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In angiosperms, pollen tube guidance allows sperm cell delivery to the female gametes within the ovule, which are deeply embedded in a flower. However, when an ovary includes multiple pollen tubes and ovules, it is unclear how each ovule is fertilized one-to-one by a pollen tube. Here, our two-photon imaging revealed the pollen tube dynamics in living ovaries. The number of pollen tubes and ovule maturity affected the target selection among multiple ovules. On the inner surface of the septum epidermis within the transmitting tract, pollen tube behavior and emergence were regulated by the ovular sporophytic signals. In funicular guidance, the second pollen tube was strictly repelled by the FERONIA and LORELEI-dependent gametophytic signal, especially more than 45 minutes after the first pollen tube had passed. Such highly spatiotemporal regulation mechanisms in the one-to-one pollen tube guidance may allow angiosperms to produce more offspring in nature.

Key message Biolistic delivery into pollen. Abstract In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.

To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.g. through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation, and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulfite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils and fertilized Arabidopsis thaliana fruits—tissues that have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colors was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In addition, autofluorescence attributed to chlorophyll hampers the observation of fluorescent proteins in plants. For a long time, various clearing reagents have been used to make plants transparent. However, conventional clearing reagents diminish fluorescent signals; therefore, it has not been possible to observe the cellular and intracellular structures with fluorescent proteins. Reagents were developed that can clear plant tissues by removing chlorophyll while maintaining fluorescent protein stability. A detailed protocol is provided here for the optical clearing of plant tissues using clearing reagents, ClearSee (CS) or ClearSeeAlpha (CSA). The preparation of cleared plant tissues involves three steps: fixation, washing, and clearing. Fixation is a crucial step in maintaining the cellular structures and intracellular stability of fluorescent proteins. The incubation time for clearing depends on the tissue type and species. In Arabidopsis thaliana, the time required for clearing with CS was 4 days for leaves and roots, 7 days for seedlings, and 1 month for pistils. CS also required a relatively short time of 4 days to make the gametophytic leaves of Physcomitrium patens transparent. In contrast, pistils in tobacco and torenia produced brown pigment due to oxidation during CS treatment. CSA reduced the brown pigment by preventing oxidation and could make tobacco and torenia pistils transparent, although it took a relatively long time (1 or 2 months). CS and CSA were also compatible with staining using chemical dyes, such as DAPI (4',6-diamidino-2-phenylindole) and Hoechst 33342 for DNA and Calcofluor White, SR2200, and Direct Red 23 for the cell wall. This method can be useful for whole-plant imaging to reveal intact morphology, developmental processes, plant-microbe interactions, and nematode infections.

During double fertilization in angiosperms, two male gametes (sperm cells), are released from a pollen tube into the receptive region between two female gametes; the egg cell and the central cell of the ovule. The sperm cells fertilize the egg cell and the central cell in a one-to-one manner to yield a zygote and an endosperm, respectively. The one-to-one distribution of the sperm cells to the two female gametes is strictly regulated, possibly via communication among the four gametes. Polyspermy block is the mechanism by which fertilized female gametes prevent fertilization by a secondary sperm cell, and has been suggested to operate in the egg cell rather than the central cell. However, whether the central cell also has the ability to avoid polyspermy during double fertilization remains unclear. Here, we assessed the one-to-one fertilization mechanism of the central cell by laser irradiation of the female gametes and live cell imaging of the fertilization process in Arabidopsis thaliana. We successfully disrupted an egg cell within the ovules by irradiation using a femtosecond pulse laser. In the egg-disrupted ovules, the central cell predominantly showed single fertilization by one sperm cell, suggesting that neither the egg cell nor its fusion with one sperm cell is necessary for one-to-one fertilization (i.e., monospermy) of the central cell. In addition, using tetraspore mutants possessing multiple sperm cell pairs in one pollen, we demonstrated that normal double fertilization was observed even when excess sperm cells were released into the receptive region between the female gametes. In ovules accepting four sperm cells, the egg cell never fused with more than one sperm cell, whereas half of the central cells fused with more than one sperm cell (i.e., polyspermy) even 1 h later. Our results suggest that the central cell can block polyspermy during double fertilization, although the central cell is more permissive to polyspermy than the egg cell. The potential contribution of polyspermy block by the central cell is discussed in terms of how it is involved in the one-to-one distribution of the sperm cells to two distinct female gametes.

All flowering plants exhibit a unique type of sexual reproduction called ‘double fertilization’ in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from Lilium longiflorum generative cells using proteomic analysis and have found that the protein is an ortholog of Arabidopsis DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2). The flowering plant DMP9 proteins analyzed in this study were predicted to have four transmembrane domains and be specifically expressed in both generative and sperm cells. Knockdown of DMP9 resulted in aborted seeds due to single fertilization of the central cell. Detailed imaging of DMP9-knockdown sperm cells during in vivo and semi-in vitro double fertilization revealed that DMP9 is involved in gamete interaction that leads to correct double fertilization.

After meiosis, an unequal cell division generates the male gamete lineage in flowering plants. The generative cell will undergo a second division, giving rise to the two gametes, i.e. the sperm cells. The other cell will develop into the vegetative cell that plays a crucial role in pollen tube formation and sperm delivery. Recently, the vegetative cell has been suggested to be important for programming of the chromatin state in sperm cells and/or the resulting fertilization products. Blocking the initial unequal division genetically, we first highlight that the default differentiation state after male meiosis is a vegetative fate, which is consistent with earlier work. We find that uni-nucleated mutant microspores differentiated as wild-type vegetative cells, including chromatin remodeling and the transcriptional activation of transposable elements. Moreover, live-cell imaging revealed that this vegetative cell is sufficient for the correct guidance of the pollen tube to the female gametes. Hence, we conclude that vegetative cell differentiation and function does not depend on the formation or presence of the actual gametes but rather on external signals or a cell-autonomous pace keeper., Summary: Cell biological analyses in Arabidopsis show that the vegetative cell differentiates without the presence of the actual gametes, and is solely sufficient for pollen tube germination, guidance, ovule penetration and pollen tube discharge.

A new plant ovule enlargement triggered by pollen tube contents, accompanied by seed coat initiation without fertilization., In angiosperms, pollen tubes carry two sperm cells toward the egg and central cells to complete double fertilization. In animals, not only sperm but also seminal plasma is required for proper fertilization. However, little is known regarding the function of pollen tube content (PTC), which is analogous to seminal plasma. We report that the PTC plays a vital role in the prefertilization state and causes an enlargement of ovules without fertilization. We termed this phenomenon as pollen tube–dependent ovule enlargement morphology and placed it between pollen tube guidance and double fertilization. Additionally, PTC increases endosperm nuclei without fertilization when combined with autonomous endosperm mutants. This finding could be applied in agriculture, particularly in enhancing seed formation without fertilization in important crops.

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.

Key message New gametic homozygous mutants. In angiosperms, a haploid male gamete (sperm cell) fuses with a haploid female gamete (egg cell) during fertilization to form a zygote carrying paternally and maternally derived chromosomes. Several fertilization-defective mutants in Arabidopsis thaliana, including a generative cell-specific 1 (gcs1)/hapless 2 mutant, the sperm cells of which are unable to fuse with female gametes, can only be maintained as heterozygous lines due to the infertile male or female gametes. Here, we report successful generation of a gcs1 homozygous mutant by heat-inducible removal of the GCS1 transgene. Using the gcs1 homozygous mutant as male, the defect in gamete fusion was observed with great frequency; in our direct observation by semi-in vivo fertilization assay using ovules, 100 % of discharged sperm cells in culture failed to show gamete fusion. More than 70 % of ovules in the pistil received a second pollen tube as attempted fertilization recovery. Moreover, gcs1 mutant sperm cells could fertilize female gametes at a low frequency in the pistil. This strategy to generate homozygous fertilization-defective mutants will facilitate novel approaches in plant reproduction research. Electronic supplementary material The online version of this article (doi:10.1007/s00497-015-0256-4) contains supplementary material, which is available to authorized users.