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Main Conclusion: A novel genomic map of the apogamous gametophyte of the fern Dryopteris affinis unlocks oldest hindrance with this complex plant group, to gain insight into evo-devo approaches. The gametophyte of the fern Dryopteris affinis ssp. affinis represents a good model to explore the molecular basis of vegetative and reproductive development, as well as stress responses. Specifically, this fern reproduces asexually by apogamy, a peculiar case of apomixis whereby a sporophyte forms directly from a gametophytic cell without fertilization. Using RNA-sequencing approach, we have previously annotated more than 6000 transcripts. Here, we selected 100 of the inferred proteins homolog to those of Arabidopsis thaliana, which were particularly interesting for a detailed study of their potential functions, protein-protein interactions, and distance trees. As expected, a plethora of proteins associated with gametogenesis and embryogenesis in angiosperms, such as FERONIA (FER) and CHROMATING REMODELING 11 (CHR11) were identified, and more than a dozen candidates potentially involved in apomixis, such as ARGONAUTE family (AGO4, AGO9, and AGO 10), BABY BOOM (BBM), FASCIATED STEM4 (FAS4), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), and MATERNAL EFFECT EMBRYO ARREST29 (MEE29). In addition, proteins involved in the response to biotic and abiotic stresses were widely represented, as shown by the enrichment of heat-shock proteins. Using the String platform, the interactome revealed that most of the protein-protein interactions were predicted based on experimental, database, and text mining datasets, with MULTICOPY SUPPRESSOR OF IRA4 (MSI4) showing the highest number of interactions: 16. Lastly, some proteins were studied through distance trees by comparing alignments with respect to more distantly or closely related plant groups. This analysis identified DCL4 as the most distant protein to the predicted common ancestor. New genomic information in relation to gametophyte development, including apomictic reproduction, could expand our current vision of evo-devo approaches., (© 2024. The Author(s).)

Ferns and lycophytes have received scant molecular attention in comparison to angiosperms. The advent of high-throughput technologies allowed an advance towards a greater knowledge of their elusive genomes. In this work, proteomic analyses of heart-shaped gametophytes of two ferns were performed: the apomictic Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades . In total, a set of 218 proteins shared by these two gametophytes were analyzed using the STRING database, and their proteome associated with metabolism, genetic information processing, and responses to abiotic stress is discussed. Specifically, we report proteins involved in the metabolism of carbohydrates, lipids, and nucleotides, the biosynthesis of amino acids and secondary compounds, energy, oxide-reduction, transcription, translation, protein folding, sorting and degradation, and responses to abiotic stresses. The interactome of this set of proteins represents a total network composed of 218 nodes and 1792 interactions, obtained mostly from databases and text mining. The interactions among the identified proteins of the ferns D. affinis and D. oreades , together with the description of their biological functions, might contribute to a better understanding of the function and development of ferns as well as fill knowledge gaps in plant evolution.

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades . Most proteins are predicted to localize to the cytoplasm, the chloroplast, or the nucleus, and are linked to enzymatic, binding, and structural activities. A subset of 145 proteins are involved in growth, reproduction, phytohormone signaling and biosynthesis, and gene expression, including homologs of SHEPHERD (SHD), HEAT SHOCK PROTEIN 90-5 (CR88), TRP4, BOBBER 1 (BOB1), FLAVONE 3'-O-METHYLTRANSFERASE 1 (OMT1), ZEAXANTHIN EPOXIDASE (ABA1), GLUTAMATE DESCARBOXYLASE 1 (GAD), and dsRNA-BINDING DOMAIN-LIKE SUPERFAMILY PROTEIN (HLY1). Nearly 25% of the annotated proteins are associated with responses to biotic and abiotic stimuli. As for biotic stress, the proteins PROTEIN SGT1 HOMOLOG B (SGT1B), SUPPRESSOR OF SA INSENSITIVE2 (SSI2), PHOSPHOLIPASE D ALPHA 1 (PLDALPHA1), SERINE/THREONINE-PROTEIN KINASE SRK2E (OST1), ACYL CARRIER PROTEIN 4 (ACP4), and NONHOST RESISTANCE TO P. S. PHASEOLICOLA1 (GLPK) are worth mentioning. Regarding abiotic stimuli, we found proteins associated with oxidative stress: SUPEROXIDE DISMUTASE[CU-ZN] 1 (CSD1), and GLUTATHIONE S-TRANSFERASE U19 (GSTU19), light intensity SERINE HYDROXYMETHYLTRANSFERASE 1 (SHM1) and UBIQUITIN-CONJUGATING ENZYME E2 35 (UBC35), salt and heavy metal stress included MITOCHONDRIAL PHOSPHATE CARRIER PROTEIN 3 (PHT3;1), as well as drought and thermotolerance: LEA7, DEAD-BOX ATP-DEPENDENT RNA HELICASE 38 (LOS4), and abundant heat-shock proteins and other chaperones. In addition, we identified interactomes using the STRING platform, revealing protein-protein associations obtained from co-expression, co-occurrence, text mining, homology, databases, and experimental datasets. By focusing on ferns, this proteomic study increases our knowledge on plant development and evolution, and may inspire future applications in crop species.

The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades . The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA 3 and GA 4 , salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA 4 , the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA 3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fernández, Grossmann, Gagliardini, Feito, Rivera, Rodríguez, Quintanilla, Quesada, Cañal and Grossniklaus.)

Establishing the embryonic body plan of multicellular organisms relies on precisely orchestrated cell divisions coupled with pattern formation, which, in animals, are regulated by Polycomb group (PcG) proteins. The conserved Polycomb Repressive Complex 2 (PRC2) mediates H3K27 trimethylation and comes in different flavors in Arabidopsis. The PRC2 catalytic subunit MEDEA is required for seed development; however, a role for PRC2 in embryonic patterning has been dismissed. Here, we demonstrate that embryos derived from medea eggs abort because MEDEA is required for patterning and cell lineage determination in the early embryo. Similar to PcG proteins in mammals, MEDEA regulates embryonic patterning and growth by controlling cell-cycle progression through repression of CYCD1;1, which encodes a core cell-cycle component. Thus, Arabidopsis embryogenesis is epigenetically regulated by PcG proteins, revealing that the PRC2-dependent modulation of cell-cycle progression was independently recruited to control embryonic cell proliferation and patterning in animals and plants., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

In plants, transgenerational inheritance of some epialleles has been demonstrated but it remains controversial whether epigenetic variation is subject to selection and contributes to adaptation. Simulating selection in a rapidly changing environment, we compare phenotypic traits and epigenetic variation between Arabidopsis thaliana populations grown for five generations under selection and their genetically nearly identical ancestors. Selected populations of two distinct genotypes show significant differences in flowering time and plant architecture, which are maintained for at least 2-3 generations in the absence of selection. While we cannot detect consistent genetic changes, we observe a reduction of epigenetic diversity and changes in the methylation state of about 50,000 cytosines, some of which are associated with phenotypic changes. Thus, we propose that epigenetic variation is subject to selection and can contribute to rapid adaptive responses, although the extent to which epigenetics plays a role in adaptation is still unclear.

The communication of changes in the extracellular matrix to the interior of the cell is crucial for a cell's function. The extracellular peptides of the RAPID ALKALINIZATION FACTOR (RALF) family have been identified as ligands of receptor-like kinases of the Cr RLK1L subclass, but the exact mechanism of their perception is unclear. We found that Arabidopsis RALF4 and RALF19 redundantly regulate pollen tube integrity and growth, and that their function depends on pollen-expressed proteins of the LEUCINE-RICH REPEAT EXTENSIN (LRX) family, which play a role in cell wall development but whose mode of action is not understood. The LRX proteins interact with RALFs, monitoring cell wall changes, which are communicated to the interior of the pollen tube via the Cr RLK1L pathway to sustain normal growth., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not well understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis . For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a "species-specific transcriptome database" (SSTDB). In total 1,397 protein clusters with 5,865 unique peptide sequences were identified (13 decoy proteins out of 1,410, protFDR 0.93% on protein cluster level). We show that using the SSTDB for protein identification increases the number of identified peptides almost four times compared to using only the publically available VPDB. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy. With the increasing availability of genomic data from non-model species, similar proteogenomics approaches will improve the sensitivity in protein identification for species only distantly related to models.

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed., (© 2016 The author(s). All Rights Reserved.)

Species-specific gamete recognition is a key premise to ensure reproductive success and the maintenance of species boundaries. During plant pollen tube (PT) reception, gametophyte interactions likely allow the species-specific recognition of signals from the PT (male gametophyte) by the embryo sac (female gametophyte), resulting in PT rupture, sperm release, and double fertilization. This process is impaired in interspecific crosses between Arabidopsis thaliana and related species, leading to PT overgrowth and a failure to deliver the sperm cells. Here we show that ARTUMES (ARU) specifically regulates the recognition of interspecific PTs in A. thaliana. ARU, identified in a genome-wide association study (GWAS), exclusively influences interspecific--but not intraspecific--gametophyte interactions. ARU encodes the OST3/6 subunit of the oligosaccharyltransferase complex conferring protein N-glycosylation. Our results suggest that glycosylation patterns of cell surface proteins may represent an important mechanism of gametophyte recognition and thus speciation.

TBP-Associated Factors (TAFs) are components of complexes like TFIID, TFTC, SAGA/STAGA and SMAT that are important for the activation of transcription, either by establishing the basic transcription machinery or by facilitating histone acetylation. However, in Drosophila embryos several TAFs were shown to be associated with the Polycomb Repressive Complex 1 (PRC1), even though the role of this interaction remains unclear. Here we show that in Arabidopsis TAF13 interacts with MEDEA and SWINGER, both members of a plant variant of Polycomb Repressive Complex 2 (PRC2). PRC2 variants play important roles during the plant life cycle, including seed development. The taf13 mutation causes seed defects, showing embryo arrest at the 8-16 cell stage and over-proliferation of the endosperm in the chalazal region, which is typical for Arabidopsis PRC2 mutants. Our data suggest that TAF13 functions together with PRC2 in transcriptional regulation during seed development., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

In flowering plants, double fertilization of the female gametes, the egg and the central cell, initiates seed development to give rise to a diploid embryo and the triploid endosperm. In the absence of fertilization, the FERTILIZATION-INDEPENDENT SEED Polycomb Repressive Complex 2 (FIS-PRC2) represses this developmental process by histone methylation of certain target genes. The FERTILIZATION-INDEPENDENT SEED (FIS) class genes MEDEA (MEA) and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) encode two of the core components of this complex. In addition, DNA methylation establishes and maintains the repression of gene activity, for instance via DNA METHYLTRANSFERASE1 (MET1), which maintains methylation of symmetric CpG residues. Here, we demonstrate that Arabidopsis MET1 interacts with MEA in vitro and in a yeast two-hybrid assay, similar to the previously identified interaction of the mammalian homologues DNMT1 and EZH2. MET1 and MEA share overlapping expression patterns in reproductive tissues before and after fertilization, a prerequisite for an interaction in vivo. Importantly, a much higher percentage of central cells initiate endosperm development in the absence of fertilization in mea-1/MEA; met1-3/MET1 as compared to mea-1/MEA mutant plants. In addition, DNA methylation at the PHERES1 and MEA loci, imprinted target genes of the FIS-PRC2, was affected in the mea-1 mutant compared with wild-type embryos. In conclusion, our data suggest a mechanistic link between two major epigenetic pathways involved in histone and DNA methylation in plants by physical interaction of MET1 with the FIS-PRC2 core component MEA. This concerted action is relevant for the repression of seed development in the absence of fertilization., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Genomic imprinting is exclusive to mammals and seed plants and refers to parent-of-origin-dependent, differential transcription. As previously shown in mammals, studies in Arabidopsis have implicated DNA methylation as an important hallmark of imprinting. The current model suggests that maternally expressed imprinted genes, such as MEDEA (MEA), are activated by the DNA glycosylase DEMETER (DME), which removes DNA methylation established by the DNA methyltransferase MET1. We report the systematic functional dissection of the MEA cis-regulatory region, resulting in the identification of a 200-bp fragment that is necessary and sufficient to mediate MEA activation and imprinted expression, thus containing the imprinting control region (ICR). Notably, imprinted MEA expression mediated by this ICR is independent of DME and MET1, consistent with the lack of any significant DNA methylation in this region. This is the first example of an ICR without differential DNA methylation, suggesting that factors other than DME and MET1 are required for imprinting at the MEA locus.

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators' sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ(9) and a 16:0-ACP Δ(4) desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.

In flowering plants, signaling between the male pollen tube and the synergid cells of the female gametophyte is required for fertilization. In the Arabidopsis thaliana mutant feronia (fer), fertilization is impaired; the pollen tube fails to arrest and thus continues to grow inside the female gametophyte. FER encodes a synergid-expressed, plasma membrane-localized receptor-like kinase. We found that the FER protein accumulates asymmetrically in the synergid membrane at the filiform apparatus. Interspecific crosses using pollen from Arabidopsis lyrata and Cardamine flexuosa on A. thaliana stigmas resulted in a fer-like phenotype that correlates with sequence divergence in the extracellular domain of FER. Our findings show that the female control of pollen tube reception is based on a FER-dependent signaling pathway, which may play a role in reproductive isolation barriers.

In mammals and seed plants, a subset of genes is regulated by genomic imprinting where an allele's activity depends on its parental origin. The parental conflict theory suggests that genomic imprinting evolved after the emergence of an embryo-nourishing tissue (placenta and endosperm), resulting in an intragenomic parental conflict over the allocation of nutrients from mother to offspring. It was predicted that imprinted genes, which arose through antagonistic co-evolution driven by a parental conflict, should be subject to positive darwinian selection. Here we show that the imprinted plant gene MEDEA (MEA), which is essential for seed development, originated during a whole-genome duplication 35 to 85 million years ago. After duplication, MEA underwent positive darwinian selection consistent with neo-functionalization and the parental conflict theory. MEA continues to evolve rapidly in the out-crossing species Arabidopsis lyrata but not in the self-fertilizing species Arabidopsis thaliana, where parental conflicts are reduced. The paralogue of MEA, SWINGER (SWN; also called EZA1), is not imprinted and evolved under strong purifying selection because it probably retained the ancestral function of the common precursor gene. The evolution of MEA suggests a late origin of genomic imprinting within the Brassicaceae, whereas imprinting is thought to have originated early within the mammalian lineage.

The imprinted Arabidopsis Polycomb group (PcG) gene MEDEA (MEA), which is homologous to Enhancer of Zeste [E(Z)], is maternally required for normal seed development. Here we show that, unlike known mammalian imprinted genes, MEA regulates its own imprinted expression: It down-regulates the maternal allele around fertilization and maintains the paternal allele silent later during seed development. Autorepression of the maternal MEA allele is direct and independent of the MEA-FIE (FERTILIZATION-INDEPENDENT ENDOSPERM) PcG complex, which is similar to the E(Z)-ESC (Extra sex combs) complex of animals, suggesting a novel mechanism. A complex network of cross-regulatory interactions among the other known members of the MEA-FIE PcG complex implies distinct functions that are dynamically regulated during reproduction.

The 26S proteasome plays a central role in the degradation of regulatory proteins involved in a variety of developmental processes. It consists of two multisubunit protein complexes: the proteolytic core protease and the regulatory particle (RP). The function of most RP subunits is poorly understood. Here, we describe mutants in the Arabidopsis thaliana RPN1 subunit, which is encoded by two paralogous genes, RPN1a and RPN1b. Disruption of RPN1a caused embryo lethality, while RPN1b mutants showed no obvious abnormal phenotype. Embryos homozygous for rpn1a arrested at the globular stage with defects in the formation of the embryonic root, the protoderm, and procambium. Cyclin B1 protein was not degraded in these embryos, consistent with cell division defects. Double mutant plants (rpn1a/RPN1a rpn1b/rpn1b) produced embryos with a phenotype indistinguishable from that of the rpn1a single mutant. Thus, despite their largely overlapping expression patterns in flowers and developing seeds, the two isoforms do not share redundant functions during gametogenesis and embryogenesis. However, complementation of the rpn1a mutation with the coding region of RPN1b expressed under the control of the RPN1a promoter indicates that the two RPN1 isoforms are functionally equivalent. Overall, our data indicate that RPN1 activity is essential during embryogenesis, where it might participate in the destruction of a specific set of protein substrates.

The maternally expressed Arabidopsis thaliana Polycomb group protein MEDEA (MEA) controls expression of the MADS-box gene PHERES1 (PHE1). Here, we show that PHE1 is mainly paternally expressed but maternally repressed and that this maternal repression of PHE1 breaks down in seeds lacking maternal MEA activity. Because Polycomb group proteins control parental imprinting in mammals as well, the independent recruitment of similar protein machineries for the imprinting of genes is a notable example of convergent evolution.

The caspase family proteases are principal components of the apoptotic pathway. In this study we demonstrate that caspase-1-like proteases and interleukin-1 beta are important for death induced by various stimuli in cell lines, primary fibroblasts and primary sensory neurons. Furthermore, we show by immunohistochemistry that during the cell death process endogenous caspase-1-like proteases translocate into the nucleus. This translocation is stimulated by interleukin-1 receptor activation. Translocation of caspase-1-like proteases and cell death can be partially prevented by blocking the interleukin-1 receptor with the interleukin-1 receptor antagonist. This finding offers for the first time a mechanistic explanation for the protective effect of the interleukin-1 receptor antagonist against cell death. Furthermore, our data suggest that caspase-1-like proteases have a function in the nucleus which is necessary for completion of the cell death program. In cultured DRG neurons from embryonic mice the combined inhibition of caspases and the interleukin-1 receptor have an additive effect and fully prevent semaphorin III-induced neuronal death. This shows that endogenous caspases work together with IL-1 beta in Semaphorin III-induced neuronal death. We hypothesize that the cell death process involves a double activation step, probably including an interleukin-1 autocrine loop. This model can explain our finding that combined inhibition of caspases and interleukin-1 receptor is necessary to strongly inhibit the cell death process.

Semaphorin III has been described to function as a guidance molecule directing growing axons to their target. However, its effect on the neuron cell body has not been characterized. Semaphorin III has a highly dynamic expression pattern, which generally corroborates a chemorepellent guidance function, but also suggests additional functions, different from axon guidance. A number of studies show that some sensory neurons are eliminated, while their axons are still pathfinding. In this study we have investigated whether Semaphorin III also influences the survival of sensory neurons. We here present evidence that Semaphorin III can function in vitro as selective death factor for NGF-dependent sensory neurons. Semaphorin III induces a type of cell death that is characterized by slow onset, cell body shrinking, nuclear condensation, and TUNEL-positive staining of dying neurons. These are all hallmarks of neuronal apoptosis. We also show evidence that neurons can modulate the response to Semaphorin III. The novel function described here may also be relevant in vivo, contributing to active elimination of neurons during development or after injury.

We compared excitatory synaptic transmission between hippocampal pyramidal cells in dissociated hippocampal cell cultures and in area CA3 of hippocampal slice cultures derived from wild-type mice and mice with a genetic deletion of the presynaptic growth associated protein GAP-43. The basal frequency and amplitude of action potential-dependent and -independent spontaneous excitatory postsynaptic currents were similar in both groups. The probability that any two CA3 pyramidal cells in wild-type or GAP-43 knockout (-/-) slice cultures were synaptically connected was assessed with paired recordings and was not different. Furthermore, unitary synaptic responses were similar in the two genotypes. Bath application of phorbol 12,13-diacetate (0.6-3 microM) elicited a comparable increase in the frequency of miniature excitatory synaptic currents in wild-type and GAP-43 (-/-) cultures. This effect was blocked by the protein kinase C inhibitor, bisindolylmaleimide I (1.2 microM). Finally, 3 microM phorbol 12,13-diacetate potentiated the amplitude of unitary synaptic currents to a comparable extent in wild-type and GAP-43 (-/-) slice cultures. We conclude that GAP-43 is not required for normal excitatory synaptic transmission or the potentiation of presynaptic glutamate release mediated by activation of protein kinase C in the hippocampus.

We used transgenic mice expressing a dominant negative mutation of interleukin-1 beta converting enzyme (ICE) (C285G) in a model of transient focal ischemia in order to investigate the role of ICE in ischemic brain damage. Transgenic mutant ICE mice (n = 11) and wild-type littermates (n = 9) were subjected to 3 h of middle cerebral artery occlusion followed by 24 h of reperfusion. Cerebral infarcts and brain swelling were reduced by 44% and 46%, respectively. Neurological deficits were also significantly reduced. Regional CBF, blood pressure, core temperature, and heart rate did not differ between groups when measured for up to 1 h after reperfusion. Increases in immunoreactive IL-1 beta levels, observed in ischemic wild-type brain at 30 min after reperfusion, were 77% lower in the mutant strain, indicating that proIL-1 beta cleavage is inhibited in the mutants. DNA fragmentation was reduced in the mutants 6 and 24 h after reperfusion. Hence, endogenous expression of an ICE inhibitor confers resistance to cerebral ischemia and brain swelling. Our results indicate that downregulation of ICE expression might provide a useful therapeutic target in cerebral ischemia.

The interleukin 1beta converting enzyme (ICE) family plays a pivotal role in programmed cell death and has been implicated in stroke and neurodegenerative diseases. During reperfusion after filamentous middle cerebral artery occlusion, ICE-like cleavage products and tissue immunoreactive interleukin 1beta (IL-1beta) levels increased in ischemic mouse brain. Ischemic injury decreased after intracerebroventricular injections of ICE-like protease inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK), acetyl-Tyr-Val-Ala-Asp-chloromethylketone, or a relatively selective inhibitor of CPP32-like caspases, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, but not a cathepsin B inhibitor, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone. z-VAD.FMK decreased ICE-like cleavage products and tissue immunoreactive IL-1beta levels in ischemic mouse brain and reduced tissue damage when administered to rats as well. Only z-VAD.FMK and acetyl-Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling, and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone did not attenuate the ischemia-induced increase in tissue IL-1beta levels. The three cysteine protease inhibitors significantly improved behavioral deficits, thereby showing that functional recovery of ischemic neuronal tissue can follow blockade of enzymes associated with apoptotic cell death. Finally, we examined the effect of z-VAD.FMK on excitotoxicity and found that it protected against alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced or to a lesser extent N-methyl-D-aspartate-induced excitotoxic brain damage. Thus, ICE-like and CPP32-like caspases contribute to mechanisms of cell death in ischemic and excitotoxic brain injury and provide therapeutic targets for stroke and neurodegenerative brain damage.

To explore the role of the interleukin (IL)-1 beta converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor. Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal-induced apoptosis. Transgenic mice expressing the fused mutant ICE-lacZ gene under the control of the neuron specific enolase promoter appeared neurologically normal. These mice are deficient in processing pro-IL-1 beta, indicating that mutant ICEC285G blocks ICE function. Dorsal root ganglial neurons isolated from transgenic mice were resistant to trophic factor withdrawal-induced apoptosis. In addition, the neurons isolated from newborn ICE knockout mice are similarly resistant to trophic factor withdrawal-induced apoptosis. After permanent focal ischemia by middle cerebral artery occlusion, the mutant ICEC285G transgenic mice show significantly reduced brain injury as well as less behavioral deficits when compared to the wild-type controls. Since ICE is the only enzyme with IL-1 beta convertase activity in mice, our data indicates that the mutant ICEC285G inhibits ICE, and hence mature IL-1 beta production, and through this mechanism, at least in part, inhibits apoptosis. Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

We report here the isolation and characterization of a new member of the ice/ced-3 family of cell death genes, named ich-3. The predicted amino acid sequence of Ich-3 protein shares 54% identity with murine interleukin-1beta converting enzyme (ICE). Overexpression of ich-3 in Rat-1 and HeLa cells induces apoptosis, which can be inhibited by CrmA and Bcl-2. The mRNA and proteins of ich-3 are dramatically induced in vivo upon stimulation with lipopolysaccharide, an inducer of septic shock. The ich-3 gene product can be cleaved by cytotoxic T cells granule serine protease granzyme B, suggesting that Ich-3 may mediate apoptosis induced by granzyme B. Ich-3 does not process proIL-1beta directly but does promote proIL-1beta processing by ICE. These results suggest that Ich-3 may play a very important role in apoptosis and inflammatory responses and may be an upstream regulator of ICE.

Interleukin-1 beta converting enzyme (ICE) is a mammalian homolog of CED-3, a protein required for programmed cell death in the nematode Caenorhabditis elegans. The activity of ICE can be specifically inhibited by the product of crmA, a cytokine response modifier gene encoded by cowpox virus. Microinjection of the crmA gene into chicken dorsal root ganglion neurons was found to prevent cell death induced by deprivation of nerve growth factor. Thus, ICE is likely to participate in neuronal death in vertebrates.

The low affinity of beta-adrenergic receptors for agonists described on intact cells at 37 degrees C has usually been interpreted in terms of reduced accessibility of agonists (which are usually hydrophilic) for sequestered receptors. We challenged this hypothesis by eliminating the plasma membrane barrier with low doses of the detergent digitonin. In human mononuclear leukocytes (MNL) permeabilized with digitonin, sequestered receptors became accessible to hydrophilic ligands such as agonists, but the affinity was still low. Then we investigated the relationship between low affinity agonist binding and sequestration using concanavalin A, which blocks sequestration. Even when sequestration was blocked, the affinity of the beta-adrenergic receptors for agonists was low. We conclude that: (a) low affinity agonist binding is independent of receptor sequestration; (b) the receptors which undergo conformational change are those that are sequestered; (c) the low affinity appears before sequestration occurs. This receptor conformational change could be the first step in agonist-induced desensitization.