A single helix repression domain is functional across diverse eukaryotes.

The corepressor TOPLESS (TPL) and its paralogs coordinately regulate a large number of genes critical to plant development and immunity. As in many members of the larger pan-eukaryotic Tup1/TLE/Groucho corepressor family, TPL contains a Lis1 Homology domain (LisH), whose function is not well understood. We have previously found that the LisH in TPL-and specifically the N-terminal 18 amino acid alpha-helical region (TPL-H1)-can act as an autonomous repression domain. We hypothesized that homologous domains across diverse LisH-containing proteins could share the same function. To test that hypothesis, we built a library of H1s that broadly sampled the sequence and evolutionary space of LisH domains, and tested their activity in a synthetic transcriptional repression assay in Saccharomyces cerevisiae. Using this approach, we found that repression activity was highly conserved and likely the ancestral function of this motif. We also identified key residues that contribute to repressive function. We leveraged this new knowledge for two applications. First, we tested the role of mutations found in somatic cancers on repression function in two human LisH-containing proteins. Second, we validated function of many of our repression domains in plants, confirming that these sequences should be of use to synthetic biology applications across many eukaryotes.

The CLAMP protein links the MSL complex to the X chromosome during Drosophila dosage compensation.

The Drosophila male-specific lethal (MSL) dosage compensation complex increases transcript levels on the single male X chromosome to equal the transcript levels in XX females. However, it is not known how the MSL complex is linked to its DNA recognition elements, the critical first step in dosage compensation. Here, we demonstrate that a previously uncharacterized zinc finger protein, CLAMP (chromatin-linked adaptor for MSL proteins), functions as the first link between the MSL complex and the X chromosome. CLAMP directly binds to the MSL complex DNA recognition elements and is required for the recruitment of the MSL complex. The discovery of CLAMP identifies a key factor required for the chromosome-specific targeting of dosage compensation, providing new insights into how subnuclear domains of coordinate gene regulation are formed within metazoan genomes.

The Molecular Dialog between Flowering Plant Reproductive Partners Defined by SNP-Informed RNA-Sequencing.

The molecular interactions between reproductive cells are critical for determining whether sexual reproduction between individuals results in fertilization and can result in barriers to interspecific hybridization. However, it is a challenge to define the complete molecular exchange between reproductive partners because parents contribute to a complex mixture of cells during reproduction. We unambiguously defined male- and female-specific patterns of gene expression during Arabidopsis thaliana reproduction using single nucleotide polymorphism-informed RNA-sequencing analysis. Importantly, we defined the repertoire of pollen tube-secreted proteins controlled by a group of MYB transcription factors that are required for sperm release from the pollen tube to the female gametes, a critical barrier to interspecific hybridization. Our work defines the pollen tube gene products that respond to the pistil and are required for reproductive success; moreover, we find that these genes are highly evolutionarily plastic both at the level of coding sequence and expression across A. thaliana accessions.

Correction: Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil.

[This corrects the article DOI: 10.1371/journal.pgen.1000621.].

Transporters involved in pH and K+ homeostasis affect pollen wall formation, male fertility, and embryo development.

Flowering plant genomes encode multiple cation/H+ exchangers (CHXs) whose functions are largely unknown. AtCHX17, AtCHX18, and AtCHX19 are membrane transporters that modulate K+ and pH homeostasis and are localized in the dynamic endomembrane system. Loss of function reduced seed set, but the particular phase(s) of reproduction affected was not determined. Pollen tube growth and ovule targeting of chx17chx18chx19 mutant pollen appeared normal, but reciprocal cross experiments indicate a largely male defect. Although triple mutant pollen tubes reach ovules of a wild-type pistil and a synergid cell degenerated, half of those ovules were unfertilized or showed fertilization of the egg or central cell, but not both female gametes. Fertility could be partially compromised by impaired pollen tube and/or sperm function as CHX19 and CHX18 are expressed in the pollen tube and sperm cell, respectively. When fertilization was successful in self-pollinated mutants, early embryo formation was retarded compared with embryos from wild-type ovules receiving mutant pollen. Thus CHX17 and CHX18 proteins may promote embryo development possibly through the endosperm where these genes are expressed. The reticulate pattern of the pollen wall was disorganized in triple mutants, indicating perturbation of wall formation during male gametophyte development. As pH and cation homeostasis mediated by AtCHX17 affect membrane trafficking and cargo delivery, these results suggest that male fertility, sperm function, and embryo development are dependent on proper cargo sorting and secretion that remodel cell walls, plasma membranes, and extracellular factors.

Pollen Tube Discharge Completes the Process of Synergid Degeneration That Is Initiated by Pollen Tube-Synergid Interaction in Arabidopsis.

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.

Interactions between pollen tube and pistil control pollen tube identity and sperm release in the Arabidopsis female gametophyte.

Flowering plants have immotile sperm that develop within the pollen cytoplasm and are delivered to female gametes by a pollen tube, a highly polarized extension of the pollen cell. In many flowering plant species, including seed crop plants, hundreds of pollen tubes grow towards a limited number of ovules. This system should ensure maximal fertilization of ovules and seed production; however, we know very little about how signalling between the critical cells is integrated to orchestrate delivery of two functional sperm to each ovule. Recent studies suggest that the pollen tube changes its gene-expression programme in response to growth through pistil tissue and that this differentiation process is critical for pollen tube attraction by the female gametophyte and for release of sperm. Interestingly, these two signalling systems, called pollen tube guidance and pollen tube reception, are also species-preferential. The present review focuses on Arabidopsis pollen tube differentiation within the pistil and addresses the idea that pollen tube differentiation defines pollen tube identity and recognition by female cells. We review recent identification of genes that may control pollen tube-female gametophyte recognition and discuss how these may be involved in blocking interspecific hybridization.

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

HAP2(GCS1)-dependent gamete fusion requires a positively charged carboxy-terminal domain.

HAP2(GCS1) is a deeply conserved sperm protein that is essential for gamete fusion. Here we use complementation assays to define major functional regions of the Arabidopsis thaliana ortholog using HAP2(GCS1) variants with modifications to regions amino(N) and carboxy(C) to its single transmembrane domain. These quantitative in vivo complementation studies show that the N-terminal region tolerates exchange with a closely related sequence, but not with a more distantly related plant sequence. In contrast, a distantly related C-terminus is functional in Arabidopsis, indicating that the primary sequence of the C-terminus is not critical. However, mutations that neutralized the charge of the C-terminus impair HAP2(GCS1)-dependent gamete fusion. Our results provide data identifying the essential functional features of this highly conserved sperm fusion protein. They suggest that the N-terminus functions by interacting with female gamete-expressed proteins and that the positively charged C-terminus may function through electrostatic interactions with the sperm plasma membrane.

Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil.

Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell-cell interaction.

Repression by the Arabidopsis TOPLESS corepressor requires association with the core mediator complex.

The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli are poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle module subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

Engineering Synthetic Signaling in Plants.

Synthetic signaling is a branch of synthetic biology that aims to understand native genetic regulatory mechanisms and to use these insights to engineer interventions and devices that achieve specified design parameters. Applying synthetic signaling approaches to plants offers the promise of mitigating the worst effects of climate change and providing a means to engineer crops for entirely novel environments, such as those in space travel. The ability to engineer new traits using synthetic signaling methods will require standardized libraries of biological parts and methods to assemble them; the decoupling of complex processes into simpler subsystems; and mathematical models that can accelerate the design-build-test-learn cycle. The field of plant synthetic signaling is relatively new, but it is poised for rapid advancement. Translation from the laboratory to the field is likely to be slowed, however, by the lack of constructive dialogue between researchers and other stakeholders.

Accelerating structure-function mapping using the ViVa webtool to mine natural variation.

Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of these data remain inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project (http://1001genomes.org). ViVa facilitates data mining on the gene, gene family, or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well-studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well-studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less-studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project ( http://snp-seek.irri.org/) and human genetic variation ( https://www.ncbi.nlm.nih.gov/clinvar/).

Synthetic hormone-responsive transcription factors can monitor and re-program plant development.

Developmental programs sculpt plant morphology to meet environmental challenges, and these same programs have been manipulated to increase agricultural productivity (Doebley et al., 1997; Khush, 2001). Hormones coordinate these programs, creating chemical circuitry (Vanstraelen and Benková, 2012) that has been represented in mathematical models (Refahi et al., 2016; Prusinkiewicz et al., 2009); however, model-guided engineering of plant morphology has been limited by a lack of tools (Parry et al., 2009; Voytas and Gao, 2014). Here, we introduce a novel set of synthetic and modular hormone activated Cas9-based repressors (HACRs) in Arabidopsis thaliana that respond to three hormones: auxin, gibberellins and jasmonates. We demonstrate that HACRs are sensitive to both exogenous hormone treatments and local differences in endogenous hormone levels associated with development. We further show that this capability can be leveraged to reprogram development in an agriculturally relevant manner by changing how the hormonal circuitry regulates target genes. By deploying a HACR to re-parameterize the auxin-induced expression of the auxin transporter PIN-FORMED1 (PIN1), we decreased shoot branching and phyllotactic noise, as predicted by existing models (Refahi et al., 2016; Prusinkiewicz et al., 2009).

Three MYB transcription factors control pollen tube differentiation required for sperm release.

In flowering plants, immotile sperm cells develop within the pollen grain and are delivered to female gametes by a pollen tube. Upon arrival at the female gametophyte, the pollen tube stops growing and releases sperm cells for successful fertilization. Several female signaling components essential for pollen tube reception have been identified; however, male components remain unknown. We show that the expression of three closely related MYB transcription factors is induced in pollen tubes by growth in the pistil. Pollen tubes lacking these three transcriptional regulators fail to stop growing in synergids, specialized cells flanking the egg cell that attract pollen tubes and degenerate upon pollen tube arrival. myb triple-mutant pollen tubes also fail to release their sperm cargo. We define a suite of pollen tube-expressed genes regulated by these critical MYBs and identify transporters, carbohydrate-active enzymes, and small peptides as candidate molecular mediators of pollen tube-female interactions necessary for flowering plant reproduction. Our data indicate that de novo transcription in the pollen tube nucleus during growth in the pistil leads to pollen tube differentiation required for release of sperm cells.

Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule.

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.