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Heterochromatin is essential for genomic integrity and stability in eukaryotes. The mechanisms that regulate meiotic heterochromatin formation remain largely undefined. Here, we show that the catalytic subunit (POL2A) of Arabidopsis DNA polymerase epsilon (POL ε) is required for proper formation of meiotic heterochromatin. The POL2A N terminus interacts with the GHKL adenosine triphosphatase (ATPase) MORC1 (Microrchidia 1), and POL2A is required for MORC1’s localization on meiotic heterochromatin. Mutations affecting the POL2A N terminus cause aberrant morphology of meiotic heterochromatin, which is also observed in morc1 . Moreover, the POL2A C-terminal zinc finger domain (ZF1) specifically binds to histone H3.1-H4 dimer or tetramer and is important for meiotic heterochromatin condensation. Interestingly, we also found similar H3.1-binding specificity for the mouse counterpart. Together, our results show that two distinct domains of POL2A, ZF1 and N terminus bind H3.1-H4 and recruit MORC1, respectively, to induce a continuous process of meiotic heterochromatin organization. These activities expand the functional repertoire of POL ε beyond its classic role in DNA replication and appear to be conserved in animals and plants.

Meiotic recombination is initiated by the SPORULATION 11 (SPO11)–triggered formation of double-strand breaks (DSBs) that usually occur in open chromatin with active transcriptional features in many eukaryotes. However, gene transcription at DSB sites appears to be detrimental for repair, but the regulatory mechanisms governing transcription at meiotic DSB sites are largely undefined in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3 to 9 homologs SUVH2 and SUVH9. N-SIM (structured illumination microscopy) observation shows that the colocalization of SUVH2 with the meiotic DSB marker γ-H2AX is dependent on POL2A. RNA-seq of male meiocytes demonstrates that POL2A and SUVH2 jointly repress the expression of 865 genes, which have several known characteristics associated with meiotic DSB sites. Bisulfite-seq and small RNA-seq of male meiocytes support the idea that the silencing of these genes by POL2A and SUVH2/9 is likely independent of CHH methylation or 24-nt siRNA accumulation. Moreover,pol2a suvh2 suvh9triple mutants have more severe defects in meiotic recombination and fertility compared with eitherpol2aorsuvh2 suvh9. Our results not only identify a epigenetic regulatory mechanism for gene silencing in male meiocytes but also reveal roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis.

Meiosis is a specialized cell division that generates gametes and is essential for sexual reproduction. Studying meiosis in plants, like the model flowering plant Arabidopsis thaliana, contributes to our understanding of the fundamental biology of reproductive biology and has practical implications for improving economically important crop species. In this chapter, we provide a detailed protocol for capillary collection of Arabidopsis male meiocytes followed by total RNA extraction, RNA-Seq, and bioinformatics analysis of small-RNAs (sRNAs) including analysis of sRNA cluster that correlate with genomic features.

During meiosis, crossovers (COs) are typically required to ensure faithful chromosomal segregation. Despite the requirement for at least one CO between each pair of chromosomes, closely spaced double COs are usually underrepresented due to a phenomenon called CO interference. Like Mus musculus and Saccharomyces cerevisiae, Arabidopsis thaliana has both interference-sensitive (Class I) and interference-insensitive (Class II) COs. However, the underlying mechanism controlling CO distribution remains largely elusive. Both AtMUS81 and AtFANCD2 promote the formation of Class II CO. Using both AtHEI10 and AtMLH1 immunostaining, two markers of Class I COs, we show that AtFANCD2 but not AtMUS81 is required for normal Class I CO distribution among chromosomes. Depleting AtFANCD2 leads to a CO distribution pattern that is intermediate between that of wild-type and a Poisson distribution. Moreover, in Atfancm, Atfigl1, and Atrmi1 mutants where increased Class II CO frequency has been reported previously, we observe Class I CO distribution patterns that are strikingly similar to Atfancd2. Surprisingly, we found that AtFANCD2 plays opposite roles in regulating CO frequency in Atfancm compared with either in Atfigl1 or Atrmi1. Together, these results reveal that although AtFANCD2, AtFANCM, AtFIGL1, and AtRMI1 regulate Class II CO frequency by distinct mechanisms, they have similar roles in controlling the distribution of Class I COs among chromosomes.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate the genetic dissection of mechanisms that control meiotic recombination.

Summary Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase‐promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin‐ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis.We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis.Meiocytes from apc8‐1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8‐1, including abnormal sister chromatid segregation and microtubule morphology.Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C‐mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.

Meiotic recombination ensures accurate chromosome segregation and results in genetic diversity in sexually reproducing eukaryotes. Over the last few decades, the genetic regulation of meiotic recombination has been extensively studied in many organisms. However, the role of endogenous meiocyte-specific small RNAs (ms-sRNAs; 21-24 nucleotide [nt]) and their involvement in meiotic recombination are unclear. Here, we sequenced the total small RNA (sRNA) and messenger RNA populations from meiocytes and leaves of wild type Arabidopsis (Arabidopsis thaliana) and meiocytes of spo11-1, a mutant defective in double-strand break formation, and we discovered 2,409 ms-sRNA clusters, 1,660 of which areSPORULATION 11-1 (AtSPO11-1)-dependent. Unlike mitotic small interfering RNAs that are enriched in intergenic regions and associated with gene silencing, ms-sRNAs are significantly enriched in genic regions and exhibit a positive correlation with genes that are preferentially expressed in meiocytes (i.e. Arabidopsis SKP1-LIKE1 and RAD51), in a fashion unrelated to DNA methylation. We also found that AtSPO11-1-dependent sRNAs have distinct characteristics compared with ms-sRNAs and tend to be associated with two known types of meiotic recombination hotspot motifs (i.e. CTT-repeat and A-rich motifs). These results reveal different meiotic and mitotic sRNA landscapes and provide new insights into how sRNAs relate to gene expression in meiocytes and meiotic recombination.

Meiotic recombination contributes to the maintenance of the association between homologous chromosomes (homologs) and ensures the accurate segregation of homologs during anaphase I, thus facilitating the redistribution of alleles among progeny. Meiotic recombination is initiated by the programmed formation of DNA double strand breaks, the repair of which requires DNA synthesis, but the role of DNA synthesis proteins during meiosis is largely unknown. Here, we hypothesized that the lagging strand-specific DNA Polymerase δ (POL δ) might be required for meiotic recombination, based on a previous analysis of DNA Replication Factor1 that suggested a role for lagging strand synthesis in meiotic recombination. In Arabidopsis (Arabidopsis thaliana), complete mutation of the catalytic subunit of POL δ, encoded by AtPOLD1, leads to embryo lethality. Therefore, we used a meiocyte-specific knockdown strategy to test this hypothesis. Reduced expression of AtPOLD1 in meiocytes caused decreased fertility and meiotic defects, including incomplete synapsis, the formation of multivalents, chromosome fragmentation, and improper segregation. Analysis of meiotic crossover (CO) frequencies showed that AtPOLD1(RNAi) plants had significantly fewer interference-sensitive COs than the wild type, indicating that AtPOL δ participates in type I CO formation. AtPOLD1(RNAi) atpol2a double mutant meiocytes displayed more severe meiotic phenotypes than those of either single mutant, suggesting that the function of AtPOLD1 and AtPOL2A is not identical in meiotic recombination. Given that POL δ is highly conserved among eukaryotes, we hypothesize that the described role of POL δ here in meiotic recombination likely exists widely in eukaryotes.

Cohesin, a multisubunit protein complex, is required for holding sister chromatids together during mitosis and meiosis. The recruitment of cohesin by the sister chromatid cohesion 2/4 (SCC2/4) complex has been extensively studied in Saccharomyces cerevisiae mitosis, but its role in mitosis and meiosis remains poorly understood in multicellular organisms, because complete loss-of-function of either gene causes embryonic lethality. Here, we identified a weak allele of Atscc2 (Atscc2-5) that has only minor defects in vegetative development but exhibits a significant reduction in fertility. Cytological analyses of Atscc2-5 reveal multiple meiotic phenotypes including defects in chromosomal axis formation, meiosis-specific cohesin loading, homolog pairing and synapsis, and AtSPO11-1-dependent double strand break repair. Surprisingly, even though AtSCC2 interacts with AtSCC4 in vitro and in vivo, meiosis-specific knockdown of AtSCC4 expression does not cause any meiotic defect, suggesting that the SCC2-SCC4 complex has divergent roles in mitosis and meiosis. SCC2 homologs from land plants have a unique plant homeodomain (PHD) motif not found in other species. We show that the AtSCC2 PHD domain can bind to the N terminus of histones and is required for meiosis but not mitosis. Taken together, our results provide evidence that unlike SCC2 in other organisms, SCC2 requires a functional PHD domain during meiosis in land plants., Author summary Cohesin is required to hold sister chromatids together during mitosis and meiosis. The recruitment of cohesin, mediated by the sister chromatid cohesion 2/4 (SCC2/4) complex, has been extensively studied in yeast mitosis. Because complete loss-of-function of either gene causes embryonic lethality in multicellular organisms, its role in mitosis and meiosis remains poorly understood. Here, we show that Arabidopsis SCC2 functions in meiosis in an AtSCC4-independent manner. We also demonstrate that SCC2 in land plants has a PHD domain not found in animal or fungal homologs and is critical for meiotic function but not mitosis.

Meiosis halves diploid chromosome numbers to haploid levels that are essential for sexual reproduction in most eukaryotes. Meiotic recombination ensures the formation of bivalents between homologous chromosomes (homologs) and their subsequent proper segregation. It also results in genetic diversity among progeny that influences evolutionary responses to selection. Moreover, crop breeding depends upon the action of meiotic recombination to rearrange elite traits between parental chromosomes. An understanding of the molecular mechanisms that drive meiotic recombination is important for both fundamental research and practical applications. This review emphasizes advances made during the past 5 years, primarily in Arabidopsis and rice, by summarizing newly characterized genes and proteins and examining the regulatory mechanisms that modulate their action.

Meiosis is a critical process for sexual reproduction. During meiosis, genetic information on homologous chromosomes is shuffled through meiotic recombination to produce gametes with novel allelic combinations. Meiosis and recombination are orchestrated by several mechanisms including regulation by small RNAs (sRNAs). Our previous work in Arabidopsis (Arabidopsis thaliana) meiocytes showed that meiocyte-specific sRNAs (ms-sRNAs) have distinct characteristics, including positive association with the coding region of genes that are transcriptionally upregulated during meiosis. Here, we characterized the ms-sRNAs in two important crops, soybean (Glycine max) and cucumber (Cucumis sativus). Ms-sRNAs in soybean have the same features as those in Arabidopsis, suggesting that they may play a conserved role in eudicots. We also investigated the profiles of microRNAs (miRNAs) and phased secondary small interfering RNAs in the meiocytes of all three species. Two conserved miRNAs, miR390 and miR167, are highly abundant in the meiocytes of all three species. In addition, we identified three novel cucumber miRNAs. Intriguingly, our data show that the previously identified phased secondary small interfering RNA pathway involving soybean-specific miR4392 is more abundant in meiocytes. These results showcase the conservation and divergence of ms-sRNAs in flowering plants, and broaden our understanding of sRNA function in crop species.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate genetic dissection of mechanisms that control meiotic recombination.

Ribosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo nonallelic exchanges. During meiosis, DNA double-strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR), which subsequently ensures genetic exchange and chromosome disjunction. In Arabidopsis (Arabidopsis thaliana), we demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications and meiotic cohesin and allows only very limited meiosis-specific DSB formation. DNA lesions within the rDNA arrays are repaired in an RAD51-independent but LIG4-dependent manner, establishing that nonhomologous end-joining maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats, we validate our findings and demonstrate that the rDNA constitutes an HR-refractory genome environment.

Ribosomal RNA genes are arranged in large arrays with hundreds of rDNA units in tandem. These highly repetitive DNA elements pose a risk to genome stability since they can undergo non-allelic exchanges. During meiosis DNA double strand breaks (DSBs) are induced as part of the regular program to generate gametes. Meiotic DSBs initiate homologous recombination (HR) which subsequently ensures genetic exchange and chromosome disjunction.InArabidopsis thalianawe demonstrate that all 45S rDNA arrays become transcriptionally active and are recruited into the nucleolus early in meiosis. This shields the rDNA from acquiring canonical meiotic chromatin modifications, meiotic cohesin and meiosis-specific DSBs. DNA breaks within the rDNA arrays are repaired in a RAD51-independent, but LIG4-dependent manner, establishing that it is non-homologous end joining (NHEJ) that maintains rDNA integrity during meiosis. Utilizing ectopically integrated rDNA repeats we validate our findings and demonstrate that the rDNA constitutes a HR-refractory genome environment.

Author(s): Barsh, Gregory S; Bhalla, Needhi; Cole, Francesca; Copenhaver, Gregory P; Lacefield, Soni; Libuda, Diana E

During meiosis, reciprocal exchange between homologous chromosomes occurs as a result of crossovers (COs). CO frequency varies within genomes and is subject to genetic, epigenetic and environmental control. As robust measurement of COs is limited by their low numbers, typically 1-2 per chromosome, we adapted flow cytometry for use with Arabidopsis transgenic fluorescent protein-tagged lines (FTLs) that express eCFP, dsRed or eYFP fluorescent proteins in pollen. Segregation of genetically linked transgenes encoding fluorescent proteins of distinct colors can be used to detect COs. The fluorescence of up to 80,000 pollen grains per individual plant can be measured in 10-15 min using this protocol. A key element of CO control is interference, which inhibits closely spaced COs. We describe a three-color assay for the measurement of CO frequency in adjacent intervals and calculation of CO interference. We show that this protocol can be used to detect changes in CO frequency and interference in the fancm zip4 double mutant. By enabling high-throughput measurement of CO frequency and interference, these methods will facilitate genetic dissection of meiotic recombination control.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres., National Institutes of Health (U.S.) (Grant GM088313)

Meiotic recombination ensures the fertility of gametes and creates novel genetic combinations. Although meiotic crossover (CO) frequency is under homeostatic control, CO frequency is also plastic in nature and can respond to environmental conditions. Most investigations have focused on temperature and recombination, but other external and internal stimuli also have important roles in modulating CO frequency. Even less is understood about the molecular mechanisms that underly these phenomenon, but recent work has begun to advance our knowledge in this field. In this review, we identify and explore potential mechanisms including changes in: the synaptonemal complex, chromatin state, DNA methylation, and RNA splicing.

PRDM9 directs human meiotic crossover hot spots to intergenic sequence motifs, whereas budding yeast hot spots overlap regions of low nucleosome density (LND) in gene promoters. To investigate hot spots in plants, which lack PRDM9, we used coalescent analysis of genetic variation in Arabidopsis thaliana. Crossovers increased toward gene promoters and terminators, and hot spots were associated with active chromatin modifications, including H2A.Z, histone H3 Lys4 trimethylation (H3K4me3), LND and low DNA methylation. Hot spot-enriched A-rich and CTT-repeat DNA motifs occurred upstream and downstream, respectively, of transcriptional start sites. Crossovers were asymmetric around promoters and were most frequent over CTT-repeat motifs and H2A.Z nucleosomes. Pollen typing, segregation and cytogenetic analysis showed decreased numbers of crossovers in the arp6 H2A.Z deposition mutant at multiple scales. During meiosis, H2A.Z forms overlapping chromosomal foci with the DMC1 and RAD51 recombinases. As arp6 reduced the number of DMC1 or RAD51 foci, H2A.Z may promote the formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hot spots within eukaryotes and PRDM9 is a derived state within vertebrates. © 2013 Nature America, Inc. All rights reserved.

"Tell me, what is it you plan to do with your one wild and precious life?" The question that concludes Mary Oliver's poem, The Summer Day, reminds us not only of a poet inspired by her observations of the natural world but also of Jane Gitschier, a scientist, author, musician, and mother whose relationship with and observations of those around her have contributed so much to our community. This editorial is intended to commemorate and celebrate Jane's series of more than 40 interviews published by PLOS Genetics, spanning ten years of publishing, about a billion years of evolution—from Archea to Brassica, from prions to mammals—and a set of themes and ideas that make the word "eclectic" seem puny by comparison. To us, Jane is the Terry Gross of science writing (or, for the younger, more podcast-oriented crowd, Marc Maron), striking a perfect balance in both tone and subject matter. Her conversations are convivial but erudite, illuminating her subject's personality as well as their societal and scientific contributions. Who knew that Mark Ptashne used to listen to Paul Robeson sing at his house [1], that David Botstein used to conduct the Harvard glee club [2], or that Elaine Strass used to be a concert pianist [3]? Reading through Jane's interviews is like being a fly on the wall for a scientific century of genetic ideas and opinions, from the role of lateral gene transfer in evolution (Ford Doolittle [4]) to a "neo-Lamarckian view of biology," which is how Jane describes the impact of Susan Lindquist's work on rapid evolutionary change driven by chaperones and remodeling proteins [5]. Technology also features prominently in Jane's interviews—the nitty-gritty details of the first nitrocellulose blots and the first microarrays are tremendously fun to read in the words of Sir Edwin Southern [6] and Pat Brown [7]. But one of the things we enjoy most about Jane's interviews is that they're not confined to working scientists. Authors (Nicholas Wade [8] and James Schwartz [9]), architects (Rafael Vinoly [10]), CEOs (Anne Wojcicki [11]), and jurists (Judge John E Jones, III [12]) contribute to and, more importantly, expand, the relationship of the genetics community to the rest of the world. Jane explores it all in a way that makes us wish—or perhaps more accurately, feel like—we were there with her during the interviews. In popular culture terms, Jane is someone with whom we would all like to drink a beer (or glass of wine). Correspondence from Jane often includes Mary Oliver's "wild and precious life" quote in the signature, which may help to explain why Jane's own life has been and continues to be as eclectic as her choice of subjects. Jane's fascination with human genetics and Mendelian disease began during her postdoc at Genentech with Dick Lawn and colleagues and catalyzed her career as a faculty member at the University of California, San Francisco (UCSF) at a time when there was not even a Department of Genetics, let alone Human Genetics (there still isn't, but that's another story). A PubMed search for Gitschier retrieves scientific contributions that read like the table of contents for a human genetics textbook (hemophilia, Menkes' disease, diabetes insipidus, pantothenate kinase–associated neurodegeneration), along with five papers on the genetics of absolute pitch. Indeed, music and musicality feature prominently not only in Jane's interviews and her science but also in her life. Now an emeritus faculty member at UCSF, Jane's current projects include music composition, publishing a book (of her interviews), and going to architecture school. Wild and precious indeed. Recordings of Jane's interviews have been archived for future use by the American Philosophical Society, which should make for some interesting listening 100 years from now. In addition, all the written interviews will remain as a collection at PLOS, open and freely available to all. In 2010, PLOS Genetics published an interview of Jane with herself [13] that concludes with the following: Gitschier: OK, last question. Do you have a favorite interview? Gitschier: …The truth is that each person's story is remarkable in its own way. I can hear all of their voices. I am so grateful to these people who put their trust in me. This project has been one of the most rewarding experiences of my life. Ours too! Precious, discerning, illuminating, and rewarding.

Whole-genome duplication through the formation of diploid gametes is a major route for polyploidization, speciation, and diversification in plants. The prevalence of polyploids in adverse climates led us to hypothesize that abiotic stress conditions can induce or stimulate diploid gamete production. In this study, we show that short periods of cold stress induce the production of diploid and polyploid pollen in Arabidopsis (Arabidopsis thaliana). Using a combination of cytological and genetic analyses, we demonstrate that cold stress alters the formation of radial microtubule arrays at telophase II and consequently leads to defects in postmeiotic cytokinesis and cell wall formation. As a result, cold-stressed male meiosis generates triads, dyads, and monads that contain binuclear and polynuclear microspores. Fusion of nuclei in binuclear and polynuclear microspores occurs spontaneously before pollen mitosis I and eventually leads to the formation of diploid and polyploid pollen grains. Using segregation analyses, we also found that the majority of cold-induced dyads and triads are genetically equivalent to a second division restitution and produce diploid gametes that are highly homozygous. In a broader perspective, these findings offer insights into the fundamental mechanisms that regulate male gametogenesis in plants and demonstrate that their sensitivity to environmental stress has evolutionary significance and agronomic relevance in terms of polyploidization.

Cell division requires proper spindle assembly; a surveillance pathway, the spindle assembly checkpoint (SAC), monitors whether the spindle is normal and correctly attached to kinetochores. The SAC proteins regulate mitotic chromosome segregation by affecting CDC20 (Cell Division Cycle 20) function. However, it is unclear whether CDC20 regulates meiotic spindle assembly and proper homolog segregation. Here, we show that the Arabidopsis thaliana CDC20.1 gene is indispensable for meiosis and male fertility. We demonstrate that cdc20.1 meiotic chromosomes align asynchronously and segregate unequally and the metaphase I spindle has aberrant morphology. Comparison of the distribution of meiotic stages at different time points between the wild type and cdc20.1 reveals a delay of meiotic progression from diakinesis to anaphase I. Furthermore, cdc20.1 meiocytes exhibit an abnormal distribution of a histone H3 phosphorylation mark mediated by the Aurora kinase, providing evidence that CDC20.1 regulates Aurora localization for meiotic chromosome segregation. Further evidence that CDC20.1 and Aurora are functionally related was provided by meiosis-specific knockdown of At- Aurora1 expression, resulting in meiotic chromosome segregation defects similar to those of cdc20.1 . Taken together, these results suggest a critical role for CDC20.1 in SAC-dependent meiotic chromosome segregation.

About a year ago, PLOS implemented a new process intended to further the overarching principle that data used in the work we publish should be accessible and reusable. The motivation goes hand-in-hand with both our open access ethos and the scientific method itself: the validity of a conclusion depends on the ability to reproduce the underlying results. In theory, PLOS’ new data policy, in which “all data underlying the findings described [must be] fully available,” is not so new; in practice, though, it may be perceived as burdensome, complicated, and/or inefficient. The policy and its purpose has been discussed extensively among PLOS Genetics Editorial Board members with regard to its potential impact on editors, authors, the community, and research subjects. The purpose of this editorial is 2-fold: to acknowledge and discuss aspects of the “data-sharing” process that are especially challenging, and to provide additional clarification and guidance in the context of a few scenarios that are especially relevant, all from the perspective of working scientists who read, evaluate, and contribute to research based on genetics and genomics. We also suggest a way forward that builds on what already works at PLOS: consulting with multiple stakeholders whose interests intersect to develop consensus. An issue that is often problematic for PLOS Genetics authors is the sheer volume of data generated by large-scale phenotyping and/or genotyping studies. Whether from a confocal microscope, a radiofrequency detector in an MRI, or a CCD in a DNA sequencing instrument, processing, filtering, and compression of digital data is inherent across many areas of modern biology. It is often neither practical nor wise to archive and distribute the primary output of digital detectors; indeed, the question of what constitutes “raw data” is a moving target. A second problematic issue arises from ethical concerns associated with human research subjects. The potential to identify research subjects based on genomic information has received considerable attention and has fostered the development of controlled access mechanisms, in which researchers must seek approval from data access committees. As with any new set of regulations, there is a risk of creating more problems than are solved; from an editorial perspective, we recognize that authors must commit significant resources to deposit data into controlled access repositories, and that the process of extracting data can also be problematic. Regulatory burdens also create conflicts since the legitimate concerns of human subject review boards and governmental agencies to protect privacy are not necessarily prima facie aligned with open access policy. In short, the system is not working efficiently. Finally, for research that is not publicly funded, there can be legitimate reasons to restrict data sharing. Personal genomics companies, agricultural genetics corporations, and domestic animal breeders all support worthwhile research, but may have responsibilities to shareholders and/or contributors that restrict the ability to fully share the underlying data. In addition, there are efforts in some countries to protect data and genetic resources as part of a national interest. While we acknowledge and understand the underlying rationale for governmental and/or commercial restrictions on data sharing, these represent an inherent conflict with the principles of open access.

For most eukaryotes, sexual reproduction is a fundamental process that requires meiosis. In turn, meiosis typically depends on a reciprocal exchange of DNA between each pair of homologous chromosomes, known as a crossover (CO), to ensure proper chromosome segregation. The frequency and distribution of COs are regulated by intrinsic and extrinsic environmental factors, but much more is known about the molecular mechanisms governing the former compared to the latter. Here we show that elevated temperature induces meiotic hyper-recombination in Arabidopsis thaliana and we use genetic analysis with mutants in different recombination pathways to demonstrate that the extra COs are derived from the major Type I interference sensitive pathway. We also show that heat-induced COs are not the result of an increase in DNA double-strand breaks and that the hyper-recombinant phenotype is likely specific to thermal stress rather than a more generalized stress response. Taken together, these findings provide initial mechanistic insight into how environmental cues modulate plant meiotic recombination and may also offer practical applications., Author summary Meiosis is the cell division used by sexually reproducing species to produce sperm and egg cells. During meiosis, programmed Double Strand Breaks (DSBs) occur on each chromosome, which allows DNA to be exchanged between chromosome pairs, resulting in crossovers (COs). COs are necessary to ensure faithful chromosome segregation during meiosis, and thus fertility, but are also an important source of genetic variation. As such, CO formation is tightly regulated. Despite this, CO frequency can be altered by external factors, such as temperature. In Arabidopsis thaliana, COs are formed through two pathways: interference-sensitive (Type I) and interference-insensitive (Type II). An increase in temperature results in an increase in CO frequency. Using a pollen based assay, we show that COs are formed in the Type I pathway, which accounts for approximately 85% of the COs in Arabidopsis. To investigate whether temperature-dependent COs are the result of additional DSBs, we used immunological staining to examine protein foci, which mark the sites of DSBs. We discovered that temperature likely increases CO frequency by shifting alternative repair outcomes, called non-crossovers, to favor additional COs, rather than by increasing DSBs. Lastly, we found that temperature is not a general stress response, as plants subject to salt stress did not exhibit an increase in CO frequency. Our results may prove valuable in aiding plant breeding by enhancing our ability to rapidly introgress suites of elite traits from wild-plants into their crop relatives, a method that is particularly attractive as it does not require genetic modifications.

During meiosis, chromatin undergoes extensive changes to facilitate recombination, homolog pairing, and chromosome segregation. To investigate the relationship between chromatin organization and meiotic processes, we used formaldehyde-assisted isolation of regulatory elements (FAIRE) to map open chromatin during the transition from mitosis to meiosis in the budding yeast Saccharomyces cerevisiae. We found that meiosis-induced opening of chromatin is associated with meiotic DSB hotpots. The positive association between open chromatin and DSB hotspots is most prominent 3 h into meiosis, when the early meiotic genes DMC1 and HOP1 exhibit maximum transcription and the early recombination genes SPO11 and RAD51 are strongly up-regulated. While the degree of chromatin openness is positively associated with the occurrence of recombination hotspots, many hotspots occur outside of open chromatin. Of particular interest, many DSB hotspots that fell outside of meiotic open chromatin nonetheless occurred in chromatin that had recently been open during mitotic growth. Finally, we find evidence for meiosis-specific opening of chromatin at the regions adjacent to boundaries of subtelomeric sequences, which exhibit specific crossover control patterns hypothesized to be regulated by chromatin.

During angiosperm reproduction, one of the two synergid cells within the female gametophyte undergoes cell death prior to fertilization. The pollen tube enters the female gametophyte by growing into the synergid cell that undergoes cell death and releases its two sperm cells within the degenerating synergid cytoplasm to effect double fertilization. In Arabidopsis (Arabidopsis thaliana) and many other species, synergid cell death is dependent upon pollination. However, the mechanism by which the pollen tube causes synergid cell death is not understood. As a first step toward understanding this mechanism, we defined the temporal relationship between pollen tube arrival at the female gametophyte and synergid cell death in Arabidopsis. Using confocal laser scanning microscopy, light microscopy, transmission electron microscopy, and real-time observation of these two events in vitro, we demonstrate that synergid cell death initiates after the pollen tube arrives at the female gametophyte but before pollen tube discharge. Our results support a model in which a signaling cascade triggered by pollen tube-synergid cell contact induces synergid cell death in Arabidopsis.