Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

The histone variant H2A.W defines heterochromatin and promotes chromatin condensation in Arabidopsis.

Histone variants play crucial roles in gene expression, genome integrity, and chromosome segregation. We report that the four H2A variants in Arabidopsis define different genomic features, contributing to overall genomic organization. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. Similarities in conserved motifs between H2A.W and another H2A variant in metazoans suggest that plants and animals share common mechanisms for heterochromatin condensation.

Evidence of a novel silencing effect on transgenes in the Arabidopsis thaliana sperm cell.

We encountered unexpected transgene silencing in Arabidopsis thaliana sperm cells; transgenes encoding proteins with no specific intracellular localization (cytoplasmic proteins) were silenced transcriptionally or posttranscriptionally. The mRNA of cytoplasmic protein transgenes tagged with a fluorescent protein gene was significantly reduced, resulting in undetectable fluorescent protein signals in the sperm cell. Silencing of the cytoplasmic protein transgenes in the sperm cell did not affect the expression of either its endogenous homologous genes or cotransformed transgenes encoding a protein with targeted intracellular localization. This transgene silencing in the sperm cell persisted in mutants of the major gene silencing machinery including DNA methylation. The incomprehensible, yet real, transgene silencing phenotypes occurring in the sperm cell could mislead the interpretation of experimental results in plant reproduction, and this Commentary calls attention to that risk and highlights details of this novel cytoplasmic protein transgene silencing.

F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus (FA) facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study, we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in FA formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the FA and loss of polar AtLURE1.2 distribution toward the FA. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male-female communication in the synergid cell.

Dehydrin client proteins identified using phage display affinity selected libraries processed with Paired-End PhAge Sequencing (PEPA-Seq).

The LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs' presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function. To this end, we used phage display affinity selection to screen libraries derived from Arabidopsis thaliana seed mRNA with recombinant orthologous LEAPs from Arabidopsis and soybean (Glycine max). Subsequent high throughput sequencing of DNA from affinity-purified phage was performed to characterize the entire sub-population of phage retained by each LEAP orthologue. This entailed cataloging in-frame fusions, elimination of false positives, and aligning the hits on the CtP scaffold to reveal domains of respective CtPs that bound to orthologous LEAPs. This approach (Paired-end PhAge Sequencing, or PEPA-Seq) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two DHNs orthologues (LEA14 and GmPm12) compared to BSA (unrelated binding control). The veracity of LEAP:CtP binding for one of the CtPs (LEA14 and GmPM12 self-association) was independently assessed using temperature related intensity change (TRIC) analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation (BiFC) assays. The results provide insights into the involvement of the DHN Y-segments and K-domains in protein binding.

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.

Prominent caudal shift of the lumbar plexus roots in spines with 18 thoracolumbar vertebrae.

PURPOSE: It remains unclear whether concomitant changes in the thoracolumbar (TL) vertebrae and lumbar plexus roots seen in experimental embryology are present in humans with different vertebral formulas, particularly in humans with 18 TL vertebrae. We thus investigated the human lumbar plexus root changes occurring in spines with an additional TL vertebra (18TL). METHODS: The lumbosacral plexus was macroscopically dissected in TL anomaly cases found in 161 computed tomography examinations. TL anomalies were distinguished as simple abnormalities in total TL count and abnormal TL trade-offs, i.e., exchanges between the last thoracic and first lumbar vertebrae, and were analyzed separately. RESULTS: One additional TL vertebra (7C_18TL_5S) was observed in 4/159 cases (2.5%), excluding cases with cervical and sacral abnormalities. Different from the unclear shifts of nerve roots in cases with 16TL and 17TL trade-offs, the 18TL trade-off tended to involve a caudal shift at the cranial limit, without event change at the caudal limit. In addition, only one nerve segment shift was reconfirmed with a change in two vertebral segments from 16 to 18 TL vertebrae. CONCLUSIONS: We revealed that concomitant changes in the lumbar plexus roots and vertebrae in humans with 18TL vertebrae may become more pronounced than those in humans with 16 or 17TL vertebrae, by approaching the typical mammalian TL formula (19TL). This study showed that the TL formula can be used to estimate changes in the lumbar plexus roots, which may assist in the planning of nerve-sparing spinal and pelvic surgery.

Integrated anatomical practice combining cadaver dissection and matched cadaver CT data processing and analysis.

PURPOSE: With the increasing significance of diagnostic imaging in clinical practice, long-term anatomical education and training is required to ensure that students can reliably distinguish anatomical structures and interpret images. To improve students' motivation and prospects for learning imaging anatomy, we developed an integrated anatomical practice program combining cadaveric dissection with cadaver CT data processing and analysis during undergraduate students' dissection courses. METHODS: Workstations imported with post-mortem CT data of dissected cadavers and various forms of clinical CT/MRI data were set in the dissection room. Medical students had free access to the imaging data during cadaver dissection, and they were challenged to process and analyze the data for submission of voluntary imaging reports on their topics of interest. Finally, we surveyed the integrated anatomical education of 481 medical students. RESULTS: The positive response rate to the integrated anatomical practice was 74.9%, and 79.4% of the students answered that this form of practice offered a suitable introduction to anatomical imaging. The usefulness of this approach in understanding the 2- to 3D arrangement of the human body and enhancing interest in anatomy was also confirmed. The submission rate of voluntary imaging reports also increased annually and is currently 97.4%. CONCLUSION: Our integrated anatomical practice only allowed students to actively browse CT images and facilitated imaging processing and analysis of their region of interest. This practice may improve students' long-term ability to analyze images and deepen their understanding. A competitive imaging contest may help improve students' motivation when they begin learning imaging anatomy.

Cellular dynamics of double fertilization and early embryogenesis in flowering plants.

Flowering plants (angiosperms) perform a unique double fertilization in which two sperm cells fuse with two female gamete cells in the embryo sac to develop a seed. Furthermore, during land plant evolution, the mode of sexual reproduction has been modified dramatically from motile sperm in the early-diverging land plants, such as mosses and ferns as well as some gymnosperms (Ginkgo and cycads) to nonmotile sperm that are delivered to female gametes by the pollen tube in flowering plants. Recent studies have revealed the cellular dynamics and molecular mechanisms for the complex series of double fertilization processes and elucidated differences and similarities between animals and plants. Here, together with a brief comparison with animals, we review the current understanding of flowering plant zygote dynamics, covering from gamete nuclear migration, karyogamy, and polyspermy block, to zygotic genome activation as well as asymmetrical division of the zygote. Further analyses of the detailed molecular and cellular mechanisms of flowering plant fertilization should shed light on the evolution of the unique sexual reproduction of flowering plants.

A shared cis-regulatory module activates transcription in the suspensor of plant embryos.

The mechanisms controlling the transcription of gene sets in specific regions of a plant embryo shortly after fertilization remain unknown. Previously, we showed that G564 mRNA, encoding a protein of unknown function, accumulates to high levels in the giant suspensor of both Scarlet Runner Bean (SRB) and Common Bean embryos, and a cis-regulatory module containing three unique DNA sequences, designated as the 10-bp, Region 2, and Fifth motifs, is required for G564 suspensor-specific transcription [Henry KF, et al. (2015) Plant Mol Biol 88:207-217; Kawashima T, et al. (2009) Proc Natl Acad Sci USA 106:3627-3632]. We tested the hypothesis that these motifs are also required for transcription of the SRB GA 20-oxidase gene, which encodes a gibberellic acid hormone biosynthesis enzyme and is coexpressed with G564 at a high level in giant bean suspensors. We used deletion and gain-of-function experiments in transgenic tobacco embryos to show that two GA 20-oxidase DNA regions are required for suspensor-specific transcription, one in the 5' UTR (+119 to +205) and another in the 5' upstream region (-341 to -316). Mutagenesis of sequences in these two regions determined that the cis-regulatory motifs required for G564 suspensor transcription are also required for GA 20-oxidase transcription within the suspensor, although the motif arrangement differs. Our results demonstrate the flexibility of motif positioning within a cis-regulatory module that activates gene transcription within giant bean suspensors and suggest that G564 and GA 20-oxidase comprise part of a suspensor gene regulatory network.

Postmortem evaluation of neuromuscular damages more extensive than the surgical intervention area after iliac crest bone graft.

Several complications may occur following iliac bone grafting, one of the common sites for autologous bone harvesting. Of these, it is difficult to localize the damage in neurological complications due to the presence of several nerves in a similar distribution area with variations among individuals. To minimize these complications, conventional clinical anatomical studies using normal human cadavers have estimated the theoretical neurological damage area corresponding to the surgical intervention area. We report a case of neuromuscular damage in a 93-year-old woman who had an iliac crest defect after a bone graft, based on the virtual and physical dissections with histological confirmations.In this study, the patient was confirmed to have severe neuromuscular complications with major complications including a hernia protruding through the iliac defect. One of the two ilioinguinal nerves was extracted with the hernia sac through the iliac defect, and its distal part was completely damaged. The iliohypogastric nerve, which was far from the defect foramen, also showed remarkable fibrosis and demyelination, affected by the degeneration of the transversus abdominis muscles.The present anatomical findings show that the area of eventual neuromuscular damage should be estimated to larger than the conventionally predicted area of direct nerve damage, which is usually concomitant with the surgical intervention area.

A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm.

Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion in an in vitro culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control.

Epigenetic reprogramming in plant sexual reproduction.

Epigenetic reprogramming consists of global changes in DNA methylation and histone modifications. In mammals, epigenetic reprogramming is primarily associated with sexual reproduction and occurs during both gametogenesis and early embryonic development. Such reprogramming is crucial not only to maintain genomic integrity through silencing transposable elements but also to reset the silenced status of imprinted genes. In plants, observations of stable transgenerational inheritance of epialleles have argued against reprogramming. However, emerging evidence supports that epigenetic reprogramming indeed occurs during sexual reproduction in plants and that it has a major role in maintaining genome integrity and a potential contribution to epiallelic variation.

Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote.

The asymmetric cell division of the zygote is the initial and crucial developmental step in most multicellular organisms. In flowering plants, whether zygote polarity is inherited from the preexisting organization in the egg cell or reestablished after fertilization has remained elusive. How dynamically the intracellular organization is generated during zygote polarization is also unknown. Here, we used a live-cell imaging system with Arabidopsis zygotes to visualize the dynamics of the major elements of the cytoskeleton, microtubules (MTs), and actin filaments (F-actins), during the entire process of zygote polarization. By combining image analysis and pharmacological experiments using specific inhibitors of the cytoskeleton, we found features related to zygote polarization. The preexisting alignment of MTs and F-actin in the egg cell is lost on fertilization. Then, MTs organize into a transverse ring defining the zygote subapical region and driving cell outgrowth in the apical direction. F-actin forms an apical cap and longitudinal arrays and is required to position the nucleus to the apical region of the zygote, setting the plane of the first asymmetrical division. Our findings show that, in flowering plants, the preexisting cytoskeletal patterns in the egg cell are lost on fertilization and that the zygote reorients the cytoskeletons to perform directional cell elongation and polar nuclear migration.

Anatomical visualization of neural course and distribution of anterior ascending aortic plexus.

The aim of this study was to document the detailed anatomy of neural course and distribution on the anterior ascending aorta, to identify the high and low density areas of the anterior ascending aortic plexus for further understandings in cardiovascular surgery. The embalmed hearts of 42 elderly individuals were submacroscopically and microscopically examined, after excluding any that were macroscopically abnormal. With its origins in the anterior ascending aortic plexus, the right coronary plexus substantially innervated the right coronary artery, the right atrium and ventricle, and the sinus node. The intensive neural area extending from 10 mm lateral to the interatrial groove below the pericardial reflection as far as the right coronary artery opening contained almost all the right coronary plexus in 61.3% of patients, and more than 40.9% of the total nerve volume of the anterior ascending aortic plexus. Our findings suggest that the most superior and lateral area on the ascending aorta show the lowest neural density of right coronary component in the anterior ascending aortic plexus and the high density areas are invisible in right lateral field of view as seen in the right trans-axillary MICS approach.

Fertilization-independent Cell-fusion between the Synergid and Central Cell in the Polycomb Mutant.

In flowering plants, fertilization of the central cell gives rise to an embryo-nourishing endosperm. Recently, we reported that the endosperm absorbs the adjacent synergid cell through a cell-fusion, terminating the pollen tube guidance by a rapid inactivation of the synergid cell. Although this synergid-endosperm fusion (SE fusion) initiates soon after fertilization, it was still unknown whether the triggers of SE fusion are stimuli during fertilization or other seed developmental processes. To further dissect out the SE fusion process, we investigated the SE fusion in an Arabidopsis mutant defective for MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a subunit of the polycomb repressive complex 2 (PRC2). The mutant msi1 develops autonomous endosperm without fertilization. Time-lapse imaging revealed a rapid efflux of the synergid contents during the autonomous endosperm development, indicating that the initiation of SE fusion is under the control of some of the events triggered by fertilization of the central cell distinct from the discharge of pollen tube contents and plasma membrane fusion.

A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements.

Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.

Insights into dynamic coenocytic endosperm development: Unraveling molecular, cellular, and growth complexity.

The endosperm, a product of double fertilization, is one of the keys to the evolution and success of angiosperms in conquering the land. While there are differences in endosperm development among flowering plants, the most common form is coenocytic growth, where the endosperm initially undergoes nuclear division without cytokinesis and eventually becomes cellularized. This complex process requires interplay among networks of transcription factors such as MADS-box, auxin response factors (ARFs), and phytohormones. The role of cytoskeletal elements in shaping the coenocytic endosperm and influencing seed growth also becomes evident. This review offers a recent understanding of the molecular and cellular dynamics in coenocytic endosperm development and their contributions to the final seed size.