Yuki Hamamura

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells

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.

Distinct Dynamics of HISTONE3 Variants between the Two Fertilization Products in Plants

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.

Double fertilization – caught in the act

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.

Live-Cell Imaging Reveals the Dynamics of Two Sperm Cells during Double Fertilization in Arabidopsis thaliana

Flowering plants have evolved a unique reproductive process called double fertilization, whereby two dimorphic female gametes are fertilized by two immotile sperm cells conveyed by the pollen tube. The two sperm cells are arranged in tandem with a leading pollen tube nucleus to form the male germ unit and are placed under the same genetic controls. Genes controlling double fertilization have been identified, but whether each sperm cell is able to fertilize either female gamete is still unclear. The dynamics of individual sperm cells after their release in the female tissue remain largely unknown. In this study, we photolabeled individual isomorphic sperm cells before their release and analyzed their fate during double fertilization in Arabidopsis thaliana. We found that sperm delivery was composed of three steps. Sperm cells were projected together to the boundary between the two female gametes. After a long period of immobility, each sperm cell fused with either female gamete in no particular order, and no preference was observed for either female gamete. Our results suggest that the two sperm cells at the front and back of the male germ unit are functionally equivalent and suggest unexpected cell-cell communications required for sperm cells to coordinate double fertilization of the two female gametes.

Ca2+-Activated Reactive Oxygen Species Production by Arabidopsis RbohH and RbohJ Is Essential for Proper Pollen Tube Tip Growth

Arabidopsis RbohH and RbohJ, NADPH oxidases expressed in pollen tubes, are activated by Ca2+ via their EF-hand motifs to produce reactive oxygen species (ROS) that are essential for proper pollen tube tip growth in vivo. Positive feedback regulation involving Ca2+ and ROS production mediated by RbohH and RbohJ is proposed to shape the long tubular structure of the pollen tube. 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 Ca2+ binding EF-hand motifs, possessed Ca2+-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca2+-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca2+-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca2+. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca2+-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Sperm entry is sufficient to trigger division of the central cell but the paternal genome is required for endosperm development in Arabidopsis

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.

Live imaging of calcium spikes during double fertilization in Arabidopsis

Ca2+ 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 Ca2+ 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 Ca2+ spikes, but not oscillations. Only the events in the egg cell correlated with the plasmogamy. In contrast, the synergid cells displayed Ca2+ oscillations on pollen tube arrival. The two synergid cells showed distinct Ca2+ dynamics depending on their respective roles in tube reception. These Ca2+ dynamics in the female gametophyte seem to represent highly specific signatures that coordinate successful double fertilization in the flowering plants.

Independent Control by Each Female Gamete Prevents the Attraction of Multiple Pollen Tubes

In flowering plants, double fertilization is normally accomplished by the first pollen tube, with the fertilized ovule subsequently inhibiting the attraction of a second pollen tube. However, the mechanism of second-pollen-tube avoidance remains unknown. We discovered that failure to fertilize either the egg cell or the central cell compromised second-pollen-tube avoidance in Arabidopsis thaliana. A similar disturbance was caused by disrupting the fertilization-independent seed (FIS) class polycomb-repressive complex 2 (FIS-PRC2), a central cell- and endosperm-specific chromatin-modifying complex for gene silencing. Therefore, the two female gametes have evolved their own signaling pathways. Intriguingly, second-pollen-tube attraction induced by half-successful fertilization allowed the ovules to complete double fertilization, producing a genetically distinct embryo and endosperm. We thus propose that each female gamete independently determines second-pollen-tube avoidance to maximize reproductive fitness in flowering plants.

Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation

Neurite growth requires two guanine nucleotide-binding protein polymers of tubulins and septins. However, whether and how those cytoskeletal systems are coordinated was unknown. Here we show that the acute knockdown or knockout of the pivotal septin subunit SEPT7 from cerebrocortical neurons impairs their interhemispheric and cerebrospinal axon projections and dendritogenesis in perinatal mice, when the microtubules are severely hyperacetylated. The resulting hyperstabilization and growth retardation of microtubules are demonstrated in vitro. The phenotypic similarity between SEPT7 depletion and the pharmacological inhibition of α-tubulin deacetylase HDAC6 reveals that HDAC6 requires SEPT7 not for its enzymatic activity, but to associate with acetylated α-tubulin. These and other findings indicate that septins provide a physical scaffold for HDAC6 to achieve efficient microtubule deacetylation, thereby negatively regulating microtubule stability to an optimal level for neuritogenesis. Our findings shed light on the mechanisms underlying the HDAC6-mediated coupling of the two ubiquitous cytoskeletal systems during neural development.

An EAR-Dependent Regulatory Module Promotes Male Germ Cell Division and Sperm Fertility in Arabidopsis

The development of sperm cells in angiosperms is coordinated by the MYB transcription factor DUO1. Male germ cell division and sperm fertility are shown to depend on DAZ1 and DAZ2, two zinc finger proteins regulated by DUO1, which physically interact with the corepressor TOPLESS. The DUO1-DAZ1/DAZ2 regulatory module thus implicates repressive mechanisms in plant sperm cell development. 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.