RESUMEN
CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.
RESUMEN
In flowering plants, double fertilization of the female gametes, the egg and the central cell, initiates seed development to give rise to a diploid embryo and the triploid endosperm. In the absence of fertilization, the FERTILIZATION-INDEPENDENT SEED Polycomb Repressive Complex 2 (FIS-PRC2) represses this developmental process by histone methylation of certain target genes. The FERTILIZATION-INDEPENDENT SEED (FIS) class genes MEDEA (MEA) and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) encode two of the core components of this complex. In addition, DNA methylation establishes and maintains the repression of gene activity, for instance via DNA METHYLTRANSFERASE1 (MET1), which maintains methylation of symmetric CpG residues. Here, we demonstrate that Arabidopsis MET1 interacts with MEA in vitro and in a yeast two-hybrid assay, similar to the previously identified interaction of the mammalian homologues DNMT1 and EZH2. MET1 and MEA share overlapping expression patterns in reproductive tissues before and after fertilization, a prerequisite for an interaction in vivo. Importantly, a much higher percentage of central cells initiate endosperm development in the absence of fertilization in mea-1/MEA; met1-3/MET1 as compared to mea-1/MEA mutant plants. In addition, DNA methylation at the PHERES1 and MEA loci, imprinted target genes of the FIS-PRC2, was affected in the mea-1 mutant compared with wild-type embryos. In conclusion, our data suggest a mechanistic link between two major epigenetic pathways involved in histone and DNA methylation in plants by physical interaction of MET1 with the FIS-PRC2 core component MEA. This concerted action is relevant for the repression of seed development in the absence of fertilization.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Endospermo/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Endospermo/citología , Endospermo/genética , Endospermo/crecimiento & desarrollo , Fertilización , Impresión Genómica , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Mutación , Plantas Modificadas Genéticamente , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
In contrast to animals, the life cycle of higher plants alternates between a gamete-producing (gametophyte) and a spore-producing (sporophyte) generation. The female gametophyte of angiosperms consists of four distinct cell types, including two gametes, the egg and the central cell, which give rise to embryo and endosperm, respectively. Based on a combined subtractive hybridization and virtual subtraction approach in wheat (Triticum aestivum L.), we have isolated a class of transcription factors not found in animal genomes, the RKD (RWP-RK domain-containing) factors, which share a highly conserved RWP-RK domain. Single-cell RT-PCR revealed that the genes TaRKD1 and TaRKD2 are preferentially expressed in the egg cell of wheat. The Arabidopsis genome contains five RKD genes, at least two of them, AtRKD1 and AtRKD2, are preferentially expressed in the egg cell of Arabidopsis. Ectopic expression of the AtRKD1 and AtRKD2 genes induces cell proliferation and the expression of an egg cell marker. Analyses of RKD-induced proliferating cells exhibit a shift of gene expression towards an egg cell-like transcriptome. Promoters of selected RKD-induced genes were shown to be predominantly active in the egg cell and can be activated by RKD in a transient protoplast expression assay. The data show that egg cell-specific RKD factors control a transcriptional program, which is characteristic for plant egg cells.
Asunto(s)
Familia de Multigenes , Óvulo Vegetal/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Triticum/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proliferación Celular , Regulación de la Expresión Génica de las Plantas , Mutagénesis Insercional , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Regiones Promotoras Genéticas , Protoplastos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transcripción Genética , Transcriptoma , Triticum/genéticaRESUMEN
The screening of enhancer detector lines in Arabidopsis thaliana has identified genes that are specifically expressed in the sporophytic tissue of the ovule. One such gene is the MADS-domain transcription factor AGAMOUS-LIKE6 (AGL6), which is expressed asymmetrically in the endothelial layer of the ovule, adjacent to the developing haploid female gametophyte. Transcription of AGL6 is regulated at multiple stages of development by enhancer and silencer elements located in both the upstream regulatory region and the large first intron. These include a bipartite enhancer, which requires elements in both the upstream regulatory region and the first intron, active in the endothelium. Transcription of the AGL13 locus, which encodes the other member of the AGL6 subfamily in Arabidopsis, is also regulated by elements located in the upstream regulatory region and in the first intron. There is, however, no overlapping expression of AGL6 and AGL13 except in the chalaza of the developing ovule, as was shown using a dual gene reporter system. Phylogenetic shadowing of the first intron of AGL6 and AGL13 homologs from other Brassicaceae identified four regions of conservation that probably contain the binding sites of transcriptional regulators, three of which are conserved outside Brassicaceae. Further phylogenetic analysis using the protein-encoding domains of AGL6 and AGL13 revealed that the MADS DNA-binding domain shows considerable divergence. Together, these results suggest that AGL6 and AGL13 show signs of subfunctionalization, with divergent expression patterns, regulatory sequences and possibly functions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Intrones , Proteínas de Dominio MADS/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Secuencia Conservada , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genes Reporteros , Proteínas de Dominio MADS/genética , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Elementos Silenciadores TranscripcionalesRESUMEN
Genomic imprinting confers parent-of-origin-specific gene expression, thus non-equivalent and complementary function of parental genomes. As a consequence, genomic imprinting poses an epigenetic barrier to parthenogenesis in sexual organisms. We report aberrant imprinting in Boechera, a genus in which apomicts evolved from sexuals multiple times. Maternal activation of a MADS-box gene, a homolog of which is imprinted and paternally expressed in the sexual relative Arabidopsis, is accompanied by locus-specific DNA methylation changes in apomicts where parental imprinting seems to be relaxed.
Asunto(s)
Brassicaceae/genética , Impresión Genómica , Proteínas de Dominio MADS/genética , Partenogénesis , Proteínas de Plantas/genética , Evolución Biológica , Metilación de ADN , Epigenómica , Regulación de la Expresión Génica de las PlantasRESUMEN
Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple-sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Exocitosis , Fertilización , Polen/fisiología , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Flores/genética , Flores/metabolismo , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Datos de Secuencia Molecular , Familia de Multigenes , Óvulo Vegetal/genética , Óvulo Vegetal/metabolismo , Óvulo Vegetal/fisiología , Polen/genética , Polen/metabolismo , Señales de Clasificación de Proteína , Transcripción GenéticaRESUMEN
The development of multicellular organisms is controlled by differential gene expression whereby cells adopt distinct fates. A spatially resolved view of gene expression allows the elucidation of transcriptional networks that are linked to cellular identity and function. The haploid female gametophyte of flowering plants is a highly reduced organism: at maturity, it often consists of as few as three cell types derived from a common precursor [1, 2]. However, because of its inaccessibility and small size, we know little about the molecular basis of cell specification and differentiation in the female gametophyte. Here we report expression profiles of all cell types in the mature Arabidopsis female gametophyte. Differentially expressed posttranscriptional regulatory modules and metabolic pathways characterize the distinct cell types. Several transcription factor families are overrepresented in the female gametophyte in comparison to other plant tissues, e.g., type I MADS domain, RWP-RK, and reproductive meristem transcription factors. PAZ/Piwi-domain encoding genes are upregulated in the egg, indicating a role of epigenetic regulation through small RNA pathways-a feature paralleled in the germline of animals [3]. A comparison of human and Arabidopsis egg cells for enrichment of functional groups identified several similarities that may represent a consequence of coevolution or ancestral gametic features.
Asunto(s)
Arabidopsis/genética , Células Germinativas de las Plantas/metabolismo , Animales , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Humanos , Óvulo/metabolismo , Plantas Modificadas Genéticamente , Especificidad de la EspecieRESUMEN
In plants, members of microRNA (miRNA) families are often predicted to target the same or overlapping sets of genes. It has thus been hypothesized that these miRNAs may act in a functionally redundant manner. This hypothesis is tested here by studying the effects of elimination of all three members of the MIR164 family from Arabidopsis. It was found that a loss of miR164 activity leads to a severe disruption of shoot development, in contrast to the effect of mutation in any single MIR164 gene. This indicates that these miRNAs are indeed functionally redundant. Differences in the expression patterns of the individual MIR164 genes imply, however, that redundancy among them is not complete, and that these miRNAs show functional specialization. Furthermore, the results of molecular and genetic analyses of miR164-mediated target regulation indicate that miR164 miRNAs function to control the transcript levels, as well as the expression patterns, of their targets, suggesting that they might contribute to developmental robustness. For two of the miR164 targets, namely CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, we provide evidence for their involvement in the regulation of growth and show that their derepression in miR164 loss-of-function mutants is likely to account for most of the mutant phenotype.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , MicroARNs/fisiología , Morfogénesis/genética , Arabidopsis/anatomía & histología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , MicroARNs/genética , Hojas de la Planta/anatomía & histología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Brotes de la Planta/anatomía & histología , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrolloRESUMEN
In flowering plants, signaling between the male pollen tube and the synergid cells of the female gametophyte is required for fertilization. In the Arabidopsis thaliana mutant feronia (fer), fertilization is impaired; the pollen tube fails to arrest and thus continues to grow inside the female gametophyte. FER encodes a synergid-expressed, plasma membrane-localized receptor-like kinase. We found that the FER protein accumulates asymmetrically in the synergid membrane at the filiform apparatus. Interspecific crosses using pollen from Arabidopsis lyrata and Cardamine flexuosa on A. thaliana stigmas resulted in a fer-like phenotype that correlates with sequence divergence in the extracellular domain of FER. Our findings show that the female control of pollen tube reception is based on a FER-dependent signaling pathway, which may play a role in reproductive isolation barriers.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Flores/fisiología , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Tubo Polínico/fisiología , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/química , Brassicaceae/genética , Brassicaceae/fisiología , Membrana Celular/enzimología , Cruzamientos Genéticos , Evolución Molecular , Flores/citología , Flores/enzimología , Expresión Génica , Genes de Plantas , Germinación , Ligandos , Datos de Secuencia Molecular , Mutación , Fosforilación , Fosfotransferasas/química , Epidermis de la Planta/enzimología , Tubo Polínico/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/metabolismo , Reproducción , Semillas/crecimiento & desarrollo , Transducción de Señal , Especificidad de la EspecieRESUMEN
BACKGROUND: The embryo sac contains the haploid maternal cell types necessary for double fertilization and subsequent seed development in plants. Large-scale identification of genes expressed in the embryo sac remains cumbersome because of its inherent microscopic and inaccessible nature. We used genetic subtraction and comparative profiling by microarray between the Arabidopsis thaliana wild-type and a sporophytic mutant lacking an embryo sac in order to identify embryo sac expressed genes in this model organism. The influences of the embryo sac on the surrounding sporophytic tissues were previously thought to be negligible or nonexistent; we investigated the extent of these interactions by transcriptome analysis. RESULTS: We identified 1,260 genes as embryo sac expressed by analyzing both our dataset and a recently reported dataset, obtained by a similar approach, using three statistical procedures. Spatial expression of nine genes (for instance a central cell expressed trithorax-like gene, an egg cell expressed gene encoding a kinase, and a synergid expressed gene encoding a permease) validated our approach. We analyzed mutants in five of the newly identified genes that exhibited developmental anomalies during reproductive development. A total of 527 genes were identified for their expression in ovules of mutants lacking an embryo sac, at levels that were twofold higher than in the wild type. CONCLUSION: Identification of embryo sac expressed genes establishes a basis for the functional dissection of embryo sac development and function. Sporophytic gain of expression in mutants lacking an embryo sac suggests that a substantial portion of the sporophytic transcriptome involved in carpel and ovule development is, unexpectedly, under the indirect influence of the embryo sac.
Asunto(s)
Arabidopsis/genética , Membranas Extraembrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes de Plantas/genética , Arabidopsis/embriología , Perfilación de la Expresión Génica , Genes Homeobox/genética , Hibridación in Situ , Análisis por Micromatrices/métodos , Reproducción/genética , Factores de Transcripción/metabolismoRESUMEN
The 26S proteasome plays a central role in the degradation of regulatory proteins involved in a variety of developmental processes. It consists of two multisubunit protein complexes: the proteolytic core protease and the regulatory particle (RP). The function of most RP subunits is poorly understood. Here, we describe mutants in the Arabidopsis thaliana RPN1 subunit, which is encoded by two paralogous genes, RPN1a and RPN1b. Disruption of RPN1a caused embryo lethality, while RPN1b mutants showed no obvious abnormal phenotype. Embryos homozygous for rpn1a arrested at the globular stage with defects in the formation of the embryonic root, the protoderm, and procambium. Cyclin B1 protein was not degraded in these embryos, consistent with cell division defects. Double mutant plants (rpn1a/RPN1a rpn1b/rpn1b) produced embryos with a phenotype indistinguishable from that of the rpn1a single mutant. Thus, despite their largely overlapping expression patterns in flowers and developing seeds, the two isoforms do not share redundant functions during gametogenesis and embryogenesis. However, complementation of the rpn1a mutation with the coding region of RPN1b expressed under the control of the RPN1a promoter indicates that the two RPN1 isoforms are functionally equivalent. Overall, our data indicate that RPN1 activity is essential during embryogenesis, where it might participate in the destruction of a specific set of protein substrates.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Complejo de la Endopetidasa Proteasomal/metabolismo , Semillas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , División Celular/genética , Ciclina B/genética , Ciclina B/metabolismo , Ciclina B1 , Desarrollo Embrionario/genética , Gametogénesis/genética , Regulación de la Expresión Génica de las Plantas/genética , Genes Letales/genética , Mutación/genética , Fenotipo , Complejo de la Endopetidasa Proteasomal/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Semillas/genéticaRESUMEN
The eukaryotic origin recognition complex (ORC) is made up of six subunits and functions in nuclear DNA replication, chromatin structure, and gene silencing in both fungi and metazoans. We demonstrate that disruption of a plant ORC subunit homolog, AtORC2 of Arabidopsis (Arabidopsis thaliana), causes a zygotic lethal mutant phenotype (orc2). Seeds of orc2 abort early, typically producing embryos with up to eight cells. Nuclear division in the endosperm is arrested at an earlier developmental stage: only approximately four nuclei are detected in orc2 endosperm. The endosperm nuclei in orc2 are dramatically enlarged, a phenotype that is most similar to class B titan mutants, which include mutants in structural maintenance of chromosomes (SMC) cohesins. The highest levels of ORC2 gene expression were found in preglobular embryos, coinciding with the stage at which homozygous orc2 mutant seeds arrest. The homologs of the other five Arabidopsis ORC subunits are also expressed at this developmental stage. The orc2 mutant phenotype is partly suppressed by a mutation in the Polycomb group gene MEDEA. In double mutants between orc2 and medea (mea), orc2 homozygotes arrest later with a phenotype intermediate between those of mea and orc2 single mutants. Either alterations in chromatin structure or the release of cell cycle checkpoints by the mea mutation may allow more cell and nuclear divisions to occur in orc2 homozygous seeds.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Genes de Plantas , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Secuencia de Bases , Ciclo Celular/genética , Cromatina/genética , ADN de Plantas/genética , Proteínas de Unión al ADN/química , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Complejo de Reconocimiento del Origen , Fenotipo , Subunidades de Proteína , Semillas/crecimiento & desarrollo , Supresión GenéticaRESUMEN
Seed development in angiosperms initiates after double fertilization, leading to the formation of a diploid embryo and a triploid endosperm. The active repression of precocious initiation of certain aspects of seed development in the absence of fertilization requires the Polycomb group proteins MEDEA (MEA), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and FERTILIZATION-INDEPENDENT SEED2. Here we show that the Arabidopsis WD-40 domain protein MSI1 is present together with MEA and FIE in a 600 kDa complex and interacts directly with FIE. Mutant plants heterozygous for msi1 show a seed abortion ratio of 50% with seeds aborting when the mutant allele is maternally inherited, irrespective of a paternal wild-type or mutant MSI1 allele. Further more, msi1 mutant gametophytes initiate endosperm development in the absence of fertilization at a high penetrance. After pollination, only the egg cell becomes fertilized, the central cell starts dividing prior to fertilization, resulting in the formation of seeds containing embryos surrounded by diploid endosperm. Our results establish that MSI1 has an essential function in the correct initiation and progression of seed development.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Proteínas Represoras/fisiología , Semillas/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatografía en Gel , Cruzamientos Genéticos , Cartilla de ADN , Prueba de Complementación Genética , Heterocigoto , Mutagénesis Insercional , Ploidias , Proteínas del Grupo Polycomb , Reacción en Cadena de la Polimerasa , Proteínas Represoras/genética , ReproducciónRESUMEN
Ovules of higher plants are the precursors of seeds. Ovules emerge from placental tissue inside the gynoecium of flowers. Three elements, funiculus, chalaza, and nucellus, can be distinguished along the proximal-distal axis of the outgrowing radially symmetrical ovule primordium. The asymmetric initiation of the outer integument marks the switch to adaxial-abaxial development, which leads to the formation of a bilaterally symmetrical ovule. The putative transcriptional regulator NOZZLE (NZZ) plays a role in mediating this transition by controlling the timing of expression of the putative transcriptional regulator INNER NO OUTER (INO) in an abaxial domain of the chalaza, from where the outer integument initiates. Integument formation depends on the homeobox gene WUSCHEL (WUS), which is expressed in the nucellus and is sufficient to induce integuments non-cell autonomously from a region adjacent to its expression domain. In this study, we describe the expression pattern of the homeobox-leucine zipper gene PHABULOSA (PHB) during ovule development, demonstrating that adaxial-abaxial polarity is established from the very beginning of ovule development. Furthermore, we examined the expression pattern of PHB, INO, and WUS in ovules of plants, which are affected in integument initiation and thus defective in the transition from proximal-distal to adaxial-abaxial development. We found that NZZ is required to restrict PHB expression to the distal chalaza, from where the inner integument initiates. PHB expression is not established in the distal chalaza of two mutants, aintegumenta (ant) and wus, which fail to form integuments. Furthermore, we suggest that one mechanism by which WUS controls integument formation is by establishing the chalaza and that outer and inner integument identity determination depends on additional region-specific factors. In addition, we present evidence that NZZ is essential for the normal nucellar expression pattern of WUS. Thus, both WUS and PHB affect processes downstream of NZZ action during the transition from proximal--distal to adaxial--abaxial ovule development.