RESUMEN
In many flowering plants, petals initiate in alternate positions from first whorl sepals, suggesting possible signaling between sepal boundaries and petal initiation sites. PETAL LOSS (PTL) and RABBIT EARS (RBE) regulate petal initiation in Arabidopsis thaliana and their transcripts are expressed in sepal boundary and petal initiation sites, respectively, suggesting that PTL acts in a non-cell-autonomous manner. Here, we determined that cells expressing PTL and RBE fusion proteins did not overlap but were adjacent, confirming the non-cell-autonomous function of PTL. Genetic ablation of intersepal cells by expressing the diphtheria toxin-A chain gene driven by the PTL promoter resulted in flowers lacking petals, suggesting these cells are required for petal initiation. Transcriptome analysis combined with a PTL induction system revealed 42 genes that were upregulated under PTL activation, including UNUSUAL FLORAL ORGANS (UFO), which likely plays an important role in petal initiation. These findings suggest a molecular mechanism in which PTL indirectly regulates petal initiation and UFO mediates positional signaling between the sepal boundary and petal initiation sites.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Flores/genética , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismoRESUMEN
Meiosis is key to sexual reproduction and genetic diversity. Here, we show that the Arabidopsis cyclin-dependent kinase Cdk1/Cdk2 homolog CDKA;1 is an important regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis homolog of Hop1 (homolog pairing 1), essential for synaptonemal complex formation, as a target of CDKA;1. The phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis reductional division 1 (Red1) homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have identified the closure motif in ASY1, typical for HORMA domain proteins, and provide evidence that the phosphorylation of ASY1 regulates the putative self-polymerization of ASY1 along the chromosome axis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/metabolismo , Meiosis , Adenosina Trifosfatasas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión , Cromosomas de las Plantas/genética , Cromosomas de las Plantas/metabolismo , Quinasas Ciclina-Dependientes/genética , Proteínas de Unión al ADN/química , Mutación , Fosforilación , Unión Proteica , Multimerización de ProteínaRESUMEN
Chromosome axis-associated HORMA domain proteins (HORMADs), e.g. ASY1 in Arabidopsis, are crucial for meiotic recombination. ASY1, as other HORMADs, is assembled on the axis at early meiosis and depleted when homologous chromosomes synapse. Puzzlingly, both processes are catalyzed by AAA+ ATPase PCH2 together with its cofactor COMET. Here, we show that the ASY1 remodeling complex is temporally and spatially differently assembled. While PCH2 and COMET appear to directly interact in the cytoplasm in early meiosis, PCH2 is recruited by the transverse filament protein ZYP1 and brought to the ASY1-bound COMET assuring the timely removal of ASY1 during chromosome synapsis. Since we found that the PCH2 homolog TRIP13 also binds to the ZYP1 homolog SYCP1 in mouse, we postulate that this mechanism is conserved among eukaryotes. Deleting the PCH2 binding site of ZYP1 led to a failure of ASY1 removal. Interestingly, the placement of one obligatory crossover per homologous chromosome pair, compromised by ZYP1 depletion, is largely restored in this separation-of-function zyp1 allele suggesting that crossover assurance is promoted by synapsis. In contrast, this zyp1 allele, similar to the zyp1 null mutant, showed elevated type I crossover numbers indicating that PCH2-mediated eviction of ASY1 from the axis restricts crossover formation.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Emparejamiento Cromosómico , Animales , Ratones , Adenosina Trifosfatasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Emparejamiento Cromosómico/genética , Meiosis/genética , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/metabolismoRESUMEN
The Aurora B kinase, encoded by the AURORA 3 (AUR3) gene in Arabidopsis (Arabidopsis thaliana), is a key regulator of cell division in all eukaryotes. Aurora B has at least two central functions during cell division; it is essential for the correct, i.e. balanced, segregation of chromosomes in mitosis and meiosis by controlling kinetochore function, and it acts at the division plane, where it is necessary to complete cytokinesis. To accomplish these two spatially distinct functions, Aurora B in animals is guided to its sites of action by Borealin, inner centromere protein (INCENP), and Survivin, which, together with Aurora B, form the chromosome passenger complex (CPC). However, besides Aurora homologs, only a candidate gene with restricted homology to INCENP has been described in Arabidopsis, raising the question of whether a full complement of the CPC exists in plants and how Aurora homologs are targeted subcellularly. Here, we have identified and functionally characterized a Borealin homolog, BOREALIN RELATED (BORR), in Arabidopsis. Together with detailed localization studies including the putative Arabidopsis INCENP homolog, these results support the existence of a CPC in plants.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Arabidopsis/metabolismo , Aurora Quinasa B/metabolismo , Centrómero/metabolismo , FosforilaciónRESUMEN
The production of the sperm cells in angiosperms requires coordination of cell division and cell differentiation. In Arabidopsis thaliana, the germline-specific MYB protein DUO1 integrates these processes, but the regulatory hierarchy in which DUO1 functions is unknown. Here, we identify an essential role for two germline-specific DUO1 target genes, DAZ1 and DAZ2, which encode EAR motif-containing C2H2-type zinc finger proteins. We show that DAZ1/DAZ2 are required for germ cell division and for the proper accumulation of mitotic cyclins. Importantly, DAZ1/DAZ2 are sufficient to promote G2- to M-phase transition and germ cell division in the absence of DUO1. DAZ1/DAZ2 are also required for DUO1-dependent cell differentiation and are essential for gamete fusion at fertilization. We demonstrate that the two EAR motifs in DAZ1/DAZ2 mediate their function in the male germline and are required for transcriptional repression and for physical interaction with the corepressor TOPLESS. Our findings uncover an essential module in a regulatory hierarchy that drives mitotic transition in male germ cells and implicates gene repression pathways in sperm cell formation and fertility.
RESUMEN
In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca(2+) binding EF-hand motifs, possessed Ca(2+)-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca(2+)-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca(2+)-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca(2+). Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca(2+)-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Calcio/metabolismo , NADPH Oxidasas/fisiología , Tubo Polínico/crecimiento & desarrollo , Especies Reactivas de Oxígeno/metabolismo , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Ionomicina/farmacología , Toxinas Marinas , Datos de Secuencia Molecular , Mutación , NADPH Oxidasas/química , NADPH Oxidasas/genética , Oxazoles/farmacología , Homología de Secuencia de AminoácidoRESUMEN
For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.
Asunto(s)
Factores Quimiotácticos/metabolismo , Defensinas/metabolismo , Magnoliopsida/citología , Magnoliopsida/crecimiento & desarrollo , Tubo Polínico/crecimiento & desarrollo , Secuencia de Aminoácidos , Factores Quimiotácticos/química , Factores Quimiotácticos/farmacología , Defensinas/química , Defensinas/farmacología , Etiquetas de Secuencia Expresada , Magnoliopsida/efectos de los fármacos , Magnoliopsida/genética , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , Tubo Polínico/efectos de los fármacos , Tubo Polínico/genética , ARN de Planta/antagonistas & inhibidores , ARN de Planta/genética , ARN de Planta/metabolismo , Transcripción GenéticaRESUMEN
In many flowering plants, flowers consist of two peripheral organs, sepals and petals, occurring in outer two whorls, and two inner reproductive organs, stamens and carpels. These organs are arranged in a concentric pattern in a floral meristem, and the organ identity is established by the combined action of floral homeotic genes expressed along the whorls. Floral organ primordia arise at fixed positions in the floral meristem within each whorl. The RABBIT EARS (RBE) gene is transcribed in the petal precursor cells and primordia, and regulates petal initiation and early growth in Arabidopsis thaliana. We investigated the spatial and temporal expression pattern of a RBE protein fused to the green fluorescent protein (GFP). Expression of the GFP:RBE fusion gene under the RBE cis-regulatory genomic fragment rescues the rbe petal defects, indicating that the fusion protein is functional. The GFP signal is located to the cells where RBE is transcribed, suggesting that RBE function is cell-autonomous. Ectopic expression of GFP:RBE under the APETALA1 promoter causes the homeotic conversion of floral organs, resulting in sterile flowers. In these plants, the class B homeotic genes APETALA3 and PISTILLATA are down-regulated, suggesting that the restriction of the RBE expression to the petal precursor cells is crucial for flower development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Represoras/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Proteínas de Dominio MADS/genética , Regiones Promotoras GenéticasRESUMEN
Fertilization in flowering plants involves two sperm cells and two female gametes, the egg cell and the central cell, progenitors of the embryo and the endosperm, respectively. The mechanisms triggering zygotic development are unknown and whether both parental genomes are required for zygotic development is unclear. In Arabidopsis, previous studies reported that loss-of-function mutations in CYCLIN DEPENDENT KINASE A1 (CDKA;1) impedes cell cycle progression in the pollen leading to the production of a single sperm cell. Here, we report that a significant proportion of single cdka;1 pollen delivers two sperm cells, leading to a new assessment of the cdka;1 phenotype. We performed fertilization of wild-type ovules with cdka;1 mutant sperm cells and monitored in vivo the fusion of the male and female nuclei using fluorescent markers. When a single cdka;1 sperm was delivered, either female gamete could be fertilized leading to similar proportions of seeds containing either a single endosperm or a single embryo. When two cdka;1 sperm cells were released, they fused to each female gamete. Embryogenesis was initiated but the fusion between the nuclei of the sperm cell and the central cell failed. The failure of karyogamy in the central cell prevented incorporation of the paternal genome, impaired endosperm development and caused seed abortion. Our results thus support that the paternal genome plays an essential role during early seed development. However, sperm entry was sufficient to trigger central cell mitotic division, suggesting the existence of signaling events associated with sperm cell fusion with female gametes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/embriología , División Celular , Quinasas Ciclina-Dependientes/metabolismo , Genoma de Planta , Polen/fisiología , Transporte Activo de Núcleo Celular , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Quinasas Ciclina-Dependientes/genética , Fertilización , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Transcripción GenéticaRESUMEN
Sexual reproduction ensures propagation of species and enhances genetic diversity within populations. In flowering plants, sexual reproduction requires complicated and multi-step cell-to-cell communications among male and female cells. However, the confined nature of plant reproduction processes, which occur in the female reproductive organs and several cell layers of the pistil, limits our ability to observe these events in vivo. In this review, we discuss recent live-cell imaging in in vitro systems and the optical manipulation techniques that are used to capture the dynamic mechanisms representing molecular and cellular communications in sexual plant reproduction.
Asunto(s)
Arabidopsis/genética , Microscopía/métodos , Tubo Polínico/metabolismo , Arabidopsis/fisiología , Comunicación Celular , Fertilización , Flores , Genes de Plantas , Variación Genética , Magnoliopsida/genética , Magnoliopsida/fisiología , Óptica y Fotónica , Plantas , Transducción de SeñalRESUMEN
Live-cell imaging is a powerful method to obtain insights into cellular processes, particularly with respect to their dynamics. This is especially true for meiosis, where chromosomes and other cellular components such as the cytoskeleton follow an elaborate choreography over a relatively short period of time. Making these dynamics visible expands understanding of the regulation of meiosis and its underlying molecular forces. However, the analysis of meiosis by live-cell imaging is challenging; specifically in plants, a temporally resolved understanding of chromosome segregation and recombination events is lacking. Recent advances in live-cell imaging now allow the analysis of meiotic events in plants in real time. These new microscopy methods rely on the generation of reporter lines for meiotic regulators and on the establishment of ex vivo culture and imaging conditions, which stabilize the specimen and keep it alive for several hours or even days. In this review, we combine an overview of the technical aspects of live-cell imaging in plants with a summary of outstanding questions that can now be addressed to promote live-cell imaging in Arabidopsis and other plant species and stimulate ideas on the topics that can be addressed in the context of plant meiotic recombination.
RESUMEN
Sexual reproduction involves epigenetic reprogramming comprising DNA methylation and histone modifications. In addition, dynamics of HISTONE3 (H3) variant H3.3 upon fertilization are conserved in animals, suggesting an essential role. In contrast to H3, H3.3 marks actively transcribed regions of the genome and can be deposited in a replication-independent manner. Although H3 variants are conserved in plants, their dynamics during fertilization have remained unexplored. We overcame technical limitations to live imaging of the fertilization process in Arabidopsis thaliana and studied dynamics of the male-gamete-specific H3.3 and the centromeric Histone Three Related 12 (HTR12). The double-fertilization process in plants produces the zygote and the embryo-nourishing endosperm. We show that the zygote is characterized by replication-independent removal of paternal H3.3 and homogeneous incorporation of parental chromatin complements. In the endosperm, the paternal H3.3 is passively diluted by replication while the paternal chromatin remains segregated apart from the maternal chromatin (gonomery). Hence epigenetic regulations distinguish the two products of fertilization in plants. H3.3-replication-independent dynamics and gonomery also mark the first zygotic divisions in animal species. We thus propose the convergent selection of parental epigenetic imbalance involving H3 variants in sexually reproducing organisms.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , Variación Genética , Histonas/genética , Reproducción/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ensamble y Desensamble de Cromatina , Cruzamientos Genéticos , Genes de Plantas , Histonas/clasificación , Histonas/metabolismoRESUMEN
In flowering plants, fertilization is unique because it involves two pairs of male and female gametes, a process known as double fertilization. Here, we provide an overview of the field and a detailed review of the outstanding recent advances, including in vivo imaging of double fertilization and the identification of a signaling pathway controlling the release of the male gametes and of a protein involved in gamete membrane fusion. These recent results are stepping stones for further research; our knowledge of double fertilization is expanding as newly discovered molecular pathways are explored and new mutants are characterized. Controlling plant fertilization is essential for seed production, and molecular understanding of double fertilization will provide the tools to improve crops and breeding programs.
Asunto(s)
Flores/fisiología , Tubo Polínico/fisiología , Polinización/fisiología , Flores/genética , Modelos Biológicos , Desarrollo de la Planta , Plantas/genética , Tubo Polínico/genética , Polinización/genética , Reproducción/genética , Reproducción/fisiología , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
Plants are powerful model systems to study meiosis. Our knowledge about the cytology of plant meiosis is mainly based on the analysis of fixed material. Although highly informative, this approach is limited in understanding the dynamics of meiosis. Here, we present a step-by-step instruction for a newly developed method to follow meiosis in male meiocytes of Arabidopsis in real time by confocal laser scanning microscopy. We envision that this method can be easily translated to other plant species and especially crops (e.g., Brassica, maize, and potato).
Asunto(s)
Arabidopsis/fisiología , Meiosis/fisiología , Imagen Molecular/métodos , Imagen Molecular/normas , Fenómenos Fisiológicos de las Plantas , Flores/citología , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Células Vegetales , Imagen de Lapso de Tiempo/métodosRESUMEN
Recombinant canine interferon-γ (rc-IFNγ; Interdogâ) was exclusively approved as a therapeutic for canine atopic dermatitis. However, it has been used off-label for the treatment of canine cancer. We examined the inhibitory effect of rc-IFNγ on the growth of canine tumor cell lines and analyzed its mechanism of action. Three (CTB-p, CTB-m, and CNM-m) out of seven mammary gland tumor cell lines and two (VIMC and CoMS) out of four mast cell tumor cell lines showed remarkable growth inhibition after treatment with rc-IFNγ. However, one (CLBL-1) out of nine lymphoma cell lines showed a significant amount of cell death. Using CTB-p and CTB-m cell lines, we showed that STAT1 was essential for inducing the growth inhibitory effect of rc-IFNγ. Although rc-IFNγ induced G1 growth arrest in CTB-p cell line, treatment with rc-IFNγ did not alter the expression of cell cycle regulatory proteins. In this study, we observed direct cytotoxicity or cytostatic effects of rc-IFNγ in canine tumor cell lines. However, the detailed mechanisms responsible for these effects need to be elucidated in the future.
Asunto(s)
Enfermedades de los Perros/tratamiento farmacológico , Interferón gamma/farmacología , Proteínas Recombinantes/farmacología , Animales , Antineoplásicos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Perros , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Interferón gamma/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismoRESUMEN
Precise control of cytoskeleton dynamics and its tight coordination with chromosomal events are key to cell division. This is exemplified by formation of the spindle and execution of cytokinesis after nuclear division. Here, we reveal that the central cell cycle regulator CYCLIN DEPENDENT KINASE A;1 (CDKA;1), the Arabidopsis homologue of Cdk1 and Cdk2, partially in conjunction with CYCLIN B3;1 (CYCB3;1), is a key regulator of the microtubule cytoskeleton in meiosis. For full CDKA;1 activity, the function of three redundantly acting CDK-activating kinases (CAKs), CDKD;1, CDKD;2, and CDKD;3, is necessary. Progressive loss of these genes in combination with a weak loss-of-function mutant in CDKA;1 allowed a fine-grained dissection of the requirement of cell-cycle kinase activity for meiosis. Notably, a moderate reduction of CDKA;1 activity converts the simultaneous cytokinesis in Arabidopsis, i.e., one cytokinesis separating all four meiotic products concurrently into two successive cytokineses with cell wall formation after the first and second meiotic division, as found in many monocotyledonous species.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Citocinesis , Microtúbulos/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclina B/genética , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes/genética , Activación Enzimática , Regulación de la Expresión Génica de las Plantas , Meiosis , Microtúbulos/genética , Mutación , Plantas Modificadas Genéticamente/genética , Transducción de Señal , Factores de TiempoRESUMEN
Mitosis and meiosis both rely on cohesin, which embraces the sister chromatids and plays a crucial role for the faithful distribution of chromosomes to daughter cells. Prior to the cleavage by Separase at anaphase onset, cohesin is largely removed from chromosomes by the non-proteolytic action of WINGS APART-LIKE (WAPL), a mechanism referred to as the prophase pathway. To prevent the premature loss of sister chromatid cohesion, WAPL is inhibited in early mitosis by Sororin. However, Sororin homologs have only been found to function as WAPL inhibitors during mitosis in vertebrates and Drosophila. Here we show that SWITCH 1/DYAD defines a WAPL antagonist that acts in meiosis of Arabidopsis. Crucially, SWI1 becomes dispensable for sister chromatid cohesion in the absence of WAPL. Despite the lack of any sequence similarities, we found that SWI1 is regulated and functions in a similar manner as Sororin hence likely representing a case of convergent molecular evolution across the eukaryotic kingdom.
Asunto(s)
Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/fisiología , Cromátides/metabolismo , Meiosis/fisiología , Proteínas Nucleares/fisiología , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolución Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F(1) plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.
Asunto(s)
Arabidopsis/citología , Células Germinativas/citología , Mitocondrias/metabolismo , Polen/citología , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cruzamientos Genéticos , Fertilización , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Mutación/genética , Plantas Modificadas Genéticamente , Transporte de ProteínasRESUMEN
In animals, microtubules and centrosomes direct the migration of gamete pronuclei for fertilization. By contrast, flowering plants have lost essential components of the centrosome, raising the question of how flowering plants control gamete nuclei migration during fertilization. Here, we use Arabidopsis thaliana to document a novel mechanism that regulates F-actin dynamics in the female gametes and is essential for fertilization. Live imaging shows that F-actin structures assist the male nucleus during its migration towards the female nucleus. We identify a female gamete-specific Rho-GTPase that regulates F-actin dynamics and further show that actin-myosin interactions are also involved in male gamete nucleus migration. Genetic analyses and imaging indicate that microtubules are dispensable for migration and fusion of male and female gamete nuclei. The innovation of a novel actin-based mechanism of fertilization during plant evolution might account for the complete loss of the centrosome in flowering plants.
Asunto(s)
Actinas/metabolismo , Arabidopsis/metabolismo , Fertilización , Arabidopsis/citología , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Óvulo Vegetal/metabolismo , Polen/metabolismo , Transporte de Proteínas , Imagen de Lapso de Tiempo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Ca(2+) waves and oscillation are key signalling elements during the fertilization process of animals, and are involved, for example, in egg activation. In the unique double fertilization process in flowering plants, both the egg cell and the neighbouring central cell fuse with a sperm cell each. Here we succeeded in imaging cytosolic Ca(2+) in these two cells, and in the two synergid cells that accompany the gametes during semi-in vivo double fertilization. Following pollen tube discharge and plasmogamy, the egg and central cells displayed transient Ca(2+) spikes, but not oscillations. Only the events in the egg cell correlated with the plasmogamy. In contrast, the synergid cells displayed Ca(2+) oscillations on pollen tube arrival. The two synergid cells showed distinct Ca(2+) dynamics depending on their respective roles in tube reception. These Ca(2+) dynamics in the female gametophyte seem to represent highly specific signatures that coordinate successful double fertilization in the flowering plants.