Imprinted gene expression occurs during seed development in plants and is associated with differential DNA methylation of parental alleles, particularly at proximal transposable elements (TEs). Imprinting variability could contribute to observed parent-of-origin effects on seed development. We investigated intraspecific variation in imprinting, coupled with analysis of DNA methylation and small RNAs, among three Arabidopsis strains with diverse seed phenotypes. The majority of imprinted genes were parentally biased in the same manner among all strains. However, we identified several examples of allele-specific imprinting correlated with intraspecific epigenetic variation at a TE. We successfully predicted imprinting in additional strains based on methylation variability. We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation. Our data demonstrate that epiallelic variation and genomic imprinting intersect to produce novel gene expression patterns in seeds.DOI: http://dx.doi.org/10.7554/eLife.03198.001

The majority (∼70%) of surface buried in protein folding is hydrocarbon, whereas in DNA helix formation, the majority (∼65%) of surface buried is relatively polar nitrogen and oxygen. Our previous quantification of salt exclusion from hydrocarbon (C) accessible surface area (ASA) and accumulation at amide nitrogen (N) and oxygen (O) ASA leads to a prediction of very different Hofmeister effects on processes that bury mostly polar (N, O) surface compared to the range of effects commonly observed for processes that bury mainly nonpolar (C) surface, e.g., micelle formation and protein folding. Here we quantify the effects of salts on folding of the monomeric DNA binding domain (DBD) of lac repressor (lac DBD) and on formation of an oligomeric DNA duplex. In accord with this prediction, no salt investigated has a stabilizing Hofmeister effect on DNA helix formation. Our ASA-based analyses of model compound data and estimates of the surface area buried in protein folding and DNA helix formation allow us to predict Hofmeister effects on these processes. We observe semiquantitative to quantitative agreement between these predictions and the experimental values, obtained from a novel separation of coulombic and Hofmeister effects. Possible explanations of deviations, including salt-dependent unfolded ensembles and interactions with other types of surface, are discussed.Hofmeister salts | m-values | thermodynamics S alts typically exert both specific (Hofmeister) and nonspecific (coulombic) effects on biomolecular processes (1-6). To manipulate and probe biopolymer processes using salts, it is extremely important to develop quantitative methods to interpret and predict these effects in terms of structure. coulombic, valencespecific effects of salt ions (due to screening of surface charges) are most significant at relatively low salt concentrations (<0.1 M). At higher concentrations (>0.1 M), ion-specific effects and relatively nonspecific osmotic effects (due to the lowering of water activity) become increasingly significant. In 1888, Franz Hofmeister discovered that the effectiveness of salts for protein precipitation generally followed a specific order, regardless of the protein being investigated (7). Since then, the so-called Hofmeister series of salt effects has been observed in physical properties of aqueous salt solutions (e.g., surface tension and surface potential) (8, 9), as well as salt effects on a variety of macromolecular processes (e.g., micelle formation, "salting out" nonpolar compounds, and protein folding) (10-13). The general ranking of ions, in decreasing order of effectiveness (best to worst) in driving processes where surface area is buried (e.g., folding and precipitation) or macroscopic surface is lost (transfer of water from the air-water interface to bulk), is as follows (14):Although it is generally accepted that interactions of salts with hydrocarbon surface are unfavorable and salt-specific, following the above order (1,3,11,(14)(15)(16), less is known about the interactions of Hofmeister sa...

RNA-directed DNA methylation (RdDM) is a biological process in which non-coding RNA molecules direct the addition of DNA methylation to specific DNA sequences. The RdDM pathway is unique to plants, although other mechanisms of RNA-directed chromatin modification have also been described in fungi and animals. To date, the RdDM pathway is best characterized within angiosperms (flowering plants), and particularly within the model plant Arabidopsis thaliana. However, conserved RdDM pathway components and associated small RNAs (sRNAs) have also been found in other groups of plants, such as gymnosperms and ferns. The RdDM pathway closely resembles other sRNA pathways, particularly the highly conserved RNAi pathway found in fungi, plants, and animals. Both the RdDM and RNAi pathways produce sRNAs and involve conserved Argonaute, Dicer and RNAdependent RNA polymerase proteins. RdDM has been implicated in a number of regulatory processes in plants. The DNA methylation added by RdDM is generally associated with transcriptional repression of the genetic sequences targeted by the pathway. Since DNA methylation patterns in plants are heritable, these changes can often be stably transmitted to progeny. As a result, one prominent role of RdDM is the stable, transgenerational suppression of transposable element (TE) activity. RdDM has also been linked to pathogen defense, abiotic stress responses, and the regulation of several key developmental transitions. Although the RdDM pathway has a number of important functions, RdDM-defective mutants in Arabidopsis thaliana are viable and can reproduce, which has enabled detailed genetic studies of the pathway. However, RdDM mutants can have a range of defects in different plant species, including lethality, altered reproductive phenotypes, TE upregulation and genome instability, and increased pathogen sensitivity. Overall, RdDM is an important pathway in plants that regulates a number of processes by establishing and reinforcing specific DNA methylation patterns, which can lead to transgenerational epigenetic effects on gene expression and phenotype. Biological functions of RdDM RdDM is involved in a number of biological processes in the plant, including stress responses, cell-to-cell communication, and the maintenance of genome stability through TE silencing. An overview of some of the biological functions performed by RdDM is shown in Fig 1.

Balance between maternal and paternal genomes within the triploid endosperm is necessary for normal seed development. The majority of endosperm genes are expressed in a 2:1 maternal:paternal ratio, reflecting genomic DNA content. Here, we find that the 2:1 transcriptional ratio is, unexpectedly, actively regulated. In A. thaliana and A. lyrata, endosperm 24-nt small RNAs are reduced in transposable elements and enriched in genes compared with the embryo. We find an inverse relationship between the parent of origin of sRNAs and mRNAs, with genes more likely to be associated with maternally than paternally biased sRNAs. Disruption of the Pol IV sRNA pathway causes a shift toward maternal allele mRNA expression for many genes. Furthermore, paternal inheritance of an RNA Pol IV mutation is sufficient to rescue seed abortion caused by excess paternal genome dosage. Thus, RNA Pol IV mediates the transcriptional balance between maternally and paternally inherited genomes in endosperm.

SUMMARYMIKC-type MADS domain proteins are key regulators of flower development in angiosperms. B sister genes constitute a clade with a close relationship to class B floral homeotic genes, and have been conserved for more than 300 million years. The loss-of-function phenotype of the A. thaliana B sister gene ABS is mild: mutants show reduced seed coloration and defects in endothelium development. This study focuses on GORDITA (GOA, formerly known as AGL63), the most closely related paralog of ABS in A. thaliana, which is thought to act redundantly with ABS. Phylogenetic trees reveal that the duplication leading to ABS and GOA occurred during diversification of the Brassicaceae, and further analyses show that GOA has evolved under relaxed selection pressure. The knockdown phenotype of GOA suggests a role for this gene in fruit longitudinal growth, while over-expression of GOA results in disorganized floral structure and addition of carpel-like features to sepals. Given the phylogeny and function of other B sister genes, our data suggest that GOA has evolved a new function as compared to ABS. Protein analysis reveals that the GOA-specific 'deviant' domain is required for protein dimerization, in contrast to other MIKC-type proteins that require the K domain for dimerization. Moreover, no shared protein interaction partners for ABS and GOA could be identified. Our experiments indicate that modification of a protein domain and a shift in expression pattern can lead to a novel gene function in a relatively short time, and highlight the molecular mechanism by which neofunctionalization following gene duplication can be achieved.

BackgroundThe floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity and is involved in the regulation of floral meristem termination in Arabidopsis. Arabidopsis ag mutants show complete homeotic conversions of stamens into petals and carpels into sepals as well as indeterminacy of the floral meristem. Gene function analysis in model core eudicots and the monocots rice and maize suggest a conserved function for AG homologs in angiosperms. At the same time gene phylogenies reveal a complex history of gene duplications and repeated subfunctionalization of paralogs.ResultsEScaAG1 and EScaAG2, duplicate AG homologs in the basal eudicot Eschscholzia californica show a high degree of similarity in sequence and expression, although EScaAG2 expression is lower than EScaAG1 expression. Functional studies employing virus-induced gene silencing (VIGS) demonstrate that knock down of EScaAG1 and 2 function leads to homeotic conversion of stamens into petaloid structures and defects in floral meristem termination. However, carpels are transformed into petaloid organs rather than sepaloid structures. We also show that a reduction of EScaAG1 and EScaAG2 expression leads to significantly increased expression of a subset of floral homeotic B genes.ConclusionsThis work presents expression and functional analysis of the two basal eudicot AG homologs. The reduction of EScaAG1 and 2 functions results in the change of stamen to petal identity and a transformation of the central whorl organ identity from carpel into petal identity. Petal identity requires the presence of the floral homeotic B function and our results show that the expression of a subset of B function genes extends into the central whorl when the C function is reduced. We propose a model for the evolution of B function regulation by C function suggesting that the mode of B function gene regulation found in Eschscholzia is ancestral and the C-independent regulation as found in Arabidopsis is evolutionarily derived.

5-Hydroxymethylcytosine (5-hmC) is an intermediate in active demethylation in metazoans, as well as a potentially stable epigenetic mark. Previous reports investigating 5-hydroxymethylcytosine in plants have reached conflicting conclusions. We systematically investigated whether 5-hmC is present in plant DNA using a range of methods. Using the model organism Arabidopsis thaliana, in addition to other plant species, we assayed the amount or distribution of 5-hydroxymethylcytosine by thin-layer chromatography, immunoprecipitation-chip, ELISA, enzymatic radiolabeling, and mass spectrometry. The failure to observe 5-hydroxymethylcytosine by thin-layer chromatography established an upper bound for the possible fraction of the nucleotide in plant DNA. Antibody-based methods suggested that there were low levels of 5-hmC in plant DNA, but these experiments were potentially confounded by cross-reactivity with the abundant base 5-methylcytosine. Enzymatic radiolabeling and mass spectrometry, the most sensitive methods for detection that we used, failed to detect 5-hydroxymethylcytosine in A. thaliana genomic DNA isolated from a number of different tissue types and genetic backgrounds. Taken together, our results led us to conclude that 5-hmC is not present in biologically relevant quantities within plant genomic DNA.

Background: Local and national initiatives to improve the learning experiences of students enrolled in Science, Technology, Engineering, and Mathematics (STEM) courses have been ongoing for a couple of decades with a heightened momentum within the last 10 years. However, recent large-scale studies have demonstrated that transmission of information is still the primary mode of instruction in STEM courses across the undergraduate curriculum. The limited impact of instructional change reform efforts can be partly explained by the one-sided focus of educational research on the development of evidence-based instructional practices and production of evidence demonstrating their impact on student learning. This has been done at the expense of understanding faculty members' instructional practices and mindsets about teaching and learning that underlie their practices. This study addresses this gap in the literature by characterizing STEM instructors' instructional intentions and reflections on their teaching performance for a week of instruction. Data was collected through semi-structured interviews with 42 STEM faculty members from one doctorate-granting institution in the USA. Results: STEM instructors in this study had teacher-centric mindsets with respect to their instructional planning (e.g., content-focused learning goals, lecture is seen as an engagement strategy). We found that these instructors mostly saw formative assessment tools as engagement strategy rather than tools to monitor student learning. Reflections on their level of satisfaction with their week of teaching focused heavily on content coverage and personal feelings and minimally considered student learning. Finally, we found that pedagogical discontent was not a driver of planned course revisions. Conclusions: This study identifies mismatches between STEM instructors' teaching mindsets and current approaches to instructional change. STEM instructors in this study paid minimal attention to student learning when considering course-level revisions and many of their reflections were anchored in their personal feelings. However, instructional reform strategies often attempt to convince faculty of a new approach by demonstrating its impact on student learning. The misalignment identified in this study further highlights the need to better characterize STEM instructors' cognition around teaching so that reform efforts can better meet them where they are.