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The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex spans the nuclear envelope and mediates nuclear mechanotransduction, the process by which mechanical signals and forces are transmitted across the nuclear envelope. In turn, the AAA+ ATPase torsinA is thought to regulate force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia, though the extent to which endogenous mechanical stresses contribute to these pathologies is unclear. Here, we use the Caenorhabditis elegans germline as a model to investigate mechanisms that maintain nuclear integrity as germ cell nuclei progress through meiotic development and migrate for gametogenesis-processes that require LINC complex function. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog. We show that decreasing OOC-5/torsinA activity prevents nuclear collapse in lamin mutants by disrupting the function of the LINC complex. At a mechanistic level, OOC-5/torsinA promotes the assembly or maintenance of the lamin-associated LINC complex and this activity is also important for interphase nuclear pore complex insertion into growing germline nuclei. These results demonstrate that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. Thus, the torsinA-LINC complex nexus might comprise a therapeutic target for certain laminopathies by preventing damage from endogenous cellular forces.

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in relapsing cases of acute myeloid leukemia, as well as in inherited myelodysplastic syndromes... Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia (AML). DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase that is recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of AML. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here, we conduct a comprehensive analysis of the Caenorhabditis elegans DDX41 ortholog, SACY-1. Biochemical analyses defined SACY-1 as a component of the C. elegans spliceosome, and genetic analyses revealed synthetic lethal interactions with spliceosomal components. We used the auxin-inducible degradation system to analyze the consequence of SACY-1 depletion on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-dependent and splicing-independent mechanisms. Altered 3′ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 in the second step of splicing. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. The transcriptome changes observed after SACY-1 depletion suggest that disruption of the spliceosome induces a stress response, which could contribute to the cellular phenotypes conferred by sacy-1 mutant alleles. Multiple sacy-1/ddx41 missense mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that their incorporation into the spliceosome is detrimental. Antagonistic variants that perturb the function of the spliceosome may be relevant to the disease-causing mutations, including DDX41, affecting highly conserved components of the spliceosome in humans.

The ubiquitin-mediated degradation of oocyte translational regulatory proteins is a conserved feature of the oocyte-to-embryo transition (OET). In the nematode Caenorhabditis elegans, multiple translational regulatory proteins, including the TRIM-NHL RNA-binding protein LIN-41/Trim71 and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11, are degraded during the OET. Degradation of each protein requires activation of the M-phase cyclin-dependent kinase CDK-1, is largely complete by the end of the first meiotic division and does not require the anaphase promoting complex (APC). However, only LIN-41 degradation requires the F-box protein SEL-10/FBW7/Cdc4p, the substrate recognition subunit of an SCF-type E3 ubiquitin ligase. This finding suggests that PUF-3 and PUF-11, which localize to LIN-41-containing ribonucleoprotein particles (RNPs), are independently degraded through the action of other factors and that the oocyte RNPs are disassembled in a concerted fashion during the OET. We develop and test the hypothesis that PUF-3 and PUF-11 are targeted for degradation by the proteasome-associated HECT-type ubiquitin ligase ETC-1/UBE3C/Hu15, which is broadly expressed in C. elegans. We find that several GFP-tagged fusion proteins that are degraded during the OET, including fusions with PUF-3, PUF-11, LIN-41, IFY-1/Securin and CYB-1/Cyclin B, are incompletely degraded when ETC-1 function is compromised. However, it is the fused GFP moiety that appears to be the critical determinant of this proteolysis defect. These findings are consistent with a conserved role for ETC-1 in promoting proteasome processivity and suggest that proteasomal processivity is an important element of the OET during which many key oocyte regulatory proteins are rapidly targeted for degradation.Article SummaryThe ubiquitin-mediated degradation of translational regulatory RNA-binding proteins is a conserved feature of the oocyte-to-embryo transition (OET). C. elegans LIN-41 is a master regulator of oogenesis and is found in a large translational regulatory ribonucleoprotein (RNP) complex with more than 1000 maternal transcripts and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11. We show that the concerted action of ubiquitin-mediated protein degradation and proteasome processivity rapidly disassemble LIN-41-containing RNPs during the OET thereby relieving repression of many maternal transcripts.

In order to understand the acidity properties of hydrated ZSM-5 zeolites having different SiO2/Al2O3 ratios, the adsorption states of the NH3 and NH4+ species on hydrated ZSM-5 zeolites were concurrently investigated by near infrared spectroscopy. In the case of hydrated H+-type ZSM-5, absorption band due to the H2O molecules directly interacting with the H+ sites (Brønsted acid sites) was observed at 5266 cm−1. When the NH3 molecules were admitted on the hydrated H/ZSM-5, absorption bands due to isolated NH3 and isolated NH4+ were simultaneously observed at 5044 and 4846 cm−1, respectively, although most of the H+ sites were occupied by H2O molecules. As the NH3 adsorption amount increased, almost isolated H2O molecules hardly changed while the NH3 and NH4+ stabilized by the hydrogen bonds shifted to 5000 and 4771 cm−1, respectively. As for the siliceous-type ZSM-5, near infrared absorption due to H2O molecules interacting with the silanol groups was mainly observed at 5285 cm−1. However, since the H2O molecules readily desorbed at r.t. due to the hydrophobic character of the siliceous zeolite surface, the NH3 adsorption behavior was almost the same as the dehydrated sample. In the case of Na+-type ZSM-5, absorption band due to H2O molecules directly interacting with the Na+ sites was observed at 5291 cm−1. When NH3 molecules were adsorbed on the hydrated Na/ZSM-5, absorption band due to the hydrogen-bonded H2O shifted to 5260 cm−1 and absorption band due to the NH3 molecules in the NH3/H2O mixed clusters was simultaneously observed at 5014 cm−1. When the NH3/H2O mixed clusters desorbed from Na/ZSM-5, an absorption band due to NH3 species interacting with the Al3+ sites (Lewis acid sites) was observed at 4957 cm−1 since the neutral Na/ZSM-5 surface might be dealuminated by the basicity of the wet NH3 clusters.

Significance Nuclear envelopathies are a complex group of human diseases caused by mutations in nuclear envelope proteins, including progeria, myopathy, and dystonia. Here, we used the Caenorhabditis elegans germline as a model system to investigate the function of the OOC-5/torsinA AAA+ ATPase, which localizes to the nuclear envelope and is mutated in early-onset DYT1 dystonia in humans. We show that OOC-5/torsinA promotes the function of the LINC complex, which spans the nuclear envelope and transmits forces to the nuclear lamina. Remarkably, decreasing the function of OOC-5/torsinA or the LINC complex prevents nuclear collapse in the absence of a functional nuclear lamina. Therapeutics targeting torsinA or the LINC complex might prevent nuclear damage from endogenous forces in certain nuclear envelopathies., The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex spans the nuclear envelope and mediates nuclear mechanotransduction, the process by which mechanical signals and forces are transmitted across the nuclear envelope. In turn, the AAA+ ATPase torsinA is thought to regulate force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia, though the extent to which endogenous mechanical stresses contribute to these pathologies is unclear. Here, we use the Caenorhabditis elegans germline as a model to investigate mechanisms that maintain nuclear integrity as germ cell nuclei progress through meiotic development and migrate for gametogenesis—processes that require LINC complex function. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog. We show that decreasing OOC-5/torsinA activity prevents nuclear collapse in lamin mutants by disrupting the function of the LINC complex. At a mechanistic level, OOC-5/torsinA promotes the assembly or maintenance of the lamin-associated LINC complex and this activity is also important for interphase nuclear pore complex insertion into growing germline nuclei. These results demonstrate that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. Thus, the torsinA–LINC complex nexus might comprise a therapeutic target for certain laminopathies by preventing damage from endogenous cellular forces.

In the nematode Caenorhabditis elegans, the conserved LIN-41 RNA-binding protein is a translational repressor that coordinately controls oocyte growth and meiotic maturation. LIN-41 exerts these effects, at least in part, by preventing the premature activation of the cyclin-dependent kinase CDK-1. Here we investigate the mechanism by which LIN-41 is rapidly eliminated upon the onset of meiotic maturation. Elimination of LIN-41 requires the activities of CDK-1 and multiple SCF (Skp1, Cul1, and F-box protein)-type E3 ubiquitin ligase subunits, including the conserved substrate adaptor protein SEL-10/Fbw7/Cdc4, suggesting that LIN-41 is a target of ubiquitin-mediated protein degradation. Within the LIN-41 protein, two nonoverlapping regions, Deg-A and Deg-B, are individually necessary for LIN-41 degradation; both contain several potential phosphodegron sequences, and at least one of these sequences is required for LIN-41 degradation. Finally, Deg-A and Deg-B are sufficient, in combination, to mediate SEL-10-dependent degradation when transplanted into a different oocyte protein. Although LIN-41 is a potent inhibitor of protein translation and M phase entry, the failure to eliminate LIN-41 from early embryos does not result in the continued translational repression of LIN-41 oocyte messenger RNA targets. Based on these observations, we propose a model for the elimination of LIN-41 by the SEL-10 E3 ubiquitin ligase and suggest that LIN-41 is inactivated before it is degraded. Furthermore, we provide evidence that another RNA-binding protein, the GLD-1 tumor suppressor, is regulated similarly. Redundant mechanisms to extinguish translational repression by RNA-binding proteins may both control and provide robustness to irreversible developmental transitions, including meiotic maturation and the oocyte-to-embryo transition.

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of theC. elegansDDX41 ortholog, SACY-1. Our results reveal general essential functions for SACY-1 in both the germline and the soma, as well as specific functions affecting germline sex determination and cell cycle control. Certainsacy-1/DDX41mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that they compromise the function of the spliceosome. Consistent with these findings,sacy-1exhibits synthetic lethal interactions with several spliceosomal components, and biochemical analyses suggest that SACY-1 is a component of theC. elegansspliceosome. We used the auxin-inducible degradation system to analyze the impact of SACY-1 on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-independent and splicing-dependent mechanisms. The observed transcriptome changes suggest that disruption of spliceosomal function induces a stress response. Altered 3’ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 as a second-step splicing factor. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing.Author SummaryMutations affecting spliceosomal proteins are frequently found in hematological malignancies. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase recurrently mutated in inherited and relapsing myelodysplastic syndromes and acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing mutations in spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of theC. elegansDDX41 ortholog, SACY-1. Our results reveal that multiplesacy-1/DDX41missense mutations, including the R525H human oncogenic variant, exhibit antimorphic activity that likely compromises the function of the spliceosome. The genomic consequences of SACY-1 depletion include splicing-splicing-independent and splicing-dependent alterations in the transcriptome.

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in relapsing cases of acute myeloid leukemia, as well as in inherited myelodysplastic syndromes..., Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia (AML). DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase that is recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of AML. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here, we conduct a comprehensive analysis of the Caenorhabditis elegans DDX41 ortholog, SACY-1. Biochemical analyses defined SACY-1 as a component of the C. elegans spliceosome, and genetic analyses revealed synthetic lethal interactions with spliceosomal components. We used the auxin-inducible degradation system to analyze the consequence of SACY-1 depletion on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-dependent and splicing-independent mechanisms. Altered 3′ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 in the second step of splicing. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. The transcriptome changes observed after SACY-1 depletion suggest that disruption of the spliceosome induces a stress response, which could contribute to the cellular phenotypes conferred by sacy-1 mutant alleles. Multiple sacy-1/ddx41 missense mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that their incorporation into the spliceosome is detrimental. Antagonistic variants that perturb the function of the spliceosome may be relevant to the disease-causing mutations, including DDX41, affecting highly conserved components of the spliceosome in humans.

In flowering plants, the female gametophyte controls pollen tube reception immediately before fertilization and regulates seed development immediately after fertilization, although the controlling mechanisms remain poorly understood. Previously, we showed that LORELEI (LRE), which encodes a putative glycosylphosphatidylinositol-anchored membrane protein, is critical for pollen tube reception by the female gametophyte before fertilization and the initiation of seed development after fertilization. Here, we show that LRE is expressed in the synergid, egg, and central cells of the female gametophyte and in the zygote and proliferating endosperm of the Arabidopsis (Arabidopsis thaliana) seed. Interestingly, LRE expression in the developing seeds was primarily from the matrigenic LRE allele, indicating that LRE expression is imprinted. However, LRE was biallelically expressed in 8-d-old seedlings, indicating that the patrigenic allele does not remain silenced throughout the sporophytic generation. Regulation of imprinted LRE expression is likely novel, as LRE was not expressed in pollen or pollen tubes of mutants defective for MET1, DDM1, RNA-dependent DNA methylation, or MSI-dependent histone methylation. Additionally, the patrigenic LRE allele inherited from these mutants was not expressed in seeds. Surprisingly, and contrary to the predictions of the parental conflict hypothesis, LRE promotes growth in seeds, as loss of the matrigenic but not the patrigenic LRE allele caused delayed initiation of seed development. Our results showed that LRE is a rare imprinted gene that functions immediately after double fertilization and supported the model that a passage through the female gametophyte establishes monoalleleic expression of LRE in seeds and controls early seed development.

An extended meiotic prophase is a hallmark of oogenesis. Hormonal signaling activates the CDK1/cyclin B kinase to promote oocyte meiotic maturation, which involves nuclear and cytoplasmic events. Nuclear maturation encompasses nuclear envelope breakdown, meiotic spindle assembly, and chromosome segregation. Cytoplasmic maturation involves major changes in oocyte protein translation and cytoplasmic organelles and is poorly understood. In the nematode Caenorhabditis elegans, sperm release the major sperm protein (MSP) hormone to promote oocyte growth and meiotic maturation. Large translational regulatory ribonucleoprotein (RNP) complexes containing the RNA-binding proteins OMA-1, OMA-2, and LIN-41 regulate meiotic maturation downstream of MSP signaling. To understand the control of translation during meiotic maturation, we purified LIN-41-containing RNPs and characterized their protein and RNA components. Protein constituents of LIN-41 RNPs include essential RNA-binding proteins, the GLD-2 cytoplasmic poly(A) polymerase, the CCR4-NOT deadenylase complex, and translation initiation factors. RNA sequencing defined messenger RNAs (mRNAs) associated with both LIN-41 and OMA-1, as well as sets of mRNAs associated with either LIN-41 or OMA-1. Genetic and genomic evidence suggests that GLD-2, which is a component of LIN-41 RNPs, stimulates the efficient translation of many LIN-41-associated transcripts. We analyzed the translational regulation of two transcripts specifically associated with LIN-41 which encode the RNA regulators SPN-4 and MEG-1. We found that LIN-41 represses translation of spn-4 and meg-1, whereas OMA-1 and OMA-2 promote their expression. Upon their synthesis, SPN-4 and MEG-1 assemble into LIN-41 RNPs prior to their functions in the embryo. This study defines a translational repression-to-activation switch as a key element of cytoplasmic maturation.

We show that every alternating pretzel knot satisfies the slice-ribbon conjecture and that every alternating pretzel link with m components ( m ≥ 2 ) is not slice in the strong sense.

In the nematode Caenorhabditis elegans, the conserved LIN-41 RNA-binding protein is a translational repressor that coordinately controls oocyte growth and meiotic maturation. LIN-41 exerts these effects, at least in part, by preventing the premature activation of the cyclin-dependent kinase CDK-1. Here we investigate the mechanism by which LIN-41 is rapidly eliminated upon the onset of meiotic maturation. Elimination of LIN-41 requires the activities of CDK-1 and multiple SCF (Skp1, Cul1, and F-box protein)-type E3 ubiquitin ligase subunits, including the conserved substrate adaptor protein SEL-10/Fbw7/Cdc4, suggesting that LIN-41 is a target of ubiquitin-mediated protein degradation. Within the LIN-41 protein, two nonoverlapping regions, Deg-A and Deg-B, are individually necessary for LIN-41 degradation; both contain several potential phosphodegron sequences, and at least one of these sequences is required for LIN-41 degradation. Finally, Deg-A and Deg-B are sufficient, in combination, to mediate SEL-10-dependent degradation when transplanted into a different oocyte protein. Although LIN-41 is a potent inhibitor of protein translation and M phase entry, the failure to eliminate LIN-41 from early embryos does not result in the continued translational repression of LIN-41 oocyte messenger RNA targets. Based on these observations, we propose a model for the elimination of LIN-41 by the SEL-10 E3 ubiquitin ligase and suggest that LIN-41 is inactivated before it is degraded. Furthermore, we provide evidence that another RNA-binding protein, the GLD-1 tumor suppressor, is regulated similarly. Redundant mechanisms to extinguish translational repression by RNA-binding proteins may both control and provide robustness to irreversible developmental transitions, including meiotic maturation and the oocyte-to-embryo transition.

In flowering plant reproduction, pollen tube reception is the signaling system that results in pollen tube discharge, synergid degeneration, and successful delivery of male gametes (two sperm cells) to the site where they can fuse with female gametes (egg cell and central cell). Some molecules required for this complex and essential signaling exchange have been identified; however, fundamental questions about the nature of the interactions between the pollen tube and the synergid cells remain to be clarified. Here, we monitor pollen tube arrival, pollen tube discharge, and synergid degeneration in Arabidopsis (Arabidopsis thaliana) wild type and in male and female gametophytic mutants that disrupt development and function of the gametophytes. By combining assays used previously to study these interactions and an assay that facilitates simultaneous analysis of pollen tube discharge and synergid degeneration, we find that synergid degeneration could be initiated without pollen tube discharge. Our data support the hypothesis that pollen tube-synergid contact, or signaling via secreted molecules, initiates receptive synergid degeneration. We also find that when pollen tubes successfully burst, they always discharge into a degenerated synergid. In addition to this pollen tube-dependent promotion of synergid degeneration, we also show that a basal developmental pathway mediates synergid degeneration in the absence of pollination. Our results are consistent with the model that a complex set of interactions between the pollen tube and synergid cells promote receptive synergid degeneration.

The adsorption states of NH3 molecules and NH4+ ions on various sites, such as hydroxyl, H+ and Na+, of ZSM-5 zeolites with different SiO2/Al2O3 ratios were investigated by near and middle infrared spectroscopy. For Na+-type and siliceous ZSM-5 zeolites, only hydrogen-bonded NH3 clusters were observed at 5021 and 4519 cm−1, due to the lack of Bronsted acid sites. The NH3 molecules interacting with the hydroxyl groups easily desorbed at room temperature (r.t.), while the molecules interacting with the Na+ sites desorbed at 473 K. For the H+-type ZSM-5, when NH3 adsorption amount was small, the NH4+ formed on the H+ sites as well as the NH3 adsorbed on the H+ (or possibly Al3+) sites were independently observed at 4846 and 5044 cm−1, respectively. As the amount of NH3 adsorption increased, the NH4+ stabilized by the 2nd layer of NH3 molecules as well as the hydrogen-bonded NH3 clusters were observed at 4782 and 5017 cm−1, respectively. The hydrogen-bonded NH3 molecules desorbed at lower than 423 K but the NH4+ ions decomposed at 573–623 K to form Bronsted acid sites.

An extended meiotic prophase is a hallmark of oogenesis. Hormonal signaling activates the CDK1/cyclin B kinase to promote oocyte meiotic maturation, which involves nuclear and cytoplasmic events. Nuclear maturation encompasses nuclear envelope breakdown, meiotic spindle assembly, and chromosome segregation. Cytoplasmic maturation involves major changes in oocyte protein translation and cytoplasmic organelles and is poorly understood. In the nematode Caenorhabditis elegans, sperm release the major sperm protein (MSP) hormone to promote oocyte growth and meiotic maturation. Large translational regulatory ribonucleoprotein (RNP) complexes containing the RNA-binding proteins OMA-1, OMA-2, and LIN-41 regulate meiotic maturation downstream of MSP signaling. To understand the control of translation during meiotic maturation, we purified LIN-41-containing RNPs and characterized their protein and RNA components. Protein constituents of LIN-41 RNPs include essential RNA-binding proteins, the GLD-2 cytoplasmic poly(A) polymerase, the CCR4-NOT deadenylase complex, and translation initiation factors. RNA sequencing defined messenger RNAs (mRNAs) associated with both LIN-41 and OMA-1, as well as sets of mRNAs associated with either LIN-41 or OMA-1. Genetic and genomic evidence suggests that GLD-2, which is a component of LIN-41 RNPs, stimulates the efficient translation of many LIN-41-associated transcripts. We analyzed the translational regulation of two transcripts specifically associated with LIN-41 which encode the RNA regulators SPN-4 and MEG-1. We found that LIN-41 represses translation of spn-4 and meg-1, whereas OMA-1 and OMA-2 promote their expression. Upon their synthesis, SPN-4 and MEG-1 assemble into LIN-41 RNPs prior to their functions in the embryo. This study defines a translational repression-to-activation switch as a key element of cytoplasmic maturation.

In flowering plants, meiocytes develop from subepidermal cells in anthers and ovules. The mechanisms that integrate gene-regulatory processes with meiotic programs during reproductive development remain poorly characterized. Here, we show that Arabidopsis thaliana plants deficient in ACTIN-RELATED PROTEIN6 (ARP6), a subunit of the SWR1 ATP-dependent chromatin-remodeling complex, exhibit defects in prophase I of female meiosis. We found that this meiotic defect is likely due to dysregulated expression of meiotic genes, particularly those involved in meiotic recombination, including DMC1 (DISRUPTED MEIOTIC cDNA1). Analysis of DMC1 expression in arp6 mutant plants indicated that ARP6 inhibits expression of DMC1 in the megasporocyte and surrounding nonsporogeneous ovule cells before meiosis. After cells enter meiosis, however, ARP6 activates DMC1 expression specifically in the megasporocyte even as it continues to inhibit DMC1 expression in the nonsporogenous ovule cells. We further show that deposition of the histone variant H2A.Z, mediated by the SWR1 chromatin-remodeling complex at the DMC1 gene body, requires ARP6. Therefore, ARP6 regulates female meiosis by determining the spatial and temporal patterns of gene expression required for proper meiosis during ovule development.

Let $K$ be the result of a 1-fusion (band sum) of a knot $k$ and a distant trivial knot in $S^3$. From results of D. Gabai and of M. G. Scharlemann, we know that the genus of $K$ is at least that of $k$ and that equality holds if and only if the band sum is, in fact, a connected sum (in which case $K$ is ambient isotopic to $k$). In this paper, we consider a generalization of this result to an $m$-fusion of a link and a distant trivial link with $m$-components.

Sexual reproduction in flowering plants is unique in multiple ways. Distinct multicellular gametophytes contain either a pair of immotile, haploid male gametes (sperm cells) or a pair of female gametes (haploid egg cell and homodiploid central cell). After pollination, the pollen tube, a cellular extension of the male gametophyte, transports both male gametes at its growing tip and delivers them to the female gametes to affect double fertilization. The pollen tube travels a long path and sustains its growth over a considerable amount of time in the female reproductive organ (pistil) before it reaches the ovule, which houses the female gametophyte. The pistil facilitates the pollen tube's journey by providing multiple, stage-specific, nutritional, and guidance cues along its path. The pollen tube interacts with seven different pistil cell types prior to completing its journey. Consequently, the pollen tube has a dynamic gene expression program allowing it to continuously reset and be receptive to multiple pistil signals as it migrates through the pistil. Here, we review the studies, including several significant recent advances, that led to a better understanding of the multitude of cues generated by the pistil tissues to assist the pollen tube in delivering the sperm cells to the female gametophyte. We also highlight the outstanding questions, draw attention to opportunities created by recent advances and point to approaches that could be undertaken to unravel the molecular mechanisms underlying pollen tube-pistil interactions.

Novel π-extended squarylium dyes have been synthesized as sensitizers for use in dye-sensitized solar cells (DSSCs). Four quaternary salts with extended π-systems, quinolinium, benz[c,d]indolium, benzopyrylium, and benzo-1-thiopyrylium, were selected as electron-rich heterocyclic components and condensed with indole-based semisquarylium bearing a carboxyl group as an anchor for their immobilization on TiO 2 to obtain four unsymmetrical squarylium dyes ( SQ1–4 ). They exhibited intense absorption in the near-infrared region in solution and on the TiO 2 surface (absorption edge: approximately 900 nm). Investigation of their electrochemical properties revealed that electron injection from the excited dyes to the TiO 2 conduction band was thermodynamically permitted. A molecular-orbital calculation indicated that the electron distribution moved from the cyclobutene core to the heterocyclic components bearing the carboxyl group by photoexcitation of SQ1–4 . The photovoltaic performances of DSSCs based on the squarylium dyes significantly depended on the structure of heterocyclic components. Among the series of squarylium dyes, SQ2–4 achieved comparatively high performance in metal-free NIR sensitizers ( η = 1.1–1.6%), in accordance with the calculation results. These results suggest that the structure of heterocyclic components strongly affects not only the absorption properties of the dyes but also the photovoltaic performances of DSSCs based on these dyes.

†‡ SUMMARY Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra-high resolution electrospray ionization (ESI), Fourier-transform ion cyclotron resonance (FT-ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C10H19NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N-methanesulfinyl 1- and 2-azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro(30 lM stimulated approximately 50% germination) and elicit accession-specific response. Although N-methanesulfinyl 2-azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin-like molecules to ensure rapid fertilization by the appropriate pollen.

In [K. Kishimoto, T. Shibuya and T. Tsukamoto, Simple-ribbon fusions and genera of links, J. Math. Soc. Japan 68 (2016) 1033–1045], we introduced a special kind of fusions, the simple-ribbon fusions. In this paper, we give a sufficient condition for simple-ribbon fusions to produce a prime knot.

Link-homotopy and self Δ-equivalence are equivalence relations on links. It was shown by J. Milnor (resp. the last author) that Milnor invariants determine whether or not a link is link-homotopic (resp. self Δ-equivalent) to a trivial link. We study link-homotopy and self Δ-equivalence on a certain component of a link with fixing the other components, in other words, homotopy and Δ-equivalence of knots in the complement of a certain link. We show that Milnor invariants determine whether a knot in the complement of a trivial link is null-homotopic, and give a sufficient condition for such a knot to be Δ-equivalent to the trivial knot. We also give a sufficient condition for knots in the complements of the trivial knot to be equivalent up to Δ-equivalence and concordance.

This study describes a successful method of somatic embryogenesis and genetic transformation using immature cotyledons of Prunus mume . Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somatic embryogenesis and genetic transformation protocols. Somatic embryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of MS basic medium supplemented with 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM 6-benzyladenine (BA). They were cultured for 30 days and then transferred to somatic embryo propagation medium containing 0.1 μM α-naphthaleneacetic acid (NAA) and 5 μM BA. It appeared that the developmental stage of the immature cotyledons used as explants was the most important factor for somatic embryogenesis; higher frequencies of somatic embryogenesis were observed when the immature cotyledons were less than 5 mm in length regardless of cultivars. For genetic transformation, the immature cotyledons were inoculated with Agrobacterium tumefaciens EHA101 harbouring a binary plasmid vector with neomycin phosphotransferase II and an intron-interrupted β-glucuronidase gene under the control of cauliflower mosaic virus 35S promoter, and three transgenic plant lines were obtained from inoculated “Sirakaga” immature cotyledons. Transgenic somatic embryos and shoots were selected using 25 mg l −1 kanamycin. Integration of transgenes in the genome of GUS-positive putative transgenic shoots was confirmed by PCR and Southern blot analyses.

In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre-5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre-5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre-5/lre-5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre-5/lre-5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre-5/lre-5 seeds. The lre-5/lre-5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI-like-GPI-anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre-5/lre-5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development.

Tetraploid sour cherry (Prunus cerasus) has an S-RNase-based gametophytic self-incompatibility (GSI) system; however, individuals can be either self-incompatible (SI) or self-compatible (SC). Unlike the situation in the Solanaceae, where self-compatibility accompanying polyploidization is often due to the compatibility of heteroallelic pollen, the genotype-dependent loss of SI in sour cherry is due to the compatibility of pollen containing two nonfunctional S haplotypes. Sour cherry individuals with the S4S6S36aS36b genotype are predicted to be SC, as only pollen containing both nonfunctional S36a and S36b haplotypes would be SC. However, we previously found that individuals of this genotype were SI. Here we describe four nonfunctional S36 variants. Our molecular analyses identified a mutation that would confer loss of stylar S function for one of the variants, and two alterations that might cause loss of pollen S function for all four variants. Genetic crosses showed that individuals possessing two nonfunctional S36 haplotypes and two functional S haplotypes have reduced self-fertilization due to a very low frequency of transmission of the one pollen type that would be SC. Our finding that the underlying mechanism limiting successful transmission of genetically compatible gametes does not involve GSI is consistent with our previous genetic model for Prunus in which heteroallelic pollen is incompatible. This provides a unique case in which breakdown of SI does not occur despite the potential to generate SC pollen genotypes.

A free self delta-trivial link is a link which can be transformed into a trivial link by trivializing a component only with self delta-moves on the component, one by one in any order. We show that if a link spans mutually disjoint disks whose singularities are arcs of the non-clasp-type and loops, then the link is free self delta-trivial. We also study free self sharp-triviality and free self pass-triviality of a link which spans mutually disjoint disks whose singularities are mutually disjoint simple arcs of the clasp-type.

Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, the S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead, the genotype dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. We previously determined that sour cherry has non-functional S-haplotypes for the S 1 -, S 6 - and S 13 -haplotypes that are also present in diploid sweet cherry (P. avium L.). The mutations underlying these non-functional S-haplotypes have been determined to be structural alterations of either the S-RNase or SFB. Based on these structural alterations we designed derived cleaved amplified polymorphic sequence (dCAPS) markers and S-haplotype specific primer pairs that took advantage of either the length polymorphisms between S-haplotypes, differential S-haplotype sequences, or differential restriction enzyme cut sites. These primer pairs can discriminate among the mutant and wild-type S-haplotypes thereby enabling the identification of the S-haplotypes present in a sour cherry individual. This information can be used to determine whether the individual is either SC or SI. In a sour cherry breeding program, the ability to discriminate between SI and SC individuals at the seedling stage so that SI individuals can be discarded prior to field planting, dramatically increases the program’s efficiency and cost-effectiveness.

This study describes a successful method for somatic embryogenesis and plant regeneration from immature cotyledons of Prunus mume 'Nanko'. It appeared that the developmental stage of cotyledons that were cultured in vitro was the most important factor for embryogenesis. The highest frequency of embryogenesis was obtained from immature cotyledons harvested about 80 days after flowering on WPM containing 3% sorbitol, 1 μM 2,4-D and 1 μM BA after 60 days of culture in the dark at 25°C. Embryos could be germinated and grown to shoots by culturing them to WPM containing 3% sorbitol, 5 μM BA and 0.7% agar under the light at 25°C. Secondary somatic embryogenesis was observed when the somatic embryos were cultured on MS containing 3% sucrose, 0.1 μM NAA and 5 μM BA, 0.7% agar in the dark at 25°C.

We previously identified both self-incompatible and self-compatible plants in a natural population of self-incompatible Petunia axillaris subsp. axillaris, and found that all the self-compatible plants studied carried either SC1- or SC2-haplotype. Genetic crosses showed that SC2 was identical to S17 identified from another natural population of P. axillaris, except that its pollen function was defective, and that the pollen-part mutation in SC2 was tightly linked to the S-locus. Recent identification of the S-locus F-box gene (SLF) as the gene that controls pollen specificity in S-RNase-based self-incompatibility has prompted us to examine the molecular basis of this pollen-part mutation. We cloned and sequenced the S17-allele of SLF of P. axillaris, named PaSLF17, and found that SC2SC2 plants contained extra restriction fragments that hybridized to PaSLF17 in addition to all of those observed in S17S17 plants. Moreover, these additional fragments co-segregated with SC2. We used the SC2-specific restriction fragments as templates to clone an allele of PaSLF by PCR. To determine the identity of this allele, named PaSLFx, primers based on its sequence were used to amplify PaSLF alleles from genomic DNA of 40 S-homozygotes of P. axillaris, S1S1 through S40S40. Sequence comparison revealed that PaSLFx was completely identical with PaSLF19 obtained from S19S19. We conclude that the S-locus of SC2 contained both S17-allele and the duplicated S19-allele of PaSLF. SC2 is the first naturally occurring pollen-part mutation of a solanaceous species that was shown to be associated with duplication of the pollen S. This finding lends support to the proposal, based on studies of irradiation-generated pollen-part mutants of solanaceous species, that duplication, but not deletion, of the pollen S, causes breakdown of pollen function.

We give a sufficient condition for an almost alternating link diagram to represent a non-splittable link. The main theorem gives us a way to see if a given almost alternating link diagram represents a splittable link without increasing numbers of crossings of diagrams in the process. Moreover, we show that almost alternating links with more than two components are non-trivial., Comment: 31 pages, 27 figures

The majority of flowering plants produce perfect flowers that contain both the male and female reproductive organs in close proximity; consequently, they would have a strong tendency to self-fertilize if there were no mechanisms to prevent them from doing so. Because inbreeding can result in reduced

Although Petunia axillaris subsp. axillaris is described as a self-incompatible taxon, some of the natural populations we have identified in Uruguay are composed of both self-incompatible and self-compatible plants. Here, we studied the self-incompatibility (SI) behavior of 50 plants derived from such a mixed population, designated U83, and examined the cause of the breakdown of SI. Thirteen plants were found to be self-incompatible, and the other 37 were found to be self-compatible. A total of 14 S -haplotypes were represented in these 50 plants, including two that we had previously identified from another mixed population, designated U1. All the 37 self-compatible plants carried either an S C1 - or an S C2 -haplotype. S C1 S C1 and S C2 S C2 homozygotes were generated by self-pollination of two of the self-compatible plants, and they were reciprocally crossed with 40 self-incompatible S -homozygotes ( S 1 S 1 through S 40 S 40 ) generated from plants identified from three mixed populations, including U83. The S C1 S C1 homozygote was reciprocally compatible with all the genotypes examined. The S C2 S C2 homozygote accepted pollen from all but the S 17 S 17 homozygote (identified from the U1 population), but the S 17 S 17 homozygote accepted pollen from the S C2 S C2 homozygote. cDNAs encoding S C2 - and S 17 -RNases were cloned and sequenced, and their nucleotide sequences were completely identical. Analysis of bud-selfed progeny of heterozygotes carrying S C1 or S C2 showed that the SI behavior of S C1 and S C2 was identical to that of S C1 and S C2 homozygotes, respectively. All these results taken together suggested that the S C2 -haplotype was a mutant form of the S 17 -haplotype, with the defect lying in the pollen function. The possible nature of the mutation is discussed.

In [Simple ribbon fusions and genera for links, J. Math. Soc. Japan 68 (2016) 1033–1045], we introduced special types of fusions, so-called simple-ribbon fusions on links, and showed that they never decrease the genera of links. In this paper, we also gave a geometric condition for a simple-ribbon fusion to preserve a link type, and it turns out that a “complicated” simple-ribbon fusion may preserve a link type if the link is split. In this paper, we show that only “clearly trivial” simple-ribbon fusion preserves a link type if the link is non-split. This enables us to give a lower bound for the difference between genera of knots related by a simple-ribbon fusion.

We study the fourth skein module of 3-manifolds, based on the skein relation b0L0 + b1L1 + b2L2 + b3L3 = 0 and a framing relation L(1) = aL (a, b0, b3 invertible). We give necessary conditions for trivial links to be linearly independent in the module. We show how elements of the skein module behave under the n-move and we compute the values for (2, n)-torus links and twist knots as elements of the skein module. Using mutants and rotors, we find different links which represent the same element in the skein module. We also show that algebraic links (in the sense of Conway) and closed 3-braids are linear combinations of trivial links. We introduce the concept of n-algebraic tangles (and links) and analyze the skein module for 3-algebraic links. As a by product we prove the Montesinos-Nakanishi 3-moves conjecture for 3-algebraic links (including 3-bridge links). For links in S3, the structure of our skein module suggests the existence of three new polynomial invariants of unoriented framed (or unframed) links. One of them would generalize the Kauffman polynomial of links and another one could be used to analyze amphicheirality of links (and may work better than the Kauffman polynomial). In the conclusion, we speculate that our new knot invariants are related to a deformation of the symplectic quotient of braid groups.

Reproductive isolation was studied in four syntopic species of Petunia sensu Jussieu (Solanaceae) at a site in Rio Grande do Sul State, Brazil. Reciprocal artificial crossing experiments confirmed that a genetic barrier exists between Petunia (P. axillaris and P. integrifolia) and Calibrachoa (C. parviflora and C. heterophylla), and also between C. parviflora and C. heterophylla. Petunia axillaris (white, nocturnally scented flower) is genetically compatible with the syntopic and allotopic P. integrifolia (coloured, unscented flower). Reproductive isolation appears to be maintained by the two species having diAerent pollinators: nocturnally active hawkmoths (Manduca contracta and M. diAssa subsp. petuniae) pollinate P. axillaris while a diurnally active bee (Hexantheda sp.) pollinates P. integrifolia. Flowers of P. integrifolia exhibit diurnal opening and closing movements synchronous with the activity period of the bee. Other than a probable nectar robber (a carpenter bee, Xylocopa augusti), no insect visited flowers of P. axillaris in the day. Amounts of floral nectar in P. axillaris and P. integrifolia were within the range of hawkmoth- and beepollinated flowers, respectively. Reproductive isolating mechanisms in the genus Petunia sensu Jussieu are discussed. # 2001 Annals of Botany Company

For Solanaceae type self-incompatibility, discrimination between self and nonself pollen by the pistil is controlled by the highly polymorphic S-RNase gene. To date, the mechanism generating the allelic diversity of this gene is largely unknown. Natural populations offer a good opportunity to address this question because they likely contain different alleles that share recent common progenitors. We identified 19 S haplotypes from a natural population ofPetunia inflata in Argentina, used reverse transcriptase-polymerase chain reaction to obtain cDNAs for 15 alleles of the S-RNase gene, and sequenced all the cDNAs. Phylogenetic studies revealed that five of these alleles and two previously identified alleles form a major clade, and that the 5′ region ofS 19 allele was derived from an ancestor allele closely related to S 2, whereas its 3′ region was derived from an ancestor allele closely related to S 8. A similar evolutionary relationship was found among S 3,S 12, andS 15 alleles. These findings suggest that intragenic recombination contributed to the generation of the allelic diversity of the S-RNase gene. Two additional findings emerged from the sequence comparisons. First, the nucleotide sequence of theS 1 allele identified in this work is completely identical to that of the previously identifiedS 1 allele of a different origin. Second, in the two hypervariable regions HVa and HVb, thought to be involved in determining S allele specificity,S 6 andS 9 alleles differ only by four nucleotides, all in HVb, resulting in two amino acid differences. The implications of these findings are discussed.

Petunia inflata has been used as a model to study solanaceous type self-incompatibility (SI), an RNase-mediated self/non-self recognition mechanism. Pistil proteins that co-segregate with S -alleles (termed S-proteins or S-RNases) were identified, and their role in SI was demonstrated by gain-of-function and loss-of-function experiments. Several lines of evidence strongly suggest that the S -locus contains another gene, termed the pollen S -gene, whose allelic products presumably interact with S-RNases to trigger the SI response. Thus, ultimate understanding of the molecular/biochemical basis of SI requires the identification of the pollen S -gene. A number of approaches that have been used to accomplish this objective are discussed. Differential display and subtractive hybridization have yielded the most promising results: cDNAs corresponding to 13 pollen-expressed genes that lie within 1 cM of the S-RNase gene have been identified. They will be used as markers to identify large S -linked genomic fragments which will then be examined for the presence of the pollen S -gene by plant transformation.