RESUMO
Accurate placement of extracellular materials is a critical part of cellular development. To study how cells achieve this accuracy, we use formation of pollen apertures as a model. In Arabidopsis (Arabidopsis thaliana), three regions on the pollen surface lack deposition of pollen wall exine and develop into apertures. In developing pollen, Arabidopsis INAPERTURATE POLLEN1 (INP1) protein acts as a marker for the preaperture domains, assembling there into three punctate lines. To understand the mechanism of aperture formation, we studied the dynamics of INP1 expression and localization and its relationship with the membrane domains at which it assembles. We found that INP1 assembly occurs after meiotic cytokinesis at the interface between the plasma membrane and the overlying callose wall, and requires the normal callose wall formation. Sites of INP1 localization coincide with positions of protruding membrane ridges in proximity to the callose wall. Our data suggest that INP1 is a late-acting factor involved in keeping specific membrane domains next to the callose wall to prevent formation of exine at these sites.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Pólen/metabolismo , Arabidopsis/ultraestrutura , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Fluorescência , Modelos Biológicos , Mutação/genética , Pólen/ultraestruturaRESUMO
Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures-openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores-the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ploidias , Pólen/genética , Arabidopsis/crescimento & desenvolvimento , Parede Celular/genética , Citocinese/genética , Meiose/genética , Proteínas Mutantes/genética , Pólen/crescimento & desenvolvimento , Especificidade da EspécieRESUMO
Pollen wall exine is usually deposited non-uniformly on the pollen surface, with areas of low exine deposition corresponding to pollen apertures. Little is known about how apertures form, with the novel Arabidopsis INP1 (INAPERTURATE POLLEN1) protein currently being the only identified aperture factor. In developing pollen, INP1 localizes to three plasma membrane domains and underlies formation of three apertures. Although INP1 homologs are found across angiosperms, they lack strong sequence conservation. Thus, it has been unclear whether they also act as aperture factors and whether their sequence divergence contributes to interspecies differences in aperture patterns. To explore the functional conservation of INP1 homologs, we used mutant analysis in maize and tested whether homologs from several other species could function in Arabidopsis. Our data suggest that the INP1 involvement in aperture formation is evolutionarily conserved, despite the significant divergence of INP1 sequences and aperture patterns, but that additional species-specific factors are likely to be required to guide INP1 and to provide information for aperture patterning. To determine the regions in INP1 necessary for its localization and function, we used fragment fusions, domain swaps, and interspecific protein chimeras. We demonstrate that the central portion of the protein is particularly important for mediating the species-specific functionality.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Pólen/crescimento & desenvolvimento , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Evolução Biológica , Pólen/genética , Pólen/metabolismo , Alinhamento de Sequência , Especificidade da EspécieRESUMO
The epigenetic activity of transposable elements (TEs) can influence the regulation of genes; though, this regulation is confined to the genes, promoters, and enhancers that neighbor the TE. This local cis regulation of genes therefore limits the influence of the TE's epigenetic regulation on the genome. TE activity is suppressed by small RNAs, which also inhibit viruses and regulate the expression of genes. The production of TE heterochromatin-associated endogenous small interfering RNAs (siRNAs) in the reference plant Arabidopsis thaliana is mechanistically distinct from gene-regulating small RNAs, such as microRNAs or trans-acting siRNAs (tasiRNAs). Previous research identified a TE small RNA that potentially regulates the UBP1b mRNA, which encodes an RNA-binding protein involved in stress granule formation. We demonstrate that this siRNA, siRNA854, is under the same trans-generational epigenetic control as the Athila family LTR retrotransposons from which it is produced. The epigenetic activation of Athila elements results in a shift in small RNA processing pathways, and new 21-22 nucleotide versions of Athila siRNAs are produced by protein components normally not responsible for processing TE siRNAs. This processing results in siRNA854's incorporation into ARGONAUTE1 protein complexes in a similar fashion to gene-regulating tasiRNAs. We have used reporter transgenes to demonstrate that the UPB1b 3' untranslated region directly responds to the epigenetic status of Athila TEs and the accumulation of siRNA854. The regulation of the UPB1b 3' untranslated region occurs both on the post-transcriptional and translational levels when Athila TEs are epigenetically activated, and this regulation results in the phenocopy of the ubp1b mutant stress-sensitive phenotype. This demonstrates that a TE's epigenetic activity can modulate the host organism's stress response. In addition, the ability of this TE siRNA to regulate a gene's expression in trans blurs the lines between TE and gene-regulating small RNAs.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Epigênese Genética/genética , MicroRNAs/genética , RNA Interferente Pequeno/genética , Retroelementos/genética , Estresse Fisiológico/genética , Regiões 3' não Traduzidas/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Humanos , MicroRNAs/metabolismo , Mutação , Especificidade de Órgãos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Pólen/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genéticaRESUMO
Pollen apertures, the characteristic gaps in pollen wall exine, have emerged as a model for studying the formation of distinct plasma membrane domains. In each species, aperture number, position, and morphology are typically fixed; across species they vary widely. During pollen development, certain plasma membrane domains attract specific proteins and lipids and become protected from exine deposition, developing into apertures. However, how these aperture domains are selected is unknown. Here, we demonstrate that patterns of aperture domains in Arabidopsis are controlled by the members of the ancient ELMOD protein family, which, although important in animals, has not been studied in plants. We show that two members of this family, MACARON (MCR) and ELMOD_A, act upstream of the previously discovered aperture proteins and that their expression levels influence the number of aperture domains that form on the surface of developing pollen grains. We also show that a third ELMOD family member, ELMOD_E, can interfere with MCR and ELMOD_A activities, changing aperture morphology and producing new aperture patterns. Our findings reveal key players controlling early steps in aperture domain formation, identify residues important for their function, and open new avenues for investigating how diversity of aperture patterns in nature is achieved.
Zooming in on cells reveals patterns on their outer surfaces. These patterns are actually a collection of distinct areas of the cell surface, each containing specific combinations of molecules. The outer layers of pollen grains consist of a cell wall, and a softer cell membrane that sits underneath. As a pollen grain develops, it recruits certain fats and proteins to specific areas of the cell membrane, known as 'aperture domains'. The composition of these domains blocks the cell wall from forming over them, leading to gaps in the wall called 'pollen apertures'. Pollen apertures can open and close, aiding reproduction and protecting pollen grains from dehydration. The number, location, and shape of pollen apertures vary between different plant species, but are consistent within the same species. In the plant species Arabidopsis thaliana, pollen normally develops three long and narrow, equally spaced apertures, but it remains unclear how pollen grains control the number and location of aperture domains. Zhou et al. found that mutations in two closely related A. thaliana proteins ELMOD_A and MCR alter the number and positions of pollen apertures. When A. thaliana plants were genetically modified so that they would produce different levels of ELMOD_A and MCR, Zhou et al. observed that when more of these proteins were present in a pollen grain, more apertures were generated on the pollen surface. This finding suggests that the levels of these proteins must be tightly regulated to control pollen aperture numbers. Further tests revealed that another related protein, called ELMOD_E, also has a role in domain formation. When artificially produced in developing pollen grains, it interfered with the activity of ELMOD_A and MCR, changing pollen aperture shape, number, and location. Zhou et al. identified a group of proteins that help control the formation of domains in the cell membranes of A. thaliana pollen grains. Further research will be required to determine what exactly these proteins do to promote formation of aperture domains and whether similar proteins control domain development in other organisms.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pólen/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Parede Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Genes de Plantas , Morfogênese , Mutação , Homologia de Sequência de AminoácidosRESUMO
Pollen apertures are an interesting model for the formation of specialized plasma-membrane domains. The plant-specific protein INP1 serves as a key aperture factor in such distantly related species as Arabidopsis, rice and maize. Although INP1 orthologues probably play similar roles throughout flowering plants, they show substantial sequence divergence and often cannot substitute for each other, suggesting that INP1 might require species-specific partners. Here, we present a new aperture factor, INP2, which satisfies the criteria for being a species-specific partner for INP1. Both INP proteins display similar structural features, including the plant-specific DOG1 domain, similar patterns of expression and mutant phenotypes, as well as signs of co-evolution. These proteins interact with each other in a species-specific manner and can restore apertures in a heterologous system when both are expressed but not when expressed individually. Our findings suggest that the INP proteins form a species-specific functional module that underlies formation of pollen apertures.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Pólen/anatomia & histologia , Pólen/crescimento & desenvolvimento , Pólen/genética , Zea mays/crescimento & desenvolvimento , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Parede Celular/genética , Parede Celular/metabolismo , Produtos Agrícolas/anatomia & histologia , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Mutação , Oryza/anatomia & histologia , Oryza/genética , Fenótipo , Proteínas de Plantas/genética , Especificidade da Espécie , Zea mays/anatomia & histologia , Zea mays/genéticaRESUMO
In most plant species, surfaces of pollen grains display characteristic patterns of apertures, formed by the gaps in the pollen wall exine. The aperture patterns are species-specific and tend to be very precise, with pollen of each species usually developing a certain number of apertures placed at distinct positions and acquiring specific morphology. The precision with which pollen apertures are produced suggests that developing pollen grains possess robust mechanisms that allow them to specify particular membrane domains as the future-aperture sites and to protect these sites from exine deposition. Recently, we demonstrated that formation of apertures in Arabidopsis depends on certain membrane domains attracting a novel protein, INP1, that assembles into punctate lines and helps to anchor these membrane domains to the overlying callose wall. Here we show that in the absence of male meiosis the ability of INP1 to assemble into lines at the pollen surface is compromised. However, INP1 still arrives to the pollen surface and mediates the interactions between the plasma membrane and the callose wall, potentially contributing to the formation of grossly abnormal patterns on pollen surface.