RESUMEN
Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , División Celular , Ciclina B1 , Microtúbulos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras , Ciclina B1/genética , Ciclina B1/metabolismo , Microtúbulos/metabolismo , Mitosis/genéticaRESUMEN
The spatiotemporal pattern of deposition, final amount, and relative abundance of oleic acid (cis-ω-9 C18:1) and its derivatives in the different lipid fractions of the seed of Arabidopsis (Arabidopsis thaliana) indicates that omega-9 monoenes are synthesized at high rates in this organ. Accordingly, we observed that four Δ9 stearoyl-ACP desaturase (SAD)-coding genes (FATTY ACID BIOSYNTHESIS2 [FAB2], ACYL-ACYL CARRIER PROTEIN5 [AAD5], AAD1, and AAD6) are transcriptionally induced in seeds. We established that the three most highly expressed ones are directly activated by the WRINKLED1 transcription factor. We characterized a collection of 30 simple, double, triple, and quadruple mutants affected in SAD-coding genes and thereby revealed the functions of these desaturases throughout seed development. Production of oleic acid by FAB2 and AAD5 appears to be critical at the onset of embryo morphogenesis. Double homozygous plants from crossing fab2 and aad5 could never be obtained, and further investigations revealed that the double mutation results in the arrest of embryo development before the globular stage. During later stages of seed development, these two SADs, together with AAD1, participate in the elaboration of the embryonic cuticle, a barrier essential for embryo-endosperm separation during the phase of invasive embryo growth through the endosperm. This study also demonstrates that the four desaturases redundantly contribute to storage lipid production during the maturation phase.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Oxigenasas de Función Mixta/genética , Semillas/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Oxigenasas de Función Mixta/metabolismo , Mutación , Plantas Modificadas Genéticamente , Semillas/genética , Semillas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Floral organ abscission is a separation process in which sepals, petals, and stamens detach from the plant at abscission zones. Here, we investigated the collective role of three amino-acid-loop-extension (TALE) homeobox genes ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1), KNAT6 (for KNOTTED LIKE from Arabidopsis thaliana) and KNAT2, which form a module that patterns boundaries under the regulation of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) co-activators. These TALE homeodomain transcription factors were shown to maintain boundaries in the flower, functioning as a unit to coordinate the growth, patterning, and activity of abscission zones. Together with BOP1 and BOP2, ATH1 and its partners KNAT6 and KNAT2 collectively contribute to the differentiation of lignified and separation layers of the abscission zone. The genetic interactions of BOP1/2 and ATH1 with INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) were also explored. We showed that BOP1/2 co-activators and ATH1 converge with the IDA signalling pathway to promote KNAT6 and KNAT2 expression in the abscission zone and cell separation. ATH1 acts as a central regulator in floral organ abscission as it controls the expression of other TALE genes in abscission zone cells.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/metabolismo , Aminoácidos/metabolismo , Inflorescencia/genética , Flores , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismoRESUMEN
Plant tissue architecture and organ morphogenesis rely on the proper orientation of cell divisions. Previous attempts to predict division planes from cell geometry in plants mostly focused on 2D symmetric divisions. Using the stereotyped division patterns of Arabidopsis thaliana early embryogenesis, we investigated geometrical principles underlying plane selection in symmetric and in asymmetric divisions within complex 3D cell shapes. Introducing a 3D computational model of cell division, we show that area minimization constrained on passing through the cell centroid predicts observed divisions. Our results suggest that the positioning of division planes ensues from cell geometry and gives rise to spatially organized cell types with stereotyped shapes, thus underlining the role of self-organization in the developing architecture of the embryo. Our data further suggested the rule could be interpreted as surface minimization constrained by the nucleus position, which was validated using live imaging of cell divisions in the stomatal cell lineage.
Asunto(s)
Arabidopsis/embriología , División Celular/fisiología , Forma de la Célula/fisiología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Linaje de la Célula , Núcleo Celular/metabolismo , Simulación por Computador , Modelos EstadísticosRESUMEN
Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoterCaMV35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoterNapin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/metabolismo , Aspartatoamoníaco Ligasa/metabolismo , Nitrógeno/metabolismo , Semillas/enzimología , Semillas/metabolismo , Aminoácidos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Aspartatoamoníaco Ligasa/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Floema/enzimología , Floema/genética , Floema/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Semillas/genéticaRESUMEN
The evolution of plant reproductive strategies has led to a remarkable diversity of structures, especially within the flower, a structure characteristic of the angiosperms. In flowering plants, sexual reproduction depends notably on the development of the gynoecium that produces and protects the ovules. In Arabidopsis thaliana, ovule initiation is promoted by the concerted action of auxin with CUC1 (CUP-SHAPED COTYLEDON1) and CUC2, two genes that encode transcription factors of the NAC family (NAM/ATAF1,2/CUC). Here we highlight an additional role for CUC2 and CUC3 in Arabidopsis thaliana ovule separation. While CUC1 and CUC2 are broadly expressed in the medial tissue of the gynoecium, CUC2 and CUC3 are expressed in the placental tissue between developing ovules. Consistent with the partial overlap between CUC1, CUC2 and CUC3 expression patterns, we show that CUC proteins can physically interact, both in yeast cells and in planta. We found that the cuc2;cuc3 double mutant specifically harbours defects in ovule separation, producing fused seeds that share the seed coat, and suggesting that CUC2 and CUC3 promote ovule separation in a partially redundant manner. Functional analyses show that CUC transcription factors are also involved in ovule development in Cardamine hirsuta. Additionally we show a conserved expression pattern of CUC orthologues between ovule primordia in other phylogenetically distant species with different gynoecium architectures. Taken together these results suggest an ancient role for CUC transcription factors in ovule separation, and shed light on the conservation of mechanisms involved in the development of innovative structures.
Asunto(s)
Óvulo Vegetal/crecimiento & desarrollo , Óvulo Vegetal/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cardamine/crecimiento & desarrollo , Cardamine/metabolismo , Regulación de la Expresión Génica de las Plantas , Magnoliopsida/crecimiento & desarrollo , Magnoliopsida/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo-vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP-labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co-localisation studies using GFP-CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast-associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP-CESA from doughnut-shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP-CESA density diminished, whereas GFP-CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP-CESA in clathrin-containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose-deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Pared Celular/metabolismo , Celulosa/metabolismo , Glucosiltransferasas/genética , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , División Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Pared Celular/ultraestructura , Clatrina/metabolismo , Citocinesis , Genes Reporteros , Glucosiltransferasas/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Isoenzimas , Microscopía Confocal , Microtúbulos/ultraestructura , Modelos Biológicos , Raíces de Plantas/citología , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión , Plantones/citología , Plantones/genética , Plantones/metabolismoRESUMEN
Vascular development is embedded into the developmental context of plant organ differentiation and can be divided into the consecutive phases of vascular patterning and differentiation of specific vascular cell types (phloem and xylem). To date, only very few genetic determinants of phloem development are known. Here, we identify OCTOPUS (OPS) as a potentiator of phloem differentiation. OPS is a polarly localised membrane-associated protein that is initially expressed in provascular cells, and upon vascular cell type specification becomes restricted to the phloem cell lineage. OPS mutants display a reduction of cotyledon vascular pattern complexity and discontinuous phloem differentiation, whereas OPS overexpressers show accelerated progress of cotyledon vascular patterning and phloem differentiation. We propose that OPS participates in vascular differentiation by interpreting longitudinal signals that lead to the transformation of vascular initials into differentiating protophloem cells.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Genes de Plantas , Microscopía Confocal/métodos , Mutación , Fenotipo , Floema/metabolismo , Fenómenos Fisiológicos de las Plantas , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas , Factores de TiempoRESUMEN
Cytochrome c oxidase is the last respiratory complex of the electron transfer chain in mitochondria and is responsible for transferring electrons to oxygen, the final acceptor, in the classical respiratory pathway. The essentiality of this step makes it that depletion in complex IV leads to lethality, thereby impeding studies on complex IV assembly and respiration plasticity in plants. Here, we characterized Arabidopsis (Arabidopsis thaliana) embryo-lethal mutant lines impaired in the expression of the CYTOCHROME C OXIDASE DEFICIENT1 (COD1) gene, which encodes a mitochondria-localized PentatricoPeptide Repeat protein. Although unable to germinate under usual conditions, cod1 homozygous embryos could be rescued from immature seeds and developed in vitro into slow-growing bush-like plantlets devoid of a root system. cod1 mutants were defective in C-to-U editing events in cytochrome oxidase subunit2 and NADH dehydrogenase subunit4 transcripts, encoding subunits of respiratory complex IV and I, respectively, and consequently lacked cytochrome c oxidase activity. We further show that respiratory oxygen consumption by cod1 plantlets is exclusively associated with alternative oxidase activity and that alternative NADH dehydrogenases are also up-regulated in these plants. The metabolomics pattern of cod1 mutants was also deeply altered, suggesting that alternative metabolic pathways compensated for the probable resulting restriction in NADH oxidation. Being the first complex IV-deficient mutants described in higher plants, cod1 lines should be instrumental to future studies on respiration homeostasis.
Asunto(s)
Arabidopsis/enzimología , Complejo IV de Transporte de Electrones/genética , Regulación de la Expresión Génica de las Plantas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Respiración de la Célula , Transporte de Electrón , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Regulación Enzimológica de la Expresión Génica , Metabolómica , Mitocondrias/enzimología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NADH Deshidrogenasa/genética , NADH Deshidrogenasa/metabolismo , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Consumo de Oxígeno , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genéticaRESUMEN
Arabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular-stage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.
Asunto(s)
Arabidopsis/enzimología , Galactanos/metabolismo , Galactosiltransferasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Mucoproteínas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/embriología , Arabidopsis/genética , Pared Celular/metabolismo , Galactosiltransferasas/genética , Mucoproteínas/genética , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , TransgenesRESUMEN
Plant leaves and flowers are positioned along the stem in a regular pattern. This pattern, which is referred to as phyllotaxis, is generated through the precise emergence of lateral organs and is controlled by gradients of the plant hormone auxin. This pattern is actively maintained during stem growth through controlled cell proliferation and elongation. The formation of new organs is known to depend on changes in cell wall chemistry, in particular the demethylesterification of homogalacturonans, one of the main pectic components. Here we report a dual function for the homeodomain transcription factor BELLRINGER (BLR) in the establishment and maintenance of the phyllotactic pattern in Arabidopsis. BLR is required for the establishment of normal phyllotaxis through the exclusion of pectin methylesterase PME5 expression from the meristem dome and for the maintenance of phyllotaxis through the activation of PME5 in the elongating stem. These results provide new insights into the role of pectin demethylesterification in organ initiation and cell elongation and identify an important component of the regulation mechanism involved.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Regulación de la Expresión Génica de las Plantas , Morfogénesis/fisiología , Proteínas Represoras/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Hidrolasas de Éster Carboxílico/genética , Pared Celular/metabolismo , Flores/anatomía & histología , Flores/crecimiento & desarrollo , Flores/metabolismo , Regulación Enzimológica de la Expresión Génica , Ácidos Indolacéticos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Meristema/ultraestructura , Fenotipo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genéticaRESUMEN
BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of seed germination acting downstream of the master repressor PHYTOCROME INTERACTING FACTOR3-LIKE 5 (PIL5). Among others, PIL5 induces the expression of the genes encoding the two DELLA proteins GA INSENSITIVE 1 (GAI) and REPRESSOR OF ga1-3 (RGA). RESULTS: Based on the properties of gai-t6 and rga28 mutant seeds, we show here that the absence of RGA severely increases dormancy, while lack of GAI only partially compensates RGA inactivation. In addition, the germination properties of the dag1rga28 double mutant are different from those of the dag1 and rga28 single mutants, suggesting that RGA and DAG1 act in independent branches of the PIL5-controlled germination pathway. Surprisingly, the dag1gai-t6 double mutant proved embryo-lethal, suggesting an unexpected involvement of (a possible complex between) DAG1 and GAI in embryo development. CONCLUSIONS: Rather than overlapping functions as previously suggested, we show that RGA and GAI play distinct roles in seed germination, and that GAI interacts with DAG1 in embryo development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Germinación , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Alelos , Arabidopsis/crecimiento & desarrollo , Desarrollo Embrionario , Epistasis Genética , Mutación , Fenotipo , Semillas/crecimiento & desarrolloRESUMEN
Sphingolipids are a class of structural membrane lipids involved in membrane trafficking and cell polarity. Functional analysis of the ceramide synthase family in Arabidopsis thaliana demonstrates the existence of two activities selective for the length of the acyl chains. Very-long-acyl-chain (C > 18 carbons) but not long-chain sphingolipids are essential for plant development. Reduction of very-long-chain fatty acid sphingolipid levels leads in particular to auxin-dependent inhibition of lateral root emergence that is associated with selective aggregation of the plasma membrane auxin carriers AUX1 and PIN1 in the cytosol. Defective targeting of polar auxin carriers is characterized by specific aggregation of Rab-A2(a)- and Rab-A1(e)-labeled early endosomes along the secretory pathway. These aggregates correlate with the accumulation of membrane structures and vesicle fragmentation in the cytosol. In conclusion, sphingolipids with very long acyl chains define a trafficking pathway with specific endomembrane compartments and polar auxin transport protein cargoes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Vías Secretoras/fisiología , Esfingolípidos , Secuencia de Aminoácidos , Animales , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Brefeldino A/metabolismo , Polaridad Celular , Ceramidas/química , Ceramidas/metabolismo , Endosomas/metabolismo , Inhibidores Enzimáticos/metabolismo , Fumonisinas/metabolismo , Humanos , Ácidos Indolacéticos/metabolismo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Inhibidores de la Síntesis de la Proteína/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Esfingolípidos/química , Esfingolípidos/metabolismoRESUMEN
To ensure an even segregation of chromosomes during somatic cell division, eukaryotes rely on mitotic spindles. Here, we measured prime characteristics of the Arabidopsis mitotic spindle and built a three-dimensional dynamic model using Cytosim. We identified the cell-cycle regulator CYCLIN-DEPENDENT KINASE B1 (CDKB1) together with its cyclin partner CYCB3;1 as key regulators of spindle morphology in Arabidopsis. We found that the augmin component ENDOSPERM DEFECTIVE1 (EDE1) is a substrate of the CDKB1;1-CYCB3;1 complex. A non-phosphorylatable mutant rescue of ede1 resembled the spindle phenotypes of cycb3;1 and cdkb1 mutants and the protein associated less efficiently with spindle microtubules. Accordingly, reducing the level of augmin in simulations recapitulated the phenotypes observed in the mutants. Our findings emphasize the importance of cell-cycle-dependent phospho-control of the mitotic spindle in plant cells and support the validity of our model as a framework for the exploration of mechanisms controlling the organization of the eukaryotic spindle.
RESUMEN
The preprophase band (PPB) is a transient ring of microtubules that forms before mitosis in land plants, and delineates the cytokinetic division plane established at telophase. It is one of the few derived traits specific to embryophytes, in which it is involved in the spatial control of cell division. Here we show that loss of function of Physcomitrella patens PpTON1 strongly affects development of the moss gametophore, phenocopying the developmental syndrome observed in Arabidopsis ton1 mutants: mutant leafy shoots display random orientation of cell division and severe defects in cell elongation, which are correlated with absence of PPB formation and disorganization of the cortical microtubule array in interphase cells. In hypomorphic Ppton1 alleles, PPB are still formed, whereas elongation defects are observed, showing the dual function of TON1 in organizing cortical arrays of microtubules during both interphase and premitosis. Ppton1 mutation has no impact on development of the protonema, which is consistent with the documented absence of PPB formation at this stage, apart from alteration of the gravitropic response, uncovering a new function of TON1 proteins in plants. Successful reciprocal cross-complementation between Physcomitrella and Arabidopsis shows conservation of TON1 function during land plant evolution. These results establish the essential role of the PPB in division plane specification in a basal land plant lineage, and provide new information on the function of TON1. They point to an ancient mechanism of cytoskeletal control of division plane positioning and cell elongation in land plants.
Asunto(s)
Bryopsida/enzimología , Bryopsida/crecimiento & desarrollo , Fosfoproteínas Fosfatasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Bryopsida/ultraestructura , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Microscopía Electrónica de Rastreo , Microtúbulos/metabolismo , Mutación , Fenotipo , Fosfoproteínas Fosfatasas/genéticaRESUMEN
Plant cytokinesis, which fundamentally differs from that in animals, requires the outward expansion of a plasma membrane precursor named the cell plate. How the transition from a cell plate to a plasma membrane occurs remains poorly understood. Here, we report that the acquisition of plasma membrane identity occurs through lateral patterning of the phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 at the newly formed cell plate membrane. There, the phosphoinositide phosphatase SAC9 emerges as a key regulator, colocalizing with and regulating the function of the microtubule-associated protein MAP65-3 at the cell plate leading zone. In sac9-3 mutant, the polar distribution of PI(4,5)P2 at the cell plate is altered, leading to ectopic recruitment of the cytokinesis apparatus and formation of an additional cell plate insertion site. We propose that at the cell plate, SAC9 drives the depletion of PI(4,5)P2, which acts as a polar cue to spatially separate cell plate expansion from the acquisition of plasma membrane identity during final step of cytokinesis.
Asunto(s)
Citocinesis , Microtúbulos , Animales , Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Ciclo Celular , Citoplasma/metabolismo , Membrana Celular/metabolismoRESUMEN
Unraveling the mechanisms that govern division plane orientation is a major challenge to understand plant development. In this respect, the Arabidopsis early embryo is a model system of choice since embryogenesis is relatively simple and cell division planes orientation is highly predictable. Here we present an integrated set of protocols to study 3D cell division patterns in early-stage Arabidopsis embryos that combine both cellular and sub-cellular localization of selected protein markers with spatial organization of cells, cytoskeleton, and nuclei.
Asunto(s)
Arabidopsis , Proteínas de Arabidopsis , División Celular , Microtúbulos , Desarrollo de la PlantaRESUMEN
In many plant tissues, division plane orientation within cell files is highly predictable since all cells divide almost perpendicular to the cell file axis. Many mutations can affect division plane orientation, and the quantification of the deviation from the expected transverse orientation in various genetic backgrounds is thus an important issue.While several software tools have been proposed for the quantification of cellular morphology in plant tissues, none of them allowed investigating division plane orientation. We propose here a complete method for measuring orientation of division planes in 2D, using an open-source ImageJ plugin named "Cell File Angles." The method comprises the staining of cell wall within whole mount roots with the calcofluor dye, the acquisition of 3D Z-stacks of the stained roots, and the measurement of cell wall orientation using image processing algorithms and semi-automated analysis.
Asunto(s)
Arabidopsis , División Celular , Algoritmos , Procesamiento de Imagen Asistido por Computador , Raíces de Plantas , Programas InformáticosRESUMEN
Noise plays a major role in cellular processes and in the development of tissues and organs. Several studies have examined the origin, the integration or the accommodation of noise in gene expression, cell growth and elaboration of organ shape. By contrast, much less is known about variability in cell division plane positioning, its origin and links with cell geometry, and its impact on tissue organization. Taking advantage of the first-stereotyped-then-variable division patterns in the embryo of the model plant Arabidopsis thaliana, we combined 3D imaging and quantitative cell shape and cell lineage analysis together with mathematical and computer modeling to perform a large-scale, systematic analysis of variability in division plane orientation. Our results reveal that, paradoxically, variability in cell division patterns of Arabidopsis embryos is accompanied by a progressive reduction of heterogeneity in cell shape topology. The paradox is solved by showing that variability operates within a reduced repertoire of possible division plane orientations that is related to cell geometry. We show that in several domains of the embryo, a recently proposed geometrical division rule recapitulates observed variable patterns, suggesting that variable patterns emerge from deterministic principles operating in a variable geometrical context. Our work highlights the importance of emerging patterns in the plant embryo under iterated division principles, but also reveal domains where deviations between rule predictions and experimental observations point to additional regulatory mechanisms.
Asunto(s)
Arabidopsis , Arabidopsis/genética , División Celular , Desarrollo Embrionario , Simulación por Computador , ComputadoresRESUMEN
In human cells and in Saccharomyces cerevisiae, BLAP75/Rmi1 acts together with BLM/Sgs1 and TopoIIIalpha/Top3 to maintain genome stability by limiting crossover (CO) formation in favour of NCO events, probably through the dissolution of double Holliday junction intermediates (dHJ). So far, very limited data is available on the involvement of these complexes in meiotic DNA repair. In this paper, we present the first meiotic study of a member of the BLAP75 family through characterisation of the Arabidopsis thaliana homologue. In A. thaliana blap75 mutants, meiotic recombination is initiated, and recombination progresses until the formation of bivalent-like structures, even in the absence of ZMM proteins. However, chromosome fragmentation can be detected as soon as metaphase I and is drastic at anaphase I, while no second meiotic division is observed. Using genetic and imunolocalisation studies, we showed that these defects reflect a role of A. thaliana BLAP75 in meiotic double-strand break (DSB) repair -- that it acts after the invasion step mediated by RAD51 and associated proteins and that it is necessary to repair meiotic DSBs onto sister chromatids as well as onto the homologous chromosome. In conclusion, our results show for the first time that BLAP75/Rmi1 is a key protein of the meiotic homologous recombination machinery. In A. thaliana, we found that this protein is dispensable for homologous chromosome recognition and synapsis but necessary for the repair of meiotic DSBs. Furthermore, in the absence of BLAP75, bivalent formation can happen even in the absence of ZMM proteins, showing that in blap75 mutants, recombination intermediates exist that are stable enough to form bivalent structures, even when ZMM are absent.