Exploring Novel Polytubey Reproduction Pathways Utilizing Cumulative Genetic Tools.

In the anthers and ovaries of flowers, pollen grains and embryo sacs are produced with uniform cell compositions. This stable gametogenesis enables elaborate interactions between male and female gametophytes after pollination, forming the highly successful sexual reproduction system in flowering plants. As most ovules are fertilized with a single pollen tube, the resulting genome set in the embryo and endosperm is determined in a single pattern by independent fertilization of the egg cell and central cell by two sperm cells. However, if ovules receive four sperm cells from two pollen tubes, the expected options for genome sets in the developing seeds would more than double. In wild-type Arabidopsis thaliana plants, around 5% of ovules receive two pollen tubes. Recent studies have elucidated the abnormal fertilization in supernumerary pollen tubes and sperm cells related to polytubey, polyspermy, heterofertilization and fertilization recovery. Analyses of model plants have begun to uncover the mechanisms underlying this new pollen tube biology. Here, we review unusual fertilization phenomena and propose several breeding applications for flowering plants. These arguments contribute to the remodeling of plant reproduction, a challenging concept that alters typical plant fertilization by utilizing the current genetic toolbox.

Migration of lipiodol into lateral ventricles after embolization of cerebral arteriovenous malformation: a case report.

N-butyl cyanoacrylate (NBCA) has been used to embolise brain arteriovenous malformations (AVMs) for over 30 years. It is a mixed with lipiodol in varying proportions. We report a 22-year-old male with intraventricular hemorrhage from a ruptured intranidal AVM aneurysm in the left temporal lobe. The intranidal aneurysm and the nidus were successfully embolized using a 20% NBCA and lipiodol mixture without any complications according to computed tomography (CT) immediately after treatment. Scattered high-density spots were observed in both lateral ventricles on CT 5 days after embolization, suggesting migration of lipiodol. We speculated that the aneurysm was a pseudoaneurysm whose wall protruded into the inferior horn of the left lateral ventricle, and the lipiodol in the NBCA migrated into the ventricles after the thin part of the wall ruptured. The patient developed pyrexia due to chemical meningitis, which responded to steroid treatment for one month.

ARP2/3-independent WAVE/SCAR pathway and class XI myosin control sperm nuclear migration in flowering plants.

After eukaryotic fertilization, gamete nuclei migrate to fuse parental genomes in order to initiate development of the next generation. In most animals, microtubules control female and male pronuclear migration in the zygote. Flowering plants, on the other hand, have evolved actin filament (F-actin)-based sperm nuclear migration systems for karyogamy. Flowering plants have also evolved a unique double-fertilization process: two female gametophytic cells, the egg and central cells, are each fertilized by a sperm cell. The molecular and cellular mechanisms of how flowering plants utilize and control F-actin for double-fertilization events are largely unknown. Using confocal microscopy live-cell imaging with a combination of pharmacological and genetic approaches, we identified factors involved in F-actin dynamics and sperm nuclear migration in Arabidopsis thaliana (Arabidopsis) and Nicotiana tabacum (tobacco). We demonstrate that the F-actin regulator, SCAR2, but not the ARP2/3 protein complex, controls the coordinated active F-actin movement. These results imply that an ARP2/3-independent WAVE/SCAR-signaling pathway regulates F-actin dynamics in female gametophytic cells for fertilization. We also identify that the class XI myosin XI-G controls active F-actin movement in the Arabidopsis central cell. XI-G is not a simple transporter, moving cargos along F-actin, but can generate forces that control the dynamic movement of F-actin for fertilization. Our results provide insights into the mechanisms that control gamete nuclear migration and reveal regulatory pathways for dynamic F-actin movement in flowering plants.

Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation.

Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.

Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana.

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.

Multiple BiP genes of Arabidopsis thaliana are required for male gametogenesis and pollen competitiveness.

Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes. Arabidopsis thaliana has three BiP genes. BIP1 and BIP2 are ubiquitously expressed. BIP3 encodes a less well conserved BiP paralog, and it is expressed only under ER stress conditions in the majority of organs. Here, we report that all BiP genes are expressed and functional in pollen and pollen tubes. Although the bip1 bip2 double mutation does not affect pollen viability, the bip1 bip2 bip3 triple mutation is lethal in pollen. This result indicates that lethality of the bip1 bip2 double mutation is rescued by BiP3 expression. A decrease in the copy number of the ubiquitously expressed BiP genes correlates well with a decrease in pollen tube growth, which leads to reduced fitness of mutant pollen during fertilization. Because an increased protein secretion activity is expected to increase the protein folding demand in the ER, the multiple BiP genes probably cooperate with each other to ensure ER homeostasis in cells with active secretion such as rapidly growing pollen tubes.

Fertilization recovery system is dependent on the number of pollen grains for efficient reproduction in plants.

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.

Fertilization recovery after defective sperm cell release in Arabidopsis.

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.

Techniques for universal evaluation of astringency of green tea infusion by the use of a taste sensor system.

A practical method for universal evaluation of the astringency of green tea infusion by a taste sensor system was established. The use of EGCg aqueous solution as a standard enabled analysis with high accuracy and reproducibility. The sensor output was converted into taste-intensity on the basis of Weber's and Weber-Fechner laws, which was named the "EIT(ast)" value ("EIT" and "ast" are abbreviations for "Estimated Intensity of Taste" and "astringency" respectively). It was clarified that green tea infusion is to be classified into eight grades on the EIT(ast) scale. Furthermore, the high correlation of the EIT(ast) value with the human gustatory sense and the high stability of the taste sensor were proved.

Callose (beta-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth.

BACKGROUND: Callose (beta-1,3 glucan) separates developing pollen grains, preventing their underlying walls (exine) from fusing. The pollen tubes that transport sperm to female gametes also contain callose, both in their walls as well as in the plugs that segment growing tubes. Mutations in CalS5, one of several Arabidopsis beta-1,3 glucan synthases, were previously shown to disrupt callose formation around developing microspores, causing aberrations in exine patterning, degeneration of developing microspores, and pollen sterility. RESULTS: Here, we describe three additional cals5 alleles that similarly alter exine patterns, but instead produce fertile pollen. Moreover, one of these alleles (cals5-3) resulted in the formation of pollen tubes that lacked callose walls and plugs. In self-pollinated plants, these tubes led to successful fertilization, but they were at a slight disadvantage when competing with wild type. CONCLUSION: Contrary to a previous report, these results demonstrate that a structured exine layer is not required for pollen development, viability or fertility. In addition, despite the presence of callose-enriched walls and callose plugs in pollen tubes, the results presented here indicate that callose is not required for pollen tube functions.