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.
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.
Long-term holding and precise handling of growing plant tissues during in vitro cultivation has been a major hurdle for experimental studies related to plant development and reproduction. In the present review, we introduce two of our newly developed poly(dimethylsiloxane)-based microdevices: a T-shaped microchannel device for pollen tube chemoattraction and a microcage array for long-term live imaging of ovules. Their design, usage and advantages are described, and future prospects of experimental approaches to plant reproduction using such microdevices are discussed.
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.
Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS-ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi-in vitro, whereas normal PT growth was shown in its sense control in vitro/semi-in vitro. PT growth was impaired in a manner dependent on the dose of AS-ODNs against both ANX1 and ANX2 above 10 ?m. The treatment with AS-ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS-ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS-ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS-ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.
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.
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 ?-tubulin deacetylase HDAC6 reveals that HDAC6 requires SEPT7 not for its enzymatic activity, but to associate with acetylated ?-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.
In mammals and plants, parental genomic imprinting restricts the expression of specific loci to one parental allele. Imprinting in mammals relies on sex-dependent de novo deposition of DNA methylation during gametogenesis but a comparable mechanism was not shown in plants. Rather, paternal silencing by the maintenance DNA methyltransferase 1 (MET1) and maternal activation by the DNA demethylase DEMETER (DME) cause maternal expression. However, genome-wide studies suggested other DNA methylation-dependent imprinting mechanisms. Here, we show that de novo RNA-directed DNA methylation (RdDM) regulates imprinting at specific loci expressed in endosperm. RdDM in somatic tissues is required to silence expression of the paternal allele. By contrast, the repression of RdDM in female gametes participates with or without DME requirement in the activation of the maternal allele. The contrasted activity of DNA methylation between male and female gametes appears sufficient to prime imprinted maternal expression. After fertilization, MET1 maintains differential expression between the parental alleles. RdDM depends on small interfering RNAs (siRNAs). The involvement of RdDM in imprinting supports the idea that sources of siRNAs such as transposons and de novo DNA methylation were recruited in a convergent manner in plants and mammals in the evolutionary process leading to selection of imprinted loci.
In land plants, the cell plate partitions the daughter cells at cytokinesis. The cell plate initially forms between daughter nuclei and expands centrifugally until reaching the plasma membrane. The centrifugal development of the cell plate is driven by the centrifugal expansion of the phragmoplast microtubule array, but the molecular mechanism underlying this expansion is unknown. Here, we show that the phragmoplast array comprises stable microtubule bundles and dynamic microtubules. We find that the dynamic microtubules are nucleated by ?-tubulin on stable bundles. The dynamic microtubules elongate at the plus ends and form new bundles preferentially at the leading edge of the phragmoplast. At the same time, they are moved away from the cell plate, maintaining a restricted distribution of minus ends. We propose that cycles of attachment of ?-tubulin complexes onto the microtubule bundles, microtubule nucleation and bundling, accompanied by minus-end-directed motility, drive the centrifugal development of the phragmoplast.
Pollen tube guidance is controlled by multiple complex interactions with the female tissues. Here, we show that pollen tubes of Torenia fournieri are regulated by a stylar tissue in a length-dependent manner to receive and respond to attractant LURE peptides secreted from synergid cells. We developed an immunostaining method to visualize LURE peptides bound at the plasma membrane of the tip region of the pollen tube. Using this method, we found that LURE peptides bound specifically to pollen tubes growing through a cut style. The peptides also bound to pollen tubes growing through a shorter style, which were not competent to respond to these peptides. These observations suggested a possibility that acquisition of the LURE peptide reception ability and acquisition of full competency are separable processes. RNA-Seq suggested that the transcription profile of pollen tubes was affected by both the length of the style and the cultivation period, consistently with physiological changes in binding activity and LURE response ability. The database generated from de novo RNA-Seq of Torenia pollen tubes was shown to be useful to identify pollen tube proteins by mass spectrometry. Our studies provide insight and an effective platform for protein identification to understand pollen tube guidance.
For over a century, plant fertilization has been thought to depend on the fertility of a single pollen tube. However, we reported recently a "fertilization recovery system" in flowering plants that actively rescues failed fertilization of a defective mutant pollen tube by attracting a second, functional pollen tube. In typical flowering plants, two synergid cells beside the egg cell attract pollen tubes, one of which degenerates upon pollen tube discharge. We observed that fertilization was rescued when the second synergid cell accepted a wild-type pollen tube. Our results suggest that flowering plants precisely control the number of pollen tubes that arrive at each ovule and use a fertilization recovery mechanism to maximize the likelihood of successful seed set. Restricted pollination experiments showed that if sufficient pollen grains are provided, ovules attract a second pollen tube for recovery. These results support our previous finding that a long period of time is required for ovules to complete the system.
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.
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.
Volvocine green algae represent the "evolutionary time machine" model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as "rotational asymmetry of cells" so that the cells become components of the individual and "cytoplasmic bridges between protoplasts in developing embryos" to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual.
Double fertilization is a flowering plant mechanism whereby two immotile sperm cells fertilize two different female gametes. One of the two sperm cells fertilizes the egg cell to produce the embryo and the other fertilizes the central cell to produce the endosperm. Despite the biological and agricultural significance of double fertilization, the mechanism remains largely unknown owing to difficulties associated with the embedded structure of female gametes in the maternal tissue. However, molecular genetic approaches combined with novel live-cell imaging techniques have begun to clarify the actual behavior of the sperm cells, which is different from that described by previous hypotheses. In this review article, we discuss the mechanism of double fertilization based on the dynamics of the two sperm cells in Arabidopsis.
During sexual reproduction in higher angiosperms, the pollen tubes are directed to the ovules in the pistil to deliver sperm cells. This pollen tube attraction is highly species specific, and a group of small secreted proteins, TfCRPs, are necessary for this process in Torenia fournieri.
Pollen tube guidance is the mechanism whereby the direction of pollen tube growth is controlled by female cells of the pistil. Some key genes and molecules have recently been identified as being involved in pollen tube guidance. In this review article, we discuss the molecular basis of pollen tube guidance, especially in Arabidopsis thaliana, by summarizing recent progress in various plant species. Attractant molecules and receptors for gametophytic pollen tube guidance are the focus of this article.
In Arabidopsis, DEMETER (DME) DNA demethylase contributes to reprogramming of the epigenetic state of the genome in the central cell. However, other aspects of the active DNA demethylation processes remain elusive. Here we show that Arabidopsis SSRP1, known as an HMG domain-containing component of FACT histone chaperone, is required for DNA demethylation and for activation and repression of many parentally imprinted genes in the central cell. Although loss of DNA methylation releases silencing of the imprinted FWA-GFP, double ssrp1-3;met1-3 mutants surprisingly showed limited activation of maternal FWA-GFP in the central cell, and only became fully active after several nuclear divisions in the endosperm. This behavior was in contrast to the dme-1;met1 double mutant in which hypomethylation of FWA-GFP by met1 suppressed the DNA demethylation defect of dme-1. We propose that active DNA demethylation by DME requires SSRP1 function through a distinctly different process from direct DNA methylation control.
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.
Mitochondrial DNA (mtDNA) is generally packaged into the mitochondrial nucleoid (mt-nucleoid) by a high-mobility group (HMG) protein. Glom is an mtDNA-packaging HMG protein in Physarum polycephalum. Here we identified a new mtDNA-packaging protein, Glom2, which had a region homologous with yeast Mgm101. Glom2 could bind to an entire mtDNA and worked synergistically with Glom for condensation of mtDNA in vitro. Down-regulation of Glom2 enhanced the alteration of mt-nucleoid morphology and the loss of mtDNA induced by down-regulation of Glom, and impaired mRNA accumulation of some mtDNA-encoded genes. These data suggest that Glom2 may organize the mt-nucleoid coordinately with Glom.
Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.
The pollen tube attractant peptide LUREs of Torenia fournieri are diffusible peptides that attract pollen tubes in vitro. Here, we report a method enabling the direct visualization of a LURE peptide without inhibiting its attraction activity by conjugating it with the Alexa Fluor 488 fluorescent dye. After purifying and refolding the recombinant LURE2 with a polyhistidine tag, its amino groups were targeted for conjugation with the Alexa Fluor dye. Labeling of LURE2 was confirmed by its fluorescence and mass spectrometry. In our in vitro assay using gelatin beads, Alexa Fluor 488-labeled LURE2 appeared to have the same activity as unlabeled LURE2. Using the labeled LURE2, the relationship between the spatiotemporal change of distribution and activity of LURE2 was examined. LURE2 attracted pollen tubes when embedded in gelatin beads, but hardly at all when in agarose beads. Direct visualization suggested that the significant difference between these conditions was the retention of LURE2 in the gelatin bead, which might delay diffusion of LURE2 from the bead. Direct visualization of LURE peptide may open the way to studying the spatiotemporal dynamics of LURE in pollen tube attraction.
Sexual reproduction in flowering plants requires pollen-tube guidance, which is thought to be mediated by chemoattractants derived from target ovules. To date, however, no convincing evidence has been reported of a particular molecule being the true attractant. Emerging data indicate that two synergid cells, which are on either side of the egg cell, emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen-tube guidance. Recently, it was demonstrated that LUREs (LURE1 and LURE2), cysteine-rich polypeptides secreted from the synergid cell, are the key molecules in pollen-tube guidance. In this review, we summarize the mechanism of pollen-tube guidance, with special focus on gametophytic guidance and the attractants.
Plant fertilization is achieved through the involvement of various pollen-pistil interactions. Self-/non-self-recognition in pollination is important to avoid inbreeding, and directional and sustainable control of pollen tube growth is critical for the pollen tube to deliver male germ cells. Recently, various secreted peptides (polypeptides) have been reported to be involved in cell-cell communication of pollen-pistil interactions. These include determinants of self-incompatibility, factors for pollen germination and tube growth, and pollen tube attractants. Interestingly, many of them are cysteine-rich peptides/polypeptides (CRPs). In this review, I focus on the peptides involved in pollen-pistil interactions and discuss properties of peptide signaling in each step from pollination to fertilization.
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.
Genes directly involved in male/female and host/parasite interactions are believed to be under positive selection. The flowering plant Arabidopsis thaliana has more than 300 defensin-like (DEFL) genes, which are likely to be involved in both natural immunity and cell-to-cell communication including pollen-pistil interactions. However, little is known of the relationship between the molecular evolution of DEFL genes and their functions. Here, we identified a recently evolved cluster of DEFL genes in A. thaliana and demonstrated that these DEFL (cysteine-rich peptide [CRP810_1]) peptides, named AtLURE1 peptides, are pollen tube attractants guiding pollen tubes to the ovular micropyle. The AtLURE1 genes formed the sole species-specific cluster among DEFL genes compared to its close relative, A. lyrata. No evidence for positive selection was detected in AtLURE1 genes and their orthologs, implying neutral evolution of AtLURE1 genes. AtLURE1 peptides were specifically expressed in egg-accompanying synergid cells and secreted toward the funicular surface through the micropyle. Genetic analyses showed that gametophytic mutants defective in micropylar guidance (myb98, magatama3, and central cell guidance) do not express AtLURE1 peptides. Downregulation of the expression of these peptides impaired precise pollen tube attraction to the micropylar opening of some populations of ovules. Recombinant AtLURE1 peptides attracted A. thaliana pollen tubes at a higher frequency compared to A. lyrata pollen tubes, suggesting that these peptides are species-preferential attractants in micropylar guidance. In support of this idea, the heterologous expression of a single AtLURE1 peptide in the synergid cell of Torenia fournieri was sufficient to guide A. thaliana pollen tubes to the T. fournieri embryo sac and to permit entry into it. Our results suggest the unique evolution of AtLURE1 genes, which are directly involved in male-female interaction among the DEFL multigene family, and furthermore suggest that these peptides are sufficient to overcome interspecific barriers in gametophytic attraction and penetration.
The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RESPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIP-quantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.
DNA methylation maintains genome stability and regulates gene expression . In mammals, DNA methylation is reprogrammed in the germline from one generation to the next . In plants, it was considered that patterns of DNA methylation are stably maintained through sexual reproduction [3-6]. However, a recent report showed discrete variations of DNA methylation profiles from mother to daughter plants . The mechanisms that explain these variations have remained unknown. Here, we report that maintenance DNA methyltransferases are barely expressed during Arabidopsis female gametogenesis. In contrast, after fertilization both maintenance and de novo DNA methyltransferases are expressed strongly in the embryo. Embryogenesis is marked by increased de novo DNA methylation, reaching levels that are further maintained in the adult plant. The accumulation of these epigenetic marks after fertilization silences a methylation-sensitive fluorescent reporter. De novo DNA methylation in the embryo provides a mechanism that could account for the gradual remethylation of experimentally demethylated genomes [8, 9]. In conclusion, we uncover that DNA methylation activity fluctuates during sexual reproduction. This cycle likely explains variations of genome-wide patterns of DNA methylation across generations in Arabidopsis [7, 10] and enables a limited degree of reprogramming of the epigenome.
In animal fertilization, multiple sperms typically arrive at an egg cell to "win the race" for fertilization. However, in flowering plants, only one of many pollen tubes, conveying plant sperm cells, usually arrives at each ovule that harbors an egg cell. Plant fertilization has thus been thought to depend on the fertility of a single pollen tube. Here we report a fertilization recovery phenomenon in flowering plants that actively rescues the failure of fertilization of the first mutant pollen tube by attracting a second, functional pollen tube. Wild-type (WT) ovules of Arabidopsis thaliana frequently (?80%) accepted two pollen tubes when entered by mutant pollen defective in gamete fertility. In typical flowering plants, two synergid cells on the side of the egg cell attract pollen tubes, one of which degenerates upon pollen tube discharge. By semi-in vitro live-cell imaging we observed that fertilization was rescued when the second synergid cell accepted a WT pollen tube. Our results suggest that flowering plants precisely control the number of pollen tubes that arrive at each ovule and employ a fertilization recovery mechanism to maximize the likelihood of successful seed set.
Leaf primordia with high division and developmental competencies are generated around the periphery of stem cells at the shoot apex. Arabidopsis ASYMMETRIC-LEAVES2 (AS2) protein plays a key role in the regulation of many genes responsible for flat symmetric leaf formation. The AS2 gene, expressed in leaf primordia, encodes a plant-specific nuclear protein containing an AS2/LOB domain with cysteine repeats (C-motif). AS2 proteins are present in speckles in and around the nucleoli, and in the nucleoplasm of some leaf epidermal cells. We used the tobacco cultured cell line BY-2 expressing the AS2-fused yellow fluorescent protein to examine subnuclear localization of AS2 in dividing cells. AS2 mainly localized to speckles (designated AS2 bodies) in cells undergoing mitosis and distributed in a pairwise manner during the separation of sets of daughter chromosomes. Few interphase cells contained AS2 bodies. Deletion analyses showed that a short stretch of the AS2 amino-terminal sequence and the C-motif play negative and positive roles, respectively, in localizing AS2 to the bodies. These results suggest that AS2 bodies function to properly distribute AS2 to daughter cells during cell division in leaf primordia; and this process is controlled at least partially by signals encoded by the AS2 sequence itself.
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