orthologues were isolated by degenerate RT-PCR and the gene sequence and structure examined; gene expression was characterized using in situ hybridization and the function assessed using virus-induced gene silencing. KEY RESULTS: In opium poppy, a basal eudicot, the AGAMOUS orthologue is alternatively spliced to produce encoded products that vary at the C-terminus, termed PapsAG-1 and PapsAG-2. Both transcripts are expressed at high levels in stamens and carpels. The functional implications of this alternative transcription were examined using virus-induced gene silencing and the results show that PapsAG-1 has roles in stamen and carpel identity, reflecting those found for Arabidopsis AG. In contrast, PapsAG-2, while displaying redundancy in these functions, has a distinctive role in aspects of carpel development reflected in septae, ovule and stigma defects seen in the loss-of-function line generated. CONCLUSIONS: These results describe the first explicit functional analysis of an AG-clade gene in a basal eudicot; illustrate one of the few examples of the functional consequences of alternative splicing in transcription factors and reveal the importance of alternative transcription, as well as gene duplication, as a driving force in evolution.
Asunto(s)
Flores/crecimiento & desarrollo , Genes de Plantas/genética , Papaver/genética , Papaver/metabolismo , Proteínas de Plantas/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Plantas/química , ADN de Plantas/genética , Flores/citología , Flores/genética , Flores/metabolismo , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Especificidad de Órganos , Papaver/citología , Papaver/crecimiento & desarrollo , Fenotipo , Filogenia , Proteínas de Plantas/genética , ARN Mensajero/genética , ARN de Planta/genética , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Root hairs are cellular protuberances extending from the root surface into the soil; there they provide access to immobile inorganic ions such as phosphate, which are essential for growth. Their cylindrical shape results from a polarized mechanism of cell expansion called tip growth in which elongation is restricted to a small area at the surface of the hair-forming cell (trichoblast) tip. Here we identify proteins that spatially control the sites at which cell growth occurs by isolating Arabidopsis mutants (scn1) that develop ectopic sites of growth on trichoblasts. We cloned SCN1 and showed that SCN1 is a RhoGTPase GDP dissociation inhibitor (RhoGDI) that spatially restricts the sites of growth to a single point on the trichoblast. We showed previously that localized production of reactive oxygen species by RHD2/AtrbohC NADPH oxidase is required for hair growth; here we show that SCN1/AtrhoGDI1 is a component of the mechanism that focuses RHD2/AtrbohC-catalysed production of reactive oxygen species to hair tips during wild-type development. We propose that the spatial organization of growth in plant cells requires the local RhoGDI-regulated activation of the RHD2/AtrbohC NADPH oxidase.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Catión/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Raíces de Plantas/citología , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Catión/genética , Clonación Molecular , Inhibidores de Disociación de Guanina Nucleótido/genética , Humanos , Morfogénesis , Mutación/genética , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fenotipo , Raíces de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Supresión Genética/genética , Proteínas de Unión al GTP rho/metabolismo , Inhibidor alfa de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Mutations at the LYS3 locus in barley have multiple effects on grain development, including an increase in embryo size and a decrease in endosperm starch content. The gene underlying LYS3 was identified by genetic mapping and mutations in this gene were identified in all four barley lys3 alleles. LYS3 encodes a transcription factor called Prolamin Binding Factor (PBF). Its role in controlling embryo size was confirmed using wheat TILLING mutants. To understand how PBF controls embryo development, we studied its spatial and temporal patterns of expression in developing grains. The PBF gene is expressed in both the endosperm and the embryos, but the timing of expression in these organs differs. PBF expression in wild-type embryos precedes the onset of embryo enlargement in lys3 mutants, suggesting that PBF suppresses embryo growth. We predicted the down-stream target genes of PBF in wheat and found them to be involved in a wide range of biological processes, including organ development and starch metabolism. Our work suggests that PBF may influence embryo size and endosperm starch synthesis via separate gene control networks.
RESUMEN
In the post-genomic era, it is necessary to adapt methods for gene expression and functional analyses to more high-throughput levels of processing. mRNA in situ hybridization (ISH) remains a powerful tool for obtaining information regarding a gene's temporal and spatial expression pattern and can therefore be used as a starting point to define the function of a gene or a whole set of genes. We have deconstructed 'traditional' ISH techniques described for a range of organisms and developed protocols for ISH that adapt and integrate a degree of automation to standardized and shortened protocols. We have adapted this technique as a high-throughput means of gene expression analysis on wax-embedded plant tissues and also on whole-mount tissues. We have used wax-embedded wheat grains and Arabidopsis floral meristems and whole-mount Arabidopsis roots as test systems and show that it is capable of highly parallel processing.
Asunto(s)
Hibridación in Situ/métodos , Plantas/genética , Arabidopsis/genética , Flores/genética , Expresión Génica , Hibridación in Situ/instrumentación , Raíces de Plantas/genética , ARN Mensajero/genética , ARN de Planta/genética , Triticum/genéticaRESUMEN
EuAP2 genes are well-known for their role in flower development, a legacy of the founding member of this subfamily of transcription factors, whose mutants lacked petals in Arabidopsis. However, studies of euAP2 genes in several species have accumulated evidence highlighting the diverse roles of euAP2 genes in other aspects of plant development. Here, we emphasize other developmental roles of euAP2 genes in various species and suggest a shift from regarding euAP2 genes as just flowering genes to consider the global role they may be playing in plant development. We hypothesize that their almost universal expression profile and pleiotropic effects of their mutation suggest their involvement in fundamental plant development processes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Flores , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Homeodominio , Mutación , Desarrollo de la Planta/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flores/genética , Flores/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismoRESUMEN
The expression pattern of genes can identify the cells in which the respective proteins are active during development. As a step towards defining the genetic network that controls the development of roots, a high-throughput method of whole-mount in situ hybridization has been developed that does not require expensive equipment and allows the definition of the expression patterns of 137 transcription factor genes in young developing roots. Of the 137 transcription factors, 81.8% were expressed in the root while 18.2% showed no detectable expression. In all three proximal distal zones (meristem, elongation, and differentiation) of the root, 52.6% were expressed whereas 21.2% were expressed in only two zones. Eight percent of the genes were expressed in a single proximal distal zone. Cell-specific gene expression patterns were also detected. This rapid approach identified potential key regulators of cell differentiation and provides important spatial information for the expression patterns of a large number of transcriptional regulators that function during root development.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Expresión Génica , Genes de Plantas , Hibridación in Situ , Familia de Multigenes , Raíces de Plantas/metabolismoRESUMEN
A consistent challenge in studying the evolution of developmental processes has been the problem of explicitly assessing the function of developmental control genes in diverse species. In recent years, virus-induced gene silencing (VIGS) has proved to be remarkably adaptable and efficient in silencing developmental control genes in species across the angiosperms. Here we describe proven protocols for Nicotiana benthamiana and Papaver somniferum, representing a core and basal eudicot species.
Asunto(s)
Nicotiana/genética , Papaver/genética , Virus de Plantas/genética , Interferencia de ARN , Agrobacterium tumefaciens/virología , Regulación de la Expresión Génica de las Plantas , Técnicas de Silenciamiento del Gen/métodos , Vectores Genéticos , Crecimiento y Desarrollo/genética , Papaver/crecimiento & desarrollo , Papaver/virología , Hojas de la Planta/virología , Nicotiana/crecimiento & desarrollo , Nicotiana/virología , Transformación GenéticaRESUMEN
MADS-box genes are crucial regulators of floral development, yet how their functions have evolved to control different aspects of floral patterning is unclear. To understand the extent to which MADS-box gene functions are conserved or have diversified in different angiosperm lineages, we have exploited the capability for functional analyses in a new model system, Papaver somniferum (opium poppy). P. somniferum is a member of the order Ranunculales, and so represents a clade that is evolutionarily distant from those containing traditional model systems such as Arabidopsis, Petunia, maize or rice. We have identified and characterized the roles of several candidate MADS-box genes in petal specification in poppy. In Arabidopsis, the APETALA3 (AP3) MADS-box gene is required for both petal and stamen identity specification. By contrast, we show that the AP3 lineage has undergone gene duplication and subfunctionalization in poppy, with one gene copy required for petal development and the other responsible for stamen development. These differences in gene function are due to differences both in expression patterns and co-factor interactions. Furthermore, the genetic hierarchy controlling petal development in poppy has diverged as compared with that of Arabidopsis. As these are the first functional analyses of AP3 genes in this evolutionarily divergent clade, our results provide new information on the similarities and differences in petal developmental programs across angiosperms. Based on these observations, we discuss a model for how the petal developmental program has evolved.
Asunto(s)
Flores/genética , Regulación de la Expresión Génica de las Plantas , Papaver/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Secuencia Conservada , Cartilla de ADN , Evolución Molecular , Flores/ultraestructura , Genes Homeobox , Genes de Plantas , Hibridación in Situ , Proteínas de Dominio MADS/genética , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Papaver/clasificación , Papaver/ultraestructura , Petunia/genética , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
OEP16, a channel protein of the outer membrane of chloroplasts, has been implicated in amino acid transport and in the substrate-dependent import of protochlorophyllide oxidoreductase A. Two major clades of OEP16-related sequences were identified in land plants (OEP16-L and OEP16-S), which arose by a gene duplication event predating the divergence of seed plants and bryophytes. Remarkably, in angiosperms, OEP16-S genes evolved by gaining an additional exon that extends an interhelical loop domain in the pore-forming region of the protein. We analysed the sequence, structure and expression of the corresponding Arabidopsis genes (atOEP16-S and atOEP16-L) and demonstrated that following duplication, both genes diverged in terms of expression patterns and coding sequence. AtOEP16-S, which contains multiple G-box ABA-responsive elements (ABREs) in the promoter region, is regulated by ABI3 and ABI5 and is strongly expressed during the maturation phase in seeds and pollen grains, both desiccation-tolerant tissues. In contrast, atOEP-L, which lacks promoter ABREs, is expressed predominantly in leaves, is induced strongly by low-temperature stress and shows weak induction in response to osmotic stress, salicylic acid and exogenous ABA. Our results indicate that gene duplication, exon gain and regulatory sequence evolution each played a role in the divergence of OEP16 homologues in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Exones , Duplicación de Gen , Canales Iónicos/genética , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Canales Iónicos/química , Canales Iónicos/metabolismo , Datos de Secuencia Molecular , Oryza/genética , Pisum sativum/genética , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Semillas/genética , Semillas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Ácido Nucleico , Nicotiana/genética , Factores de TranscripciónRESUMEN
Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are resistant to conventional genetic approaches. However, VIGS has been shown to be effective in only a few, closely related plant species. Tobacco rattle virus (TRV), a bipartite RNA virus, has a wide host range and so in principle could serve as an efficient vector for VIGS in a diverse array of plant species. Here we show that a vector based on TRV sequences is effective at silencing the endogenous phytoene desaturase (PapsPDS) gene in Papaver somniferum (opium poppy). We show that this vector does not compromise the growth or reproduction of poppy and the plants did not display viral symptoms. The silencing of PapsPDS resulted in a significant reduction in PapsPDS mRNA and a concomitant photobleached phenotype. The ability to rapidly assay gene function in P. somniferum will be valuable in manipulation of the opiate pathway in this pharmaceutically important species. We suggest that our vacuum infiltration method used to deliver TRV-based vectors into poppy is a promising approach for expanding VIGS to diverse angiosperm species in which traditional delivery methods fail to induce VIGS. Furthermore, these studies demonstrate the utility of TRV-VIGS for probing gene function in a basal eudicot species that is phylogenetically distant from model plant species.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas/genética , Genes de Plantas/fisiología , Papaver/genética , Papaver/metabolismo , Virus de Plantas/fisiología , Mapeo Cromosómico , Cromosomas de las Plantas , Flores/genética , Flores/metabolismo , Flores/efectos de la radiación , Vectores Genéticos/genética , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Papaver/enzimología , Papaver/virología , Fotoblanqueo , Virus de Plantas/genéticaRESUMEN
The cereal caryopsis is a complex tissue in which maternal and endosperm tissues follow distinct but coordinated developmental programs. Because of the hexaploid genome in wheat (Triticum aestivum), the identification of genes involved in key developmental processes by genetic approaches has been difficult. To bypass this limitation, we surveyed 888 genes that are expressed during caryopsis development using a novel high-throughput mRNA in situ hybridization method. This survey revealed novel distinct spatial expression patterns that either reflected the ontogeny of the developing caryopsis or indicated specialized cellular functions. We have identified both known and novel genes whose expression is cell cycle-dependent. We have identified the crease region as important in setting up the developmental patterning, because the transition from proliferation to differentiation spreads from this region to the rest of the endosperm. A comparison of this set of genes with the rice (Oryza sativa) genome shows that approximately two-thirds have rice counterparts but also suggests considerable divergence with regard to proteins involved in grain filling. We found that the wheat genes had significant homology with 350 Arabidopsis thaliana genes. At least 25 of these are already known to be essential for seed development in Arabidopsis, but many others remain to be characterized.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Triticum/crecimiento & desarrollo , Triticum/genética , Ciclo Celular , División Celular , Genoma de Planta , Ploidias , Transcripción Genética , Triticum/citologíaRESUMEN
The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.
Asunto(s)
Alelos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Flores/fisiología , Proteínas de Unión al ARN/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/fisiología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , TransgenesRESUMEN
BACKGROUND: In situ hybridisation can provide cellular, and in some cases sub-cellular, resolution of mRNA levels within multicellular organisms and is widely used to provide spatial and temporal information on gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time. Whole-mount and reverse transcriptase-PCR (RT-PCR) based protocols increase throughput, but can compromise both specificity and resolution. With the advent of genome-wide analysis of gene expression, there is an urgent need to develop high-throughput in situ methods that also provide high resolution. RESULTS: Here we describe the development of a method for performing high-throughput in situ hybridisations that retains both the high resolution and the specificity of the best manual versions. This refined semi-automated protocol has the potential for determining the spatial and temporal expression patterns of hundreds of genes in parallel on a variety of tissues. We show how tissue sections can be organized on microscope slides in a manner that allows the screening of multiple probes on each slide. Slide handling, hybridisation and processing steps have been streamlined providing a capacity of at least 200 probes per week (depending on the tissue type). The technique can be applied easily to different species and tissue types, and we illustrate this with wheat seed and Arabidopsis floral meristems, siliques and seedlings. CONCLUSION: The approach has the high specificity and high resolution of previous in situ methods while allowing for the analysis of several genes expression patterns in parallel. This method has the potential to provide an analysis of gene expression patterns at the genome level.