SUMMARYThe endosperm is a terminal seed tissue that is destined to support embryo development. In most angiosperms, the endosperm develops initially as a syncytium to facilitate rapid seed growth. The transition from the syncytial to the cellularized state occurs at a defined time point during seed development. Manipulating the timing of endosperm cellularization through interploidy crosses negatively impacts on embryo growth, suggesting that endosperm cellularization is a critical step during seed development. In this study, we show that failure of endosperm cellularization in fertilization independent seed 2 (fis2) and endosperm defective 1 (ede1) Arabidopsis mutants correlates with impaired embryo development. Restoration of endosperm cellularization in fis2 seeds by reducing expression of the MADS-box gene AGAMOUS-LIKE 62 (AGL62) promotes embryo development, strongly supporting an essential role of endosperm cellularization for viable seed formation. Endosperm cellularization failure in fis2 seeds correlates with increased hexose levels, suggesting that arrest of embryo development is a consequence of failed nutrient translocation to the developing embryo. Finally, we demonstrate that AGL62 is a direct target gene of FIS Polycomb group repressive complex 2 (PRC2), establishing the molecular basis for FIS PRC2-mediated endosperm cellularization.

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae. Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

Polycomb group (PcG) proteins act as evolutionary conserved epigenetic mediators of cell identity because they repress transcriptional programs that are not required at particular developmental stages. Each tissue is likely to have a specific epigenetic profile, which acts as a blueprint for its developmental fate. A hallmark for Polycomb Repressive Complex 2 (PRC2) activity is trimethylated lysine 27 on histone H3 (H3K27me3). In plants, there are distinct PRC2 complexes for vegetative and reproductive development, and it was unknown so far whether these complexes have target gene specificity. The FERTILIZATION INDEPENDENT SEED (FIS) PRC2 complex is specifically expressed in the endosperm and is required for its development; loss of FIS function causes endosperm hyperproliferation and seed abortion. The endosperm nourishes the embryo, similar to the physiological function of the placenta in mammals. We established the endosperm H3K27me3 profile and identified specific target genes of the FIS complex with functional roles in endosperm cellularization and chromatin architecture, implicating that distinct PRC2 complexes have a subset of specific target genes. Importantly, our study revealed that selected transposable elements and protein coding genes are specifically targeted by the FIS PcG complex in the endosperm, whereas these elements and genes are densely marked by DNA methylation in vegetative tissues, suggesting that DNA methylation prevents targeting by PcG proteins in vegetative tissues.

Our understanding of the origin of animals has been transformed by characterizing their most closely related, unicellular sisters: the choanoflagellates, filastereans, and ichthyosporeans. Together with animals, these lineages make up the Holozoa [1, 2]. Many traits previously considered "animal specific" were subsequently found in other holozoans [3, 4], showing that they evolved before animals, although exactly when is currently uncertain because several key relationships remain unresolved [2, 5]. Here we report the morphology and transcriptome sequencing from three novel unicellular holozoans: Pigoraptor vietnamica and Pigoraptor chileana, which are related to filastereans, and Syssomonas multiformis, which forms a new lineage with Corallochytrium in phylogenomic analyses. All three species are predatory flagellates that feed on large eukaryotic prey, and all three also appear to exhibit complex life histories with several distinct stages, including multicellular clusters. Examination of genes associated with multicellularity in animals showed that the new filastereans contain a cell-adhesion gene repertoire similar to those of other species in this group. Syssomonas multiformis possessed a smaller complement overall but does encode genes absent from the earlier-branching ichthyosporeans. Analysis of the T-box transcription factor domain showed expansion of T-box transcription factors based on combination with a non-T-box domain (a receiver domain), which has not been described outside of vertebrates. This domain and other domains we identified in all unicellular holozoans are part of the two-component signaling system that has been lost in animals, suggesting the continued use of this system in the closest relatives of animals and emphasizing the importance of studying loss of function as well as gain in major evolutionary transitions.

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Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Highlights d The origin of apicomplexans from algae occurred at least three times independently d Piridium and Platyproteum form distinct lineages of obligate animal parasites d They both retain cryptic plastids that are highly convergent with apicomplexans

Background: The membrane-associated progesterone receptor (MAPR) family consists of heme-binding proteins containing a cytochrome b5 (cytb5) domain characterized by the presence of a MAPR-specific interhelical insert region (MIHIR) between helices 3 and 4 of the canonical cytb5-domain fold. Animals possess three MAPR families (PGRMClike, Neuferricin and Neudesin). Results: Here we show that all animal MAPR families were already present in the common ancestor of the Opisthokonta (comprising animals and fungi as well as related protistan taxa). All three MAPR genes acquired extensions C-terminal to the cytb5 domain, either before or with the evolution of animals. The archetypical MAPR protein, progesterone receptor membrane component 1 (PGRMC1), contains phosphorylated tyrosines Y139 and Y180. The combination of Y139/Y180 appeared in the common ancestor of Cnidaria and bilaterally symmetrical animals, along with an early embryological organizer and synapsed neurons, and is strongly conserved in all bilateral animals. A predicted protein interaction motif in the PGRMC1 MIHIR is potentially regulated by Y139 phosphorylation. A multilayered model of animal MAPR function acquisition includes some pre-metazoan functions (e.g., heme binding and cytochrome P450 interactions) and some acquired animal-specific functions that involve regulation of strongly conserved protein interaction motifs acquired by early-branching animals. Conclusions: This study provides a conceptual framework for future studies, against which PGRMC1's multiple functions can perhaps be stratified and functionally dissected. In accompanying papers we show that mutational perturbation of PGRMC1 phosphorylation status of the Y180 motif is associated with dramatic changes cell pasticity assayed by protein abundances, cell morphology, mitochondrial function, genomic stability, and epigenetic status, with pathways analysis associating Y180 mutation with processes related to organizer function. These combined works reveal .