RESUMEN
Formation of pollen wall exine is preceded by the development of several transient layers of extracellular materials deposited on the surface of developing pollen grains. One such layer is primexine (PE), a thin, ephemeral structure that is present only for a short period of time and is difficult to visualize and study. Recent genetic studies suggested that PE is a key factor in the formation of exine, making it critical to understand its composition and the dynamics of its formation. In this study, we used high-pressure frozen/freeze-substituted samples of developing Arabidopsis (Arabidopsis thaliana) pollen for a detailed transmission electron microscopy analysis of the PE ultrastructure throughout the tetrad stage of pollen development. We also analyzed anthers from wild-type Arabidopsis and three mutants defective in PE formation by immunofluorescence, carefully tracing several carbohydrate epitopes in PE and nearby anther tissues during the tetrad and the early free-microspore stages. Our analyses revealed likely sites where these carbohydrates are produced and showed that the distribution of these carbohydrates in PE changes significantly during the tetrad stage. We also identified tools for staging tetrads and demonstrate that components of PE undergo changes resembling phase separation. Our results indicate that PE behaves like a much more dynamic structure than has been previously appreciated and clearly show that Arabidopsis PE creates a scaffolding pattern for formation of reticulate exine.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Arabidopsis/ultraestructura , Microscopía Electrónica de Transmisión , Polen/ultraestructuraRESUMEN
Exine, the sporopollenin-based outer layer of the pollen wall, forms through an unusual mechanism involving interactions between two anther cell types: developing pollen and tapetum. How sporopollenin precursors and other components required for exine formation are delivered from tapetum to pollen and assemble on the pollen surface is still largely unclear. Here, we characterized an Arabidopsis (Arabidopsis thaliana) mutant, thin exine2 (tex2), which develops pollen with abnormally thin exine. The TEX2 gene (also known as REPRESSOR OF CYTOKININ DEFICIENCY1 (ROCK1)) encodes a putative nucleotide-sugar transporter localized to the endoplasmic reticulum. Tapetal expression of TEX2 is sufficient for proper exine development. Loss of TEX2 leads to the formation of abnormal primexine, lack of primary exine elements, and subsequent failure of sporopollenin to correctly assemble into exine structures. Using immunohistochemistry, we investigated the carbohydrate composition of the tex2 primexine and found it accumulates increased amounts of arabinogalactans. Tapetum in tex2 accumulates prominent metabolic inclusions which depend on the sporopollenin polyketide biosynthesis and transport and likely correspond to a sporopollenin-like material. Even though such inclusions have not been previously reported, we show mutations in one of the known sporopollenin biosynthesis genes, LAP5/PKSB, but not in its paralog LAP6/PKSA, also lead to accumulation of similar inclusions, suggesting separate roles for the two paralogs. Finally, we show tex2 tapetal inclusions, as well as synthetic lethality in the double mutants of TEX2 and other exine genes, could be used as reporters when investigating genetic relationships between genes involved in exine formation.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Biopolímeros/metabolismo , Carotenoides/metabolismo , Proteínas Portadoras/genética , Polen/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Polen/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Polen/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Pared Celular/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Genes de Plantas , Morfogénesis , Mutación , Homología de Secuencia de AminoácidoRESUMEN
Pollen grains show an enormous variety of aperture systems. What genes are involved in the aperture formation pathway and how conserved this pathway is in angiosperms remains largely unknown. INAPERTURATE POLLEN1 (INP1) encodes a protein of unknown function, essential for aperture formation in Arabidopsis, rice and maize. Yet, because INP1 sequences are quite divergent, it is unclear if their function is conserved across angiosperms. Here, we conducted a functional study of the INP1 ortholog from the basal eudicot Eschscholzia californica (EcINP1) using expression analyses, virus-induced gene silencing, pollen germination assay, and transcriptomics. We found that EcINP1 expression peaks at the tetrad stage of pollen development, consistent with its role in aperture formation, which occurs at that stage, and showed, via gene silencing, that the role of INP1 as an important aperture factor extends to basal eudicots. Using germination assays, we demonstrated that, in Eschscholzia, apertures are dispensable for pollen germination. Our comparative transcriptome analysis of wild-type and silenced plants identified over 900 differentially expressed genes, many of them potential candidates for the aperture pathway. Our study substantiates the importance of INP1 homologs for aperture formation across angiosperms and opens up new avenues for functional studies of other aperture candidate genes.
RESUMEN
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.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Oryza/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Polen/anatomía & histología , Polen/crecimiento & desarrollo , Polen/genética , Zea mays/crecimiento & desarrollo , Arabidopsis/anatomía & histología , Arabidopsis/genética , Pared Celular/genética , Pared Celular/metabolismo , Productos Agrícolas/anatomía & histología , Productos Agrícolas/genética , Productos Agrícolas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Genotipo , Mutación , Oryza/anatomía & histología , Oryza/genética , Fenotipo , Proteínas de Plantas/genética , Especificidad de la Especie , Zea mays/anatomía & histología , Zea mays/genéticaRESUMEN
Pollen grains are covered by the complex extracellular structure, called exine, which in most species is deposited on the pollen surface non-uniformly. Certain surface areas receive fewer exine deposits and develop into regions whose structure and morphology differ significantly from the rest of pollen wall. These regions are known as pollen apertures. Across species, pollen apertures can vary in their numbers, positions, and morphology, generating highly diverse patterns. The process of aperture formation involves establishment of cell polarity, formation of distinct plasma membrane domains, and deposition of extracellular materials at precise positions. Thus, pollen apertures present an excellent model for studying the development of cellular domains and formation of patterns at the single-cell level. Until very recently, the molecular mechanisms underlying the specification and formation of aperture sites were completely unknown. Here, we review recent advances in understanding of the molecular processes involved in pollen aperture formation, focusing on the molecular players identified through genetic approaches in the model plant Arabidopsis. We discuss a potential working model that describes the process of aperture formation, including specification of domains, creation of their defining features, and protection of these regions from exine deposition.
Asunto(s)
Arabidopsis/citología , Polen/citología , Membrana Celular/metabolismoRESUMEN
Pollen provides an excellent system to study pattern formation at the single-cell level. Pollen surface is covered by the pollen wall exine, whose deposition is excluded from certain surface areas, the apertures, which vary between the species in their numbers, positions, and morphology. What determines aperture patterns is not understood. Arabidopsis thaliana normally develops three apertures, equally spaced along the pollen equator. However, Arabidopsis mutants whose pollen has higher ploidy and larger volume develop four or more apertures. To explore possible mechanisms responsible for aperture patterning, we developed a mathematical model based on the Gierer-Meinhardt system of equations. This model was able to recapitulate aperture patterns observed in the wild-type and higher-ploidy pollen. We then used this model to further explore geometric and kinetic factors that may influence aperture patterns and found that pollen size, as well as certain kinetic parameters, like diffusion and decay of morphogens, could play a role in formation of aperture patterns. In conjunction with mathematical modeling, we also performed a forward genetic screen in Arabidopsis and discovered two mutants with aperture patterns that had not been previously observed in this species but were predicted by our model. The macaron mutant develops a single ring-like aperture, matching the unusual ring-like pattern produced by the model. The doughnut mutant forms two pore-like apertures at the poles of the pollen grain. Further tests on these novel mutants, motivated by the modeling results, suggested the existence of an area of inhibition around apertures that prevents formation of additional apertures in their vicinity. This work demonstrates the ability of the theoretical model to help focus experimental efforts and to provide fundamental insights into an important biological process.
Asunto(s)
Arabidopsis , Modelos Biológicos , Morfogénesis , Mutación , Polen , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Biología Computacional , Simulación por Computador , Cinética , Morfogénesis/genética , Morfogénesis/fisiología , Mutación/genética , Mutación/fisiología , Polen/genética , Polen/crecimiento & desarrollo , Polen/fisiologíaRESUMEN
Certain regions on the surfaces of developing pollen grains exhibit very limited deposition of pollen wall exine. These regions give rise to pollen apertures, which are highly diverse in their patterns and specific for individual species. Arabidopsis thaliana pollen develops three equidistant longitudinal apertures. The precision of aperture formation suggests that, to create them, pollen employs robust mechanisms that generate distinct cellular domains. To identify players involved in this mechanism, we screened natural Arabidopsis accessions and discovered one accession, Martuba, whose apertures form abnormally due to the disruption of the protein kinase D6PKL3. During pollen development, D6PKL3 accumulates at the three plasma membrane domains underlying future aperture sites. Both D6PKL3 localization and aperture formation require kinase activity. Proper D6PKL3 localization is also dependent on a polybasic motif for phosphoinositide interactions, and we identified two phosphoinositides that are specifically enriched at the future aperture sites. The other known aperture factor, INAPERTURATE POLLEN1, fails to aggregate at the aperture sites in d6pkl3 mutants, changes its localization when D6PKL3 is mislocalized, and, in turn, affects D6PKL3 localization. The discovery of aperture factors provides important insights into the mechanisms cells utilize to generate distinct membrane domains, develop cell polarity, and pattern their surfaces.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Polen/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Mutación , Polen/genéticaRESUMEN
Background and Aims: Pollen grains of flowering plants display a fascinating diversity of forms, including diverse patterns of apertures, the specialized areas on the pollen surface that commonly serve as the sites of pollen tube initiation and, therefore, might play a key role in reproduction. Although many aperture patterns exist in angiosperms, pollen with three apertures (triaperturate) constitutes the predominant pollen type found in eudicot species. The aim of this study was to explore whether having three apertures provides selective advantages over other aperture patterns in terms of pollen survival, germination and reproductive success, which could potentially explain the prevalence of triaperturate pollen among eudicots. Methods: The in vivo pollen germination, pollen tube growth, longevity and competitive ability to sire seeds were compared among pollen grains of Arabidopsis thaliana with different aperture numbers. For this, an arabidopsis pollen aperture series was used, which included the triaperturate wild type, as well as mutants without an aperture (inaperturate) and with more than three apertures. Key Results: Aperture number appears to influence pollen grain performance. In most germination and longevity experiments, the triaperturate and inaperturate pollen grains performed better than pollen with higher aperture numbers. In mixed pollinations, in which triaperturate and inaperturate pollen were forced to compete with each other, the triaperturate pollen outperformed the inaperturate pollen. Conclusions: Triaperturate pollen grains might provide the best trade-off among various pollen performance traits, thus explaining the prevalence of this morphological trait in the eudicot clade.
Asunto(s)
Arabidopsis/fisiología , Polen/fisiología , Arabidopsis/anatomía & histología , Germinación/fisiología , Polen/anatomía & histología , Tubo Polínico/crecimiento & desarrollo , Polinización , Reproducción , Semillas/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Polen/metabolismo , Arabidopsis/ultraestructura , Pared Celular/metabolismo , Pared Celular/ultraestructura , Fluorescencia , Modelos Biológicos , Mutación/genética , Polen/ultraestructuraRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Polen/crecimiento & desarrollo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Evolución Biológica , Polen/genética , Polen/metabolismo , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Pared Celular/metabolismo , Meiosis , Microdominios de Membrana/metabolismo , Polen/citología , Polen/metabolismo , Mutación/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ploidias , Polen/genética , Arabidopsis/crecimiento & desarrollo , Pared Celular/genética , Citocinesis/genética , Meiosis/genética , Proteínas Mutantes/genética , Polen/crecimiento & desarrollo , Especificidad de la EspecieRESUMEN
Pollen grains protect the sperm cells inside them with the help of the unique cell wall, the exine, which exhibits enormous morphological variation across plant taxa, assembling into intricate and diverse species-specific patterns. How this complex extracellular structure is faithfully deposited at precise sites and acquires precise shape within a species is not understood. Here, we describe the isolation and characterization of the novel Arabidopsis thaliana gene INAPERTURATE POLLEN1 (INP1), which is specifically involved in formation of the pollen surface apertures, which arise by restriction of exine deposition at specific sites. Loss of INP1 leads to the loss of all three apertures in Arabidopsis pollen, and INP1 protein exhibits a unique tripartite localization in developing pollen, indicative of its direct involvement in specification of aperture positions. We also show that aperture length appears to be sensitive to INP1 dosage and INP1 misexpression can affect global exine patterning. Phenotypes of some inp1 mutants indicate that Arabidopsis apertures are initiated at three nonrandom positions around the pollen equator. The identification of INP1 opens up new avenues for studies of how formation of distinct cellular domains results in the production of different extracellular morphologies.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Pared Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Polen/genética , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Mapeo Cromosómico , Clonación Molecular , Dosificación de Gen , Modelos Biológicos , Mutagénesis Insercional , Especificidad de Órganos , Fenotipo , Plantas Modificadas Genéticamente , Polen/citología , Polen/crecimiento & desarrollo , Polen/metabolismo , Polimorfismo de Nucleótido Simple , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión , Especificidad de la EspecieRESUMEN
Exine, the outer plant pollen wall, has elaborate species-specific patterns, provides a protective barrier for male gametophytes, and serves as a mediator of strong and species-specific pollen-stigma adhesion. Exine is made of sporopollenin, a material remarkable for its strength, elasticity, and chemical durability. The chemical nature of sporopollenin, as well as the developmental mechanisms that govern its assembly into diverse patterns in different species, are poorly understood. Here, we describe a simple yet effective genetic screen in Arabidopsis (Arabidopsis thaliana) that was undertaken to advance our understanding of sporopollenin synthesis and exine assembly. This screen led to the recovery of mutants with a variety of defects in exine structure, including multiple mutants with novel phenotypes. Fifty-six mutants were selected for further characterization and are reported here. In 14 cases, we have mapped defects to specific genes, including four with previously demonstrated or suggested roles in exine development (MALE STERILITY2, CYP703A2, ANTHER-SPECIFIC PROTEIN6, TETRAKETIDE α-PYRONE REDUCTASE/DIHYDROFLAVONOL-4-REDUCTASE-LIKE1), and a number of genes that have not been implicated in exine production prior to this screen (among them, fatty acid ω-hydroxylase CYP704B1, putative glycosyl transferases At1g27600 and At1g33430, 4-coumarate-coenzyme A ligase 4CL3, polygalacturonase QUARTET3, novel gene At5g58100, and nucleotide-sugar transporter At5g65000). Our study illustrates that morphological screens of pollen can be extremely fruitful in identifying previously unknown exine genes and lays the foundation for biochemical, developmental, and evolutionary studies of exine production.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Biopolímeros/metabolismo , Carotenoides/metabolismo , Polen/fisiología , Proteínas de Arabidopsis/metabolismo , Biopolímeros/genética , Carotenoides/genética , Sistema Enzimático del Citocromo P-450/genética , Regulación de la Expresión Génica de las Plantas , Mutación , FenotipoRESUMEN
Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production.
Asunto(s)
Aciltransferasas/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Sintasas Poliquetidas/metabolismo , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Tipificación del Cuerpo/genética , Chalcona/química , Cromatografía Líquida de Alta Presión , Mapeo Cromosómico , Ácidos Grasos/metabolismo , Flavanonas/biosíntesis , Flavanonas/química , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Hidroxilación , Espectrometría de Masas , Datos de Secuencia Molecular , Familia de Multigenes , Mutación/genética , Especificidad de Órganos/genética , Polen/citología , Polen/enzimología , Polen/genética , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Especificidad por SustratoRESUMEN
We isolated lap3-1 and lap3-2 mutants in a screen for pollen that displays abnormal stigma binding. Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine is thinner, weaker, and is missing some connections between their roof-like tectum structures. We describe the mapping and identification of LAP3 as a novel gene that contains a repetitive motif found in beta-propeller enzymes. Insertion mutations in LAP3 lead to male sterility. To investigate possible roles for LAP3 in pollen development, we assayed the metabolite profile of anther tissues containing developing pollen grains and found that the lap3-2 defect leads to a broad range of metabolic changes. The largest changes were seen in levels of a straight-chain hydrocarbon nonacosane and in naringenin chalcone, an obligate compound in the flavonoid biosynthesis pathway.